【作者】 张旭；

【导师】 张宏伟；

【作者基本信息】 郑州大学 ， 外科学（肝胆胰腺外科）（专业学位）， 2018， 硕士

【摘要】 目的研究长链非编码RNA-MEG3(lncRNA-MEG3,lncR-MEG3)在急性胰腺炎中的临床意义,及其同系物LncR-RGD1566401对大鼠胰腺外分泌细胞的凋亡作用。方法LncR-RGD1566401是位于大鼠染色体6q32的母系印迹基因,长度为1,915bp,是母系表达基因3(MEG3)的同源类似物。用携带有lncR-RGD1566401目的基因的慢病毒(Lenti-LncRNA)和特异性lncR-RGD1566401抑制剂(lncRNA Smart Silencer)转染AR42J细胞系24h,转染空病毒(Lenti-NC)和特异性lncR-RGD1566401阴性抑制剂(lncRNA smart silencer negative control)作为对照;以100 nmol/L雨蛙肽(Caerulin)体外诱导正常培养AR42J及转染后AR42J的凋亡,使用药物诱导凋亡24h以后,采用流式细胞技术(Flow cytometry,FCM)检测各组AR42J细胞的凋亡率,使用实时荧光定量RT-PCR法检测各组lncR-RGD1566401的表达量,而后采用蛋白质印迹法(Western blot)检测各组凋亡蛋白活性Caspase-3和P53表达量的变化。同时收集郑州大学人民医院2016年1月至2017年1月的急性胰腺炎患者63例,其中重症急性胰腺炎(serve acute pancreatitis,SAP)21例,轻症急性胰腺炎(mild acute pancreatitis,MAP)42例。采用实时荧光定量聚合酶链反应(qRT-PCR)检测患者入院后24h血清lncR-MEG3的表达水平,比较各组间lncR-MEG3、C反应蛋白(CRP)、D-二聚体(D-Dimer)浓度差异。使用SPSS22.0软件进行统计学分析获得的数据,然后制作受试者工作特征曲线(ROC曲线),进一步评价该血清学指标对SAP的诊断效能。结果雨蛙肽体外诱导AR42J凋亡的凋亡率(19.34±0.54)%较对照组(7.23±0.33)%明显升高,lncR-RGD1566401表达量较对照组升高(5.43±0.31)倍,活性Caspase-3和P53表达量明显升高;转染携带有lncR-RGD1566401目的基因的慢病毒组lncR-RGD1566401表达量较空病毒组升高(8.24±0.22)倍(p<0.05);AR42J凋亡率(38.20±0.37)%较空病毒组(22.60±0.96)%明显升高(p<0.05);活性Caspase-3和P53表达量明显升高(p<0.05);应用特异性lncR-RGD1566401抑制剂组较阴性对照组lncR-RGD1566401表达量降低(0.54±0.14)倍,(p<0.05),AR42J凋亡率(13.60±0.33)%较阴性对照组(19.60±0.46)%显著降低,(p<0.05),P53表达量和凋亡蛋白活性Caspase-3明显降低(p<0.05)。在收集的临床血清样本中,患者血清lncR-MEG3的表达水平在SAP组较MAP组显著降低(p<0.05),CRP、D-Dimer浓度显著升高(p<0.05);lncR-MEG3、CRP和D-Dimer联合检测应用于诊断急性重症胰腺炎时的曲线下面积(AUC)高于CRP和D-Dimer联合的AUC,也高于lncR-MEG3、CRP、D-Dimer单独使用时的AUC。结论1.lncR-RGD1566401与大鼠AR42J细胞凋亡相关;2.上调lncR-RGD1566401表达水平,AR42J细胞凋亡率增加;下调lncR-RGD1566401表达量,AR42J细胞凋亡率降低;3.MAP组患者血清lncR-MEG3表达量较SAP组lncR-MEG3表达量升高明显;4.联合lncR-MEG3、CRP、D-Dimer指标诊断急性重症胰腺炎的诊断效能显著高于单独指标或CRP与D-Dimer联合指标;5.lncR-MEG3表达水平可以在早期反映AP患者病情严重程度,lncR-MEG3可能是潜在的作为区分MAP和SAP的血清学生物标志物。更多还原

【Abstract】 Objectives:To explore the clinical significance of RNA-MEG3(lncRNA-MEG3,lncR-MEG3)in acute pancreatitis and the effect of its homologous analogue LncR-RGD1566401 on apoptosis of pancreatic acinar cells(AR42J)in rat acute pancreatitis model.Methods:LncR-RGD1566401 is a maternal blotting gene located in rat chromosome 6q32,with a length of 1,915 bp,and it is a homologous analogue of maternal expression gene 3(MEG3).The GFP LncR-RGD1566401 overexpressed Lentivirus and special LncRNA Smart Silencer were designed and transfected into AR42 J repectively for 24 h.The apoptosis of AR42 J in different groups was established by inducing cells with 100 nmol/L Caerulin for 24 h.The flow cytometry was used to detect the apoptosis of AR42 J in different groups.Real time RT-PCR was used to detect the expression level of lncR-RGD1566401 in AR42 J cells of different groups after transfection.The expression of Caspase3 and P53 was detected by western blot.In this retrospective study,63 patients of acute pancreatitis were consecutively included between Jun 2016 and Jun 2017,including 21 cases of mild acute pancreatitis,severe acute pancreatitis in 42 cases.All patients collected venous blood in 24 hours after admission.Real time RT-PCR was used to detect the expression level of lncR-MEG3 in acute pancreatitis.To analyze the variability among lncR-MEG3、C-reactionprotein(CRP)and D-Dimer in different groups.Using SPSS 22.0 software for statistical analysis,and draw the ROC curve.Results:The apoptosis rate [(19.34±0.54)%] of Caerulin-induced AR42 J elevated compared with the control group[(7.23±0.33)%].The expression of lncR-RGD1566401 in the AR42 J cells was increased by(5.43±0.31)times in the experimental group after dealing with Caerulin and the level of Caspase-3 and P53 were increased obviously.LncR-RGD1566401 level was elevated by(8.24±0.22)times after AR42 J cells were transfected with lenti-LncRNA as compared with the vehicle groups(p<0.05).The AR42 J cells showed more tendency to apoptosis [(38.20±0.37)%] than the vehicle groups[(22.60±0.96)%,p<0.05] and the level of Caspase-3 and P53 were increased obviously.The AR42 J cells that were transfected with Silencer-LncRNA expressed the lower level of lncR-RGD1566401 about [(0.54±0.14),p<0.05]times and less apoptosis [(13.60±0.33)%] than the Silencer-NC groups [(19.60±0.46)%,p<0.05].The level of lncR-MEG3 in MAP group is significantly increased compared with SAP group(p<0.05).The level of CRP and D-Dimer in SAP group is increased obviously compared with MAP group(p>0.05).Combined detection of lncR-MEG3,CRP and D-Dimer in prognosis of severe acute pancreatitis positive rate is higher than detection of CRP and D-Dimer or single positive rate.Conclusion:1.LncR-RGD1566401 is associated with the apoptosis of AR42J;2.Up-regulation of LncR-RGD1566401 can promote the apoptosis of AR42 J,while down-regulation of LncR-RGD1566401 can inhibit the apoptosis of AR42J;3.Patients with MAP have a higher expression level of circulating lncR-MEG3 than SAP.4.Combined detection of lncR-MEG3,CRP and D-Dimer in prognosis of severe acute pancreatitis positive rate is higher than detection of CRP and D-Dimer or single positive rate.5.The level of lncR-MEG3 can be a potential biomarker related to inflammation and used to diagnose SAP from AP.更多还原