负载自体骨髓间充质干细胞人羊膜贴片移植对切除翼状胬肉患者损伤角膜的保护  

Transplantation of human amniotic membrane loaded with autologous bone marrow mesenchymal stem cells in treatment of corneal wound in a pterygium excision patient

作　　者：毕薇薇[1] 郭立斌[1] 李志刚[1] 

机构地区：[1]四平市中心医院中心实验室,吉林省四平市136000

出　　处：《中国组织工程研究与临床康复》2008年第43期8445-8448,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:近年来国内外对单纯羊膜移植和羊膜负载细胞移植治疗角膜损伤的研究逐渐增多,在动物实验中已取得确切疗效,但临床应用报道甚少。目的:拟制备人羊膜负载的自体骨髓间充质干细胞贴片,观察贴片移植后角膜损伤的改善。设计、时间及地点:细胞组织工程学体内观察,于2008-02在四平市中心医院中心实验室完成。对象:同期四平市中心医院收治的1例翼状胬肉患者。羊膜取自正常剖腹分娩产妇的胎盘。方法:无菌条件下钝性分离人羊膜与绒毛膜,胰蛋白酶消化去除羊膜表面上皮细胞,将硝酸纤维素膜剪成适合眼部大小的圆形支架贴于基底面,将膜片浸泡在α-MEM培养基中,上皮面向上,4℃保存备用。采集翼状胬肉患者自体骨髓,Ficoll法密度梯度分离骨髓单个核细胞,于37℃、体积分数为0.05的CO2培养箱中孵育扩增,将鉴定后的第3代骨髓间充质干细胞按2×105密度接种至羊膜上,加入含10%血清的α-MEM培养基,细胞长满后进行移植。洗涤负载骨髓间充质干细胞的人羊膜贴片至无橙红液体,通过眼科手术去除病变组织后,将羊膜贴片细胞面向上覆盖于翼状胬肉患者角膜病变表面,缝合固定后,另缝合一层单纯羊膜用于保护移植细胞。主要观察指标:人羊膜负载的骨髓间充质干细胞鉴定结果,贴片移植效果。结果:接种到人羊膜上4h细胞开始贴壁,培养24h细胞变为梭形且数量增多,48h后细胞紧密排列,倒置显微镜下人羊膜上的骨髓间充质干细胞覆盖面积大于80%,锥虫蓝染色细胞存活率大于90%,免疫荧光染色细胞CD44和CD90阳性率均大于90%。人羊膜负载的骨髓间充质干细胞贴片移植后1周内,羊膜贴片无溶解和脱落,患者眼表透明,无水肿;移植后8个月内无新生血管生成。结论:骨髓间充质干细胞接种到人羊膜上生长状态良好,将该贴片移植至切除翼状胬肉患者后短期内未发生溶解和脱落BACKGROUND： Recently, amniotic membrane transplantation and amniotic membrane loaded cell transplantation were studied extensively. The reliable curative effect was obtained in animals. Studies in clinic were few. OBJECTIVE： To study preparation of human amniotic membrane loaded autologous bone marrow mesenchymal stem cells, and to observe the improvement of cornea injury after transplantation. DESIGN, TIME AND SETTING： Cell tissue engineering in vivo experiment was performed at the Central Laboratory, Siping Center Hospital in February 2008. PARTICIPANTS： One patient with pterygium at Siping Center Hospital was enrolled. Amniotic membrane was obtained from placenta of normal delivery parturients. METHODS： Human amniotic membrane and chorion were collected sterilely. Surface epithelial cells of amniotic membrane were removed by trypsin digestion. Nitrocellulose filter was cut into round scaffold for the size of eye, and adhered to the basal surface. Membrane was immersed in a -MEM medium, with epithelium surface upward, and placed at 4 ℃. Bone marrow was obtained from a patient ,;uffered from pterygium. Single nucleus cells were isolated and purified by Ficoll density and gradient centrifuge, cultivated by culture medium at 37 ℃ in a 0.05 volume fraction CO： incubator. At the third passage, BMSCs were incubated in amniotic membrane at a density of 2×10^5 with α -MEM medium containing 10% serum. After cell confluence, transplantation was performed. Human amniotic membrane patch loaded with autologous bone marrow mesenchymal stem cells were washed till orange red liquid disappeared. Lesion tissues were removed by ophthalmologic operation. Amniotic membrane patch with cell surface upward was co vered on corneal lesion surface in the patient with pterygium. After suturing, another simple amniotic membrane was sutured to protect transplanted cells. MAIN OUTCOME MEASURES： Identification results of human amniotic membrane loaded autologous bone marrow mesenchymal stem cells; patch

关 键 词：人骨髓间充质干细胞 羊膜 角膜 贴片 

分 类 号：R394.2[医药卫生—医学遗传学]