靶向干扰ghrelin基因对急性胰腺炎胰腺腺泡细胞炎症与钙通路的调节机制研究
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摘要
实践及研究表明,急性胰腺炎(acute pancreatitis, AP)的发病机制是个复杂的、多因素参与的病理生理过程,其发病机制至今尚未完全阐明。新近提出的“炎症介质或细胞因子学说”和“钙超载学说”是近年关注的热点,在这些学说中有何种机制发挥重要作用呢?本课题组的前期动物实验表明ghrelin在大鼠AP胰腺组织中表达增高,且其表达水平随着AP病情程度的加重而增加[1]。既往已有动物实验研究证实外源性给予ghrelin可以减少AP炎症介质和炎症因子的释放,也可以引起胰腺腺泡细胞内钙离子增高。由于ghrelin的广泛分布及其所具有的多种生物学功能,我们推测内源性ghrelin在AP的发生发展中可能发挥了重要作用,而其作用机理是否与此类似?本文拟对此展开研究。
第一部分体外急性胰腺炎胰腺腺泡细胞模型构建及细胞模型内ghrelin的表达
目的：应用雨蛙肽或脂多糖诱导大鼠AR42J胰腺腺泡细胞,以构建体外AP胰腺腺泡细胞模型；研究ghrelin在正常AR42J细胞以及在雨蛙肽诱导AR42J细胞构建的AP胰腺腺泡细胞模型内的表达情况。方法：应用不同浓度雨蛙肽(0,10-8,10-7,10-6mol/L)或脂多糖(0,1,10,100mg/L)刺激大鼠AR42J细胞24h,采用底物酶法测定细胞上清液淀粉酶含量,RT-PCR方法测定细胞内核转录因子B (nuclear factor kappa B, NF-kB)p65、磷酸化IkB-α、L型钙通道(L-type calcium channel, Cav1.2,Cav1.3)、P/Q型钙通道(P/Q-type calcium channel, Cav2.1)、N型钙通道(N-type calcium channel, Cav2.2)、T型钙通道(T-type calcium channel,Cav3.1, Cav3.2)、 ghrelin mRNA表达水平,Western Blot方法测定细胞内NF-kB p65、磷酸化IkB-α、Cav1.2、Cav1.3、ghrelin蛋白表达水平,Elisa方法测定细胞培养上清液白介素(interleukin, IL)-1β、IL-6、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)蛋白表达水平,Fluo-4-am钙离子荧光探针联合激光共聚焦扫描显微镜测定细胞内钙离子浓度([Ca2+]i)。
结果：1.不同浓度雨蛙肽或脂多糖干预AR42J细胞24h后,细胞培养液上清中淀粉酶含量与对照组相比无显著差异(P＞0.05)。2.与对照组相比,不同浓度雨蛙肽作用24小时后,AR42J细胞NF-kB p65、IKB-α, Cav1.2、 Cav1.3、Cav2.1、Cav3.1、Cav3.2mRNA (F=9.259,15.370,19.189,14.735,42.459,23.000,16.63, P<0.05)及NF-kB p65、IkB-α、Cav1.2、Cav1.3蛋白表达水平(F=7.325,11.341,7.862,42.616,P＜0.05)均明显增高；部分因子组内比较(NF-kB p65、Cav1.3、Cav2.1、Cav3.2mRNA及NF-kB p65、 IkB-α、Cav1.2、Cav1.3)呈剂量依赖性增高(P<0.05), Cav2.2mRNA表达无明显变化(F=2.121,P=0.138＞0.05)；不同浓度脂多糖作用24小时后的AR42J细胞上述基因mRNA表达水平均无明显变化(F=0.552,0.174,0.491,0.182,0.185,0.509,0.125,0.350,P＞0.05)。3.不同浓度雨蛙肽作用24小时后,AR42J细胞培养上清液中IL-1β、IL-6蛋白表达水平较对照组均明显增高,并呈剂量依赖性(F=102.185,349.787,P＜0.05)；不同浓度脂多糖对上述炎症因子蛋白表达无显著影响(F=1.720,0.514;P=0.203,0.679>0.05); Elisa检测各组AR42J细胞TNF-a的OD值过低,无法计算出相应的蛋白浓度。4.不同浓度雨蛙肽作用24小时后,AR42J细胞内[Ca2+]i较对照组均显著增高,且不同浓度雨蛙肽组间[Ca2+]i比较呈剂量依赖性增高(F=421.939,P＜0.05)。5.雨蛙肽处理组AR42J细胞ghrelin mRNA和蛋白表达水平较对照组明显升高(F=45.128,67.2358,P＜0.05),并呈剂量依赖性(P<0.05)。
结论：1.雨蛙肽和脂多糖刺激AR42J细胞前后,细胞培养上清液淀粉酶无明显变化；2.雨蛙肽刺激能引起AR42J细胞多种炎症因子和钙通道基因mRNA、蛋白表达水平以及[Ca2+]i升高,提示AP胰腺腺泡细胞模型构建成功；3.脂多糖刺激对AR42J细胞多种炎症介质和钙通道基因mRNA及蛋白表达无显著影响；4. ghrelin在大鼠胰腺腺泡细胞AR42J中存在高表达,雨蛙肽刺激AR42J细胞后ghrelin mRNA和蛋白表达水平较对照组升高。
第二部分ghrelin miRNA慢病毒干扰载体的构建、筛选及对AR42J细胞的靶向干扰效果
目的：筛选有效的大鼠ghrelin miRNA序列,构建ghrelin miRNA慢病毒载体；应用构建的慢病毒ghrelin miRNA载体转染AR42J细胞,观察其对细胞内ghrelin基因表达的抑制情况。
方法：1.针对ghrelin基因构建四个干扰质粒：pcDNATM6.2-X207-1-3, pcDNATM6.2-X207-2-1, pcDNATM6.2-X207-3-1, pcDNATM6.2-X207-4-1;构建高表达载体pCDNA3.1(+);干扰载体和高表达载体共转染HEK293细胞,qPCR检测目的基因的表达情况,筛选出最佳干扰载体X207-1-3；使用BP重组系统和LR重组系统将目的片段从miRNA载体重组到慢病毒载体pLenti6.3/v5DEST上,筛选阳性克隆并测序验证；用构建的慢病毒表达载体pLenti6.3-X207-1和包装质粒共转染293T细胞,包装病毒,收集病毒原液,超速离心浓缩,并测定滴度。2.将AR42J细胞分为5组：空白对照组、阴性对照组(MOI=100)、ghrelin-miRNA A组(MOI=50)、ghrelin-miRNA B组(MOI=80)、 ghrelin-miRNA C组(MOI=100)；应用RT-PCR和Western Blot方法分别检测各组细胞内ghrelin mRNA和蛋白的表达水平。
