背景 寻求理想的种子细胞是解决角膜移植供体匮乏的研究热点。骨髓间充质干细胞(BMSCs)已被成功地在体内或体外诱导为角膜上皮细胞或视网膜神经节样细胞，但尚无诱导为角膜内皮细胞的报道。 目的 探讨BMSCs移植于角膜内皮表面后的存活和增生情况，进而为BMSCs体内诱导为角膜内皮细胞的研究奠定基础。方法 健康成年恒河猴4只，分为实验组(3只)和对照组(1只)，均取右眼为实验眼。采用密度梯度离心联合贴壁法分离培养BMSCs，培养至第3代，通过流式细胞仪检测细胞表面分子、透射电子显微镜观察细胞超微结构以及对细胞进行脂肪细胞诱导等方法鉴定体外培养的BMSCs，同时用含0.01 g/L 5-溴脱氧尿嘧啶核苷(BrdU)的完全培养基增生培养对细胞进行标记备用。实验组与对照组均用7 mm环钻钻取恒河猴右眼角膜植片，撕除角膜内皮细胞层，实验组采用离心沉淀法将BrdU标记的BMSCs移植于角膜植片，而对照组未进行细胞移植，最后将角膜植片原位缝合回植床。分别于术后1、2、3个月摘取术眼角膜植片进行扫描电子显微镜检查，观察移植后细胞分布及连接情况，同时行BrdU免疫组织化学染色。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12 ～ 16 d细胞90％融合，细胞呈梭形，平行或漩涡状排列。培养细胞的CD29表达阳性率为94.26％，CD45表达阳性率为4.02％，CD34表达阳性率为7.51％。透射电子显微镜显示核质比增大，细胞质内可见丰富的线粒体、高尔基体及粗面内质网。培养的细胞通过脂肪细胞诱导液培养3周后，油红O染色细胞质被染为橘红色，细胞核染为蓝色。实验组术后1个月，右眼角膜植片内皮表面细胞呈短梭形，术后2个月呈梭形及多角形，术后3个月多数细胞呈多角形，细胞数量增多，细胞连接较前紧密，均为单细胞层;角膜植片内表面可见BrdU抗体染色阳性的细胞;对照组扫描电子显微镜检测角膜植片内皮表面无细胞生长，弹性纤维裸露，BrdU染色阴性。 结论 BMSCs通过离心沉淀法移植于角膜内表面可以存活并增生为单细胞层。%Background The quest to look for seed cells is a hot spot of cornea transplant research in solving the problem of the lack of donor. Bone marrow mesenchymal stem cells(BMSCs) have been successfully induced into retinal ganglion cells(RGCs) in vivo,but the successful induction of BMSCs into corneal endothelial cells has not been reported.Objective This experiment was to study the transplantation of BMSCs on corneal endothelial surface using the splitting Descemet's membrane. Methods Four healthy adult rhesus monkeys were divided into the experimental group ( 3 monkeys) and control group ( 1 monkey). Mesenchymal stem cells (MSCs) were isolated from bone marrow by density gradient centrifugation combined with adhering means. The cultured cells were identified by flow cytometry and its ability to differentiate was determined by allowing them to differentiate into adipocytes in vitro and labeled by 5-bromodeoxyuridine ( BrdU ) for subsequent identification. Corneal grafts of 7 mm in size with tearing of the Descemet' s membrane were prepared in the experimental group and control group. After labeling by 5-bromodeoxyuridine( BrdU ) ,cultured cells were transplanted onto the endothelial surface of cornea grafts in the experimental group, but no cultured cells were seeded in the graft of the control group. The corneal grafts were then sutured in situ, and were removed 1,2 or 3 months after operation to examine the distribution and connection between transplanted cells and their morphologic changes under the electron microscope. Results High purity MSCs were harvested by density gradient centrifugation combined with adhering method. Cultured cells reached confluency after 12 to 16 days, presenting with a spindle shape and parallel or swirling arrangement. Flow cytometry analysis showed that 94.26％ of cells were positive for CD29,7. 51％ for CD34 and 4. 02％ for CD45. Larger nuclei filled with plastosomes, golgiosomes and rough endoplasmic reticula were found on the graft under the transmission electron microscope( TEM ). After 3 weeks, MSCs were differentiated into adipocytes where Oil Red O staining resulted in an orange-red staining in the cytoplasm and blue staining in the nuclei. The transplanted cells attached loosely on the endothelial surface of the corneal graft and came in contact with each other in one month. The shape of the cells appeared as spindle-shaped and polygonal after 2 months and became tightly packed after 3 months. The positive cells retained the BrdU label and presented with brown nuclei. No endothelia cells grew in the cornea graft in the control group, with an absence of BrdU labeling. Conclusions Mesenchymal stem cells can be transplanted onto the corneal endothelial surface successfully and form a monolayer using the centrifugation method, and present with good survival and proliferation ability.
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