Ca_v3.1在盐酸利多卡因致SH-SY5Y细胞损伤中的作用研究
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摘要
随着局部麻醉的广泛应用,局麻药的神经毒性损伤及全身毒性反应已引起人们的广泛关注。特别是局麻药所致的外周神经损伤,给病人生产生活带来明显不良影响。局麻药神经损伤的预防及其治疗成为临床麻醉中的重要课题。
临床上,椎管内麻醉后,部分病人可出现短暂性神经病学综合征(transient neurological syndrome, TNS),表现为烧灼样、持续固定、痉挛性、放射性疼痛的症状。部分患者甚至可发生严重的神经功能永久性损害如马尾综合征。一般认为,局麻药所致的神经损伤与其剂量、浓度及脊神经在局麻药中暴露的时间呈正相关。但对于局麻药诱导的神经损伤机制目前尚不明确,可能与多种因素有关。
细胞内钙超载是局麻药神经损伤的重要机制之一。细胞外Ca2＋可通过细胞膜上的电压依赖性与受体依赖性等Ca2＋通道进入细胞内,激活细胞内钙依赖性酶,诱导细胞凋亡,产生神经损伤。同时,进入细胞内的Ca2＋尚可诱发细胞内钙依赖性钙释放,使细胞内钙库释放大量Ca2＋,进一步加重细胞内钙超载,最终导致细胞凋亡,产生神经损伤。
电压依赖性钙通道分为高电压依赖性钙通道(high voltage activated calcium channel, HAV)和低电压依赖性钙通道(low voltage activated calcium channel, LAV). Cav1和Cav2属于HAV,包括L型,N型,P/Q型和R型。低电压依赖性钙通道即T型钙通道则由Cav3编码,包括三种亚型(Cav3.1,Cav3.2, Cav3.3),其在静息电位下(-65mV～-50mV)即可激活,且具有快速激活,缓慢失活的特点；同时,T型钙通道还具有“窗口电流”,即在T型钙通道稳定激活曲线和稳定失活曲线之间有一相互重叠部分。HVA和LVA在慢性神经病理性疼痛中均有重要作用,与神经损伤有关。低电压依赖性钙通道即T型钙通道,是调节神经元兴奋性和神经递质分泌的重要启动子,也是调节细胞内钙信号的关键因素。目前关于T型钙通道在局麻药神经损伤中的作用尚不清楚。
p38丝裂原活化蛋白激酶(p38mitogen-activated protein kinases,p38MAPK)激活是局麻药神经损伤的另一重要途径,细胞外Ca2＋进入细胞内,激活钙依赖的钙调蛋白酶(Ca2+/calmodulin-dependent protein kinase type Ⅱ, Ca2+/CaMK II),进
‘步激活凋亡信号调节相关激酶1(Apoptosis signal-regulating kinase1,ASK1),诱导p38MAPK的激活,形成Ca2+/CaMKⅡ-ASK1-p38MAPK通路,p38MAPK的激活则可诱导神经细胞发生不依赖于半胱胺酸蛋白酶蛋白(Caspase)的凋亡反应。
本课题采用SH-SY5Y细胞体外培养模型,通过免疫印迹和real-time PCR等方法,鉴定SH-SY5Y细胞表面T型钙通道各亚型的表达,并观察10mM盐酸利多卡因处理24h后,SH-SY5Y细胞表面T型钙通道的表达变化；采用RNAi技术,构建T型钙通道低表达的SH-SY5Y细胞株,并观察抑制SH-SY5Y细胞表面T型钙通道表达后,能否减轻利多卡因对该细胞的损伤作用,并检测p38MAPK的表达,探讨T型钙通道是否通过激活p38MAPK产生细胞毒性损伤作用。
第1章盐酸利多卡因对SH-SY5Y细胞表面Cav3.1表达的影响
实验1T型钙通道在SH-SY5Y细胞上的表达
目的通过免疫印迹和real-time PCR方法检测T型钙通道各亚型(Cav3.1、Cav3.2、Cav3.3) mRNA和蛋白质在SH-SY5Y细胞上的表达,鉴定SH-SY5Y细胞表面T型钙通道表达亚型。
方法SH-SY5Y细胞体外培养,采用免疫印迹技术和real-time PCR技术,检测Cav3.1、Cav3.2和Cav3.3在SH-SY5Y细胞上的表达。将免疫印迹X胶片扫描,采用quantity one图像分析软件分析目标条带的吸光度值,以β-actin条带吸光度值为参照,两者比值为目标蛋白的表达水平。real-time PCR定量的方法以2-ΔΔCt(Ct代表循环阈值)表示基因的表达量,计算公式为ΔΔCt=[Ct(待测组目标基因)-Ct (β-actin)]-[Ct(对照组目标基因)-Ct (β-actin)]。
统计学处理采用SPSS10.0统计学软件进行数据分析,计量资料以均数±标准差(χ-±s)表示,组间比较采用单因素方差分析(one-way ANOVA),多重比较采用LSD法,方差不齐时则分别采用Welch法和Dunnett' s T3法,P<0.05为差异有统计学意义。
结果免疫印迹法检测发现,在SH-SY5Y细胞上T型钙通道的三种亚型Cav3.1、 Cav3.2及Cav3.3蛋白均有表达,但表达量差异有统计学意义(F=777.113,P=0.000). Cav3.1蛋白在SH-SY5Y细胞上表达最丰富,而Cav3.2及Cav3.3蛋白的表达水平比Cav3.1蛋白表达水平降低,尤以Cav3.2的表达量为最少。Cav3.1、 Cav3.2、Cav3.3mRNA在SH-SY5Y细胞上均有表达,但表达量不一,Cav3.1mRNA的表达量最多,Cav3.2表达量则最少(F=194898.000,P=0.000)。
结论T型钙通道三种亚型Cav3.1、Cav3.2、Cav3.3在SH-SY5Y细胞上均有表达,但以Ca,,3.1表达为主。
实验2盐酸利多卡因对SH-SY5Y细胞Cav3.1表达的影响
目的Cav3.1是SH-SY5Y细胞上表达最为丰富的T型钙通道亚型,因此,SH-SY5Y细胞上低电压依赖性钙通道的生理学特征主要由Cav3.1通道体现,本研究的目的是观察SH-SY5Y细胞在盐酸利多卡因处理24h后其表面Cav3.1表达的变化。
方法SH-SY5Y细胞离体培养,分为两组,一组正常培养,另一组细胞则用10mM盐酸利多卡因处理24h后,采用免疫印迹和real-time PCR方法检测Cav3.1的表达变化。将免疫印迹X胶片扫描,采用quantity one图像分析软件分析目标条带的吸光度值,以β-actin条带吸光度值为参照,两者比值为目标蛋白的表达水平。real-time PCR定量的方法以2-ΔΔCt (Ct代表循环阈值)表示基因的表达量,计算公式为ΔΔCt=[Ct(待测组目标基因)-Ct (β-actin)]-[Ct(对照组目标基因)-Ct (β-actin)]。
统计学处理采用SPSS10.0统计学软件进行数据分析,计量资料以均数±标准差(χ-±s)表示,Cav3.1mRNA及蛋白表达采用配对t检验,P<0.05为差异有统计学意义。
结果与对照组比较,10mM盐酸利多卡因处理组Cav3.1蛋白表达量增加,两组比较有统计学意义(t=9.932, P=0.000); Cav3.1mRNA表达量也增加,两组比较有统计学意义(t=12.573,p=0.000)。
结论10mM盐酸利多卡因处理SH-SY5Y细胞24h后,细胞表面Cav3.1蛋白及mRNA表达增加。
第2章pshRNA-CACNA1G-SH-SY5Y细胞株的构建与鉴定
目的T型钙通道目前尚无特异性强的抑制剂,这成为阻碍T型钙通道研究进展的重要原因。随着基因干扰和基因工程技术的发展,靶向干扰目的基因的表达成为当前研究的重要工具,本研究的目的是通过RNAi技术,靶向干扰CACNA1G(Cav3.1基因),构建pshRNA-CACNA1G-SH-SY5Y细胞,以达到抑制Cav3.1基因和蛋白质的表达,为后续研究提供实验模型。
方法根据RNAi设计原则和Genbank上Cav3.1(CACNAIG)基因序列(序列号NM018896),选择3个干扰靶点,同时设计一个阴性序列。选择pSUPERretro-puro为载体,经退火、酶切、连接、转化等步骤,所得产物测序鉴定；同时大提质粒,293FT细胞、慢病毒包装、收集病毒液,并将病毒转染到SH-SY5Y细胞,经免疫印迹和real-time PCR鉴定,构建pshRNA-CACNA1G-SH-SY5Y细胞株。Cav3.1蛋白及基因表达以均数±标准差(x±s)表示,组间比较采用单因素方差分析(one-way ANOVA),多重比较采用LSD法,方差不齐时则分别采用Welch法和Dunnett's T3法,P<0.05为差异有统计学意义。
结果经DNA测序,三个干扰序列和阴性序列均顺利插入pSUPERretro-puro质粒中。免疫印迹和real-time PCR鉴定结果表明,三个干扰序列RNAi1、RNAi2、 RNAi3均能明显抑制pshRNA-CACNA1G-SH-SY5Y细胞上CACNA1G基因(Cav3.1)的表达,尤其以RNAi2转染效率最高,而阴性序列则对Cav3.1表达则无影响。
结论经免疫印迹和real-time PCR鉴定,成功构建pshRNA-CACNA1G-SH-SY5Y(RNAi2)细胞株,为后续实验提供细胞模型。
第3章盐酸利多卡因对pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞的影响
目的通过上述实验,我们建立了Cav3.1低表达的pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞株,为探讨Cav3.1钙通道在盐酸利多卡因致神经细胞损伤中的作用提供实验模型。本实验目的是观察10mM盐酸利多卡因对Cav3.1低表达的pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞的影响,探讨Cav3.1在盐酸利多卡因致神经细胞损伤中的作用。
方法细胞共分为6组,即对照组分别为SH-SY5Y组,pNC-puro-CACNAIG-SH-SY5Y(阴性序列组,NC组),pshRNA-CACNA1G-SH-SY5Y (RNAi2)组(RNAi2组)；实验组则为上述三组分别用终浓度为10mM盐酸利多卡因处理24h,即SH-SY5Y+利多卡因组(SH-SY5Y+Lido组),阴性序列+利多卡因组(NC+Lido组),RNAi2+利多卡因组(RNAi2+Lido组)。药物处理24h后在倒置显微镜下观察细胞形态；MTT法检测细胞存活率；流式细胞仪检测细胞凋亡率和细胞内Ca2+浓度([Ca2+]free);免疫印迹法检测Cav3.1蛋白及Caspase-3蛋白表达；
统计学处理采用SPSS10.0统计学软件进行分析,计量资料以均数±标准差(χ-±s)表示,细胞存活率、细胞凋亡率、Cav3.1蛋白及Caspase-3蛋白表达及[Ca2+]free采用两因素析因设计方差分析,单独效应分析时组间比较采用单因素方差分析(one-way ANOVA),多重比较采用LSD法,方差不齐时则分别采用Welch法和Dunnett's T3法,P<0.05为差异有统计学意义。
结果
(1)细胞形态倒置显微镜下观察各组细胞形态学变化,见图3-1。三个对照组即SH-SY5Y组、NC组、RNAi2组细胞生长情况良好,细胞呈梭型,突起丰富,细胞呈网状生长。实验组分别为SH-SY5Y+Lido组、NC+Lido组和RNAi2+Lido组,SH-SY5Y+Lido组、NC+Lido组细胞蜷缩,突起消失,细胞漂浮；RNAi2+Lido组细胞虽也表现为部分细胞漂浮,胞体蜷缩,但仍然观察到部分细胞突起,部分细胞形态完整。
