观察在体灌注SCL基因重组慢病毒对豚鼠糖尿病膀胱中ICC功能的影响
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摘要
目的探究豚鼠糖尿病膀胱病(DCP)在经尿道灌注干细胞白血病(SCL)基因重组慢病毒后所引发的Cajal样间质细胞(ICC)电位活动的改变。方法购买健康雄性豚鼠90只,按照200 mg/kg单次腹腔注射1%的链脲佐菌素(STZ),正常喂养12周后,筛选出符合DCP模型标准的的豚鼠并将其随机分为3组:实验组、阳性对照组、空白对照组。实验组经尿道灌注0.2 m L滴度为2×107IU的SCL基因重组慢病毒,阳性对照组经尿道灌注0.2 mL空慢病毒,空白对照组经尿道灌注0.2 mL PBS液。每组在转染后2、7、14、28 d这4个时间节点每次处死3只豚鼠,迅速摘取膀胱,选择胶原酶V消融法分离提取初代ICC并继续培养。72 h后运用膜片钳技术检测ICC自发及ATP诱发电位活动。结果体外通过胶原酶消融法可分离培养DCP豚鼠膀胱中ICC,运用细胞膜片钳技术观察到在无刺激条件下检测不到ICC的内向电流,而在ATP(100μmol/L)条件激发下可检测到ICC的内向电流。实验组2 d时与其他两组内向电流无明显变化,实验组SCL慢病毒转染DCP膀胱7、14、28 d时可检测到ICC内向电流比其他两组高(P<0.05),14 d达到峰值350.0±29.9pA,随后降低。阳性对照组与空白对照组随着DCP膀胱持续受损,内向电流逐步降低(P<0.05)。结论 SCL基因重组慢病毒经尿道灌注可成功转染豚鼠DCP膀胱,使受损的膀胱ICC电流幅度逐步增高,说明SCL基因对DCP膀胱中受损ICC功能有部分改善作用。
Objective To investigate the change of potential activity of interstitial cells of Cajal(ICC) induced by guinea pigs with diabetic urinary bladder disease(DCP) after transfection of stem cell leukemia(SCL) gene recombinant lentiviral. Methods A total of 90 healthy male guinea pigs were purchased and injected with 1% of streptozotocin(STZ) intraperitoneally at 200 mg/kg.After 12 weeks of normal feeding,guinea pigs that met the DCP model criteria were selected and randomly divided into three groups: experimental group,positive control group,blank control group.The experimental group was transfused through the urethra with 0.2 m L of recombinant SCL gene lentivirus with a titer of 2 ×107 IU.The experimental group was transfused through the urethra with 0.2 m L of recombinant SCL gene lentivirus with a titer of 2×107 IU.The positive control group was perfused with 0.2 m L of empty lentivirus through the urethra,and the control group was perfused with 0.2 m L of PBS via the urethra.In each group,3 guinea pigs were sacrificed at each of the four time points of 2,7,14,28 d after transfection.The bladder was quickly removed,and the collagenase V ablation method was chosen to separate and extract the primary ICC and continue the culture.Seventy-two hours later,patch clamp techniques were used to detect ICC spontaneous and ATP evoked potentials. Results The ICC of DCP guinea pig bladder was isolated and cultured in vitro by collagenase ablation method.It was observed by cell patch clamp technique that the inward current of ICC was not detected under the condition of no stimulation,and the inward current of ICC could be detected under the excitation of ATP(100 μmol/L).At day 2,there was no significant change in the inward current between the experimental group and the other two groups.The inward current of ICC was higher in the experimental group than in the other two groups at 7,14,28 d after transfection of the SCL lentiviruses in the experimental group(P<0.05),and the peak value of 14 d reached 350 + 29.9 pA,and then decreased.In the positive control group and blank control group,with the continuous damage of DCP bladder,the inward current gradually decreased(P<0.05).Conclusion The transfected DCP bladder of guinea pigs can be successfully transfected with SCL gene recombinant lentivirus via urethral perfusion,and the ICC current amplitude of the damaged bladder is gradually increased,which indicates that the SCL gene may partially improve the damaged ICC function of DCP bladder.
