骨髓间充质干细胞对角膜缘干细胞微环境的影响

The impact of the bone marrow-derived mesenchymal stem cell on the microenvironment of the human limbal stem cell

背景角膜缘干细胞功能缺乏（LSCD）时不仅损伤了角膜缘干细胞（LSCs），其基质微环境也被破坏，治疗LSCD应包括补充LSCs和恢复微环境两个方面。目前改善LSCD者微环境的方法尚少，迫切需要建立更适合LSCs体外生长的微环境。目的探讨骨髓间充质干细胞（BMSCs）能否成为修复角膜缘微环境的理想细胞，及其作为饲养细胞在人LSCs体外扩增过程中改善微环境的可能机制。方法体外培养人BMSCs并传3代，用流式细胞仪检测培养细胞的表面抗原CD45、CD71、CD90、CD105和HLA-DR的表达，并将其定向诱导分化为成骨细胞和脂肪细胞。将人BMSCs经丝裂霉素c（MMC）作用后成为饲养细胞。用供体人角膜缘组织分离培养LSCs，分别与BMSCs饲养细胞（BMSCs饲养组）、Swiss-3T3成纤维细胞饲养细胞（Swiss-3T3饲养组）共培养或单独培养（无饲养细胞组），比较3个组LSCs的集落生成率（CFE）。将BMSCs饲养组的LSCs继续进行培养，用流式细胞仪对培养的细胞进行鉴定。逆转录PCR（RT—PCR）法分析人BMSCs中相关细胞因子的表达，以评估其改善角膜缘基质微环境的可能机制。结果体外培养的BMSCs均质性好，间质细胞标志物CD71、CD90、CDl05在获得的BMSCs上均呈高表达，而造血细胞标志物CD45和HLA-DR抗原均呈低表达。共培养12d后，BMSCs饲养组、Swiss-3T3饲养组以及无饲养细胞组LSCs的CFE分别为3．67％±O．58％、4．30％±1．54％、0．20％±0．10％，3个组间总体差异有统计学意义（F=15．420，P=0．040）；BMSCs饲养组与Swiss=3T3饲养组的CFE差异无统计学意义（P=0．456）。BMSCs饲养组与无饲养细胞组间、Swiss-3T3饲养组与无饲养细胞组间CFE的差异均有统计学意义（P=0．005、0．002）。经流式细胞学检测，BMSCs饲养组LSCs的ABCG2抗原阳性。RT-PCR反应后琼脂糖凝胶电泳结果显示，碱性成纤维细胞生长因子（bFGF）、干细胞因子（SCF）、N．钙黏连蛋白（N-cad）在BMSCs中呈阳性表达。结论人BMSCs能够改善LSCs生长的基质微环境，提高其增生能力，是LSCs体外培养饲养细胞的理想来源。

Background When limbal stem cell deficiency （LSCD） occurs, not only the limbal stem cells （LSCs） were damaged,but also the LSCs matrix microenvironment was under destruction. The treatment of LSCI） include both replenishing of stem cells and restoration of microenvironment. So far, the method to improve the microenvironment of LSCD still exist limitation and urgently need to establish more appropriate microenvironment for the LSCs growth in vitro. Objective This study was to investigate whether the human bone marrow-derived mesenchymal stem cells （BMSCs） can be used as the ideal cells to repair limbal microenvironment and its possible mechanism of repairing limbal microenvironment during the human LSCs amplification in vitro as feeder cells. Methods BMSCs were cultured and passaged in vitro, and flow cytometry was used to assay the expressions of CD45,CD71,CDg0, CD105 and HLA-DR and directionally induced BMSCs to the osteoblasts and adipocytes. BMSCs were treated using mitomycin C （MMC） to use as the feeder cells. LSCs were separately co-cultured with BMSCs, Swiss-3T3 feeder ceils and free-feeder cells,and colony-forming efficiency （CFE） of the LSCs was compared among different co-cultured groups. LSCs were then cultured sequentially and identified by flow cytometry. Expression of cytokines in BMSCs was confirmed by reverse transcriptional polymerase chain reaction （RT-PCR）. Results Cuhured BMSCs showed a good homogeneity, with a lot of expressions of interstitial cell markers such as CD71, CD90,CD105 and less expressions of hematopoietic cell markers including CD45 and HLA-DR. After separately co- cultured with feeder cells for 12 days, the CFE of the LSCs co-cultured with BMSCs, Swiss-3T3 and no feeder cells was 3.67% -＋0.58% ,4. 30% ~ 1.54% and 0.20% ~0. 10% , showing a statistical significant difference among the three groups （F = 15. 420,P = 0. 040）. There was no statistically significant difference in the C FE of the LSCs between the BMSCs feeder group and the Swiss-3T3 feeder cells group（P = 0. 456） ,between the BMSCs feeder group and the free-feeder cells group or the Swiss-3T3 co-culture group and the free-feeder cells group （P = 0. 005,0. 002 ）. LSCs presented with a positive response for ABCG2 antigen in the co-cultured with BMSCs group. Basic fibroblast growth factor（bFGF） ,stem cell factor （SCF） and N-cadherin（N-cad） were positively expressed in the BMSCs as feeder ceils. Conclusions Human BMSCs-derived feeder cells can improve the growth of the stromal microenvironment of the LSCs and enhance their proliferation ability. Human BMSCs are suitable for engineering of epithelial sheets.