(2)细胞活力MTT法检测细胞经盐酸利多卡因处理24h后细胞活力,对照组细胞活力分别为(100.0±0.0)%,(98.3±5.9)%,(96.5±5.9)%；而SH-SY5Y+Lido组、NC+Lido组及RNAi2+Lido组细胞活力明显降低,分别为(50.0±2.4)%,(50.6±6.6)%,(65.2±3.0)%。经析因方差分析,结果显示利多卡因处理因素各组间差异有统计学意义(F=798.768,P=0.000),基因序列的插入因素各组间差异有统计学意义(F=7.141,P=0.003),利多卡因处理因素及基因序列插入因素间有交互作用(F=14.896,P=0.000)。进一步分析单独效应,结果显示在固定非利多卡因处理因素时,基因序列插入因素各水平条件下均无统计学意义(F=0.829,P=0.456),在固定利多卡因处理因素下,SH-SY5Y+Lido组和NC+Lido组及RNAi2+Lido组间差异有统计学意义(F=22.267,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=2500.000,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=175.377,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组计较差异有统计学意义(F=142.746,P=0.000),见表3-1,图3-2。
(3)细胞凋亡率三个对照组细胞凋亡率分别为(7.0±1.4)%、(6.7±1.0)%、(6.8±1.5)%,各对照组间比较差异无统计学意义；10mM盐酸利多卡因处理24h后,SH-SY5Y+Lido组和NC+Lido组细胞凋亡率分别为(61.5±2.7)%、(61.7±2.7)%,10mM盐酸利多卡因处理RNAi2组细胞后细胞凋亡率为(46.5±5.2)%。经析因方差分析,结果显示利多卡因处理因素各组间差异有统计学意义(F=2871.057,P=0.000),插入基因序列因素各组间差异有统计学意义(F=29.358,P=0.000),利多卡因处理因素及基因序列插入因素间有交互效应(F=29.380,P=0.000)。进一步分析单独效应,结果显示在固定非利多卡因处理因素时,基因序列插入因素各水平条件下差异均无统计学意义(F=0.096,P=0.909),在固定利多卡因处理因素下,SH-SY5Y+Lido组和NC+Lido组及RNAi2+Lido组间差异有统计学意义(F=33.082,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=1875.947,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=2231.557,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组计较差异有统计学意义(F'-327.044,P-0.000),见表3-2,图3-3。
(4)Cav3.1的表达SH-SY5Y组细胞Cav3.1表达与NC组细胞表达无统计学意义,但RNAi2组细胞Cav3.1表达减少,10mM盐酸利多卡因处理24h后,SH-SY5Y+Lido组细胞和NC+Lido组细胞Cav3.1表达增加,RNAi2+Lido组细胞Ca,,3.1表达比三个对照组均有增加,但与SH-SY5Y+Lido组细胞和NC+Lido组细胞比较,其增加的幅度降低,见图3-4。统计学分析表明,插入基因序列主效应有统计学意义(F=93.860,P=0.005),利多卡因处理效应也有统计学意义(F=973.876,P=0.000),但RNA干扰与利多卡因处理这两个因素之间无交互效应(F=2.042,P=0.147),见表3-3。
(5) Caspase-3的表达三个对照组细胞活化的Caspase-3(Cleaved-caspase-3)表达较低,而非活化的Caspase-3(Procaspase-3)的表达较多,三个对照组间比较差异无统计学意义(F=0.304,P=0.742)；与此相反,10mmM盐酸利多卡因处理24h后,三个实验组活化的Cleaved-caspase-3表达增加,而非活化的Procaspase-3表达则减少。统计学分析表明,对Procaspase-3及Cleaved-caspase-3的表达,利多卡因处理因素主效应有统计学意义(F=1150.570,P=0.000和F=582.441P=0.000),插入基因序列因素主效应也有统计学意义(F=91.081,P=0.000和F=32.372,P=0.000),利多卡因处理与基因序列插入这两个因素之间有交互效应(F=92.318,P=0.000和F=33.529,P=0.000)。单独效应分析表明,固定无利多卡因处理因素时,SH-SY5Y组、NC组、RNAi2组procaspase-3和cleavedcaspase-3表达差异无统计学意义(F=0.304,P=0.742和F=0.932,P=0.416)。固定利多卡因处理因素时,SH-SY5Y+Lido组、NC+Lido组、RNAi2+Lido组procaspase-3和cleavedcaspase-3表达差异有统计学意义(F=227.787,P=0.000和F=57.604,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=672.256,P=0.000和F=216.216,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=671.226,P=0.000和F=415.059,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组比较差异有统计学意义(F=672.256,P=0.000和F=216.216,P=0.000)见表3-4,3-5。(6)[Ca2+]free
三个对照组细胞内[Ca2+]free分别为(417±13)nM、(413±16) nM、(408±15)nM。各对照组间比较无统计学差异；10mM盐酸利多卡因处理24h后,SH-SY5Y+Lido组和NC+Lido组细胞[Ca+]free分别为(608±15) nM、(607±13) nM,10mM盐酸利多卡因处理RNAi2组细胞[Ca2+]free为(520±12)nM。经析因方差分析,结果显示利多卡因处理因素各组间差异有显著性(F=1217.976,P=0.000),插入基因序列因素各组间差异有统计学意义(F=43.549,P=0.000),利多卡因处理因素及基因序列插入因素间有交互效应(F=32.035,P=0.000)。进一步分析单独效应,结果显示在固定非利多卡因处理因素时,基因序列插入因素各水平条件下均无显著性(F=0.514,P=0.608),在固定利多卡因处理因素下,SH-SY5Y+Lido组和NC+Lido组及RNAi2+Lido组间差异有统计学意义(F=81.695,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=538.156,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=503.833,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组计较差异有统计学意义(F=207.950,P=0.000),见表3-6,图3-6。
结论10mM盐酸利多卡因处理24h后,SH-SY5Y组、NC组及RNAi2组细胞Cav3.1表达增加,但RNAi2组表达增加幅度低于SH-SY5Y组和NC组细胞。经盐酸利多卡因处理后,各组细胞形态学发生改变,细胞活力降低、细胞凋亡增加、Caspase-3表达增加,[Ca2+]free增加；但RNAi2组细胞损伤的程度低于SH-SY5Y组和NC组细胞,提示盐酸利多卡因对pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞的损伤作用减轻。
第4章Cav3.1钙通道通过p38MAPK通路致SH-SY5Y细胞损伤
目的观察10mM盐酸利多卡因处理pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞24h后,p38MAPK表达情况,探讨抑制Cav3.1表达在盐酸利多卡因对SH-SY5Y细胞损伤中的保护作用是否通过抑制p38MAPK激活途径,为盐酸利多卡因神经损伤的防治提供理论依据。
方法细胞共分为6组,即对照组分别为SH-SY5Y组,阴性序列NC组(pNC-puro-CACNA1G-SH-SY5Y), RNAi2组[pshRNA-CACNA1G-SH-SY5Y (RNAi2)]组；实验组则为上述三组分别用终浓度为10mM盐酸利多卡因处理24h,即SH-SY5Y+利多卡因组(SH-SY5Y+Lido组),阴性序列NC±利多卡因组(NC+Lido组),RNAi2+利多卡因组(RNAi3+Lido组)。药物处理24h后在倒置显微镜下观察细胞形态；MTT法检测细胞存活率；流式细胞仪检测细胞凋亡率；免疫印迹法检测p38和p-p38MAPK蛋白的表达；
统计学处理采用SPSS10.0统计学软件进行分析,计量资料以均数±标准差(i±s)表示,细胞存活率、细胞凋亡率、p38和p-p38MAPK蛋白表达采用两因素析因设计方差分析,单独效应分析时组间比较采用单因素方差分析(one-way ANOVA),多重比较采用LSD法,方差不齐时则分别采用Welch法和Dunnett's T3法,P<0.05为差异有统计学意义。
结果细胞形态、细胞活力、细胞凋亡率及细胞凋亡情况同第3章实验结果。各组p38MAPK表达比较差异无统计学意义,主效应(F=0.071,P=0.791和F=0.061,P=0.941)及二者之间的交互效应(F=0.363,P=0.699)均无统计学意义。对p-p38MAPK表达的影响,盐酸利多卡因处理因素主效应(F=911.349,P=0.000)和插入序列因素主效应(F=53.082,P=0.000)及二者之间的交互效应(F=49.737,P=0.000)均有统计学意义。单独效应分析表明,固定非利多卡因处理因素时,插入基因序列对p-p38表达无影响(F=0.145,P=0.866)。固定利多卡因处理因素时,SH-SY5Y+Lido组、NC+Lido组及RNAi2+Lido组p-p38MAPK表达比较差异有统计学意义(F=59.673,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=264.266,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=520.990,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组计较差异有统计学意义(F=278.400,P=0.000)见图4-1,表4-1,4-2。
结论pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞靶向抑制Cav3.1表达后,10mM盐酸利多卡因对其损伤作用减轻,且这种减轻作用可能与p-p38MAPK表达减少有关。
With the wide application of local anesthesia, neurotoxicity and systemic toxicity reaction of local anesthetics have aroused more and more people's attention. Especially peripheral nerve damage induced by local anesthetics, as will bring obvious adverse affect for the patient lives and production.