Objective To investigate the change of potential activity of interstitial cells of Cajal(ICC) induced by guinea pigs with diabetic urinary bladder disease(DCP) after transfection of stem cell leukemia(SCL) gene recombinant lentiviral. Methods A total of 90 healthy male guinea pigs were purchased and injected with 1% of streptozotocin(STZ) intraperitoneally at 200 mg/kg.After 12 weeks of normal feeding,guinea pigs that met the DCP model criteria were selected and randomly divided into three groups: experimental group,positive control group,blank control group.The experimental group was transfused through the urethra with 0.2 m L of recombinant SCL gene lentivirus with a titer of 2 ×107 IU.The experimental group was transfused through the urethra with 0.2 m L of recombinant SCL gene lentivirus with a titer of 2×107 IU.The positive control group was perfused with 0.2 m L of empty lentivirus through the urethra,and the control group was perfused with 0.2 m L of PBS via the urethra.In each group,3 guinea pigs were sacrificed at each of the four time points of 2,7,14,28 d after transfection.The bladder was quickly removed,and the collagenase V ablation method was chosen to separate and extract the primary ICC and continue the culture.Seventy-two hours later,patch clamp techniques were used to detect ICC spontaneous and ATP evoked potentials. Results The ICC of DCP guinea pig bladder was isolated and cultured in vitro by collagenase ablation method.It was observed by cell patch clamp technique that the inward current of ICC was not detected under the condition of no stimulation,and the inward current of ICC could be detected under the excitation of ATP(100 μmol/L).At day 2,there was no significant change in the inward current between the experimental group and the other two groups.The inward current of ICC was higher in the experimental group than in the other two groups at 7,14,28 d after transfection of the SCL lentiviruses in the experimental group(P<0.05),and the peak value of 14 d reached 350 + 29.9 pA,and then decreased.In the positive control group and blank control group,with the continuous damage of DCP bladder,the inward current gradually decreased(P<0.05).Conclusion The transfected DCP bladder of guinea pigs can be successfully transfected with SCL gene recombinant lentivirus via urethral perfusion,and the ICC current amplitude of the damaged bladder is gradually increased,which indicates that the SCL gene may partially improve the damaged ICC function of DCP bladder.
引文
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[3] Wang J P,Ding G F,Wang Q Z.Interstitial cells of Cajal mediate excitatory sympathetic neurotransmission in guinea pig prostate[J].Cell&Tissue Research,2013,352(3):479-486.
[4] Ward S M,Sanders K M,Hirst G D S.Role of interstitial cells of Cajal in neural control of gastrointestinal smooth muscles[J].Neurogastroenterology&Motility,2004,16(1):112-117.
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[11]李维仁,王勤章,丁国富,等.豚鼠糖尿病膀胱模型的建立[J].现代泌尿外科杂志,2008(5):368-370.Li W R,Wang Q Z,Ding G F,et al.A guinea pig model of diabetic cystopathy.[J].Modern Department of Urology,2008(5):368-370.
[12]罗广承,何志华,罗建珍,等.构建糖尿病膀胱病豚鼠模型及其尿动力学评价[J].中国组织工程研究,2014,18(07):1063-1068.Luo G C,He Z H,Luo J Z,et al.Establishment of diabetic cystopathy guinea pig model and its urodynamic evaluation[J].Chinese Journal of Tissue Engineering Research,2014,18(07):1063-1068.
[13]魏艳青,王江平,王勤章,等.经尿道灌注慢病毒转染豚鼠膀胱的研究[J].天津医药,2015,43(11):1275-1277.Wei Y Q,Wang J P,Wang Q Z,et al.The study of transfection through perfusing bladder of guinea pig with lentivirus[J].Tianjin Medicine,2015,43(11):1275-1277.
[14]于建超,王江平,李应龙,等.人干细胞白血病基因重组慢病毒载体的构建及其在Cajal样间质细胞中的表达[J].中国现代医学杂志,2017,27(6):10-16.Yu J C,Wang J P,Li Y L,et al.Construction of recombinant human stem cell leukemia gene lentiviral vector and its expression in Cajal-like interstitial cells[J].China Journal of Modern Medicine,2017,27(6):10-16.
[15]王江平,王勤章,丁国富,等.膀胱二聚体Cajal间质细胞的超微结构及其对ATP的电反应[J].神经解剖学杂志,2012,28(6):617-620.Wang J P,Wang Q Z,Ding G F,et al.Ultrastructure and responses to ATP of dimmer interstitial cells of Cajal in bladder[J].Chinese Journal of Neuroanatomy,2012,28(6):617-620.
