恒河猴骨髓間充質(zhì)干細(xì)胞移植代替損傷的角膜內(nèi)皮細(xì)胞的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-14 13:22
【摘要】： 目的:將體外培養(yǎng)的恒河猴骨髓間充質(zhì)干細(xì)胞(BMSCs)移植于角膜內(nèi)皮表面,觀察細(xì)胞形態(tài)及功能變化,以探討骨髓間充質(zhì)干細(xì)胞體內(nèi)誘導(dǎo)為角膜內(nèi)皮細(xì)胞并發(fā)揮其功能的可行性。 方法:恒河猴4只分為實(shí)驗(yàn)組(3只)和對(duì)照組(1只)。采用密度梯度離心聯(lián)合貼壁法分離培養(yǎng)BMSCs,通過(guò)流式細(xì)胞儀檢測(cè)細(xì)胞表面分子及對(duì)細(xì)胞進(jìn)行脂肪細(xì)胞誘導(dǎo)等方法以鑒定體外培養(yǎng)的BMSCs,采用5-溴脫氧尿嘧啶核苷(Brdu)對(duì)培養(yǎng)的BMSCs進(jìn)行標(biāo)記,通過(guò)離心沉淀法將BMSCs移植于撕除后彈力層及角膜內(nèi)皮層的恒河猴角膜植片內(nèi)皮面并原位縫合于植床,術(shù)后觀察角膜植片的透明度,于術(shù)后一月、二月、三月摘取術(shù)眼角膜植片進(jìn)行病理切片、抗Brdu單克隆抗體免疫組化染色及電鏡檢查,觀察移植細(xì)胞的分布情況、細(xì)胞之間的連接、形態(tài)變化以及細(xì)胞表面分子的變化。 結(jié)果:密度梯度離心聯(lián)合貼壁法可以獲得較高純度的BMSCs,體外培養(yǎng)24小時(shí)有少量細(xì)胞貼壁,48小時(shí)細(xì)胞貼壁率約5%,12～16天細(xì)胞90%匯合,細(xì)胞呈梭形,平行或漩渦狀排列;CD29表達(dá)陽(yáng)性率94.26%,CD45表達(dá)陽(yáng)性率4.02%,CD34表達(dá)陽(yáng)性率7.51%,培養(yǎng)的細(xì)胞通過(guò)脂肪細(xì)胞誘導(dǎo)液培養(yǎng)3周后,油紅O染色細(xì)胞漿被染為橘紅色,細(xì)胞核染為藍(lán)色;術(shù)后實(shí)2周左右實(shí)驗(yàn)組及對(duì)照組角膜植片均水腫渾濁,術(shù)后7-8周后實(shí)驗(yàn)組角膜植片保持一定的透明性,而對(duì)照組角膜水腫嚴(yán)重,發(fā)生大泡性角膜病變;術(shù)后角膜植片掃描電鏡顯示:移植術(shù)后一月,移植的細(xì)胞散狀分布于角膜植片內(nèi)皮面,呈短梭形;術(shù)后二月,部分細(xì)胞連成片狀,細(xì)胞呈梭形及多角形;術(shù)后三月,細(xì)胞較均勻生長(zhǎng)于角膜植片內(nèi)皮面,細(xì)胞呈多角形,細(xì)胞間相互連接較緊密;Brdu免疫組織化學(xué)染色可見(jiàn)角膜植片內(nèi)皮表面細(xì)胞染色陽(yáng)性,細(xì)胞核被染為褐色。 結(jié)論:通過(guò)密度梯度離心聯(lián)合貼壁法可以獲得純度較高的骨髓間充質(zhì)干細(xì)胞,BMSCs通過(guò)離心沉淀法移植到角膜內(nèi)皮面后,在體內(nèi)誘導(dǎo)為角膜內(nèi)皮樣細(xì)胞,并發(fā)揮一定的功能。
[Abstract]:Aim: to observe the morphological and functional changes of mesenchymal stem cells (BMSCs) from rhesus monkey bone marrow mesenchymal stem cells (BMSCs) in vitro and to investigate the feasibility of inducing them into corneal endothelial cells in vivo. Methods: four rhesus monkeys were divided into experimental group (3) and control group (1). BMSCs were isolated and cultured by density gradient centrifugation combined with adherent method. Cell surface molecules were detected by flow cytometry and adipocytes were induced to identify BMSCs in vitro. 5-bromodeoxyuridine (5-bromodeoxyuridine) (Brdu) was used to identify BMSCs. The cultured BMSCs was labeled, BMSCs was transplanted into the corneal graft of rhesus monkey by centrifugal precipitation and was sutured in situ. The transparency of corneal graft was observed in 1 month and 2 months after operation. The corneal grafts were harvested in March for pathological section, immunohistochemical staining against Brdu monoclonal antibody and electron microscopy to observe the distribution of the transplanted cells, the connections between the cells, the morphological changes and the changes of cell surface molecules. Results: high purity BMSCs could be obtained by density gradient centrifugation combined with adherent method. After 24 hours of culture in vitro, a small number of cells adhered to the wall for 48 hours. The cell adhesion rate was about 5 / 1212 / 16 days, and the cells were fusiform. The positive rate of CD29 expression in parallel or whirlpool arrangement was 94.26% and 4.02% respectively. The positive rate of CD34 expression was 7.51%. After cultured in adipocyte inducer for 3 weeks, the cytoplasm of oil-red O staining cells was stained orange and the nucleus was blue. The corneal grafts in the experimental group and the control group were edema and turbidity about 2 weeks after operation. The corneal grafts in the experimental group remained transparent 7 to 8 weeks after operation, while the corneal edema in the control group was serious and the corneal lesions occurred. Scanning electron microscopy (SEM) showed that the transplanted cells were scattered in the corneal grafts at one month after transplantation, and appeared to be short fusiform. Two months after the operation, some cells were flaky, and the cells were fusiform and polygonal; 3 months after the operation, the cells were in the shape of spindle and polygon. The cells grew homogeneously on the surface of cornea grafts, and the cells were polygonal. Brdu immunohistochemical staining showed that the endothelial cells were positive and the nuclei were brown. Conclusion: BMSCs with high purity can be obtained by density gradient centrifugation combined with adherent method. BMSCs can be induced to be corneal endothelioid cells in vivo and play a certain function after being transplanted into corneal endothelium by centrifugal precipitation method.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R772.2