In clinic, some patients have transient neurological syndrome manifested burning, radiating, convulsive and continuous pain after intravertebral anesthesia; and some patients even show cauda equina syndrome, one more serious disorder of nervous functions. Generally, nerve damage resulted by local anesthetics is related with the dose, concentration, and the time of the nerve exposure to the local anesthetics. The mechanisms of the nerve damage induced by local anesthetics remain unclear, it maybe related with many factors.
Cell calcium overload is one of the important mechanisms of the local anesthetics nerve injury. Extracellular calcium ions can enter into cell through the voltage-dependent calcium ion channels or receptor-dependent calcium ion channels, activate cells calcium-dependent enzyme and apoptosis, and induce the nerve damage. At the same time, calcium ion entered into the cell can produce calcium induced calcium release(CICR), cause intracellular calcium overload, result in cell apoptosis and nerve damage.
Another important mechanism of the local anesthetics nerve damage is the p38mitogen-activated protein kinase (p38MAPK). Extracellular calcium ions enter into cells, activate the calcium-dependent protease, further activate apoptosis signal-regulating kinase1(ASK1), induce p38MAPK activation, activate Ca2+/CaMK Ⅱ-ASK1-p38MAPK pathways, p38MAPK activation can induce Caspase-independent apoptosis.
This study adopt SH-SY5Y cell model, detected the subtype of T-type calcium channel expression by western blotting and real-time PCR, and observed the changes of the T-type channel expression with lidocaine hydrochloride treatment for24h. Then we constructed pshRNA-CACNA1G-SH-SY5Y (RNAi2) with RNA interfere and measured the effects of lidocaine hydrochloride on the pshRNA-CACNA1G-SH-SY5Y (RNAi2) and the expression of p38MAPK to test the mechanism of p38MAPK related to the local anesthethics nerve injury.
Chapter1Effects of lidocaine hydrochloride on the expression of Cav3.1in SH-SY5Y cells
Experiment1Expression of T-type calcium channel in SH-SY5Y cells
Objective To observe the mRNA and protein expression of the T-type calcium channel (Cav3.1, Cav3.2and Cav3.3) in SH-SY5Y cells and identify the subtype of the T-type calcium channel in SH-SY5Y cells.
Method We cultured SH-SY5Y cells in vitro, measured the expression of protein and mRNA of Cav3.1, Cav3.2and Cav3.3with western blotting (WB) and real-time PCR. The films of WB were scanned, Quantity one, one picture analysis software, was adopted to analysis absorbance of the target band, the ratio of absorbance referred to the band of β-actin was regard as the expression of protein. Expression of mRNA was calculated with the formula of2-ΔΔCt, Ct was the threshold of cycle,Δ ΔCt=[Ct (target gene of experiment)-Ct (β-actin)]-[Ct (target gene of control)-Ct (β-actin)]. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Completely randomized single-factor analysis of variance was made by LSD (homogeneous variance) and Dunnet's T3or Welch (heterogeneity of variance). P<0.05was regard as the significant difference.
Results We found that all three subtype, Cav3.1, Cav3.2and Cav3.3mRNA or protein, were detected in SH-SY5Y cells. However, Cav3.1was the primary subtype, the expression of Cav3.1protein (F=777.113,P=0.000) and mRNA (F=194898.000,P=0.000) was more than Cav3.2and Cav3.3.
Conclusion Cav3.1, Cav3.2and Cav3.3protein and mRNA were detected in SH-SY5Y cells, and Cav3.1was the primary subtype.
Experiment2Effects of lidocaine hydrochloride on the expression of Cav3.1in SH-SY5Y cells
Objective Cav3.1was the main subtype in SH-SY5Y cells, the physiology characteristics of T-type calcium were reflected mainly by Cav3.1in SH-SY5Y cells. This study was to observe the changes of Cav3.1protein and mRNA in SH-SY5Y cells treated with lidocaine hydrochloride for24h.
Methods SH-SY5Y cells cultured in vitro were divided into two group, one group SH-SY5Y cells were treated with lOmM lidocaine hydrochloride for24h and another were not treated with lidocaine hydrochloride. Cav3.1were detected with the western blotting and real-time PCR. The films of WB were scanned, Quantity one, one picture analysis software, was adopt to analysis absorbance of the target band, the ratio of absorbance referred to the band of β-actin was regard as the expression of protein. Expression of mRNA was calculated with the formula of2-ΔΔCt, Ct was the threshold of cycle, ΔΔCt=[Ct (target gene of experiment)-Ct (β-actin)]-[Ct (target gene of control)-Ct (β-actin)]. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Paired t-test was used to treated the data of Cav3.1mRNA and protein expression. P<0.05was regarded as the significance difference.
Results Compared with Control group, SH-SY5Y cells treated with10mM lidocaine hydrochloride for24h can up-regulate Cav3.1protein (t=9.932, P=0.000) and mRNA expression (t=12.573, P=0.000)
Conclusion SH-SY5Y cells treated with10mM lidocaine hydrochloride for24h can up-regulate Cav3.1protein and mRNA expression.
Chapter2Construction and identification of pshRNA-CACNAIG-SH-SY5Y
Objective Up to now there were no specific antagonist of T-type calcium channel, which has become the main hinder of T-type calcium channel research.With the development of gene interference and genetic engineering, gene interference to silence the tagart gene has become one important method in science research. This study constructed the pshRNA-CACNA1G-SH-SY5Y cells with the RNAi to silence the Cav3.1gene, and test this cells with western blotting and real-time PCR.
Method Three oligonucleotides of shRNA interferenc sequence tagarting to CACNA1G and one negative control oligonucleotide were designed. Those olgonucletides after annealed were jointed with vector enzyme cutting, and were transfered into Top10, The recombinant plasmid were measured by DNA sequencing after confirmed by enzyme digestion analysis. The recombinant plasmid then was transferred into SH-SY5Y cell after a series of experimental procedure. Cav3.1gene and protein expression of shRNA-CACNA1G-SH-SY5Y was identified by real-time PCR and western blotting. Data were record as mean±standard deviation(x±s), SPSS10.0software was used to analysis the data. Completely randomized single-factor analysis of variance was made by LSD (homogeneous variance) and Dunnett's T3(heterogeneity of variance). P<0.05was regarded as the significance difference.
Results The three oligonucleotides of shRNA interference sequence and negative control sequence were successfully insertted pSUPERretro-puro by the DNA sequencing. All three shRNA interference sequence can silence the Cav3.1gene and down-regulate the Cav3.1protein expression, especially the RNAi(2), with the highest efficiency of interference, however, negative control had no effects on the Cav3.1gene and protein.
Conclusion We successfully constructed shRNA-CACNA1G-SH-SY5Y cells identified with western blotting and real-time PCR and provided one cell model with silenced Cav3.1gene for the further experiments.