结果：1.qPCR检测4个miRNA序列对ghrelin基因的沉默效应,其中以pcDNATM6.2-X207-1-3下调最为明显(86%)。2.选择沉默效应最明显(86%)的序列包装慢病毒,病毒滴度为1×108TU/ml。3. ghrelin miRNA能被重组慢病毒高效地转染入AR42J细胞,转染效率高达80%,RT-PCR和Western Blot结果显示靶基因慢病毒转染C组(MOI=100)细胞内ghrelin mRNA和蛋白表达的干扰效果最明显(P＜0.05)。
结论：成功构建慢病毒ghrelin miRNA载体,并包装出高滴度病毒,为后续实验奠定基础；构建的慢病毒ghrelin miRNA载体可以有效沉默AR42J细胞内的ghrelin基因。
第三部分靶向干扰ghrelin基因对急性胰腺炎胰腺腺泡细胞炎症通路及钙通路的影响
目的：研究靶向抑制ghrelin表达对雨蛙肽刺激AR42J细胞构建的AP胰腺腺泡细胞模型内炎症通路及钙通路的变化。
方法：1.将AR42J细胞分为4组：空白对照组、雨蛙肽组、阴性对照+雨蛙肽组、ghrelin-miRNA+雨蛙肽组。2.应用RT-PCR和Western Blot方法分别检测各组细胞内NF-kB p65、磷酸化IkB-α、Cav1.2、Cav1.3. Cav2.1mRNA及蛋白的表达,Elisa方法检测各组细胞上清液IL-1β、IL-6、TNF-α蛋白的表达。3. Calcium CrimsonTM, AM钙离子荧光探针标记联合激光共聚焦扫描显微镜测定各组[Ca2+]i。
结果：与雨蛙肽组和阴性对照+雨蛙肽组相比,ghrelin-miRNA+雨蛙肽组AR42J细胞：NF-KB p65、磷酸化IKB-αmRNA表达水平(F=51.255,9.266；P<0.05)和蛋白表达水平显著升高(F=7.533,18.876；P<0.05),细胞上清液IL-1β、IL-6蛋白表达水平显著升高(F=334.554,336.462；P＜0.05)；Cav1.2、Cav1.3、Cav2.1mRNA表达水平(F=37.208,55.651,42.640；P<0.05)及Cavl.2、Cav1.3蛋白表达水平明显降低(F=6.894,64.239; P<0.05);[Ca2+]i明显降低(F=61.358,P<0.05)。
结论：靶向抑制AP胰腺腺泡细胞内ghrelin基因的表达,一方面可以上调细胞内炎症因子的表达,另一方面可以减轻其细胞内钙超载。
第四部分全基因组基因芯片分析靶向干扰ghrelin基因对急性胰腺炎胰腺腺泡细胞基因表达谱特征的影响
目的：筛选靶向抑制ghrelin表达的AP胰腺腺泡细胞内基因表达差异,并进一步分析相关信号通路,以探讨其分子生物学机制。
方法：1.将AR42J细胞分为2组：ghrelin-miRNA+雨蛙肽组、阴性对照+雨蛙肽组。2.实验干预后收集细胞提取RNA,并进行RNA质量鉴定。将总RNA合成为ds-DNA后进行标记,并与NimbleGen大鼠全基因组基因芯片杂交,运用Axon GenePix4000B基因芯片扫描仪进行扫描。3.所有基因水平文件输入Agilent GeneSpring GX11.5.1软件进行聚类分级,进一步进行GO分析和通路分析。
结果：1. NimbleGen全基因组基因芯片筛选出ghrelin基因沉默及雨蛙肽诱导的AR42J细胞差异表达基因上调或下调2倍以上基因共2938个,其中表达上调基因1435个,表达下调基因1503个。2.GO分析结果显示差异表达基因在biological process分类中,与各种代谢过程相关的基因出现最多,其次为生物调节、进化过程、细胞过程调节等；在cellular component分类中,改变最为显著的差异表达基因主要位于细胞及细胞内部分、细胞核、膜型细胞器等细胞成分中；在molecular function分类中,与结合相关的基因差异最多,包括蛋白结合、金属离子结合、阳离子结合、核苷酸结合、小分子结合等。3. Pathway分析结果显示表达上调基因涉及31个信号通路,表达下调基因涉及30个信号通路。4.通过筛查与炎症和钙通路相关的差异表达基因,结果显示,与CAMP/Ca2+信号通路相关的差异表达基因有60个,与炎症通路相关的差异表达基因有199个。
结论：通过芯片结果、Pathway及GO分析等初步筛查发现：ghrelin对AP胰腺腺泡细胞的作用机制可能与细胞内代谢过程及离子、蛋白等结合功能有关；相关通路可能包括MAPK信号通路、细胞间粘附分子信号通路、焦点连接、Wnt信号通路、P53信号通路等。
Practice and studies have shown that the pathogenesis of acute pancreatitis (AP) was a pathophysiological process involved in complex and multi-factor. And its pathogenesis has not been fully elucidated. The theories of "Inflammatory mediators or cytokines" and "calcium overload" are the focus of attention in the recent years. What mechanism will play an important role in these theories? Our previous animal experiments showed that expression of ghrelin was increased in the rats'pancreatic tissue of AP, and its expression levels were increased with the severity of AP. Previous studies have verified that exogenous of ghrelin could reduce the release of inflammatory mediators and inflammatory factors in AP, furthermore, it could also increased calcium in pancreatic acinar cells. Because ghrelin is widely distributed and has a variety of biological functions, we hypothesize that endogenous ghrelin may play an important role in the development of AP, and whether its mechanism is similar to exogenous ghrelin? This article intends to study it.