本文編號(hào)：2182988
[Abstract]:Aim: to observe the morphological and functional changes of mesenchymal stem cells (BMSCs) from rhesus monkey bone marrow mesenchymal stem cells (BMSCs) in vitro and to investigate the feasibility of inducing them into corneal endothelial cells in vivo. Methods: four rhesus monkeys were divided into experimental group (3) and control group (1). BMSCs were isolated and cultured by density gradient centrifugation combined with adherent method. Cell surface molecules were detected by flow cytometry and adipocytes were induced to identify BMSCs in vitro. 5-bromodeoxyuridine (5-bromodeoxyuridine) (Brdu) was used to identify BMSCs. The cultured BMSCs was labeled, BMSCs was transplanted into the corneal graft of rhesus monkey by centrifugal precipitation and was sutured in situ. The transparency of corneal graft was observed in 1 month and 2 months after operation. The corneal grafts were harvested in March for pathological section, immunohistochemical staining against Brdu monoclonal antibody and electron microscopy to observe the distribution of the transplanted cells, the connections between the cells, the morphological changes and the changes of cell surface molecules. Results: high purity BMSCs could be obtained by density gradient centrifugation combined with adherent method. After 24 hours of culture in vitro, a small number of cells adhered to the wall for 48 hours. The cell adhesion rate was about 5 / 1212 / 16 days, and the cells were fusiform. The positive rate of CD29 expression in parallel or whirlpool arrangement was 94.26% and 4.02% respectively. The positive rate of CD34 expression was 7.51%. After cultured in adipocyte inducer for 3 weeks, the cytoplasm of oil-red O staining cells was stained orange and the nucleus was blue. The corneal grafts in the experimental group and the control group were edema and turbidity about 2 weeks after operation. The corneal grafts in the experimental group remained transparent 7 to 8 weeks after operation, while the corneal edema in the control group was serious and the corneal lesions occurred. Scanning electron microscopy (SEM) showed that the transplanted cells were scattered in the corneal grafts at one month after transplantation, and appeared to be short fusiform. Two months after the operation, some cells were flaky, and the cells were fusiform and polygonal; 3 months after the operation, the cells were in the shape of spindle and polygon. The cells grew homogeneously on the surface of cornea grafts, and the cells were polygonal. Brdu immunohistochemical staining showed that the endothelial cells were positive and the nuclei were brown. Conclusion: BMSCs with high purity can be obtained by density gradient centrifugation combined with adherent method. BMSCs can be induced to be corneal endothelioid cells in vivo and play a certain function after being transplanted into corneal endothelium by centrifugal precipitation method.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R772.2
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前2條
1 李柳;誘導(dǎo)hUC-MSCs和VECs轉(zhuǎn)化為角膜內(nèi)皮樣細(xì)胞的實(shí)驗(yàn)研究[D];暨南大學(xué);2012年
2 肖中男;恒河猴血管內(nèi)皮細(xì)胞移植替代角膜內(nèi)皮細(xì)胞的實(shí)驗(yàn)研究[D];昆明醫(yī)科大學(xué);2012年
，本文編號(hào)：2182988
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