Chapter3The effects of lidocaine hydrochloride on pshRNA-CACNA1G-SH-SY5Y (RNAi2) cells
Objective We had constructed the pshRNA-CACNA1G-SH-SY5Y(RNAi2) cells with the gene interference to silence the Cav3.1gene expression, which provided the cell model to investigate the role of Cav3.1in the neurotoxicity induced by lidocaine. This study was designed to observe the effects of10mM lidocaien on the pshRNA-CACNA1G-SH-SY5Y (RNAi2) cells and to test the roles of Cav3.1in the neurotoxicity induced by lidocaine.
Methods There were three control groups followed as:SH-SY5Y group, RNAi2group [pshRNA-CACNA1G-SH-SY5Y (RNAi2)] and NC group(pNC-puro-CACNA1G-SH-SY5Y). As well as, three experiment group were SH-SY5Y+Lido group, RNAi2+Lido group and NC+Lido group, which treated with10mM lidocaine for24hours. After drug treated for24hours, the cell shape were observed with microscope, cell viability were detected with MTT,cell apoptosis rate measured with flow cytometry, Cav3.1and Caspase-3were detected with western blotting. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Two factors factorial design variance analysis (LSD for the homogeneity of variance and Welch or Dunnett's T3for the unequal variances and were adopted when separate effect analysis as necessary) was used to analysis the cell viability, cell apoptosis rate, Cav3.1and Caspase-3expression. P<0.05was regard as the significance difference.
Results
(1) Cell shape
Cell in those control groups grew well in reticulation with a spindle shape cell and many protuberance extended from the cell body. However, cells in experiment groups (SH-SY5Y+Lido group and NC+Lido group) curled up and float, protuberance disappeared. Although cell in RNi2+Lido group also appeared above conditions, cell shape was intact and some protuberance can still be found.
(2) Cell viability
After treated with lidocaine for24hours, cell viability of cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (100.0±0.0)%,(98.3±5.9)%,(96.5±5.9)%,(50.0±2.4)%,(50.6±6.6)%,(65.2±3.0)%respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=798.768, P=0.000) and sequence insert (F=7.141,P=0.003) and the interaction effect (F=14.896,P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.829, P=0.456) to the cells viability when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=22.267,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, cell viability of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=2500.000, P=0.000).When fixed negative sequence insert factor, cell viability of the cells in NC group and NC+Lido group had significant difference (F=175.377, P=0.000). When fixed the interfere sequence insert factor, cell viability of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=142.746, P=0.000), tab3-1and fig3-2.
(3) Apoptosis rate
Apoptosis rate of the cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (7.0±1.4)%,(6.7±1.0)%,(6.8±1.5)%,(61.5±2.7)%,(61.7±2.7)%,and (46.5±5.2)%respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=2871.057,P=0.000) and sequence insert(F=29.358,P=0.000) and the interaction effect (F=29.380,P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.096, P=0.909) to the apoptosis rate when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=33.082,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, apoptosis rate of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=1875.947, P=0.000). When fixed negative sequence insert factor, apoptosis rate of the cells in NC group and NC+Lido group had significant difference (F=2231.557,P=0.000). When fixed the interfere sequence insert factor, apoptosis rate of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=327.044,P=0.000), tab3-2and fig3-3.
(4) Cav3.1expression
There was no difference of the Cav3.1expression in cells that in SH-SY5Y group and NC group. Compared with the above two group, Cav3.1expression reduced in RNAi2group. After treated with10mM lidocaine hydrochloride, Cav3.1expression of the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group up regulated, but increase of Cav3.1expression of the cells in RNAi2+Lido group was less than the other two experiment group, see fig3-4. Factor analysis showed the main effects of lidocaine hydrochloride (F=93.860,P=0.005) and sequence insert (F=973.876,P=0.000) had significant difference. However, the interaction effect (F=2.042, P=0.147) of those two factors had no significant difference, see tab3-3.
(5) Caspase-3expression
The expression of cleavedcaspase-3(the activated caspase-3) or procaspase-3(the inactivated caspase-3) was no difference among the cells in the three control groups. Compared with the control groups, cleavedcaspase-3expression of the cells in experiment group increased and procaspase-3expression decreased. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the procaspase-3and cleaved caspase-3protein expression showed the main effects of lidocaine hydrochloride (F=1150.570,P=0.000and F=582.441,P=0.000) and sequence insert (F=91.081, P=0.000and F=32.372, P=0.000) and the interaction effect (F=92.318, P=0.000and F=33.529, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.304, P=0.742and F=0.932, P=0.416) to the procaspase-3and cleavedcaspase-3protein expression when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=227.787, P=0.000and F=57.604, P=0.000)among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=672.256, P=0.000and F=216.216, P=0.000). When fixed negative sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in NC group and NC+Lido group had significant difference (F=671.226, P=0.000and F=415.059, P=0.000). When fixed the interfere sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=672.256, P=0.000and F=216.216, P=0.000), tab3-4,3-5and fig3-5.
(6)[Ca2+]free
[Ca2+]free of the cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (417± 13)nM,(413±16)nM,(408±15)nM,(608±15) nM,(607±13) nM and (520±12) nM, respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=1217.976,P=0.000) and sequence insert (F=43.549,P=0.000) and the interaction effect (F=32.035, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.514,P=0.608) to the [Ca2+]free when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=81.695,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor,[Ca2+]free of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=538.156, P=0.000). When fixed negative sequence insert factor,[Ca2+]free of the cells in NC group and NC+Lido group had significant difference (F=503.833, P=0.000). When fixed the interfere sequence insert factor,[Ca2+]free of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=207.950, P=0.000), tab3-6and fig3-6.
Conclusion Cav3.1expression increased after treated with lOmM lidocaine hydrochloride for24hours. Compared with the control group, lOmM lidocaine hydrochloride caused the cell shape change, cell viability decrease,[Ca2+]free increase, cell apoptosis rate increase and cleavedcaspase-3upregulate. Cav3.1gene silence of the cells in RNAi2+Lido group can protect the cells damage from the lidocaine hydrochloride.
Chapter4SH-SY5Y cells injury induced by lidocaine hydrochloride via p38MAPK regulated by Cav3.1
Objective To ivestigate the relation of the protection effects of Cav3.1gene silence with the p38MAPK in the damage induced by lidocaine hydrochloride, we detected the expression of the p38MAPK of the SH-SY5Y cells treated with lidocaine hydrochloride for24hours.
Methods There were three control groups followed as:SH-SY5Y group, RNAi2group [pshRNA-CACNA1G-SH-SY5Y (RNAi2)] and NC group(pNC-puro-CACNA1G-SH-SY5Y). As well as, three experiment group were SH-SY5Y+Lido group, RNAi2+Lido group and NC+Lido group, which treated with10mM lidocaine for24hours. After drug treated for24hours, the cell shape were observed with microscope, cell viability were detected with MTT,cell apoptosis rate measured with flow cytometry, p38MAPK and p-p38MAPK were measured with western blotting. Data were record as mean±standard deviation(x±s), SPSS10.0software was used to analysis the data. Two factors factorial design variance analysis (LSD for the homogeneity of variance or Dunnet's T3for the unequal variances and Paired Sample t test were adopted when separate effect analysis as necessary) was used to analysis the cell viability, cell apoptosis rate, p38MAPK and p-p38MAPK protein expression. P<0.05was regarded as the significance difference.
Results Cell shape, cell viability and cell apoptosis rate were the same to the Part Ⅲ. There was no significant difference of the p38MAPK expression among the groups. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the p38MAPK protein expression showed the main effects of lidocaine hydrochloride (F=0.071, P=0.791) and sequence insert (F=0.061, P=0.941) and the interaction effect (F=0.363, P=0.699) of those two factors had no significant difference. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the p-p38MAPK protein expression showed the main effects of lidocaine hydrochloride (F=911.349,P=0.000) and sequence insert(F=53.082, P=0.000) and the interaction effect (F=49.737, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects(F=0.145, P=0.866) to the p-p38MAPK protein expression when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=59.673, P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group.When fixed no sequence insert factor, p-p38MAPK protein expression of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=264.266, P=0.000). When fixed negative sequence insert factor, p-p38MAPK protein expression of the cells in NC group and NC+Lido group had significant difference (F=520.990, P=0.000). When fixed the interfere sequence insert factor, p-p38MAPK protein expression of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=278.400, P=0.000), fig4-1and tab4-1,4-2.
Conclusion pshRNA-CACNA1G-SH-SY5Y (RNAi2) can reduce the cell injury induced by lidocaine hydrochloride. And this pretection maybe invovled with the p-p38MAPK downregulation.