Part One
Pancreatic Acinar Cell Model Building of Acute Pancreatitis in Vitro and Expression of Ghrelin in This Cell Model
Objective:Rats'pancreatic acinar cells AR42J were induced by caerulin (CAE) or lipopolysaccharide (LPS) in order to build the pancreatic acinar cell model of AP in vitro. Study the expression of ghrelin in AR42J cells as well as builted in AR42J cells induced by caerulin.
Methods:AR42J cells were induced by different concentration of CAE (0,10-8,10-7,10-6mol/L)or LPS(0,1,10,100mg/L)for24hrs.Amylase(AMS) content in cell supernatant was measured by bottom objects enzymatic. Expression of NF-kB p65, phosphorylation IkB-α, Cavl.2, Cav1.3, Cav2.1, Cav2.2Cav3.1Cav3.2and ghrelin mRNA was determined by RT-PCR. Proteins expression of NF-kB p65, phosphorylation IkB-α, Cav1.2, Cav1.3and ghrelin were determined by Western Blot. Proteins expression of IL-1β, IL-6, TNF-α in cell culture supernatant were determined by Elisa.[Ca2+]i was determined by calcium fluorescence probe Fluo-4-am loading combined with confocal laser scanning microscope (CLSM).
Results:1.There was no statistically significant difference with AMS between control group cells and cells induced by CAE or LPS(P>0.05).2. Compared to control group, expression of NF-kB p65, IkB-α, Cav1.2, Cav1.3, Cav2.1,Cav3.1,Cav3.2mRNA(F=9.259,15.370,19.189,14.735,42.459,23.000,16.63, P<0.05) and NF-kB p65, IkB-α, Cav1.2, Cav1.3proteins (F=7.325,11.341,7.862,42.616, P<0.05) were increased significantly. Comparison in each group, some factors were dose-dependently increased (NF-kB p65, Cav1.3, Cav2.1, Cav3.2mRNA and NF-kB p65, IkB-α, Cavl.2, Cavl.3proteins, P<0.05). There was no significant difference (F=2.121, P=0.138>0.05) in Cav2.2mRNA. There was no significant difference in mRNA expression of aboved genes between control group cells and cells induced by LPS(F=0.552,0.174,0.491,0.182,0.185,0.509,0.125,0.350, P>0.05).3. Proteins expression of IL-1β and IL-6were increased significantly in AR42J cells induced by CAE, which was a dose-dependent manner (F=102.185,349.787, P<0.05). However, there was no significant difference in proteins expression of IL-1β and IL-6in AR42J cells induced by LPS (F=1.720,0.514, P=0.203,0.679>0.05). Because of OD value of TNF-a was too low, the corresponding protein concentration can not be calculated.4. Compared to control group,[Ca2+]i was increased significantly in AR42J cells induced by CAE, which was a dose-dependent manner (F=21.939, P＜0.05).5. Expressions of ghrelin mRNA and protein were increased in AR42J cells induced by CAE, which was a dose-dependent manner (F=45.128,67.2358, P<0.05).
Conclusions:1. There is no significant change of AMS in cell culture supernatant between normal AR42J cells and AR42J cells induced by CAE or LPS.2. CAE can enhance mRNA and protein levels of a variety of inflammatory cytokines and calcium channel, as well as [Ca2+]i in AR42J cells. It may indicate pancreatic acinar cell model of acute pancreatitis to be built successfully.3. LPS can not affect mRNA and protein levels of a variety of inflammatory cytokines and calcium channel in AR42J cells.4. High expression of ghrelin in AR42J cells. Compared with control group, ghrelin mRNA and protein expression levels are elevated in AR42J cells induced by CAE.
Part Two
Building and Screen of Lentiviral Interference Vector of Ghrelin miRNA and Its Targeted Interference Effect in AR42J Cells
Objective:To screen the effective sequence of ghrelin miRNA of rat in order to build lentiviral vector of ghrelin miRNA. After AR42J cells were transfected with the lentiviral vector of ghrelin miRNA, we observed interference effect of ghrelin gene in cells.
