慢病毒介导HO-1基因转染对骨髓间充质干细胞体外缺血性损伤保护作用的实验研究
发布时间：2019-05-17 13:46
【摘要】：目的:通过慢病毒介导HO-1基因转染猪骨髓间充质干细胞(MSCs),并建立体外模拟缺血性损伤模型,探讨HO-1基因修饰对MSCs缺血性损伤的保护作用及其机制。从而为下一步HO-1基因修饰的MSCs移植治疗心肌缺血的研究提供实验依据。 方法:采用密度梯度离心结合贴壁法分离、培养猪MSCs,并用免疫荧光法鉴定;用慢病毒包装系统介导的HO-1基因转染猪MSCs,通过荧光显微镜观察并绘制转染HO-1基因的MSCs(HO-1-MSCs组)、未转染HO-1基因的MSCs(MSCs组)生长曲线,观察慢病毒转染对MSCs增殖的影响。PCR检测HO-1-MSCs组、MSCs组的HO-1mRNA表达;采用血清剥夺和缺氧(SOD)处理建立MSCs体外模拟缺血性损伤模型,通过流式细胞术检测评价HO-1-MSCs组、MSCs组在SOD后3h、6h及9h时的凋亡、死亡情况,并通过RT-PCR、Western blot技术检测两组在SOD各个时间点的HO-1mRNA及HO-1蛋白的表达情况。 结果:密度梯度离心结合贴壁法能有效分离、纯化猪MSCs,转染48小时后,可检测到强荧光出现,免疫荧光染色显示培养的细胞CD44、CD105表达阳性;细胞计数并绘制生长曲线结果显示,转基因的MSCs与未转基因的MSCs生长曲线没有显著差别,无统计学意义;HO-1-MSCs组较MSCs组在常氧培养条件下凋亡率(AR)(P0.05)和死亡率(DR)没有显著差别(P0.05),无统计学意义;两组MSCs在各SOD时间点的AR和DR较常氧培养条件下均显著性增高(P0.01),有统计学意义。在同一SOD时间点,HO-1-MSCs组较MSCs组的AR显著性降低(P0.01),有统计学意义。RT-PCR、Western blot结果显示:在常氧培养条件下,HO-1-MSCs组较MSCs组的HO-1mRNA和HO-1蛋白表达均显著增高(P0.01),有统计学意义;在各SOD时间点,两组MSCs的HO-1mRNA和HO-1蛋白表达较常氧均显著性增高(P0.01),有统计学意义;随着SOD时间延长,两组MSCs的HO-1mRNA和蛋白表达均逐步增强;在同一SOD时间点,HO-1-MSCs组较MSCs组的HO-1mRNA和HO-1蛋白表达均显著性增加(P0.01),有统计学意义。 结论: 1.根据黏附特性可成功分离、培养获得猪骨髓MSCs。 2.按MOI=20,慢病毒包装系统可成功将HO-1基因转染MSCs:效率肯定、相对安全,且对MSCs的增殖没有明显影响。 3.本实验结果显示:HO-1基因转染及其蛋白的稳定表达可提高缺氧、缺血环境下MSCs的生存能力,对抗体外模拟缺血性损伤引起的细胞凋亡,从而具有保护性作用。
[Abstract]:Aim: to investigate the protective effect and mechanism of HO-1 gene modification on HO-1 ischemic injury induced by lentivirus mediated transfer of (MSCs), from pig bone marrow mesenchymal stem cells (BMSCs) and the establishment of simulated ischemic injury model in vitro. In order to provide experimental basis for the next step of HO-1 gene modified MSCs transplantation in the treatment of myocardial ischemia. Methods: pig MSCs, was isolated by density gradient centrifugation combined with adherent method and identified by immunofluorescence. The HO-1 gene mediated by lentivirus packaging system was transferred into pig MSCs,. The growth curve of MSCs (HO-1-MSCs group) and MSCs (MSCs group without HO-1 gene were observed and drawn by fluorescence microscope. The effect of lentivirus transfer on the proliferation of MSCs was observed. The expression of HO-1mRNA in HO-1-MSCs group and MSCs group was detected by PCR. The model of simulated ischemic injury of MSCs in vitro was established by serum deprivation and hypoxia (SOD) treatment. The apoptosis and death of HO-1-MSCs group were evaluated by flow cytometry at 6 h and 9 h after SOD, and RT-PCR, was used to evaluate the apoptosis and death of MSCs group at 6 h and 9 h after SOD. The expression of HO-1mRNA and HO-1 proteins in the two groups at each time point of SOD was detected by Western blot. Results: density gradient centrifugation combined with adherent method could effectively isolate the purified pig MSCs, for 48 hours, and strong fluorescence could be detected, and the expression of CD44,CD105 in cultured cells was positive by immunofluorescence staining. The results of cell counting and drawing growth curve showed that there was no significant difference in the growth curve between transgenic MSCs and non-transgenic MSCs, and there was no statistical significance. There was no significant difference in apoptosis rate (AR) (P 0.05) and mortality (DR) (P 0.05) between HO-1-MSCs group and MSCs group under normoxic culture (P 0.05), but there was no significant difference in apoptosis rate (P 0.05) and mortality rate (P 0.05). The AR and DR of MSCs in both groups were significantly higher than those in normoxic culture at each time point of SOD (P 0.01). At the same time point of SOD, the AR of HO-1-MSCs group was significantly lower than that of MSCs group (P 0.01). The results of RT-PCR,Western blot showed that under normoxic culture conditions, The expression of HO-1mRNA and HO-1 protein in HO-1-MSCs group was significantly higher than that in MSCs group (P 0.01). At each SOD time point, the expression of HO-1mRNA and HO-1 protein in MSCs was significantly higher than that in normoxic group (P 0.01), and the expression of HO-1mRNA and protein in MSCs increased gradually with the prolongation of SOD time. At the same SOD time point, the expression of HO-1mRNA and HO-1 protein in HO-1-MSCs group was significantly higher than that in MSCs group (P 0.01). Conclusion: 1. According to the adhesion characteristics, pig bone marrow MSCs. could be successfully separated and cultured. 2. According to MOI=20, lentivirus packaging system, HO-1 gene can be successfully transferred into MSCs:, which is relatively safe, and has no significant effect on the proliferation of MSCs. 3. The results showed that the stable expression of HO-1 gene and its protein could improve the survival ability of MSCs under hypoxia and ischemia, and antagonize the apoptosis induced by simulated ischemic injury in vitro, so it had protective effect.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
