间充质干细胞联合热休克蛋白90α功能性基因序列治疗大鼠皮瓣缺血再灌注损伤的实验研究

发布时间：2019-01-06 16:41

【摘要】：研究目的1.研究热休克蛋白90α功能性基因序列(以下称“F-5”基因)的慢病毒载体转染间充质干细胞(Mesenchymal Stem Cells,MSC)后对间充质干细胞生物活性的影响及“F-5”基因在MSC的表达情况;2.探讨间充质干细胞联合转染“F-5”基因移植治疗大鼠皮瓣缺血再灌注损伤的临床效果。研究方法1.将构建好的“F-5”基因慢病毒载体体外转染间充质干细胞,确定最佳的病毒感染复数,在镜下观察细胞形态、绘制细胞的生长曲线、测定细胞周期、划痕实验和MMT检测转染后的间充质干细胞的生物学活性;荧光显微镜和细胞免疫荧光检测“F-5”多肽在间充质干细胞中的表达情况;Elisa分别检测细胞上清液中F-5多肽在1d、3d、5d、7d和14d的分泌情况。2.设“F-5”基因慢病毒载体转染后的间充质干细胞为实验组、空载慢病毒载体为对照组、1×PBS为空白组,分别注射移植于缺血再灌注损伤的大鼠腹部皮瓣术区;术后每天观察皮瓣的颜色、质地、毛发生长、坏死范围及针刺出血情况等,术后7 d比较各组皮瓣的成活率,并切取成活皮瓣远端组织分别进行HE染色、免疫组织化学染色检测血管内皮生长因子(VEGF)的表达,并用TUNEL法检测凋亡细胞,比较各组间的差异。结果:1.携带“F-5”基因的慢病毒载体转染间充质干细胞后,通过镜下观察的细胞形态、绘制细胞的生长曲线、测定细胞的周期、划痕实验和MMT检测并未见明显差异;通过细胞免疫荧光检测发现在转染后的间充质干细胞中的细胞质和细胞膜上均能够检测到“F-5”多肽的表达;通过Elisa检测法发现实验组从第3天开始细胞上清液中分泌的F-5-flag的量明显比其他两组分泌量多,第5天时可发现实验组比其他两组明显高出10倍的量。2.实验组皮瓣成活率为(92.38±8.67)%,而对照组为(81.57±7.38)%,空白组为(47.83+6.89)%。实验组大鼠皮瓣VEGF的表达密度为(91.25±5.92)%,对照组的为(76.37±6.42)%和空白组的为(33.37±6.62)%;各组凋亡细胞阳性表达百分指数分别为:实验组(21.4±5.3)%,对照组(23.3±3.6)%,空白组为(54.7±5.7)%。结论及意义:1.“F-5”基因慢病毒载体转染间充质干细胞后对细胞的生物活性无明显影响,“F-5”基因能够在MSC中成功表达并将F-5多肽分泌至细胞外,同时本实验为“F-5”基因在组织修复等研究工作中提供了实验基础;2.间充质干细胞联合转染“F-5”基因治疗大鼠皮瓣缺血再灌注损伤的效果明显优于单纯MSC移植的效果。
[Abstract]:Objective 1. Study on transfection of heat shock protein 90 伪 functional gene sequence (hereinafter referred to as "F-5" gene) lentivirus vector into mesenchymal stem cell (Mesenchymal Stem Cells, The effect of MSC on the biological activity of mesenchymal stem cells and the expression of "F-5" gene in MSC; 2. To investigate the clinical effect of mesenchymal stem cells (MSCs) combined with transfection of F-5 gene in the treatment of ischemia reperfusion injury of skin flap in rats. Method 1. The "F-5" gene lentivirus vector was transfected into mesenchymal stem cells in vitro, and the optimal number of virus infection was determined. The morphology of cells was observed under microscope, the growth curve of cells was drawn, and the cell cycle was measured. The biological activity of transfected mesenchymal stem cells was detected by scratch test and MMT. The expression of "F-5" polypeptide in mesenchymal stem cells was detected by fluorescence microscope and immunofluorescence, and the secretion of F-5 polypeptide in supernatant was detected by Elisa for 7 days and 14 days, respectively. Mesenchymal stem cells transfected with "F-5" gene lentivirus vector were used as experimental group, non-loaded lentivirus vector as control group and 1 脳 PBS as blank group. The color, texture, hair growth, necrotic range and acupuncture bleeding were observed every day after operation. The survival rate of each flap was compared 7 days after operation, and the distal tissue of the surviving flap was stained with HE. The expression of vascular endothelial growth factor (VEGF) was detected by immunohistochemical staining, and the apoptotic cells were detected by TUNEL method. Results: 1. After transfection of lentivirus vector carrying "F-5" gene into mesenchymal stem cells, cell morphology was observed under microscope, cell growth curve was drawn and cell cycle was measured. There was no significant difference between scratch test and MMT test. The expression of "F-5" polypeptide was detected in the cytoplasm and cell membrane of the transfected mesenchymal stem cells by immunofluorescence assay. The amount of F-5-flag secreted in the supernatant of the experimental group from the third day was obviously higher than that in the other two groups by Elisa assay. On the fifth day, the amount of F-5-flag secreted in the experimental group was 10 times higher than that in the other two groups. The survival rate of flap was (92.38 卤8.67)% in the experimental group, (81.57 卤7.38)% in the control group and (47.83.6.89)% in the blank group. The expression density of VEGF was (91.25 卤5.92)% in the experimental group, (76.37 卤6.42)% in the control group and (33.37 卤6.62)% in the blank group. The positive expression index of apoptotic cells was (21.4 卤5.3)% in the experimental group, (23.3 卤3.6)% in the control group and (54.7 卤5.7)% in the blank group. Conclusion: 1. After transfection of "F-5" gene lentivirus vector into mesenchymal stem cells, the biological activity of the cells was not significantly affected. "F-5" gene could be successfully expressed in MSC and secreted into the cells. 2. At the same time, this experiment provides the experimental basis for the study of "F-5" gene in tissue repair. 2. The effect of mesenchymal stem cells combined with transfection of "F-5" gene on ischemic reperfusion injury of rat flap was better than that of MSC transplantation alone.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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