Methods:l.Four interference plasmids were constructed: pcDNATM6.2-X207-1-3,pcDNATM6.2-X207-2-1,pcDNATM6.2-X207-3-1,pcDN ATM6.2-X207-4-1. Expression vector pCDNA3.1(+) was constructed; After HEK293cells were co-transfected with the interference vector and expression vector, expression of target gene was determined by qPCR so as to screen out the best interference vector X207-1-3. Target fragment was restructured from miRNA vector to the lentiviral vector pLenti6.3/v5DEST through BP recombination system and LR recombination system, and positive clones were screened out and sequenced. After293T cells were cotransfected with lentiviral vector pLenti6.3-X207-1and packaging plasmid, packing virus, collecting virus stoste, ultracentrifugation to concentrate, and determining the titer.2. AR42J cells were divided into5groups:blank control group, negative control group (MOI=100), ghrelin-miRNA group A (MOI=50), ghrelin-miRNA group B (MOI=80), ghrelin-miRNA C group (MOI=100). Expression of ghrelin mRNA and protein in each group was detected by RT-PCR and Western Blot.
Results:1.The four miRNA sequences to ghrelin gene silencing effection were detected by qPCR and the result showed that pcDNA TM6.2-X207-1-3was the most significant silencing effected sequence (86%).2. To pack lentivirus with the most obvious silencing effected sequence (86%) and the viral titer was1×108TU/ml.3. Ghrelin miRNA could be transfected efficiently into AR42J cells. And the transfection efficiency was up to80%. The results of RT-PCR and Western Blot showed that the most significant interference effect of ghrelin mRNA and protein expression in AR42J cells transfected by ghrelin-miRNA C group (MOI=100)(P<0.05).
Conclusions:We successfully built lentiviral vector of ghrelin-miRNA and packed high titers of virus, so as to lay the foundation for the subsequent experiments. The lentiviral vector of ghrelin-miRNA can effectively silence ghrelin gene in AR42J cells.
Part Three
Effects of Ghrelin Gene Silence on Inflammation Pathway and Calcium Channel in Pancreatic Acinar Cells of Acute Pancreatitis
Objective:Study the changes of the inflammation pathway and calcium channel by ghrelin gene targeting suppression in pancreatic acinar cell model of acute pancreatitis induced by caerulin.
Method:1. AR42J cells were divided into four groups:blank control group, CAE group, negative control (NG)+CAE group and miRNA+CAE group.2. Expression of NF-κB p65, phosphorylation IκB-α, Cav1.2, Cav1.3, Cav2.1mRNA and proteins was determined by RT-PCR and Western Blot. Proteins expression of IL-1β, IL-6and TNF-αwere determined by Elisa.3.[Ca2+]i in AR42J cells of each group was determined by Calcium Crimson TM, AM calcium fluorescent probe loading combined with confocal laser scanning microscope.
Results:Compared with CAE group and NC+CAE group, the following results of miRNA+CAE group showed that:expression of NF-KBp65and IkB-α mRNA (F=51.255,9.266, P<0.05) and proteins (F=7.533,18.876, P<0.05) was elevated; proteins expression of IL-1β, IL-6in cell culture supernatant were elevated (F=334.554,336.462, P<0.05); expression of Cav1.2, Cav1.3, Cav2.1mRNA (F=37.208,55.651,42.640, P<0.05) and Cav1.2, Cavl.3proteins (F=6.894,64.239, P<0.05) were significantly decreased;[Ca2+]i in cells was significantly decreased (F=61.358, P<0.05).
Conclusions:Targeting suppression of ghrelin gene in pancreatic acinar cells of acute pancreatitis can up-regulate the expression of inflammatory cytokines, as well as can reduce calcium overload in cells.
Part Four
Analysing the expression profiles characteristic of ghrelin gene silencing gene in pancreatic acinar cells of AP with whole genome microarray
Objective:Screen out differentially expressed genes of targeting suppression of ghrelin gene in pancreatic acinar cells of acute pancreatitis. And further analysis of the relevant signal pathway was employed to explore the mechanism of molecular biology.
Method:1. AR42J cells were divided into two groups:miRNA+CAE group and NC+CAE group.2. AR42J cells were collected to extract RNA after intervention of lentivirus and CAE. RNA quality was assessed. Synthesize total RNA into ds-DNA and mark it which was hybridized with NimbleGen rat whole genome microarray. Then, we scanned the microarray by Axon GenePix4000B gene chip scanner.3. All gene-level files were input Agilent GeneSpring GX11.5.1software to clustering classify, and further GO analysis and pathway analysis were worked.
Results:1. There are2938genes of differentially expression including more than tow times of up-regulation (1435) and down-regulation (1503).2. The results of GO analysis showed that the differentially expressed genes which were associated with various metabolic processes appeared most frequently in the kind of biological process, followed by biological regulation, evolutionary process, regulation of cell process. In the kind of cellular component, the differentially expressed genes of the most significant change mainly located in the cells, nucleus, membrane type organelles and other cellular components. In the kind of molecular function, the most amount of differentially expression genes was associated with combination, including protein binding, metal ion binding, cation binding, nucleotide binding and small molecules binding.3. The results of pathway analysis showed that31signaling pathways involved in up-regulated genes and30signaling pathways involved in down-regulated genes.4. There are60differentially expressed genes involved in CAMP/Ca2+signal pathway and199differentially expressed genes involved in inflammation pathway.