临床上,椎管内麻醉后,部分病人可出现短暂性神经病学综合征(transient neurological syndrome, TNS),表现为烧灼样、持续固定、痉挛性、放射性疼痛的症状。部分患者甚至可发生严重的神经功能永久性损害如马尾综合征。一般认为,局麻药所致的神经损伤与其剂量、浓度及脊神经在局麻药中暴露的时间呈正相关。但对于局麻药诱导的神经损伤机制目前尚不明确,可能与多种因素有关。
细胞内钙超载是局麻药神经损伤的重要机制之一。细胞外Ca2＋可通过细胞膜上的电压依赖性与受体依赖性等Ca2＋通道进入细胞内,激活细胞内钙依赖性酶,诱导细胞凋亡,产生神经损伤。同时,进入细胞内的Ca2＋尚可诱发细胞内钙依赖性钙释放,使细胞内钙库释放大量Ca2＋,进一步加重细胞内钙超载,最终导致细胞凋亡,产生神经损伤。
电压依赖性钙通道分为高电压依赖性钙通道(high voltage activated calcium channel, HAV)和低电压依赖性钙通道(low voltage activated calcium channel, LAV). Cav1和Cav2属于HAV,包括L型,N型,P/Q型和R型。低电压依赖性钙通道即T型钙通道则由Cav3编码,包括三种亚型(Cav3.1,Cav3.2, Cav3.3),其在静息电位下(-65mV～-50mV)即可激活,且具有快速激活,缓慢失活的特点；同时,T型钙通道还具有“窗口电流”,即在T型钙通道稳定激活曲线和稳定失活曲线之间有一相互重叠部分。HVA和LVA在慢性神经病理性疼痛中均有重要作用,与神经损伤有关。低电压依赖性钙通道即T型钙通道,是调节神经元兴奋性和神经递质分泌的重要启动子,也是调节细胞内钙信号的关键因素。目前关于T型钙通道在局麻药神经损伤中的作用尚不清楚。
p38丝裂原活化蛋白激酶(p38mitogen-activated protein kinases,p38MAPK)激活是局麻药神经损伤的另一重要途径,细胞外Ca2＋进入细胞内,激活钙依赖的钙调蛋白酶(Ca2+/calmodulin-dependent protein kinase type Ⅱ, Ca2+/CaMK II),进
‘步激活凋亡信号调节相关激酶1(Apoptosis signal-regulating kinase1,ASK1),诱导p38MAPK的激活,形成Ca2+/CaMKⅡ-ASK1-p38MAPK通路,p38MAPK的激活则可诱导神经细胞发生不依赖于半胱胺酸蛋白酶蛋白(Caspase)的凋亡反应。
本课题采用SH-SY5Y细胞体外培养模型,通过免疫印迹和real-time PCR等方法,鉴定SH-SY5Y细胞表面T型钙通道各亚型的表达,并观察10mM盐酸利多卡因处理24h后,SH-SY5Y细胞表面T型钙通道的表达变化；采用RNAi技术,构建T型钙通道低表达的SH-SY5Y细胞株,并观察抑制SH-SY5Y细胞表面T型钙通道表达后,能否减轻利多卡因对该细胞的损伤作用,并检测p38MAPK的表达,探讨T型钙通道是否通过激活p38MAPK产生细胞毒性损伤作用。
第1章盐酸利多卡因对SH-SY5Y细胞表面Cav3.1表达的影响
实验1T型钙通道在SH-SY5Y细胞上的表达
目的通过免疫印迹和real-time PCR方法检测T型钙通道各亚型(Cav3.1、Cav3.2、Cav3.3) mRNA和蛋白质在SH-SY5Y细胞上的表达,鉴定SH-SY5Y细胞表面T型钙通道表达亚型。
方法SH-SY5Y细胞体外培养,采用免疫印迹技术和real-time PCR技术,检测Cav3.1、Cav3.2和Cav3.3在SH-SY5Y细胞上的表达。将免疫印迹X胶片扫描,采用quantity one图像分析软件分析目标条带的吸光度值,以β-actin条带吸光度值为参照,两者比值为目标蛋白的表达水平。real-time PCR定量的方法以2-ΔΔCt(Ct代表循环阈值)表示基因的表达量,计算公式为ΔΔCt=[Ct(待测组目标基因)-Ct (β-actin)]-[Ct(对照组目标基因)-Ct (β-actin)]。
统计学处理采用SPSS10.0统计学软件进行数据分析,计量资料以均数±标准差(χ-±s)表示,组间比较采用单因素方差分析(one-way ANOVA),多重比较采用LSD法,方差不齐时则分别采用Welch法和Dunnett' s T3法,P<0.05为差异有统计学意义。
结果免疫印迹法检测发现,在SH-SY5Y细胞上T型钙通道的三种亚型Cav3.1、 Cav3.2及Cav3.3蛋白均有表达,但表达量差异有统计学意义(F=777.113,P=0.000). Cav3.1蛋白在SH-SY5Y细胞上表达最丰富,而Cav3.2及Cav3.3蛋白的表达水平比Cav3.1蛋白表达水平降低,尤以Cav3.2的表达量为最少。Cav3.1、 Cav3.2、Cav3.3mRNA在SH-SY5Y细胞上均有表达,但表达量不一,Cav3.1mRNA的表达量最多,Cav3.2表达量则最少(F=194898.000,P=0.000)。
结论T型钙通道三种亚型Cav3.1、Cav3.2、Cav3.3在SH-SY5Y细胞上均有表达,但以Ca,,3.1表达为主。
实验2盐酸利多卡因对SH-SY5Y细胞Cav3.1表达的影响
目的Cav3.1是SH-SY5Y细胞上表达最为丰富的T型钙通道亚型,因此,SH-SY5Y细胞上低电压依赖性钙通道的生理学特征主要由Cav3.1通道体现,本研究的目的是观察SH-SY5Y细胞在盐酸利多卡因处理24h后其表面Cav3.1表达的变化。
方法SH-SY5Y细胞离体培养,分为两组,一组正常培养,另一组细胞则用10mM盐酸利多卡因处理24h后,采用免疫印迹和real-time PCR方法检测Cav3.1的表达变化。将免疫印迹X胶片扫描,采用quantity one图像分析软件分析目标条带的吸光度值,以β-actin条带吸光度值为参照,两者比值为目标蛋白的表达水平。real-time PCR定量的方法以2-ΔΔCt (Ct代表循环阈值)表示基因的表达量,计算公式为ΔΔCt=[Ct(待测组目标基因)-Ct (β-actin)]-[Ct(对照组目标基因)-Ct (β-actin)]。
统计学处理采用SPSS10.0统计学软件进行数据分析,计量资料以均数±标准差(χ-±s)表示,Cav3.1mRNA及蛋白表达采用配对t检验,P<0.05为差异有统计学意义。
结果与对照组比较,10mM盐酸利多卡因处理组Cav3.1蛋白表达量增加,两组比较有统计学意义(t=9.932, P=0.000); Cav3.1mRNA表达量也增加,两组比较有统计学意义(t=12.573,p=0.000)。
结论10mM盐酸利多卡因处理SH-SY5Y细胞24h后,细胞表面Cav3.1蛋白及mRNA表达增加。
第2章pshRNA-CACNA1G-SH-SY5Y细胞株的构建与鉴定
目的T型钙通道目前尚无特异性强的抑制剂,这成为阻碍T型钙通道研究进展的重要原因。随着基因干扰和基因工程技术的发展,靶向干扰目的基因的表达成为当前研究的重要工具,本研究的目的是通过RNAi技术,靶向干扰CACNA1G(Cav3.1基因),构建pshRNA-CACNA1G-SH-SY5Y细胞,以达到抑制Cav3.1基因和蛋白质的表达,为后续研究提供实验模型。
方法根据RNAi设计原则和Genbank上Cav3.1(CACNAIG)基因序列(序列号NM018896),选择3个干扰靶点,同时设计一个阴性序列。选择pSUPERretro-puro为载体,经退火、酶切、连接、转化等步骤,所得产物测序鉴定；同时大提质粒,293FT细胞、慢病毒包装、收集病毒液,并将病毒转染到SH-SY5Y细胞,经免疫印迹和real-time PCR鉴定,构建pshRNA-CACNA1G-SH-SY5Y细胞株。Cav3.1蛋白及基因表达以均数±标准差(x±s)表示,组间比较采用单因素方差分析(one-way ANOVA),多重比较采用LSD法,方差不齐时则分别采用Welch法和Dunnett's T3法,P<0.05为差异有统计学意义。
结果经DNA测序,三个干扰序列和阴性序列均顺利插入pSUPERretro-puro质粒中。免疫印迹和real-time PCR鉴定结果表明,三个干扰序列RNAi1、RNAi2、 RNAi3均能明显抑制pshRNA-CACNA1G-SH-SY5Y细胞上CACNA1G基因(Cav3.1)的表达,尤其以RNAi2转染效率最高,而阴性序列则对Cav3.1表达则无影响。
结论经免疫印迹和real-time PCR鉴定,成功构建pshRNA-CACNA1G-SH-SY5Y(RNAi2)细胞株,为后续实验提供细胞模型。
第3章盐酸利多卡因对pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞的影响
目的通过上述实验,我们建立了Cav3.1低表达的pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞株,为探讨Cav3.1钙通道在盐酸利多卡因致神经细胞损伤中的作用提供实验模型。本实验目的是观察10mM盐酸利多卡因对Cav3.1低表达的pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞的影响,探讨Cav3.1在盐酸利多卡因致神经细胞损伤中的作用。
方法细胞共分为6组,即对照组分别为SH-SY5Y组,pNC-puro-CACNAIG-SH-SY5Y(阴性序列组,NC组),pshRNA-CACNA1G-SH-SY5Y (RNAi2)组(RNAi2组)；实验组则为上述三组分别用终浓度为10mM盐酸利多卡因处理24h,即SH-SY5Y+利多卡因组(SH-SY5Y+Lido组),阴性序列+利多卡因组(NC+Lido组),RNAi2+利多卡因组(RNAi2+Lido组)。药物处理24h后在倒置显微镜下观察细胞形态；MTT法检测细胞存活率；流式细胞仪检测细胞凋亡率和细胞内Ca2+浓度([Ca2+]free);免疫印迹法检测Cav3.1蛋白及Caspase-3蛋白表达；
统计学处理采用SPSS10.0统计学软件进行分析,计量资料以均数±标准差(χ-±s)表示,细胞存活率、细胞凋亡率、Cav3.1蛋白及Caspase-3蛋白表达及[Ca2+]free采用两因素析因设计方差分析,单独效应分析时组间比较采用单因素方差分析(one-way ANOVA),多重比较采用LSD法,方差不齐时则分别采用Welch法和Dunnett's T3法,P<0.05为差异有统计学意义。
结果
(1)细胞形态倒置显微镜下观察各组细胞形态学变化,见图3-1。三个对照组即SH-SY5Y组、NC组、RNAi2组细胞生长情况良好,细胞呈梭型,突起丰富,细胞呈网状生长。实验组分别为SH-SY5Y+Lido组、NC+Lido组和RNAi2+Lido组,SH-SY5Y+Lido组、NC+Lido组细胞蜷缩,突起消失,细胞漂浮；RNAi2+Lido组细胞虽也表现为部分细胞漂浮,胞体蜷缩,但仍然观察到部分细胞突起,部分细胞形态完整。