[Abstract]:Aim: to investigate the protective effect and mechanism of HO-1 gene modification on HO-1 ischemic injury induced by lentivirus mediated transfer of (MSCs), from pig bone marrow mesenchymal stem cells (BMSCs) and the establishment of simulated ischemic injury model in vitro. In order to provide experimental basis for the next step of HO-1 gene modified MSCs transplantation in the treatment of myocardial ischemia. Methods: pig MSCs, was isolated by density gradient centrifugation combined with adherent method and identified by immunofluorescence. The HO-1 gene mediated by lentivirus packaging system was transferred into pig MSCs,. The growth curve of MSCs (HO-1-MSCs group) and MSCs (MSCs group without HO-1 gene were observed and drawn by fluorescence microscope. The effect of lentivirus transfer on the proliferation of MSCs was observed. The expression of HO-1mRNA in HO-1-MSCs group and MSCs group was detected by PCR. The model of simulated ischemic injury of MSCs in vitro was established by serum deprivation and hypoxia (SOD) treatment. The apoptosis and death of HO-1-MSCs group were evaluated by flow cytometry at 6 h and 9 h after SOD, and RT-PCR, was used to evaluate the apoptosis and death of MSCs group at 6 h and 9 h after SOD. The expression of HO-1mRNA and HO-1 proteins in the two groups at each time point of SOD was detected by Western blot. Results: density gradient centrifugation combined with adherent method could effectively isolate the purified pig MSCs, for 48 hours, and strong fluorescence could be detected, and the expression of CD44,CD105 in cultured cells was positive by immunofluorescence staining. The results of cell counting and drawing growth curve showed that there was no significant difference in the growth curve between transgenic MSCs and non-transgenic MSCs, and there was no statistical significance. There was no significant difference in apoptosis rate (AR) (P 0.05) and mortality (DR) (P 0.05) between HO-1-MSCs group and MSCs group under normoxic culture (P 0.05), but there was no significant difference in apoptosis rate (P 0.05) and mortality rate (P 0.05). The AR and DR of MSCs in both groups were significantly higher than those in normoxic culture at each time point of SOD (P 0.01). At the same time point of SOD, the AR of HO-1-MSCs group was significantly lower than that of MSCs group (P 0.01). The results of RT-PCR,Western blot showed that under normoxic culture conditions, The expression of HO-1mRNA and HO-1 protein in HO-1-MSCs group was significantly higher than that in MSCs group (P 0.01). At each SOD time point, the expression of HO-1mRNA and HO-1 protein in MSCs was significantly higher than that in normoxic group (P 0.01), and the expression of HO-1mRNA and protein in MSCs increased gradually with the prolongation of SOD time. At the same SOD time point, the expression of HO-1mRNA and HO-1 protein in HO-1-MSCs group was significantly higher than that in MSCs group (P 0.01). Conclusion: 1. According to the adhesion characteristics, pig bone marrow MSCs. could be successfully separated and cultured. 2. According to MOI=20, lentivirus packaging system, HO-1 gene can be successfully transferred into MSCs:, which is relatively safe, and has no significant effect on the proliferation of MSCs. 3. The results showed that the stable expression of HO-1 gene and its protein could improve the survival ability of MSCs under hypoxia and ischemia, and antagonize the apoptosis induced by simulated ischemic injury in vitro, so it had protective effect.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
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