中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2014年
8期
1404-1409
,共6页
刘庆%郭永龙%郭晓令%连瑞玲%陈建苏
劉慶%郭永龍%郭曉令%連瑞玲%陳建囌
류경%곽영룡%곽효령%련서령%진건소
诱导多能干细胞%角膜内皮细胞%Transwell接触共培养
誘導多能榦細胞%角膜內皮細胞%Transwell接觸共培養
유도다능간세포%각막내피세포%Transwell접촉공배양
Induced pluripotent stem cells%Corneal endothelial cells%Transwell contactco-culture
目的：观察Transwell接触共培养促进单散人诱导多能干细胞（inducedpluripotentstemcells， iPSCs）生长及分化的作用。方法：将1～2代牛角膜内皮细胞（corneal endothelial cells， CECs）接种在Transwell 小室底面培养8 h后，应用Accutase消化及40μm过滤处理获得单散iPSCs，将其接种到已有CECs的Transwell小室内共培养14 d，前3 d使用mTeSR1培养基，第4天开始用含10％胎牛血清的低糖DMEM培养基。分别进行实时荧光定量聚合酶链式反应（ real-time fluorescence quantitative polymerase chain reaction ， qPCR ）、免疫荧光、死活细胞染色及碱性磷酸酶（ alkaline phosphatase ， ALP）染色，对iPSCs多能特性表达及分化进行鉴定。设定单散iPSCs共培养组为实验组，常规培养iPSCs组为对照组（一），非共培养单散iPSCs组为对照组（二）。结果：培养牛CECs形态呈典型的六边形铺路石样外观。人iPSCs呈克隆样生长，共培养3 d后iPSCs贴壁呈单散细胞生长，免疫荧光检测未分化标志Nanog和Oct4呈阳性。 qPCR检测Nanog、Oct4和Sox2 mRNA表达，实验组与对照组（一）比较差异无统计学意义（P＞0.05）。死活细胞染色显示，实验组死细胞明显减少，与对照组（二）比较差异有统计学意义（P＜0.01）。共培养14 d后，人iPSCs形态比较均一，呈多边形，体积增大，无明显克隆团块；ALP染色阴性；免疫荧光染色ZO-1、AQP1和CD31表达阳性，CD34和CD133表达阴性。 qPCR检测Oct4、Nanog和Sox2 mRNA表达明显下调，与对照组（一）比较差异有统计学意义（P＜0．01）。结论：与牛CECs共培养可增强人单散iPSCs活性，使iPSCs形态上向内皮样细胞转化，表达部分CECs的标志。 Transwell接触共培养模型可以促进单散iPSCs生长及分化。
目的：觀察Transwell接觸共培養促進單散人誘導多能榦細胞（inducedpluripotentstemcells， iPSCs）生長及分化的作用。方法：將1～2代牛角膜內皮細胞（corneal endothelial cells， CECs）接種在Transwell 小室底麵培養8 h後，應用Accutase消化及40μm過濾處理穫得單散iPSCs，將其接種到已有CECs的Transwell小室內共培養14 d，前3 d使用mTeSR1培養基，第4天開始用含10％胎牛血清的低糖DMEM培養基。分彆進行實時熒光定量聚閤酶鏈式反應（ real-time fluorescence quantitative polymerase chain reaction ， qPCR ）、免疫熒光、死活細胞染色及堿性燐痠酶（ alkaline phosphatase ， ALP）染色，對iPSCs多能特性錶達及分化進行鑒定。設定單散iPSCs共培養組為實驗組，常規培養iPSCs組為對照組（一），非共培養單散iPSCs組為對照組（二）。結果：培養牛CECs形態呈典型的六邊形鋪路石樣外觀。人iPSCs呈剋隆樣生長，共培養3 d後iPSCs貼壁呈單散細胞生長，免疫熒光檢測未分化標誌Nanog和Oct4呈暘性。 qPCR檢測Nanog、Oct4和Sox2 mRNA錶達，實驗組與對照組（一）比較差異無統計學意義（P＞0.05）。死活細胞染色顯示，實驗組死細胞明顯減少，與對照組（二）比較差異有統計學意義（P＜0.01）。共培養14 d後，人iPSCs形態比較均一，呈多邊形，體積增大，無明顯剋隆糰塊；ALP染色陰性；免疫熒光染色ZO-1、AQP1和CD31錶達暘性，CD34和CD133錶達陰性。 qPCR檢測Oct4、Nanog和Sox2 mRNA錶達明顯下調，與對照組（一）比較差異有統計學意義（P＜0．01）。結論：與牛CECs共培養可增彊人單散iPSCs活性，使iPSCs形態上嚮內皮樣細胞轉化，錶達部分CECs的標誌。 Transwell接觸共培養模型可以促進單散iPSCs生長及分化。
