摘要
目的探索人骨髓间充质干细胞(MSCs)的诱导分化条件,观察负载人MSCs的羊膜移植重建碱烧伤大鼠眼表的疗效。方法传三代人MSCs分组培养,不同条件刺激后种于羊膜表面。碱烧伤后1个月大鼠随机分A、B、C和D组:A组(A1、A2、A3、A4)移植不同条件诱导的人MSCs,B组移植人角膜缘干细胞(LSCs),C组移植羊膜,D组未治疗。术后观察眼表并评分,2周和1个月时行印迹细胞学(IC)检查,1个月时切片行苏木精-伊红染色,免疫组织化学和免疫荧光检测溴脱氧尿嘧啶(BrdU)、ABCG2、K3/K12、Cx43和血管内皮生长因子(VEGF)。结果术后A、B组较C、D组角膜新生血管和炎性细胞减少、透明度增加(P<0.05)。IC检查示A、B组未见杯状细胞,C、D组可见杯状细胞。A、B组均形成3层以上上皮细胞,C组3层左右,D组为1～2层。免疫组织化学和免疫荧光检查示:A、B组角膜表面可检测到BrdU、ABCG2阳性细胞;A组Cx43均为阴性,A4组中有2个标本K3/K12为弱阳性,其余A组标本均为阴性,B组K3/K12、Cx43均为阳性;A、B组VEGF均较低,C组较强,D组最强。结论人MSCs与人LSCs相同,可成功重建碱烧伤大鼠眼表。其眼表重建功能可能与抗炎和抗血管化作用有关。
Objective This study was to access the differentiation of human mesenchymal stem cells （hMSCs） cultured in vitro and whether transplantation of hMSCs could reconstruct the damaged cornea. Methods HMSCs were isolated from health donors and cultured in BrdU 10 μg/mL,EGF 8 μg/L＋BrdU 10 μg/mL,30% condition medium＋BrdU 10 μg/mL and EGF 8 μg/L＋30% condition medium＋BrdU 10 μg/mL respectively.Cultured cells were labeled with BrdU.Human limbal stem cells （hLSCs） were cultured under the same condition as control.Alkali burned cornea model was established by putting the filter with 1.0 mol/L NaOH on the cornea in 45 SD rats.After growth and expansion on amniotic membrane,cultured cells were transplanted to alkali burned cornea for 1 month in rats.The eyes transplanted with hMSCs were assigned to group A,and group B for LSCs transplantation,group C for amniotic membrane transplantation.Normal eyes without any treatment were assigned to group D.Damaged cornea was clinically scorred and impression cytology was conveyed.The rats were sacrificed 1 month after transplantation and corneal sample was collected and prepared for immuno-fluorescence study of BrdU,ABCG2,K3/K12 and Cx43.Expression of vascular endothelial growth factor（VEGF） was investigated for inflammation-related angiogenesis. Results Less vascular was observed in cornea of group A and B than that of group C and D one month after transplantation（P〈0.05）.Goblet cells were seen on the corneal surface after 1 month of alkali burn in group C and D,but there were no goblet cells in group A and B.The multiple layer epithelium was found in group A,B and C by HE staining.ABCG2 and BrdU were positive in group A and B,and K3/K 12,Cx43 were positively stained in group B instead of in group A. Conclusion MSCs and LSCs on corneal surface of rats can be detected 1 month after transplantation.Transplantation of hMSCs,like LSCs,can successfully reconstruct damaged corneal surface of rat.MSCs do not differentiate into corneal epithelium cells.The function of MSCs reconstructing rat corneal surface may be associated with the inhibition of inflammation and angiogenesis.
出处
《眼科研究》
CSCD
北大核心
2008年第11期810-813,共4页
Chinese Ophthalmic Research
基金
教育部博士点基金资助(20040001112)