[16]余运运,王勤章,王子雄,等.经尿道灌注干细胞白血病重组基因慢病毒对糖尿病膀胱病变豚鼠膀胱功能的影响[J].山东医药,2017,57(30):30-32.Yu Y Y,Wang Q Z,Wang Z X,et al.Reseach of lentivirusmediated stem cell leukemia improving bladder function of guinea pig diabetic cystopathy,2017,57(30):30-32.
[17]王勤章,李云飞,丁国富,等.豚鼠膀胱Cajal样间质细胞类型及其与钙离子振荡特性的关系[J].中华泌尿外科杂志,2010,31(9):614-617.Wang Q Z,Li Y F,Ding G F,et al.Types and spontaneous Ca2+waves of ICC in the bladder of guinea pigs[J].Chinese Journal of Department of Urology,2010,31(9):614-617.
[18] Van d A F,Roskams T,Blyweert W,et al.Identification of kit positive cells in the human urinary tract[J].Journal of Urology,2004,171(6):2492-2496.
[19] Davidson R A,Mccloskey K D.Morphology and localization of interstitial cells in the guinea pig bladder:structural relationships with smooth muscle and neurons[J].Journal of Urology,2005,173(4):1385-1390.
[20]蔡志强,丁国富,李云飞,等.体外Cajal样细胞c-kit基因m RNA在高糖环境下表达变化及意义[J].农垦医学,2010,32(1):1-4.Cai Z Q,Ding G F,Li Y F,et al.Diversity of hyperglycemia on the expressions of c-kit m RNA of interstitial cells of Cajal-like in vitro[J].Journal of Nongken Medicine,2010,32(1):1-4.
[21]于建超,王江平,李应龙,等.含人干细胞白血病基因的重组慢病毒表达载体的构建及鉴定[J].山东医药,2015,55(44):15-17.Yu J C,Wang J P,Li Y L,et al.Construction and identification of recombinant lentiviral vectors containing human stem cell leukemia gene[J].Shandong Medical Journal,2015(44):15-17.
[22]余运运.慢病毒介导干细胞白血病基因对糖尿病豚鼠膀胱病变C-kit m RNA和蛋白表达的影响[D].石河子:石河子大学,2017.
[2] Lin T L,Chen G D,Chen Y C,et al.Aging and recurrent urinary tract infections are associated with bladder dysfunction in type 2 diabetes[J].Taiwan J Obstet Gynecol,2012,51(3):381-386.
[3] Wang J P,Ding G F,Wang Q Z.Interstitial cells of Cajal mediate excitatory sympathetic neurotransmission in guinea pig prostate[J].Cell&Tissue Research,2013,352(3):479-486.
[4] Ward S M,Sanders K M,Hirst G D S.Role of interstitial cells of Cajal in neural control of gastrointestinal smooth muscles[J].Neurogastroenterology&Motility,2004,16(1):112-117.
[5] Kubota Y,Biers S M,Kohri K,et al.Effects of imatinib mesylate(Glivec?)as a c-kit tyrosine kinase inhibitor in the guineapig urinary bladder[J].Neurourology&Urodynamics,2010,25(3):205-210.
[6] Biers S M,Reynard J M,Doore T,et al.The functional effects of a c-kit tyrosine inhibitor on guinea-pig and human detrusor[J].Bju International,2010,97(3):612-616.
[7] Klemm M F,Betty E,Lang R J.Identification of the cells underlying pacemaker activity in the guineapig upper urinary tract.[J].J Physiol,2010,519(3):867-884.
[8] Shafik A,Elsibai O,Shafik A A,et al.Identification of interstitial cells of Cajal in human urinary bladder:concept of vesical pacemaker.[J].Urology,2004,64(4):809-813.
[9]李云飞,王勤章,丁国富.早期糖尿病膀胱尿动力学及cajal样细胞的变化和意义[J].临床泌尿外科杂志,2009,24(9):694-697.Li Y F,Wang Q Z,Ding G F.Changes and significances of urodynamics and cajal-like cells in diabetic cystopathy of the early stage[J].Journal of Clinical Urology,2009,24(9):694-697.
