体外诱导BMP7基因修饰的大鼠骨髓间充质干细胞向肾小管上皮样细胞分化的实验研究

发布时间：2018-05-09 05:26

  本文选题：肾小管上皮细胞缺氧复氧损伤 + 人骨形态发生蛋白7　； 参考：《南昌大学》2009年硕士论文

【摘要】： 目的:观察体外肾小管上皮细胞(Normal Rat Kidney Epithelial Cells)缺氧复氧损伤(Hypoxia- reoxygenation,H/R)微环境下慢病毒介导的重组人骨形态发生蛋白7(human Bone Morphogenetic Protein-7,hBMP-7)转染对大鼠骨髓间充质干细胞(Rat Bone Mesenchymal Stem Cells,rBMSCs)向肾小管样上皮细胞分化的影响,初步探讨以BMSCs作为hBMP-7基因运载细胞,hBMP-7和rMSCs联合修复缺氧再灌注急性肾脏损伤的可行性。 方法:1、贴壁法分离培养SD大鼠乳鼠BMSCs,经CD29、CD34、CD44、CD45鉴定BMSCs,并绘制不同代数细胞生长曲线和贴壁率曲线分析其生物学特性。选取生物活性良好的第三代(P3)、四代(P4)BMSCs作为转染BMP7载体细胞;2、构建编码绿色荧光蛋白(GFP)的重组人骨形态发生蛋白7慢病毒载体(Lv-hBMP-7-GFP),体外转染BMSCs后,MTT、Brdu标记及流式细胞分析Lv-hBMP-7-GFP转染对BMSCs生物活性的影响;3、构建大鼠肾小管上皮细胞(NRK-52E)的缺氧复氧(H/R)模型,经流式细胞分析检测H/R后细胞凋亡情况,并在Transwell培养体系中与rMSCs共培养,分别于共培养后第3d、5d、7d收集细胞,经RT-PCR检测诱导后细胞E-钙粘蛋白(E-cadherin)的表达,并经免疫组化染色及流式细胞仪测定第18型细胞角蛋白(Cytokeratin18,CK-18)的阳性表达率。 结果:1、贴壁法可以简便提取原代BMSCs并进行体外扩增培养予以纯化;2、构建的Lv-hBMP-7-GFP可以高效转染rMSCs,转染效率约为70%,转染后的rMSCs增殖活性无明显改变(0.322±0.022,0.302±0.017,0.319±0.031, 0.311±0.029)(P0.05)并可以持续稳定的分泌hBMP-7(5d后为0.329±0.043);3、H/R NRK-52E与各组rMSCs随共培养时间的延长E-cadherin和CK-18表达增加( 37.22±0.21,36.54±0.32,38.37±0.38,47.02±0.31 ) ,与单纯rMSCs培养组(2.43±0.18)、NRK-52E与rMSCs共培养组(8.52±0.27)、NRK-52E与转染Lv-BMP-7的rMSCs共培养组(8.65±0.22)比较存在显著性差异(P0.01);H/R NRK-52E与转染Lv-BMP-7的rMSCs共培养组(47.02±0.31)与其他实验组(H/R NRK-52E与rMSCs共培养组、H/R NRK-52E与转染空病毒的rMSCs共培养组、H/R NRK-52E与BMP-7因子作用下的rMSCs共培养组)相比存在统计学差异(P0.05)。 结论:体外大鼠肾小管上皮细胞缺氧复氧微环境下rMSCs可以有效向肾小管上皮样细胞分化,并且hBMP-7转染可以促进rMSCs的定向分化。研究结果可能为hBMP-7和BMSCs联合改善急性缺血再灌注所致的肾脏损伤提供新的研究方法。
[Abstract]:Objective: to observe the effect of lentivirus-mediated recombinant human bone morphogenetic protein (7(human Bone Morphogenetic protein-7hBMP-7) transfection on rat bone marrow mesenchymal stem cells (BMSCs) by normal Rat Kidney Epithelial Cells) hypoxia and reoxygenation injury (Hypoxia- reoxygenation H / R) microenvironment in vitro. Effects of renal tubuloid epithelial cell differentiation, To explore the feasibility of using BMSCs as a carrier of hBMP-7 gene, hBMP-7 and rMSCs to repair acute renal injury induced by hypoxia reperfusion. Methods Sprague-Dawley rat BMSCs were isolated and cultured by cell adhesion method. BMSCs were identified by CD29 CD34 and CD44pCD45, and their biological characteristics were analyzed by drawing different algebraic cell growth curves and adherent rate curves. A recombinant human bone morphogenetic protein-7 lentivirus vector, Lv-hBMP-7-GFPN, was constructed by selecting the third generation of P3BMSCs with good bioactivity and the fourth generation of P4BMSCs as BMP7 vector cells. The recombinant human bone morphogenetic protein-7 lentivirus vector (Lv-hBMP-7-GFPN) was transfected with BMSCs and labeled with MTTP-Brdu and flow cytometry in vitro. To analyze the effect of Lv-hBMP-7-GFP transfection on the biological activity of BMSCs, a rat renal tubular epithelial cell line NRK-52 (E) model of hypoxia reoxygenation was established. Apoptosis after H / R was detected by flow cytometry, and co-cultured with rMSCs in Transwell culture system. The cells were collected on the 3rd day and 5th day after co-culture, and the expression of E-cadherin was detected by RT-PCR. The positive rate of cytokeratin18CK-18 was detected by immunohistochemical staining and flow cytometry. Results the primary BMSCs could be easily extracted by using the cell adhesion method and purified by in vitro amplification and culture. The constructed Lv-hBMP-7-GFP could be transfected into rMSCs efficiently and the transfection efficiency was about 70. The proliferative activity of rMSCs after transfection did not change 0.322 卤0.022 卤0.302 卤0.017 0.319 卤0.031, 0.311 卤0.029 P0.05) and could be sustained and stable. The expression of E-cadherin and CK-18 increased with the prolongation of co-culture time (37.22 卤0.21 卤36.54 卤0.32 卤38.37 卤0.38 卤47.02 卤0.31). There was significant difference between rMSCs group (2.43 卤0.18) NRK-52E and rMSCs coculture group (8.52 卤0.27nRK-52E) and Lv-BMP-7 transfected rMSCs co-culture group (8.65 卤0.22). There was significant difference between HRK-52E group and rMSCs co-culture group (47.02 卤0.31) and other experimental groups (HR-NRK-52E and rMSCs co-culture group). There was a significant difference between the rMSCs co-culture group and the rMSCs co-culture group treated with BMP-7 factor (P 0.05). Conclusion: rMSCs can effectively differentiate into renal tubular epithelial-like cells under anoxic reoxygenation microenvironment of rat renal tubular epithelial cells in vitro, and hBMP-7 transfection can promote the directional differentiation of rMSCs. The results may provide a new method for the combination of hBMP-7 and BMSCs to improve renal injury induced by acute ischemia reperfusion.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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