Conclusions:Through the results of gene chip, pathway and GO analysis, we preliminarily speculate that the mechanism of ghrelin on pancreatic acinar cells of acute pancreatitis may be related with metabolic processes in cells and binding of ion, protein, etc. And the related pathways of this mechanism may include MAPK signal pathway, cell adhesion molecules signal pathway, focal adhesion, Wnt signal pathway and p53signal pathway, etc.
第一部分体外急性胰腺炎胰腺腺泡细胞模型构建及细胞模型内ghrelin的表达
目的：应用雨蛙肽或脂多糖诱导大鼠AR42J胰腺腺泡细胞,以构建体外AP胰腺腺泡细胞模型；研究ghrelin在正常AR42J细胞以及在雨蛙肽诱导AR42J细胞构建的AP胰腺腺泡细胞模型内的表达情况。方法：应用不同浓度雨蛙肽(0,10-8,10-7,10-6mol/L)或脂多糖(0,1,10,100mg/L)刺激大鼠AR42J细胞24h,采用底物酶法测定细胞上清液淀粉酶含量,RT-PCR方法测定细胞内核转录因子B (nuclear factor kappa B, NF-kB)p65、磷酸化IkB-α、L型钙通道(L-type calcium channel, Cav1.2,Cav1.3)、P/Q型钙通道(P/Q-type calcium channel, Cav2.1)、N型钙通道(N-type calcium channel, Cav2.2)、T型钙通道(T-type calcium channel,Cav3.1, Cav3.2)、 ghrelin mRNA表达水平,Western Blot方法测定细胞内NF-kB p65、磷酸化IkB-α、Cav1.2、Cav1.3、ghrelin蛋白表达水平,Elisa方法测定细胞培养上清液白介素(interleukin, IL)-1β、IL-6、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)蛋白表达水平,Fluo-4-am钙离子荧光探针联合激光共聚焦扫描显微镜测定细胞内钙离子浓度([Ca2+]i)。
结果：1.不同浓度雨蛙肽或脂多糖干预AR42J细胞24h后,细胞培养液上清中淀粉酶含量与对照组相比无显著差异(P＞0.05)。2.与对照组相比,不同浓度雨蛙肽作用24小时后,AR42J细胞NF-kB p65、IKB-α, Cav1.2、 Cav1.3、Cav2.1、Cav3.1、Cav3.2mRNA (F=9.259,15.370,19.189,14.735,42.459,23.000,16.63, P<0.05)及NF-kB p65、IkB-α、Cav1.2、Cav1.3蛋白表达水平(F=7.325,11.341,7.862,42.616,P＜0.05)均明显增高；部分因子组内比较(NF-kB p65、Cav1.3、Cav2.1、Cav3.2mRNA及NF-kB p65、 IkB-α、Cav1.2、Cav1.3)呈剂量依赖性增高(P<0.05), Cav2.2mRNA表达无明显变化(F=2.121,P=0.138＞0.05)；不同浓度脂多糖作用24小时后的AR42J细胞上述基因mRNA表达水平均无明显变化(F=0.552,0.174,0.491,0.182,0.185,0.509,0.125,0.350,P＞0.05)。3.不同浓度雨蛙肽作用24小时后,AR42J细胞培养上清液中IL-1β、IL-6蛋白表达水平较对照组均明显增高,并呈剂量依赖性(F=102.185,349.787,P＜0.05)；不同浓度脂多糖对上述炎症因子蛋白表达无显著影响(F=1.720,0.514;P=0.203,0.679>0.05); Elisa检测各组AR42J细胞TNF-a的OD值过低,无法计算出相应的蛋白浓度。4.不同浓度雨蛙肽作用24小时后,AR42J细胞内[Ca2+]i较对照组均显著增高,且不同浓度雨蛙肽组间[Ca2+]i比较呈剂量依赖性增高(F=421.939,P＜0.05)。5.雨蛙肽处理组AR42J细胞ghrelin mRNA和蛋白表达水平较对照组明显升高(F=45.128,67.2358,P＜0.05),并呈剂量依赖性(P<0.05)。
结论：1.雨蛙肽和脂多糖刺激AR42J细胞前后,细胞培养上清液淀粉酶无明显变化；2.雨蛙肽刺激能引起AR42J细胞多种炎症因子和钙通道基因mRNA、蛋白表达水平以及[Ca2+]i升高,提示AP胰腺腺泡细胞模型构建成功；3.脂多糖刺激对AR42J细胞多种炎症介质和钙通道基因mRNA及蛋白表达无显著影响；4. ghrelin在大鼠胰腺腺泡细胞AR42J中存在高表达,雨蛙肽刺激AR42J细胞后ghrelin mRNA和蛋白表达水平较对照组升高。
第二部分ghrelin miRNA慢病毒干扰载体的构建、筛选及对AR42J细胞的靶向干扰效果
目的：筛选有效的大鼠ghrelin miRNA序列,构建ghrelin miRNA慢病毒载体；应用构建的慢病毒ghrelin miRNA载体转染AR42J细胞,观察其对细胞内ghrelin基因表达的抑制情况。
方法：1.针对ghrelin基因构建四个干扰质粒：pcDNATM6.2-X207-1-3, pcDNATM6.2-X207-2-1, pcDNATM6.2-X207-3-1, pcDNATM6.2-X207-4-1;构建高表达载体pCDNA3.1(+);干扰载体和高表达载体共转染HEK293细胞,qPCR检测目的基因的表达情况,筛选出最佳干扰载体X207-1-3；使用BP重组系统和LR重组系统将目的片段从miRNA载体重组到慢病毒载体pLenti6.3/v5DEST上,筛选阳性克隆并测序验证；用构建的慢病毒表达载体pLenti6.3-X207-1和包装质粒共转染293T细胞,包装病毒,收集病毒原液,超速离心浓缩,并测定滴度。2.将AR42J细胞分为5组：空白对照组、阴性对照组(MOI=100)、ghrelin-miRNA A组(MOI=50)、ghrelin-miRNA B组(MOI=80)、 ghrelin-miRNA C组(MOI=100)；应用RT-PCR和Western Blot方法分别检测各组细胞内ghrelin mRNA和蛋白的表达水平。
