本文選題：腎小管上皮細(xì)胞缺氧復(fù)氧損傷 + 人骨形態(tài)發(fā)生蛋白7　； 參考：《南昌大學(xué)》2009年碩士論文

【摘要】： 目的:觀察體外腎小管上皮細(xì)胞(Normal Rat Kidney Epithelial Cells)缺氧復(fù)氧損傷(Hypoxia- reoxygenation,H/R)微環(huán)境下慢病毒介導(dǎo)的重組人骨形態(tài)發(fā)生蛋白7(human Bone Morphogenetic Protein-7,hBMP-7)轉(zhuǎn)染對大鼠骨髓間充質(zhì)干細(xì)胞(Rat Bone Mesenchymal Stem Cells,rBMSCs)向腎小管樣上皮細(xì)胞分化的影響,初步探討以BMSCs作為hBMP-7基因運載細(xì)胞,hBMP-7和rMSCs聯(lián)合修復(fù)缺氧再灌注急性腎臟損傷的可行性。 方法:1、貼壁法分離培養(yǎng)SD大鼠乳鼠BMSCs,經(jīng)CD29、CD34、CD44、CD45鑒定BMSCs,并繪制不同代數(shù)細(xì)胞生長曲線和貼壁率曲線分析其生物學(xué)特性。選取生物活性良好的第三代(P3)、四代(P4)BMSCs作為轉(zhuǎn)染BMP7載體細(xì)胞;2、構(gòu)建編碼綠色熒光蛋白(GFP)的重組人骨形態(tài)發(fā)生蛋白7慢病毒載體(Lv-hBMP-7-GFP),體外轉(zhuǎn)染BMSCs后,MTT、Brdu標(biāo)記及流式細(xì)胞分析Lv-hBMP-7-GFP轉(zhuǎn)染對BMSCs生物活性的影響;3、構(gòu)建大鼠腎小管上皮細(xì)胞(NRK-52E)的缺氧復(fù)氧(H/R)模型,經(jīng)流式細(xì)胞分析檢測H/R后細(xì)胞凋亡情況,并在Transwell培養(yǎng)體系中與rMSCs共培養(yǎng),分別于共培養(yǎng)后第3d、5d、7d收集細(xì)胞,經(jīng)RT-PCR檢測誘導(dǎo)后細(xì)胞E-鈣粘蛋白(E-cadherin)的表達(dá),并經(jīng)免疫組化染色及流式細(xì)胞儀測定第18型細(xì)胞角蛋白(Cytokeratin18,CK-18)的陽性表達(dá)率。 結(jié)果:1、貼壁法可以簡便提取原代BMSCs并進(jìn)行體外擴(kuò)增培養(yǎng)予以純化;2、構(gòu)建的Lv-hBMP-7-GFP可以高效轉(zhuǎn)染rMSCs,轉(zhuǎn)染效率約為70%,轉(zhuǎn)染后的rMSCs增殖活性無明顯改變(0.322±0.022,0.302±0.017,0.319±0.031, 0.311±0.029)(P0.05)并可以持續(xù)穩(wěn)定的分泌hBMP-7(5d后為0.329±0.043);3、H/R NRK-52E與各組rMSCs隨共培養(yǎng)時間的延長E-cadherin和CK-18表達(dá)增加( 37.22±0.21,36.54±0.32,38.37±0.38,47.02±0.31 ) ,與單純rMSCs培養(yǎng)組(2.43±0.18)、NRK-52E與rMSCs共培養(yǎng)組(8.52±0.27)、NRK-52E與轉(zhuǎn)染Lv-BMP-7的rMSCs共培養(yǎng)組(8.65±0.22)比較存在顯著性差異(P0.01);H/R NRK-52E與轉(zhuǎn)染Lv-BMP-7的rMSCs共培養(yǎng)組(47.02±0.31)與其他實驗組(H/R NRK-52E與rMSCs共培養(yǎng)組、H/R NRK-52E與轉(zhuǎn)染空病毒的rMSCs共培養(yǎng)組、H/R NRK-52E與BMP-7因子作用下的rMSCs共培養(yǎng)組)相比存在統(tǒng)計學(xué)差異(P0.05)。 結(jié)論:體外大鼠腎小管上皮細(xì)胞缺氧復(fù)氧微環(huán)境下rMSCs可以有效向腎小管上皮樣細(xì)胞分化,并且hBMP-7轉(zhuǎn)染可以促進(jìn)rMSCs的定向分化。研究結(jié)果可能為hBMP-7和BMSCs聯(lián)合改善急性缺血再灌注所致的腎臟損傷提供新的研究方法。
[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 鹵0.022 鹵0.302 鹵0.017 0.319 鹵0.031, 0.311 鹵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 鹵0.21 鹵36.54 鹵0.32 鹵38.37 鹵0.38 鹵47.02 鹵0.31). There was significant difference between rMSCs group (2.43 鹵0.18) NRK-52E and rMSCs coculture group (8.52 鹵0.27nRK-52E) and Lv-BMP-7 transfected rMSCs co-culture group (8.65 鹵0.22). There was significant difference between HRK-52E group and rMSCs co-culture group (47.02 鹵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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329

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