胰十二指肠同源盒基因-1在人脂肪间充质干细胞分化为胰岛分泌细胞中的作用

发布时间：2018-03-05 09:40

本文选题：胰十二指肠同源盒基因-1　切入点：人脂肪间充质干细胞　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨胰十二指肠同源盒基因-1(PDX-1)在人脂肪间充质干细胞(h ADSC)分化为胰岛分泌细胞中的作用。方法将冻存的人脂肪间充质干细胞进行复苏,并随机分为实验组与对照组,实验组:将携带胰十二指肠同源盒基因-1的病毒液(MOI(感染复数)=50)加入细胞悬液中,混合培养4小时后,去除含有病毒的培养液,更换新鲜的完全培养液,继续培养至48小时,以保证转染效果;对照组:采用不含病毒液的培养液培养48小时。然后分别采用蛋白质印迹法(Western-blotting)和细胞免疫荧光染色方法对感染结果进行检测。对感染后15天细胞利用双硫腙(DTZ)染色观察胰岛素阳性反应细胞,酶联免疫吸附试验(ELISA)检测细胞胰岛素的分泌量,细胞免疫荧光染色法检测胰岛分泌相关基因insulin 1的表达。结果(1)实验组的人脂肪间充质干细胞进行病毒感染48h后,Western blot法和免疫荧光法均可检测到PDX-1的表达,对照组的细胞则无PDX-1的表达,并且感染的细胞在15d之后仍能检测到PDX-1的表达。(2)实验组出现胰岛素阳性细胞且被染成棕红色,对照组未出现胰岛素阳性细胞。(3)感染后15d,实验组和对照组细胞在低糖环境下分泌的胰岛素量分别为(6.98±0.72)、(6.78±0.54)ng/m L,两组比较,P0.05,无统计学意义;高糖环境下分泌的胰岛素量分别为(13.38±0.66)、(6.50±0.64)ng/m L,两组比较,P0.05,有统计学意义;实验组胰岛分泌相关基因insulin 1的表达量多于对照组。结论胰十二指肠同源盒基因-1在人脂肪间充质干细胞向胰岛分泌细胞分化过程中具有促进作用,对开展胰岛移植治疗I型糖尿病具有重要意义。
[Abstract]:Objective to investigate the role of pancreaticoduodenal homologous box gene-1 PDX-1 in differentiation of human adipose mesenchymal stem cells into pancreatic islet secretory cells. Methods cryopreserved human adipose mesenchymal stem cells were resuscitated and randomly divided into experimental group and control group. In the experimental group, the virus moi containing the gene 1 of pancreaticoduodenal homologous box 1 was added to the cell suspension. After 4 hours of mixed culture, the virus-containing culture medium was removed, the fresh complete culture medium was replaced, and the culture was continued for 48 hours. To ensure the transfection effect; The control group was cultured in virus-free medium for 48 hours. Then the infection results were detected by Western blotting and cell immunofluorescence staining. The cells were stained with dithizone DTZ 15 days after infection. Color observation of insulin positive reaction cells, Enzyme linked immunosorbent assay (Elisa) was used to detect the secretion of insulin. The expression of human adipose-derived mesenchymal stem cells (HMSCs) in the experimental group was detected by Western blot assay and immunofluorescence assay after 48 h of viral infection. The cells in the control group showed no PDX-1 expression, and the infected cells could still detect the expression of PDX-1 after 15 days.) Insulin positive cells were found in the experimental group and were stained brown red. In the control group, the amount of insulin secreted by the cells of the experimental group and the control group was 6.98 卤0.72 卤0.54 ng / mL at 15 days after infection. There was no significant difference between the two groups (P 0.05). The amount of insulin secreted in high glucose environment was 6.50 卤0.64 ng / mL, respectively, which was significantly higher than that in the control group (P 0.05). The expression of pancreatic islet secretory gene insulin 1 in the experimental group was higher than that in the control group. Conclusion the pancreaticoduodenal homologous box gene 1 plays an important role in the differentiation of human adipose mesenchymal stem cells into pancreatic islet secretory cells. It is of great significance to carry out islet transplantation in the treatment of type I diabetes.
【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】：R587.1

【参考文献】