中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
2期
141-146
,共6页
骨髓间充质干细胞%角膜缘干细胞%饲养细胞%体外培养
骨髓間充質榦細胞%角膜緣榦細胞%飼養細胞%體外培養
골수간충질간세포%각막연간세포%사양세포%체외배양
Bone marrow-derived mesenchymal stem cell%Limbal stem cell%Feeder cell%Cell culture in vitro
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、CD105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％±0.58％、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.
揹景 角膜緣榦細胞功能缺乏(LSCD)時不僅損傷瞭角膜緣榦細胞(LSCs),其基質微環境也被破壞,治療LSCD應包括補充LSCs和恢複微環境兩箇方麵.目前改善LSCD者微環境的方法尚少,迫切需要建立更適閤LSCs體外生長的微環境. 目的 探討骨髓間充質榦細胞(BMSCs)能否成為脩複角膜緣微環境的理想細胞,及其作為飼養細胞在人LSCs體外擴增過程中改善微環境的可能機製. 方法 體外培養人BMSCs併傳3代,用流式細胞儀檢測培養細胞的錶麵抗原CD45、CD71、CD90、CD105和HLA-DR的錶達,併將其定嚮誘導分化為成骨細胞和脂肪細胞.將人BMSCs經絲裂黴素C(MMC)作用後成為飼養細胞.用供體人角膜緣組織分離培養LSCs,分彆與BMSCs飼養細胞(BMSCs飼養組)、Swiss-3T3成纖維細胞飼養細胞(Swiss-3T3飼養組)共培養或單獨培養(無飼養細胞組),比較3箇組LSCs的集落生成率(CFE).將BMSCs飼養組的LSCs繼續進行培養,用流式細胞儀對培養的細胞進行鑒定.逆轉錄PCR(RT-PCR)法分析人BMSCs中相關細胞因子的錶達,以評估其改善角膜緣基質微環境的可能機製. 結果 體外培養的BMSCs均質性好,間質細胞標誌物CD71、CD90、CD105在穫得的BMSCs上均呈高錶達,而造血細胞標誌物CD45和HLA-DR抗原均呈低錶達.共培養12d後,BMSCs飼養組、Swiss-3T3飼養組以及無飼養細胞組LSCs的CFE分彆為3.67％±0.58％、4.30％±1.54％、0.20％±0.10％,3箇組間總體差異有統計學意義(F=15.420,P=0.040)；BMSCs飼養組與Swiss-3T3飼養組的CFE差異無統計學意義(P=0.456).BMSCs飼養組與無飼養細胞組間、Swiss-3T3飼養組與無飼養細胞組間CFE的差異均有統計學意義(P=0.005、0.002).經流式細胞學檢測,BMSCs飼養組LSCs的ABCG2抗原暘性.RT-PCR反應後瓊脂糖凝膠電泳結果顯示,堿性成纖維細胞生長因子(bFGF)、榦細胞因子(SCF)、N-鈣黏連蛋白(N-cad)在BMSCs中呈暘性錶達.結論 人BMSCs能夠改善LSCs生長的基質微環境,提高其增生能力,是LSCs體外培養飼養細胞的理想來源.
배경 각막연간세포공능결핍(LSCD)시불부손상료각막연간세포(LSCs),기기질미배경야피파배,치료LSCD응포괄보충LSCs화회복미배경량개방면.목전개선LSCD자미배경적방법상소,박절수요건립경괄합LSCs체외생장적미배경. 목적 탐토골수간충질간세포(BMSCs)능부성위수복각막연미배경적이상세포,급기작위사양세포재인LSCs체외확증과정중개선미배경적가능궤제. 방법 체외배양인BMSCs병전3대,용류식세포의검측배양세포적표면항원CD45、CD71、CD90、CD105화HLA-DR적표체,병장기정향유도분화위성골세포화지방세포.장인BMSCs경사렬매소C(MMC)작용후성위사양세포.용공체인각막연조직분리배양LSCs,분별여BMSCs사양세포(BMSCs사양조)、Swiss-3T3성섬유세포사양세포(Swiss-3T3사양조)공배양혹단독배양(무사양세포조),비교3개조LSCs적집락생성솔(CFE).장BMSCs사양조적LSCs계속진행배양,용류식세포의대배양적세포진행감정.역전록PCR(RT-PCR)법분석인BMSCs중상관세포인자적표체,이평고기개선각막연기질미배경적가능궤제. 결과 체외배양적BMSCs균질성호,간질세포표지물CD71、CD90、CD105재획득적BMSCs상균정고표체,이조혈세포표지물CD45화HLA-DR항원균정저표체.공배양12d후,BMSCs사양조、Swiss-3T3사양조이급무사양세포조LSCs적CFE분별위3.67％±0.58％、4.30％±1.54％、0.20％±0.10％,3개조간총체차이유통계학의의(F=15.420,P=0.040)；BMSCs사양조여Swiss-3T3사양조적CFE차이무통계학의의(P=0.456).BMSCs사양조여무사양세포조간、Swiss-3T3사양조여무사양세포조간CFE적차이균유통계학의의(P=0.005、0.002).경류식세포학검측,BMSCs사양조LSCs적ABCG2항원양성.RT-PCR반응후경지당응효전영결과현시,감성성섬유세포생장인자(bFGF)、간세포인자(SCF)、N-개점련단백(N-cad)재BMSCs중정양성표체.결론 인BMSCs능구개선LSCs생장적기질미배경,제고기증생능력,시LSCs체외배양사양세포적이상래원.
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 LSCD 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,CD90,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 cells 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 Cultured 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 cocultured 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 cells.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.