(2)细胞活力MTT法检测细胞经盐酸利多卡因处理24h后细胞活力,对照组细胞活力分别为(100.0±0.0)%,(98.3±5.9)%,(96.5±5.9)%；而SH-SY5Y+Lido组、NC+Lido组及RNAi2+Lido组细胞活力明显降低,分别为(50.0±2.4)%,(50.6±6.6)%,(65.2±3.0)%。经析因方差分析,结果显示利多卡因处理因素各组间差异有统计学意义(F=798.768,P=0.000),基因序列的插入因素各组间差异有统计学意义(F=7.141,P=0.003),利多卡因处理因素及基因序列插入因素间有交互作用(F=14.896,P=0.000)。进一步分析单独效应,结果显示在固定非利多卡因处理因素时,基因序列插入因素各水平条件下均无统计学意义(F=0.829,P=0.456),在固定利多卡因处理因素下,SH-SY5Y+Lido组和NC+Lido组及RNAi2+Lido组间差异有统计学意义(F=22.267,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=2500.000,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=175.377,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组计较差异有统计学意义(F=142.746,P=0.000),见表3-1,图3-2。
(3)细胞凋亡率三个对照组细胞凋亡率分别为(7.0±1.4)%、(6.7±1.0)%、(6.8±1.5)%,各对照组间比较差异无统计学意义；10mM盐酸利多卡因处理24h后,SH-SY5Y+Lido组和NC+Lido组细胞凋亡率分别为(61.5±2.7)%、(61.7±2.7)%,10mM盐酸利多卡因处理RNAi2组细胞后细胞凋亡率为(46.5±5.2)%。经析因方差分析,结果显示利多卡因处理因素各组间差异有统计学意义(F=2871.057,P=0.000),插入基因序列因素各组间差异有统计学意义(F=29.358,P=0.000),利多卡因处理因素及基因序列插入因素间有交互效应(F=29.380,P=0.000)。进一步分析单独效应,结果显示在固定非利多卡因处理因素时,基因序列插入因素各水平条件下差异均无统计学意义(F=0.096,P=0.909),在固定利多卡因处理因素下,SH-SY5Y+Lido组和NC+Lido组及RNAi2+Lido组间差异有统计学意义(F=33.082,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=1875.947,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=2231.557,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组计较差异有统计学意义(F'-327.044,P-0.000),见表3-2,图3-3。
(4)Cav3.1的表达SH-SY5Y组细胞Cav3.1表达与NC组细胞表达无统计学意义,但RNAi2组细胞Cav3.1表达减少,10mM盐酸利多卡因处理24h后,SH-SY5Y+Lido组细胞和NC+Lido组细胞Cav3.1表达增加,RNAi2+Lido组细胞Ca,,3.1表达比三个对照组均有增加,但与SH-SY5Y+Lido组细胞和NC+Lido组细胞比较,其增加的幅度降低,见图3-4。统计学分析表明,插入基因序列主效应有统计学意义(F=93.860,P=0.005),利多卡因处理效应也有统计学意义(F=973.876,P=0.000),但RNA干扰与利多卡因处理这两个因素之间无交互效应(F=2.042,P=0.147),见表3-3。
(5) Caspase-3的表达三个对照组细胞活化的Caspase-3(Cleaved-caspase-3)表达较低,而非活化的Caspase-3(Procaspase-3)的表达较多,三个对照组间比较差异无统计学意义(F=0.304,P=0.742)；与此相反,10mmM盐酸利多卡因处理24h后,三个实验组活化的Cleaved-caspase-3表达增加,而非活化的Procaspase-3表达则减少。统计学分析表明,对Procaspase-3及Cleaved-caspase-3的表达,利多卡因处理因素主效应有统计学意义(F=1150.570,P=0.000和F=582.441P=0.000),插入基因序列因素主效应也有统计学意义(F=91.081,P=0.000和F=32.372,P=0.000),利多卡因处理与基因序列插入这两个因素之间有交互效应(F=92.318,P=0.000和F=33.529,P=0.000)。单独效应分析表明,固定无利多卡因处理因素时,SH-SY5Y组、NC组、RNAi2组procaspase-3和cleavedcaspase-3表达差异无统计学意义(F=0.304,P=0.742和F=0.932,P=0.416)。固定利多卡因处理因素时,SH-SY5Y+Lido组、NC+Lido组、RNAi2+Lido组procaspase-3和cleavedcaspase-3表达差异有统计学意义(F=227.787,P=0.000和F=57.604,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=672.256,P=0.000和F=216.216,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=671.226,P=0.000和F=415.059,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组比较差异有统计学意义(F=672.256,P=0.000和F=216.216,P=0.000)见表3-4,3-5。(6)[Ca2+]free
三个对照组细胞内[Ca2+]free分别为(417±13)nM、(413±16) nM、(408±15)nM。各对照组间比较无统计学差异；10mM盐酸利多卡因处理24h后,SH-SY5Y+Lido组和NC+Lido组细胞[Ca+]free分别为(608±15) nM、(607±13) nM,10mM盐酸利多卡因处理RNAi2组细胞[Ca2+]free为(520±12)nM。经析因方差分析,结果显示利多卡因处理因素各组间差异有显著性(F=1217.976,P=0.000),插入基因序列因素各组间差异有统计学意义(F=43.549,P=0.000),利多卡因处理因素及基因序列插入因素间有交互效应(F=32.035,P=0.000)。进一步分析单独效应,结果显示在固定非利多卡因处理因素时,基因序列插入因素各水平条件下均无显著性(F=0.514,P=0.608),在固定利多卡因处理因素下,SH-SY5Y+Lido组和NC+Lido组及RNAi2+Lido组间差异有统计学意义(F=81.695,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=538.156,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=503.833,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组计较差异有统计学意义(F=207.950,P=0.000),见表3-6,图3-6。
结论10mM盐酸利多卡因处理24h后,SH-SY5Y组、NC组及RNAi2组细胞Cav3.1表达增加,但RNAi2组表达增加幅度低于SH-SY5Y组和NC组细胞。经盐酸利多卡因处理后,各组细胞形态学发生改变,细胞活力降低、细胞凋亡增加、Caspase-3表达增加,[Ca2+]free增加；但RNAi2组细胞损伤的程度低于SH-SY5Y组和NC组细胞,提示盐酸利多卡因对pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞的损伤作用减轻。
第4章Cav3.1钙通道通过p38MAPK通路致SH-SY5Y细胞损伤
目的观察10mM盐酸利多卡因处理pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞24h后,p38MAPK表达情况,探讨抑制Cav3.1表达在盐酸利多卡因对SH-SY5Y细胞损伤中的保护作用是否通过抑制p38MAPK激活途径,为盐酸利多卡因神经损伤的防治提供理论依据。
方法细胞共分为6组,即对照组分别为SH-SY5Y组,阴性序列NC组(pNC-puro-CACNA1G-SH-SY5Y), RNAi2组[pshRNA-CACNA1G-SH-SY5Y (RNAi2)]组；实验组则为上述三组分别用终浓度为10mM盐酸利多卡因处理24h,即SH-SY5Y+利多卡因组(SH-SY5Y+Lido组),阴性序列NC±利多卡因组(NC+Lido组),RNAi2+利多卡因组(RNAi3+Lido组)。药物处理24h后在倒置显微镜下观察细胞形态；MTT法检测细胞存活率；流式细胞仪检测细胞凋亡率；免疫印迹法检测p38和p-p38MAPK蛋白的表达；
统计学处理采用SPSS10.0统计学软件进行分析,计量资料以均数±标准差(i±s)表示,细胞存活率、细胞凋亡率、p38和p-p38MAPK蛋白表达采用两因素析因设计方差分析,单独效应分析时组间比较采用单因素方差分析(one-way ANOVA),多重比较采用LSD法,方差不齐时则分别采用Welch法和Dunnett's T3法,P<0.05为差异有统计学意义。
结果细胞形态、细胞活力、细胞凋亡率及细胞凋亡情况同第3章实验结果。各组p38MAPK表达比较差异无统计学意义,主效应(F=0.071,P=0.791和F=0.061,P=0.941)及二者之间的交互效应(F=0.363,P=0.699)均无统计学意义。对p-p38MAPK表达的影响,盐酸利多卡因处理因素主效应(F=911.349,P=0.000)和插入序列因素主效应(F=53.082,P=0.000)及二者之间的交互效应(F=49.737,P=0.000)均有统计学意义。单独效应分析表明,固定非利多卡因处理因素时,插入基因序列对p-p38表达无影响(F=0.145,P=0.866)。固定利多卡因处理因素时,SH-SY5Y+Lido组、NC+Lido组及RNAi2+Lido组p-p38MAPK表达比较差异有统计学意义(F=59.673,P=0.000)。固定无基因序列插入时,SH-SY5Y组与SH-SY5Y+Lido组比较差异有统计学意义(F=264.266,P=0.000)。固定有阴性序列插入时,NC组与NC+Lido组比较差异有统计学意义(F=520.990,P=0.000),固定有干扰序列插入时,RNAi2组与RNAi2+Lido组计较差异有统计学意义(F=278.400,P=0.000)见图4-1,表4-1,4-2。
结论pshRNA-CACNA1G-SH-SY5Y (RNAi2)细胞靶向抑制Cav3.1表达后,10mM盐酸利多卡因对其损伤作用减轻,且这种减轻作用可能与p-p38MAPK表达减少有关。
With the wide application of local anesthesia, neurotoxicity and systemic toxicity reaction of local anesthetics have aroused more and more people's attention. Especially peripheral nerve damage induced by local anesthetics, as will bring obvious adverse affect for the patient lives and production.