结果：1.qPCR检测4个miRNA序列对ghrelin基因的沉默效应,其中以pcDNATM6.2-X207-1-3下调最为明显(86%)。2.选择沉默效应最明显(86%)的序列包装慢病毒,病毒滴度为1×108TU/ml。3. ghrelin miRNA能被重组慢病毒高效地转染入AR42J细胞,转染效率高达80%,RT-PCR和Western Blot结果显示靶基因慢病毒转染C组(MOI=100)细胞内ghrelin mRNA和蛋白表达的干扰效果最明显(P＜0.05)。
结论：成功构建慢病毒ghrelin miRNA载体,并包装出高滴度病毒,为后续实验奠定基础；构建的慢病毒ghrelin miRNA载体可以有效沉默AR42J细胞内的ghrelin基因。
第三部分靶向干扰ghrelin基因对急性胰腺炎胰腺腺泡细胞炎症通路及钙通路的影响
目的：研究靶向抑制ghrelin表达对雨蛙肽刺激AR42J细胞构建的AP胰腺腺泡细胞模型内炎症通路及钙通路的变化。
方法：1.将AR42J细胞分为4组：空白对照组、雨蛙肽组、阴性对照+雨蛙肽组、ghrelin-miRNA+雨蛙肽组。2.应用RT-PCR和Western Blot方法分别检测各组细胞内NF-kB p65、磷酸化IkB-α、Cav1.2、Cav1.3. Cav2.1mRNA及蛋白的表达,Elisa方法检测各组细胞上清液IL-1β、IL-6、TNF-α蛋白的表达。3. Calcium CrimsonTM, AM钙离子荧光探针标记联合激光共聚焦扫描显微镜测定各组[Ca2+]i。
结果：与雨蛙肽组和阴性对照+雨蛙肽组相比,ghrelin-miRNA+雨蛙肽组AR42J细胞：NF-KB p65、磷酸化IKB-αmRNA表达水平(F=51.255,9.266；P<0.05)和蛋白表达水平显著升高(F=7.533,18.876；P<0.05),细胞上清液IL-1β、IL-6蛋白表达水平显著升高(F=334.554,336.462；P＜0.05)；Cav1.2、Cav1.3、Cav2.1mRNA表达水平(F=37.208,55.651,42.640；P<0.05)及Cavl.2、Cav1.3蛋白表达水平明显降低(F=6.894,64.239; P<0.05);[Ca2+]i明显降低(F=61.358,P<0.05)。
结论：靶向抑制AP胰腺腺泡细胞内ghrelin基因的表达,一方面可以上调细胞内炎症因子的表达,另一方面可以减轻其细胞内钙超载。
第四部分全基因组基因芯片分析靶向干扰ghrelin基因对急性胰腺炎胰腺腺泡细胞基因表达谱特征的影响
目的：筛选靶向抑制ghrelin表达的AP胰腺腺泡细胞内基因表达差异,并进一步分析相关信号通路,以探讨其分子生物学机制。
方法：1.将AR42J细胞分为2组：ghrelin-miRNA+雨蛙肽组、阴性对照+雨蛙肽组。2.实验干预后收集细胞提取RNA,并进行RNA质量鉴定。将总RNA合成为ds-DNA后进行标记,并与NimbleGen大鼠全基因组基因芯片杂交,运用Axon GenePix4000B基因芯片扫描仪进行扫描。3.所有基因水平文件输入Agilent GeneSpring GX11.5.1软件进行聚类分级,进一步进行GO分析和通路分析。
结果：1. NimbleGen全基因组基因芯片筛选出ghrelin基因沉默及雨蛙肽诱导的AR42J细胞差异表达基因上调或下调2倍以上基因共2938个,其中表达上调基因1435个,表达下调基因1503个。2.GO分析结果显示差异表达基因在biological process分类中,与各种代谢过程相关的基因出现最多,其次为生物调节、进化过程、细胞过程调节等；在cellular component分类中,改变最为显著的差异表达基因主要位于细胞及细胞内部分、细胞核、膜型细胞器等细胞成分中；在molecular function分类中,与结合相关的基因差异最多,包括蛋白结合、金属离子结合、阳离子结合、核苷酸结合、小分子结合等。3. Pathway分析结果显示表达上调基因涉及31个信号通路,表达下调基因涉及30个信号通路。4.通过筛查与炎症和钙通路相关的差异表达基因,结果显示,与CAMP/Ca2+信号通路相关的差异表达基因有60个,与炎症通路相关的差异表达基因有199个。
结论：通过芯片结果、Pathway及GO分析等初步筛查发现：ghrelin对AP胰腺腺泡细胞的作用机制可能与细胞内代谢过程及离子、蛋白等结合功能有关；相关通路可能包括MAPK信号通路、细胞间粘附分子信号通路、焦点连接、Wnt信号通路、P53信号通路等。
Practice and studies have shown that the pathogenesis of acute pancreatitis (AP) was a pathophysiological process involved in complex and multi-factor. And its pathogenesis has not been fully elucidated. The theories of "Inflammatory mediators or cytokines" and "calcium overload" are the focus of attention in the recent years. What mechanism will play an important role in these theories? Our previous animal experiments showed that expression of ghrelin was increased in the rats'pancreatic tissue of AP, and its expression levels were increased with the severity of AP. Previous studies have verified that exogenous of ghrelin could reduce the release of inflammatory mediators and inflammatory factors in AP, furthermore, it could also increased calcium in pancreatic acinar cells. Because ghrelin is widely distributed and has a variety of biological functions, we hypothesize that endogenous ghrelin may play an important role in the development of AP, and whether its mechanism is similar to exogenous ghrelin? This article intends to study it.
