miR-210促进小鼠BMMSCs血管内皮生长因子的表达和分泌

发布时间：2018-03-23 13:17

本文选题：miR-210　切入点：BMMSCs　出处：《安徽医科大学》2017年硕士论文

【摘要】：目的研究小分子非编码RNA mi R-210对小鼠骨髓间充质干细胞(BMMSCs)表达和分泌血管内皮生长因子(VEGF)的影响,为骨组织工程血管化研究提供实验数据。方法首先体外全骨髓液贴壁法培养小鼠BMMSCs,通过构建慢病毒载体Lenti-Lac Z和Lenti-mi R-210,分别转染小鼠BMMSCs和人脐静脉内皮细胞(HUVEC);MTT法检测慢病毒载体对小鼠BMMSCs增殖情况的影响;Lenti-mi R-210/BMMSCs和Lenti-Lac Z/BMMSCs于转染慢病毒载体后培养至第7d RT-PCR检测VEGF m RNA基因表达,第14d Western blot检测VEGF蛋白表达;通过PKH26红色荧光染料对Lenti-mi R-210/HUVEC和Lenti-Lac Z/HUVEC进行染色后,进行24h Matrigel成管实验,通过荧光显微镜观察成管效果,统计两组成管长度;构建Hy Stem-HP凝胶缓释系统,agomir-210和agomir-NC与BMMSCs复合Hy Stem-HP凝胶后行裸鼠皮下注射,7d后取标本进行10%中性福尔马林固定,脱水后包埋,石蜡切片行CD31免疫荧光染色,检测CD31阳性细胞含量。使使用SPSS13.0软件进行统计分析,实验数据以均数±标准差形式呈现,t检验和方差分析处理实验数据,检验水准取α=0.05。结果全骨髓液培养后5-7d,镜下见部分细胞呈梭形或多角形,散在分布;传代后贴壁细胞呈梭形旋涡状生长,细胞形态随传代培养逐渐一致;mi R-210慢病毒载体转染小鼠BMMSCs和HUVEC,当MOI=10时,转染效率最高,MTT法检测病毒对BMMSCs增殖无影响,Lenti-mi R-210和Lenti-Lac Z两组细胞培养第7dRT-PCR检测VEGF m RNA基因表达结果,mi R-210促进小鼠BMMSCs VEGF的表达(P0.01);第14d Western blot检测两组细胞VEGF蛋白表达,mi R-210组VEGF蛋白表达明显高于对照组,与RT-PCR结果一致;Matrigel成管实验24h结果Lenti-mi R-210组与Lenti-Lac Z相比,环形管状结构明显,端端接触多,管壁更完整;统计小管长度后比较两组小管长度,差异有统计学意义(P0.05);agomir-210和agomir-NC分别与BMMSCs复合Hy Stem-HP凝胶缓释系统裸鼠皮下注射7d后标本CD31免疫荧光染色结果统计分析显示agomir-210组CD31阳性细胞含量明显高于agomir-NC组,约是agomir-NC组的3倍。结论通过慢病毒载体,转染小鼠BMMSCs,过表达目的基因mi R-210后能够有效促进小鼠BMMSCs VEGF的表达和分泌,agomir-210 Hy Stem-HP凝胶裸鼠皮下实验表明,agomir-210促进BMMSCs微血管的形成。
[Abstract]:Objective to study the effect of small molecule noncoding RNA mi R-210 on the expression and secretion of vascular endothelial growth factor (VEGF) in mouse bone marrow mesenchymal stem cells (BMMSCs). Methods BMMSCs were cultured in vitro by whole bone marrow adherent method. The lentivirus vectors Lenti-Lac Z and Lenti-mi R-210 were constructed and transfected into mouse BMMSCs and human umbilical vein endothelial cells respectively. Effect of lentivirus vector on BMMSCs proliferation in mice Lenti-mi R-210/BMMSCs and Lenti-Lac Z/BMMSCs were transfected into lentivirus vector to detect the expression of VEGF m RNA gene by RT-PCR on the 7th day. On the 14th day, Western blot was used to detect the expression of VEGF protein, Lenti-mi R-210/HUVEC and Lenti-Lac Z/HUVEC were dyed with PKH26 red fluorescent dye, then the tube formation of Lenti-mi R-210/HUVEC and Lenti-Lac Z/HUVEC was carried out for 24 hours. The tube forming effect was observed by fluorescence microscope, and the length of the two components was counted. Hy Stem-HP gel sustained release system (Hy Stem-HP gel) was constructed. The Hy Stem-HP gel was combined with agomir-NC and BMMSCs. After 7 days of subcutaneous injection of Hy Stem-HP gel, 10% neutral formalin was fixed and embedded in paraffin sections of Hy Stem-HP gel. CD31 immunofluorescence staining was performed on paraffin sections. The content of CD31 positive cells was detected. The statistical analysis was carried out by using SPSS13.0 software. The experimental data presented t test and ANOVA in the form of mean 卤standard deviation. Results after 5 to 7 days of whole bone marrow culture, some cells were spindle-shaped or polygonal and scattered, and the adherent cells grew like spindle-shaped swirls after passage. The morphology of mouse BMMSCs and HUVECs was consistent with the passage culture. When MOI = 10:00, the cells were transfected into murine BMMSCs and HUVECs. The expression of VEGF m RNA gene was detected by 7dRT-PCR in the two groups of BMMSCs proliferation and Lenti-Lac Z cell culture. The results showed that Mi R-210 promoted the expression of BMMSCs VEGF in mice (P0.01), and on day 14, Western blot was used to detect the expression of VEGF protein in both groups. The expression of VEGF protein in R- 210 group was significantly higher than that in control group. The results were consistent with the results of RT-PCR experiment for 24 h. Results compared with Lenti-Lac Z, the Lenti-mi R-210 group had obvious annular tubular structure, more end-end contact and more complete tube wall, and the length of the two groups was compared after statistics of the tubule length. The results of CD31 immunofluorescence staining showed that the content of CD31 positive cells in agomir-210 group was significantly higher than that in agomir-NC group. Conclusion the overexpression of the target gene mi R-210 can effectively promote the expression of BMMSCs VEGF and secretion of agomir-210Hy Stem-HP gel in nude mice. Conclusion the results of subcutaneous experiments show that agomir-210 can promote the formation of BMMSCs microvessels in nude mice after transfection of lentivirus vector into BMMSCs, and overexpression of the target gene mi R-210 can effectively promote the expression of BMMSCs VEGF and the secretion of agomir-210Hy Stem-HP gel.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782

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