[10] Mccloskey K D.Interstitial cells in the urinary bladderlocalization and function.[J].Neurourology&Urodynamics,2010,29(1):82-87.
[11]李维仁,王勤章,丁国富,等.豚鼠糖尿病膀胱模型的建立[J].现代泌尿外科杂志,2008(5):368-370.Li W R,Wang Q Z,Ding G F,et al.A guinea pig model of diabetic cystopathy.[J].Modern Department of Urology,2008(5):368-370.
[12]罗广承,何志华,罗建珍,等.构建糖尿病膀胱病豚鼠模型及其尿动力学评价[J].中国组织工程研究,2014,18(07):1063-1068.Luo G C,He Z H,Luo J Z,et al.Establishment of diabetic cystopathy guinea pig model and its urodynamic evaluation[J].Chinese Journal of Tissue Engineering Research,2014,18(07):1063-1068.
[13]魏艳青,王江平,王勤章,等.经尿道灌注慢病毒转染豚鼠膀胱的研究[J].天津医药,2015,43(11):1275-1277.Wei Y Q,Wang J P,Wang Q Z,et al.The study of transfection through perfusing bladder of guinea pig with lentivirus[J].Tianjin Medicine,2015,43(11):1275-1277.
[14]于建超,王江平,李应龙,等.人干细胞白血病基因重组慢病毒载体的构建及其在Cajal样间质细胞中的表达[J].中国现代医学杂志,2017,27(6):10-16.Yu J C,Wang J P,Li Y L,et al.Construction of recombinant human stem cell leukemia gene lentiviral vector and its expression in Cajal-like interstitial cells[J].China Journal of Modern Medicine,2017,27(6):10-16.
[15]王江平,王勤章,丁国富,等.膀胱二聚体Cajal间质细胞的超微结构及其对ATP的电反应[J].神经解剖学杂志,2012,28(6):617-620.Wang J P,Wang Q Z,Ding G F,et al.Ultrastructure and responses to ATP of dimmer interstitial cells of Cajal in bladder[J].Chinese Journal of Neuroanatomy,2012,28(6):617-620.
[16]余运运,王勤章,王子雄,等.经尿道灌注干细胞白血病重组基因慢病毒对糖尿病膀胱病变豚鼠膀胱功能的影响[J].山东医药,2017,57(30):30-32.Yu Y Y,Wang Q Z,Wang Z X,et al.Reseach of lentivirusmediated stem cell leukemia improving bladder function of guinea pig diabetic cystopathy,2017,57(30):30-32.
[17]王勤章,李云飞,丁国富,等.豚鼠膀胱Cajal样间质细胞类型及其与钙离子振荡特性的关系[J].中华泌尿外科杂志,2010,31(9):614-617.Wang Q Z,Li Y F,Ding G F,et al.Types and spontaneous Ca2+waves of ICC in the bladder of guinea pigs[J].Chinese Journal of Department of Urology,2010,31(9):614-617.
[18] Van d A F,Roskams T,Blyweert W,et al.Identification of kit positive cells in the human urinary tract[J].Journal of Urology,2004,171(6):2492-2496.
[19] Davidson R A,Mccloskey K D.Morphology and localization of interstitial cells in the guinea pig bladder:structural relationships with smooth muscle and neurons[J].Journal of Urology,2005,173(4):1385-1390.
[20]蔡志强,丁国富,李云飞,等.体外Cajal样细胞c-kit基因m RNA在高糖环境下表达变化及意义[J].农垦医学,2010,32(1):1-4.Cai Z Q,Ding G F,Li Y F,et al.Diversity of hyperglycemia on the expressions of c-kit m RNA of interstitial cells of Cajal-like in vitro[J].Journal of Nongken Medicine,2010,32(1):1-4.
[21]于建超,王江平,李应龙,等.含人干细胞白血病基因的重组慢病毒表达载体的构建及鉴定[J].山东医药,2015,55(44):15-17.Yu J C,Wang J P,Li Y L,et al.Construction and identification of recombinant lentiviral vectors containing human stem cell leukemia gene[J].Shandong Medical Journal,2015(44):15-17.
[22]余运运.慢病毒介导干细胞白血病基因对糖尿病豚鼠膀胱病变C-kit m RNA和蛋白表达的影响[D].石河子:石河子大学,2017.