Part One
Pancreatic Acinar Cell Model Building of Acute Pancreatitis in Vitro and Expression of Ghrelin in This Cell Model
Objective:Rats'pancreatic acinar cells AR42J were induced by caerulin (CAE) or lipopolysaccharide (LPS) in order to build the pancreatic acinar cell model of AP in vitro. Study the expression of ghrelin in AR42J cells as well as builted in AR42J cells induced by caerulin.
Methods:AR42J cells were induced by different concentration of CAE (0,10-8,10-7,10-6mol/L)or LPS(0,1,10,100mg/L)for24hrs.Amylase(AMS) content in cell supernatant was measured by bottom objects enzymatic. Expression of NF-kB p65, phosphorylation IkB-α, Cavl.2, Cav1.3, Cav2.1, Cav2.2Cav3.1Cav3.2and ghrelin mRNA was determined by RT-PCR. Proteins expression of NF-kB p65, phosphorylation IkB-α, Cav1.2, Cav1.3and ghrelin were determined by Western Blot. Proteins expression of IL-1β, IL-6, TNF-α in cell culture supernatant were determined by Elisa.[Ca2+]i was determined by calcium fluorescence probe Fluo-4-am loading combined with confocal laser scanning microscope (CLSM).
Results:1.There was no statistically significant difference with AMS between control group cells and cells induced by CAE or LPS(P>0.05).2. Compared to control group, expression of NF-kB p65, IkB-α, Cav1.2, Cav1.3, Cav2.1,Cav3.1,Cav3.2mRNA(F=9.259,15.370,19.189,14.735,42.459,23.000,16.63, P<0.05) and NF-kB p65, IkB-α, Cav1.2, Cav1.3proteins (F=7.325,11.341,7.862,42.616, P<0.05) were increased significantly. Comparison in each group, some factors were dose-dependently increased (NF-kB p65, Cav1.3, Cav2.1, Cav3.2mRNA and NF-kB p65, IkB-α, Cavl.2, Cavl.3proteins, P<0.05). There was no significant difference (F=2.121, P=0.138>0.05) in Cav2.2mRNA. There was no significant difference in mRNA expression of aboved genes between control group cells and cells induced by LPS(F=0.552,0.174,0.491,0.182,0.185,0.509,0.125,0.350, P>0.05).3. Proteins expression of IL-1β and IL-6were increased significantly in AR42J cells induced by CAE, which was a dose-dependent manner (F=102.185,349.787, P<0.05). However, there was no significant difference in proteins expression of IL-1β and IL-6in AR42J cells induced by LPS (F=1.720,0.514, P=0.203,0.679>0.05). Because of OD value of TNF-a was too low, the corresponding protein concentration can not be calculated.4. Compared to control group,[Ca2+]i was increased significantly in AR42J cells induced by CAE, which was a dose-dependent manner (F=21.939, P＜0.05).5. Expressions of ghrelin mRNA and protein were increased in AR42J cells induced by CAE, which was a dose-dependent manner (F=45.128,67.2358, P<0.05).
Conclusions:1. There is no significant change of AMS in cell culture supernatant between normal AR42J cells and AR42J cells induced by CAE or LPS.2. CAE can enhance mRNA and protein levels of a variety of inflammatory cytokines and calcium channel, as well as [Ca2+]i in AR42J cells. It may indicate pancreatic acinar cell model of acute pancreatitis to be built successfully.3. LPS can not affect mRNA and protein levels of a variety of inflammatory cytokines and calcium channel in AR42J cells.4. High expression of ghrelin in AR42J cells. Compared with control group, ghrelin mRNA and protein expression levels are elevated in AR42J cells induced by CAE.
Part Two
Building and Screen of Lentiviral Interference Vector of Ghrelin miRNA and Its Targeted Interference Effect in AR42J Cells
Objective:To screen the effective sequence of ghrelin miRNA of rat in order to build lentiviral vector of ghrelin miRNA. After AR42J cells were transfected with the lentiviral vector of ghrelin miRNA, we observed interference effect of ghrelin gene in cells.