목적：관찰Transwell접촉공배양촉진단산인유도다능간세포（inducedpluripotentstemcells， iPSCs）생장급분화적작용。방법：장1～2대우각막내피세포（corneal endothelial cells， CECs）접충재Transwell 소실저면배양8 h후，응용Accutase소화급40μm과려처리획득단산iPSCs，장기접충도이유CECs적Transwell소실내공배양14 d，전3 d사용mTeSR1배양기，제4천개시용함10％태우혈청적저당DMEM배양기。분별진행실시형광정량취합매련식반응（ real-time fluorescence quantitative polymerase chain reaction ， qPCR ）、면역형광、사활세포염색급감성린산매（ alkaline phosphatase ， ALP）염색，대iPSCs다능특성표체급분화진행감정。설정단산iPSCs공배양조위실험조，상규배양iPSCs조위대조조（일），비공배양단산iPSCs조위대조조（이）。결과：배양우CECs형태정전형적륙변형포로석양외관。인iPSCs정극륭양생장，공배양3 d후iPSCs첩벽정단산세포생장，면역형광검측미분화표지Nanog화Oct4정양성。 qPCR검측Nanog、Oct4화Sox2 mRNA표체，실험조여대조조（일）비교차이무통계학의의（P＞0.05）。사활세포염색현시，실험조사세포명현감소，여대조조（이）비교차이유통계학의의（P＜0.01）。공배양14 d후，인iPSCs형태비교균일，정다변형，체적증대，무명현극륭단괴；ALP염색음성；면역형광염색ZO-1、AQP1화CD31표체양성，CD34화CD133표체음성。 qPCR검측Oct4、Nanog화Sox2 mRNA표체명현하조，여대조조（일）비교차이유통계학의의（P＜0．01）。결론：여우CECs공배양가증강인단산iPSCs활성，사iPSCs형태상향내피양세포전화，표체부분CECs적표지。 Transwell접촉공배양모형가이촉진단산iPSCs생장급분화。
[ABSTRACT]AIM:ToinvestigatethepromotingroleofTranswellcontactco-culturesysteminthegrowthand differentiation of single-dissociated induced pluripotent stem cells (iPSCs).METHODS:Bovine corneal endothelial cells (CECs) at passage 1~2 (P1~2) were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃and 5%CO2 for 8 h.Accutase digestion and 40μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs , and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR 1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks.The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction ( qPCR ) , immunofluorescence staining, live&dead cell staining and alkaline phosphatase (ALP) staining.The group of iPSCs cultured in conventional medium was used as control group 1.The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2.RESULTS: The bovine CECs showed typical hexagonal cobblestone shape .iPSCs showed colony-like growth , while became single-dissociated cells after Tran-swell contact co-culture with bovine CECs for 3 d.The single-dissociated iPSCs positively expressed the undifferentiated markers, Nanog and Oct4.The mRNA expression levels of Nanog , Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference (P>0.05).The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 (P<0.01).After 14 d of induced differentiation co-culture , the single-dissociated iPSCs showed rather uniform polygonal morphology , increased dimension and no obvious colony existence .Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed.The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased , and had statistically significant difference compared with control group 1 (P<0.01).CONCLUSION: When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs .Transwell contact co-culture system not only enhances the growth of single-dis-sociated iPSCs , but also promotes their differentiation .
