Autophagy of MSCs was analyzed by flow cytometry and transmission electron microscopy, and the expression of beclin Ⅰ?and LC3-Ⅱ was detected by Western blot. MSCs were labeled in vivo with the fluorescent dye, CM-Dil, and subsequently transplanted into the portal veins of rats that had undergone HIRI. Liver levels of proliferating cell nuclear antigen(PCNA) were quantified by fluorescent microscopy. Serum selleck抑制剂 aminotransferase activity and the extent of HIRI were also assessed at each time point.RESULTS: HSP for 2 h reduced apoptosis of MSCs induced by H2O2 as seen by a decrease in apoptotic rate, a decrease in Bax and cytochrome C expression

and an increase in Bcl-2 expression(P < 0.001). In addition, HSP for 2 h induced autophagy of MSCs exposed to H2O2 as shown by an increase in acidic vesicular organelle-positive cells, beclin 1 and LC3-Ⅱ expression, and autophagosome formation(P < 0.05). Treatment with 3-methyladenine attenuated HSPinduced autophagy and abolished the protective effects of HSP on the apoptosis of MSCs. Rapamycin failed to have additional effects on either autophagy or apoptosis compared with HSP

alone. The phosphorylation of p38 MAPK was significantly elevated and the phosphorylation of m TOR was downregulated in heat shock pretreated MSCs. Treatment with the p38 MAPK inhibitor, SB203580, reduced HSP-induced autophagy in MSCs. In vivo studies showed that the transplantation of HSP-MSCs Protein Tyrosine激酶抑制剂 resulted in lower serum aminotransferase levels, lower Suzuki scores, improved histopathology and an increase in PCNA-positive

cells(P < 0.05).CONCLUSION: HSP effectively induces autophagy following exposure to H2O2 via the p38MAPK/m TOR pathway, which leads to enhanced MSC survival and improved MSC repair following HIRI in rats.
目的研究参苓白术散抗脂多糖(LPS)诱导肠隐窝上皮细胞(IEC-6)损伤作用及其机制。方法用200μg·mL~(-1)LPS复制模型,将IEC-6细胞分为10组:空白对照组,模型组,低(4%)、中(8%)、高(16%)剂量参苓白术散含药血清组,10%胎牛血清(FBS)阴性对照组,ROCK阻断剂Y27632组,MLCK阻断剂ML-7组,MAPK抑制剂SB203580组,ERK抑制剂PD98059组。通过MTT法检测LPS细胞毒性;用蛋白质印迹法(western BVD 523 blot)检测p38MAPK、ERK1/2通路蛋白水平;用逆转录-聚合酶链式反应(RT-PCR)技术测定肿瘤坏死因子(TNF-α)mRNA的表达;通过激光共聚焦测定细胞骨架结构蛋白纤维肌动蛋白(F-actin)的变化。结果 MTT实验中,不同浓度的LPS对IEC-6有不同程度的抑制、损伤作用。Western blot和RT-PCR结果显示,与空白对照组比较,模型组IEC-6细胞p38MAPK、磷酸化ERK1/2、TNF-αmRNA水平明显上升;而参苓白术散含药血清对其有明显的下调作用(P<0.01,P<0.05)。激光共聚焦检测F-actin实验中,模型组与空白组比较细胞微丝纤维表达显著增多;而参苓白术散含药血清组相对于模型组,细胞微丝纤维表达明显下降。结论参苓白术散含药血清对LPS诱导的IEC-6细胞损伤具有保护作用。
目的探讨血管细胞黏附分子-1(VCAM-1)过表达对小鼠间充质干细胞(MSC)迁移能力的影响,并初步探讨其作用机制。方法将正常小鼠MSC(C3)、转染空载体的小鼠MSC(C3+N)和基因修饰过表达VCAM-1的小鼠MSC(C3+VCAM-1)分别接种在Transwell培养体系培养8和12 h,以胎牛血清为趋化物质诱导MSC迁移。甲紫(结晶紫)和DAPI染色法观察并计数各组细胞的迁移细胞数和迁移率。利用丝裂原活化蛋白激酶(MAPK)通路抑制剂〔SB203580,PD98059和Jun激酶(JNK)抑制剂Ⅱ〕阻断细胞活化的信号通路,观察各组MSC迁移能力的改变。结果 MSC在Transwell体系培养8和12 h,C3+VCAM-1组细胞迁移数分别为7467±485和8795±255,迁移率分别为(14.9±1.0)%和(17.6±0.5)%,均显著高于C3组〔2731±562和4779±224;(5.5±1.1)%和(9.6±0.4)%〕和C3+N组〔2539±321和5645±1080;(5.1±0.6)%和(11.3±1.1)%〕(P<0.05,P<0.01)。加入JNK抑制剂Ⅱ可抑制MSC的迁移能力,C3+VCAM-1组迁移的细胞数为4843±167,迁移率为(9.7%±0.