恒河猴骨髓间充质干细胞移植代替损伤的角膜内皮细胞的实验研究
发布时间：2018-08-14 13:22
【摘要】： 目的:将体外培养的恒河猴骨髓间充质干细胞(BMSCs)移植于角膜内皮表面,观察细胞形态及功能变化,以探讨骨髓间充质干细胞体内诱导为角膜内皮细胞并发挥其功能的可行性。 方法:恒河猴4只分为实验组(3只)和对照组(1只)。采用密度梯度离心联合贴壁法分离培养BMSCs,通过流式细胞仪检测细胞表面分子及对细胞进行脂肪细胞诱导等方法以鉴定体外培养的BMSCs,采用5-溴脱氧尿嘧啶核苷(Brdu)对培养的BMSCs进行标记,通过离心沉淀法将BMSCs移植于撕除后弹力层及角膜内皮层的恒河猴角膜植片内皮面并原位缝合于植床,术后观察角膜植片的透明度,于术后一月、二月、三月摘取术眼角膜植片进行病理切片、抗Brdu单克隆抗体免疫组化染色及电镜检查,观察移植细胞的分布情况、细胞之间的连接、形态变化以及细胞表面分子的变化。 结果:密度梯度离心联合贴壁法可以获得较高纯度的BMSCs,体外培养24小时有少量细胞贴壁,48小时细胞贴壁率约5%,12～16天细胞90%汇合,细胞呈梭形,平行或漩涡状排列;CD29表达阳性率94.26%,CD45表达阳性率4.02%,CD34表达阳性率7.51%,培养的细胞通过脂肪细胞诱导液培养3周后,油红O染色细胞浆被染为橘红色,细胞核染为蓝色;术后实2周左右实验组及对照组角膜植片均水肿浑浊,术后7-8周后实验组角膜植片保持一定的透明性,而对照组角膜水肿严重,发生大泡性角膜病变;术后角膜植片扫描电镜显示:移植术后一月,移植的细胞散状分布于角膜植片内皮面,呈短梭形;术后二月,部分细胞连成片状,细胞呈梭形及多角形;术后三月,细胞较均匀生长于角膜植片内皮面,细胞呈多角形,细胞间相互连接较紧密;Brdu免疫组织化学染色可见角膜植片内皮表面细胞染色阳性,细胞核被染为褐色。 结论:通过密度梯度离心联合贴壁法可以获得纯度较高的骨髓间充质干细胞,BMSCs通过离心沉淀法移植到角膜内皮面后,在体内诱导为角膜内皮样细胞,并发挥一定的功能。
[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.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R772.2
本文编号：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.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R772.2
【引证文献】
相关硕士学位论文 前2条
1 李柳;诱导hUC-MSCs和VECs转化为角膜内皮样细胞的实验研究[D];暨南大学;2012年
2 肖中男;恒河猴血管内皮细胞移植替代角膜内皮细胞的实验研究[D];昆明医科大学;2012年
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