Methods:l.Four interference plasmids were constructed: pcDNATM6.2-X207-1-3,pcDNATM6.2-X207-2-1,pcDNATM6.2-X207-3-1,pcDN ATM6.2-X207-4-1. Expression vector pCDNA3.1(+) was constructed; After HEK293cells were co-transfected with the interference vector and expression vector, expression of target gene was determined by qPCR so as to screen out the best interference vector X207-1-3. Target fragment was restructured from miRNA vector to the lentiviral vector pLenti6.3/v5DEST through BP recombination system and LR recombination system, and positive clones were screened out and sequenced. After293T cells were cotransfected with lentiviral vector pLenti6.3-X207-1and packaging plasmid, packing virus, collecting virus stoste, ultracentrifugation to concentrate, and determining the titer.2. AR42J cells were divided into5groups:blank control group, negative control group (MOI=100), ghrelin-miRNA group A (MOI=50), ghrelin-miRNA group B (MOI=80), ghrelin-miRNA C group (MOI=100). Expression of ghrelin mRNA and protein in each group was detected by RT-PCR and Western Blot.
Results:1.The four miRNA sequences to ghrelin gene silencing effection were detected by qPCR and the result showed that pcDNA TM6.2-X207-1-3was the most significant silencing effected sequence (86%).2. To pack lentivirus with the most obvious silencing effected sequence (86%) and the viral titer was1×108TU/ml.3. Ghrelin miRNA could be transfected efficiently into AR42J cells. And the transfection efficiency was up to80%. The results of RT-PCR and Western Blot showed that the most significant interference effect of ghrelin mRNA and protein expression in AR42J cells transfected by ghrelin-miRNA C group (MOI=100)(P<0.05).
Conclusions:We successfully built lentiviral vector of ghrelin-miRNA and packed high titers of virus, so as to lay the foundation for the subsequent experiments. The lentiviral vector of ghrelin-miRNA can effectively silence ghrelin gene in AR42J cells.
Part Three
Effects of Ghrelin Gene Silence on Inflammation Pathway and Calcium Channel in Pancreatic Acinar Cells of Acute Pancreatitis
Objective:Study the changes of the inflammation pathway and calcium channel by ghrelin gene targeting suppression in pancreatic acinar cell model of acute pancreatitis induced by caerulin.
Method:1. AR42J cells were divided into four groups:blank control group, CAE group, negative control (NG)+CAE group and miRNA+CAE group.2. Expression of NF-κB p65, phosphorylation IκB-α, Cav1.2, Cav1.3, Cav2.1mRNA and proteins was determined by RT-PCR and Western Blot. Proteins expression of IL-1β, IL-6and TNF-αwere determined by Elisa.3.[Ca2+]i in AR42J cells of each group was determined by Calcium Crimson TM, AM calcium fluorescent probe loading combined with confocal laser scanning microscope.
Results:Compared with CAE group and NC+CAE group, the following results of miRNA+CAE group showed that:expression of NF-KBp65and IkB-α mRNA (F=51.255,9.266, P<0.05) and proteins (F=7.533,18.876, P<0.05) was elevated; proteins expression of IL-1β, IL-6in cell culture supernatant were elevated (F=334.554,336.462, P<0.05); expression of Cav1.2, Cav1.3, Cav2.1mRNA (F=37.208,55.651,42.640, P<0.05) and Cav1.2, Cavl.3proteins (F=6.894,64.239, P<0.05) were significantly decreased;[Ca2+]i in cells was significantly decreased (F=61.358, P<0.05).
Conclusions:Targeting suppression of ghrelin gene in pancreatic acinar cells of acute pancreatitis can up-regulate the expression of inflammatory cytokines, as well as can reduce calcium overload in cells.
Part Four
Analysing the expression profiles characteristic of ghrelin gene silencing gene in pancreatic acinar cells of AP with whole genome microarray
Objective:Screen out differentially expressed genes of targeting suppression of ghrelin gene in pancreatic acinar cells of acute pancreatitis. And further analysis of the relevant signal pathway was employed to explore the mechanism of molecular biology.
Method:1. AR42J cells were divided into two groups:miRNA+CAE group and NC+CAE group.2. AR42J cells were collected to extract RNA after intervention of lentivirus and CAE. RNA quality was assessed. Synthesize total RNA into ds-DNA and mark it which was hybridized with NimbleGen rat whole genome microarray. Then, we scanned the microarray by Axon GenePix4000B gene chip scanner.3. All gene-level files were input Agilent GeneSpring GX11.5.1software to clustering classify, and further GO analysis and pathway analysis were worked.
Results:1. There are2938genes of differentially expression including more than tow times of up-regulation (1435) and down-regulation (1503).2. The results of GO analysis showed that the differentially expressed genes which were associated with various metabolic processes appeared most frequently in the kind of biological process, followed by biological regulation, evolutionary process, regulation of cell process. In the kind of cellular component, the differentially expressed genes of the most significant change mainly located in the cells, nucleus, membrane type organelles and other cellular components. In the kind of molecular function, the most amount of differentially expression genes was associated with combination, including protein binding, metal ion binding, cation binding, nucleotide binding and small molecules binding.3. The results of pathway analysis showed that31signaling pathways involved in up-regulated genes and30signaling pathways involved in down-regulated genes.4. There are60differentially expressed genes involved in CAMP/Ca2+signal pathway and199differentially expressed genes involved in inflammation pathway.
Conclusions:Through the results of gene chip, pathway and GO analysis, we preliminarily speculate that the mechanism of ghrelin on pancreatic acinar cells of acute pancreatitis may be related with metabolic processes in cells and binding of ion, protein, etc. And the related pathways of this mechanism may include MAPK signal pathway, cell adhesion molecules signal pathway, focal adhesion, Wnt signal pathway and p53signal pathway, etc.
引文
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