In clinic, some patients have transient neurological syndrome manifested burning, radiating, convulsive and continuous pain after intravertebral anesthesia; and some patients even show cauda equina syndrome, one more serious disorder of nervous functions. Generally, nerve damage resulted by local anesthetics is related with the dose, concentration, and the time of the nerve exposure to the local anesthetics. The mechanisms of the nerve damage induced by local anesthetics remain unclear, it maybe related with many factors.
Cell calcium overload is one of the important mechanisms of the local anesthetics nerve injury. Extracellular calcium ions can enter into cell through the voltage-dependent calcium ion channels or receptor-dependent calcium ion channels, activate cells calcium-dependent enzyme and apoptosis, and induce the nerve damage. At the same time, calcium ion entered into the cell can produce calcium induced calcium release(CICR), cause intracellular calcium overload, result in cell apoptosis and nerve damage.
Another important mechanism of the local anesthetics nerve damage is the p38mitogen-activated protein kinase (p38MAPK). Extracellular calcium ions enter into cells, activate the calcium-dependent protease, further activate apoptosis signal-regulating kinase1(ASK1), induce p38MAPK activation, activate Ca2+/CaMK Ⅱ-ASK1-p38MAPK pathways, p38MAPK activation can induce Caspase-independent apoptosis.
This study adopt SH-SY5Y cell model, detected the subtype of T-type calcium channel expression by western blotting and real-time PCR, and observed the changes of the T-type channel expression with lidocaine hydrochloride treatment for24h. Then we constructed pshRNA-CACNA1G-SH-SY5Y (RNAi2) with RNA interfere and measured the effects of lidocaine hydrochloride on the pshRNA-CACNA1G-SH-SY5Y (RNAi2) and the expression of p38MAPK to test the mechanism of p38MAPK related to the local anesthethics nerve injury.
Chapter1Effects of lidocaine hydrochloride on the expression of Cav3.1in SH-SY5Y cells
Experiment1Expression of T-type calcium channel in SH-SY5Y cells
Objective To observe the mRNA and protein expression of the T-type calcium channel (Cav3.1, Cav3.2and Cav3.3) in SH-SY5Y cells and identify the subtype of the T-type calcium channel in SH-SY5Y cells.
Method We cultured SH-SY5Y cells in vitro, measured the expression of protein and mRNA of Cav3.1, Cav3.2and Cav3.3with western blotting (WB) and real-time PCR. The films of WB were scanned, Quantity one, one picture analysis software, was adopted to analysis absorbance of the target band, the ratio of absorbance referred to the band of β-actin was regard as the expression of protein. Expression of mRNA was calculated with the formula of2-ΔΔCt, Ct was the threshold of cycle,Δ ΔCt=[Ct (target gene of experiment)-Ct (β-actin)]-[Ct (target gene of control)-Ct (β-actin)]. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Completely randomized single-factor analysis of variance was made by LSD (homogeneous variance) and Dunnet's T3or Welch (heterogeneity of variance). P<0.05was regard as the significant difference.
Results We found that all three subtype, Cav3.1, Cav3.2and Cav3.3mRNA or protein, were detected in SH-SY5Y cells. However, Cav3.1was the primary subtype, the expression of Cav3.1protein (F=777.113,P=0.000) and mRNA (F=194898.000,P=0.000) was more than Cav3.2and Cav3.3.
Conclusion Cav3.1, Cav3.2and Cav3.3protein and mRNA were detected in SH-SY5Y cells, and Cav3.1was the primary subtype.
Experiment2Effects of lidocaine hydrochloride on the expression of Cav3.1in SH-SY5Y cells
Objective Cav3.1was the main subtype in SH-SY5Y cells, the physiology characteristics of T-type calcium were reflected mainly by Cav3.1in SH-SY5Y cells. This study was to observe the changes of Cav3.1protein and mRNA in SH-SY5Y cells treated with lidocaine hydrochloride for24h.
Methods SH-SY5Y cells cultured in vitro were divided into two group, one group SH-SY5Y cells were treated with lOmM lidocaine hydrochloride for24h and another were not treated with lidocaine hydrochloride. Cav3.1were detected with the western blotting and real-time PCR. The films of WB were scanned, Quantity one, one picture analysis software, was adopt to analysis absorbance of the target band, the ratio of absorbance referred to the band of β-actin was regard as the expression of protein. Expression of mRNA was calculated with the formula of2-ΔΔCt, Ct was the threshold of cycle, ΔΔCt=[Ct (target gene of experiment)-Ct (β-actin)]-[Ct (target gene of control)-Ct (β-actin)]. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Paired t-test was used to treated the data of Cav3.1mRNA and protein expression. P<0.05was regarded as the significance difference.
Results Compared with Control group, SH-SY5Y cells treated with10mM lidocaine hydrochloride for24h can up-regulate Cav3.1protein (t=9.932, P=0.000) and mRNA expression (t=12.573, P=0.000)
Conclusion SH-SY5Y cells treated with10mM lidocaine hydrochloride for24h can up-regulate Cav3.1protein and mRNA expression.
Chapter2Construction and identification of pshRNA-CACNAIG-SH-SY5Y
Objective Up to now there were no specific antagonist of T-type calcium channel, which has become the main hinder of T-type calcium channel research.With the development of gene interference and genetic engineering, gene interference to silence the tagart gene has become one important method in science research. This study constructed the pshRNA-CACNA1G-SH-SY5Y cells with the RNAi to silence the Cav3.1gene, and test this cells with western blotting and real-time PCR.
Method Three oligonucleotides of shRNA interferenc sequence tagarting to CACNA1G and one negative control oligonucleotide were designed. Those olgonucletides after annealed were jointed with vector enzyme cutting, and were transfered into Top10, The recombinant plasmid were measured by DNA sequencing after confirmed by enzyme digestion analysis. The recombinant plasmid then was transferred into SH-SY5Y cell after a series of experimental procedure. Cav3.1gene and protein expression of shRNA-CACNA1G-SH-SY5Y was identified by real-time PCR and western blotting. Data were record as mean±standard deviation(x±s), SPSS10.0software was used to analysis the data. Completely randomized single-factor analysis of variance was made by LSD (homogeneous variance) and Dunnett's T3(heterogeneity of variance). P<0.05was regarded as the significance difference.
Results The three oligonucleotides of shRNA interference sequence and negative control sequence were successfully insertted pSUPERretro-puro by the DNA sequencing. All three shRNA interference sequence can silence the Cav3.1gene and down-regulate the Cav3.1protein expression, especially the RNAi(2), with the highest efficiency of interference, however, negative control had no effects on the Cav3.1gene and protein.
Conclusion We successfully constructed shRNA-CACNA1G-SH-SY5Y cells identified with western blotting and real-time PCR and provided one cell model with silenced Cav3.1gene for the further experiments.
Chapter3The effects of lidocaine hydrochloride on pshRNA-CACNA1G-SH-SY5Y (RNAi2) cells
Objective We had constructed the pshRNA-CACNA1G-SH-SY5Y(RNAi2) cells with the gene interference to silence the Cav3.1gene expression, which provided the cell model to investigate the role of Cav3.1in the neurotoxicity induced by lidocaine. This study was designed to observe the effects of10mM lidocaien on the pshRNA-CACNA1G-SH-SY5Y (RNAi2) cells and to test the roles of Cav3.1in the neurotoxicity induced by lidocaine.
