【摘要】：目的:觀察Transwell接觸共培養(yǎng)促進(jìn)單散人誘導(dǎo)多能干細(xì)胞(induced pluripotent stem cells,iPSCs)生長(zhǎng)及分化的作用。方法:將1~2代牛角膜內(nèi)皮細(xì)胞(corneal endothelial cells,CECs)接種在Transwell小室底面培養(yǎng)8 h后,應(yīng)用Accutase消化及40μm過(guò)濾處理獲得單散iPSCs,將其接種到已有CECs的Transwell小室內(nèi)共培養(yǎng)14 d,前3 d使用mTeSR1培養(yǎng)基,第4天開(kāi)始用含10%胎牛血清的低糖DMEM培養(yǎng)基。分別進(jìn)行實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)(real-time fluorescence quantitative polymerase chain reaction,qPCR)、免疫熒光、死活細(xì)胞染色及堿性磷酸酶(alkaline phosphatase,ALP)染色,對(duì)iPSCs多能特性表達(dá)及分化進(jìn)行鑒定。設(shè)定單散iPSCs共培養(yǎng)組為實(shí)驗(yàn)組,常規(guī)培養(yǎng)iPSCs組為對(duì)照組(一),非共培養(yǎng)單散iPSCs組為對(duì)照組(二)。結(jié)果:培養(yǎng)牛CECs形態(tài)呈典型的六邊形鋪路石樣外觀。人iPSCs呈克隆樣生長(zhǎng),共培養(yǎng)3 d后iPSCs貼壁呈單散細(xì)胞生長(zhǎng),免疫熒光檢測(cè)未分化標(biāo)志Nanog和Oct4呈陽(yáng)性。qPCR檢測(cè)Nanog、Oct4和Sox2 mRNA表達(dá),實(shí)驗(yàn)組與對(duì)照組(一)比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。死活細(xì)胞染色顯示,實(shí)驗(yàn)組死細(xì)胞明顯減少,與對(duì)照組(二)比較差異有統(tǒng)計(jì)學(xué)意義(P0.01)。共培養(yǎng)14 d后,人iPSCs形態(tài)比較均一,呈多邊形,體積增大,無(wú)明顯克隆團(tuán)塊;ALP染色陰性;免疫熒光染色ZO-1、AQP1和CD31表達(dá)陽(yáng)性,CD34和CD133表達(dá)陰性。qPCR檢測(cè)Oct4、Nanog和Sox2 mRNA表達(dá)明顯下調(diào),與對(duì)照組(一)比較差異有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論:與牛CECs共培養(yǎng)可增強(qiáng)人單散iPSCs活性,使iPSCs形態(tài)上向內(nèi)皮樣細(xì)胞轉(zhuǎn)化,表達(dá)部分CECs的標(biāo)志。Transwell接觸共培養(yǎng)模型可以促進(jìn)單散iPSCs生長(zhǎng)及分化。
[Abstract]:Aim: to observe the effect of co-culture of Transwell on the growth and differentiation of (induced pluripotent stem cells. Methods: bovine corneal endothelial cells (corneal endothelial cells) were cultured at the bottom of Transwell chamber for 8 h, then digested with Accutase and treated with 40 渭 m filtration. The cells were inoculated into the Transwell chamber of CECs for 14 days, and mTeSR1 medium was used for the first 3 days. On the 4th day, low sugar DMEM medium containing 10% fetal bovine serum was used. The multipotent expression and differentiation of iPSCs were identified by real-time fluorescence quantitative polymerase chain reaction (real-time fluorescence quantitative polymerase chain reaction- Q PCR), immunofluorescence, live and dead cell staining and alkaline phosphatase (alkaline) staining. Single powder iPSCs co-culture group was established as experimental group, conventional cultured iPSCs group as control group (1) and non co cultured single powder iPSCs group as control group (2). Results: the morphology of cultured bovine CECs showed typical hexagonal paving stone appearance. After 3 days of co-culture, human iPSCs grew as a single cell, and Nanog and Oct4 were positive for Nanog and Oct4. The expression of Nanog Oct4 and Sox2 mRNA in the experimental group was not significantly different from that in the control group (P0.05). The dead cells in the experimental group were significantly decreased compared with the control group (P0.01). After 14 days of co-culture, the morphology of human iPSCs was homogenous, polygonal, volume increased, and there was no significant negative staining of iPSCs, and the expression of CD34 and CD133 was negative in ZO-1AQP1 and CD31 by immunofluorescence staining. QPCR showed that the expression of Oct4Nangand Sox2 mRNA was down-regulated. Compared with the control group (1), the difference was statistically significant (P0.01). Conclusion: Co-culture with bovine CECs can enhance the activity of iPSCs and transform iPSCs into endothelium-like cells in morphology. Transwell contact co-culture model of partial CECs expression can promote the growth and differentiation of monodisperse iPSCs.
【作者單位】： 暨南大學(xué)附屬第一醫(yī)院眼科;暨南大學(xué)再生醫(yī)學(xué)教育部重點(diǎn)實(shí)驗(yàn)室;暨南大學(xué)醫(yī)學(xué)院眼科研究所;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81371689) 廣東省自然科學(xué)基金資助項(xiàng)目(No.S2013010013391)
【分類號(hào)】：R329

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