Methods There were three control groups followed as:SH-SY5Y group, RNAi2group [pshRNA-CACNA1G-SH-SY5Y (RNAi2)] and NC group(pNC-puro-CACNA1G-SH-SY5Y). As well as, three experiment group were SH-SY5Y+Lido group, RNAi2+Lido group and NC+Lido group, which treated with10mM lidocaine for24hours. After drug treated for24hours, the cell shape were observed with microscope, cell viability were detected with MTT,cell apoptosis rate measured with flow cytometry, Cav3.1and Caspase-3were detected with western blotting. Data were record as mean±standard deviation (x±s), SPSS10.0software was used to analysis the data. Two factors factorial design variance analysis (LSD for the homogeneity of variance and Welch or Dunnett's T3for the unequal variances and were adopted when separate effect analysis as necessary) was used to analysis the cell viability, cell apoptosis rate, Cav3.1and Caspase-3expression. P<0.05was regard as the significance difference.
Results
(1) Cell shape
Cell in those control groups grew well in reticulation with a spindle shape cell and many protuberance extended from the cell body. However, cells in experiment groups (SH-SY5Y+Lido group and NC+Lido group) curled up and float, protuberance disappeared. Although cell in RNi2+Lido group also appeared above conditions, cell shape was intact and some protuberance can still be found.
(2) Cell viability
After treated with lidocaine for24hours, cell viability of cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (100.0±0.0)%,(98.3±5.9)%,(96.5±5.9)%,(50.0±2.4)%,(50.6±6.6)%,(65.2±3.0)%respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=798.768, P=0.000) and sequence insert (F=7.141,P=0.003) and the interaction effect (F=14.896,P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.829, P=0.456) to the cells viability when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=22.267,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, cell viability of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=2500.000, P=0.000).When fixed negative sequence insert factor, cell viability of the cells in NC group and NC+Lido group had significant difference (F=175.377, P=0.000). When fixed the interfere sequence insert factor, cell viability of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=142.746, P=0.000), tab3-1and fig3-2.
(3) Apoptosis rate
Apoptosis rate of the cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (7.0±1.4)%,(6.7±1.0)%,(6.8±1.5)%,(61.5±2.7)%,(61.7±2.7)%,and (46.5±5.2)%respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=2871.057,P=0.000) and sequence insert(F=29.358,P=0.000) and the interaction effect (F=29.380,P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.096, P=0.909) to the apoptosis rate when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=33.082,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, apoptosis rate of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=1875.947, P=0.000). When fixed negative sequence insert factor, apoptosis rate of the cells in NC group and NC+Lido group had significant difference (F=2231.557,P=0.000). When fixed the interfere sequence insert factor, apoptosis rate of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=327.044,P=0.000), tab3-2and fig3-3.
(4) Cav3.1expression
There was no difference of the Cav3.1expression in cells that in SH-SY5Y group and NC group. Compared with the above two group, Cav3.1expression reduced in RNAi2group. After treated with10mM lidocaine hydrochloride, Cav3.1expression of the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group up regulated, but increase of Cav3.1expression of the cells in RNAi2+Lido group was less than the other two experiment group, see fig3-4. Factor analysis showed the main effects of lidocaine hydrochloride (F=93.860,P=0.005) and sequence insert (F=973.876,P=0.000) had significant difference. However, the interaction effect (F=2.042, P=0.147) of those two factors had no significant difference, see tab3-3.
(5) Caspase-3expression
The expression of cleavedcaspase-3(the activated caspase-3) or procaspase-3(the inactivated caspase-3) was no difference among the cells in the three control groups. Compared with the control groups, cleavedcaspase-3expression of the cells in experiment group increased and procaspase-3expression decreased. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the procaspase-3and cleaved caspase-3protein expression showed the main effects of lidocaine hydrochloride (F=1150.570,P=0.000and F=582.441,P=0.000) and sequence insert (F=91.081, P=0.000and F=32.372, P=0.000) and the interaction effect (F=92.318, P=0.000and F=33.529, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.304, P=0.742and F=0.932, P=0.416) to the procaspase-3and cleavedcaspase-3protein expression when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=227.787, P=0.000and F=57.604, P=0.000)among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=672.256, P=0.000and F=216.216, P=0.000). When fixed negative sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in NC group and NC+Lido group had significant difference (F=671.226, P=0.000and F=415.059, P=0.000). When fixed the interfere sequence insert factor, procaspase-3and cleavedcaspase-3protein expression of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=672.256, P=0.000and F=216.216, P=0.000), tab3-4,3-5and fig3-5.
(6)[Ca2+]free
[Ca2+]free of the cells in SH-SY5Y group, NC group, RNAi2group,SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group were (417± 13)nM,(413±16)nM,(408±15)nM,(608±15) nM,(607±13) nM and (520±12) nM, respectively. Factor analysis showed the main effects of lidocaine hydrochloride (F=1217.976,P=0.000) and sequence insert (F=43.549,P=0.000) and the interaction effect (F=32.035, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects (F=0.514,P=0.608) to the [Ca2+]free when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=81.695,P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group. When fixed no sequence insert factor,[Ca2+]free of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=538.156, P=0.000). When fixed negative sequence insert factor,[Ca2+]free of the cells in NC group and NC+Lido group had significant difference (F=503.833, P=0.000). When fixed the interfere sequence insert factor,[Ca2+]free of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=207.950, P=0.000), tab3-6and fig3-6.
Conclusion Cav3.1expression increased after treated with lOmM lidocaine hydrochloride for24hours. Compared with the control group, lOmM lidocaine hydrochloride caused the cell shape change, cell viability decrease,[Ca2+]free increase, cell apoptosis rate increase and cleavedcaspase-3upregulate. Cav3.1gene silence of the cells in RNAi2+Lido group can protect the cells damage from the lidocaine hydrochloride.
Chapter4SH-SY5Y cells injury induced by lidocaine hydrochloride via p38MAPK regulated by Cav3.1
Objective To ivestigate the relation of the protection effects of Cav3.1gene silence with the p38MAPK in the damage induced by lidocaine hydrochloride, we detected the expression of the p38MAPK of the SH-SY5Y cells treated with lidocaine hydrochloride for24hours.
Methods There were three control groups followed as:SH-SY5Y group, RNAi2group [pshRNA-CACNA1G-SH-SY5Y (RNAi2)] and NC group(pNC-puro-CACNA1G-SH-SY5Y). As well as, three experiment group were SH-SY5Y+Lido group, RNAi2+Lido group and NC+Lido group, which treated with10mM lidocaine for24hours. After drug treated for24hours, the cell shape were observed with microscope, cell viability were detected with MTT,cell apoptosis rate measured with flow cytometry, p38MAPK and p-p38MAPK were measured with western blotting. Data were record as mean±standard deviation(x±s), SPSS10.0software was used to analysis the data. Two factors factorial design variance analysis (LSD for the homogeneity of variance or Dunnet's T3for the unequal variances and Paired Sample t test were adopted when separate effect analysis as necessary) was used to analysis the cell viability, cell apoptosis rate, p38MAPK and p-p38MAPK protein expression. P<0.05was regarded as the significance difference.
Results Cell shape, cell viability and cell apoptosis rate were the same to the Part Ⅲ. There was no significant difference of the p38MAPK expression among the groups. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the p38MAPK protein expression showed the main effects of lidocaine hydrochloride (F=0.071, P=0.791) and sequence insert (F=0.061, P=0.941) and the interaction effect (F=0.363, P=0.699) of those two factors had no significant difference. Factor analysis about the effects of lidocaine hydrochloride and sequence insert on the p-p38MAPK protein expression showed the main effects of lidocaine hydrochloride (F=911.349,P=0.000) and sequence insert(F=53.082, P=0.000) and the interaction effect (F=49.737, P=0.000) of those two factors had significant difference. Separate effect analysis showed sequence insert factor had no effects(F=0.145, P=0.866) to the p-p38MAPK protein expression when those cells untreated with lidocaine hydrochloride. When fixed lidocaine hydrochloride treatment factor, there was significant difference (F=59.673, P=0.000) among the cells in SH-SY5Y+Lido group, NC+Lido group and RNAi2+Lido group.When fixed no sequence insert factor, p-p38MAPK protein expression of the cells in SH-SY5Y group and SH-SY5Y+Lido group had significant difference (F=264.266, P=0.000). When fixed negative sequence insert factor, p-p38MAPK protein expression of the cells in NC group and NC+Lido group had significant difference (F=520.990, P=0.000). When fixed the interfere sequence insert factor, p-p38MAPK protein expression of the cells in RNAi2group and RNAi2+Lido group had significant difference (F=278.400, P=0.000), fig4-1and tab4-1,4-2.
Conclusion pshRNA-CACNA1G-SH-SY5Y (RNAi2) can reduce the cell injury induced by lidocaine hydrochloride. And this pretection maybe invovled with the p-p38MAPK downregulation.
引文
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[7]Kirihara Y, Saito Y, Sakura S, et al. Comparative neurotoxicity of intrathecal and epidural lidocaine in rats[J]. Anesthesiology,2003,99 (4):961-968.
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[10]Lirk P, Haller I, Colvin HP, et al. In vitro, inhibition of mitogen-activated protein kinase pathways protects against bupivacaine-and ropivacaine-induced neurotoxicity[J]. Anesth Analg,2008,106(5):1456-1464, table of contents.
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[15]Cueni L, Canepari M, Adelman JP, et al. Ca2+ signaling by T-type Ca2+ channels in neurons [J]. Pflugers Arch,2009,457(5):1161-1172.
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[20]Jevtovic-Todorovic V, Todorovic SM. The role of peripheral T-type calcium channels in pain transmission [J]. Cell Calcium,2006,40(2):197-203.
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