本文選題：組織工程角膜 + 脂肪來源間充質(zhì)干細胞��； 參考：《中國修復(fù)重建外科雜志》2014年08期

【摘要】：目的探討應(yīng)用Pax6基因轉(zhuǎn)染誘導(dǎo)脂肪來源間充質(zhì)干細胞(adipose-derived mesenchymal stem cells,ADMSCs)分化為角膜上皮樣細胞的可行性。方法取健康5~6周齡C57BL/6小鼠雙側(cè)腹股溝脂肪,分離、培養(yǎng)ADMSCs,取第3代細胞進行基因轉(zhuǎn)染。設(shè)未轉(zhuǎn)染組(A組)、pcDNA3.1空質(zhì)粒組(B組)和pcDNA3.1-Pax6重組質(zhì)粒組(C組),轉(zhuǎn)染48 h后取B、C組進行G418篩選,倒置顯微鏡下觀察細胞形態(tài)學(xué)改變;Western blot檢測各組Pax6蛋白以及角膜上皮細胞特異性標(biāo)志分子——細胞角蛋白12(cytokeratin 12,CK-12)表達;實時熒光定量PCR檢測各組CK-12mRNA表達。結(jié)果 A、B組細胞形態(tài)無改變;C組篩選出兩類不同形狀的細胞克隆,一類呈"鋪路石"狀,形態(tài)類似角膜上皮細胞,命名為篩選克隆1;另一類呈網(wǎng)狀,有3~7個細胞突起,命名為篩選克隆2。Western blot檢測示,A、B組Pax6蛋白表達均為陰性,C組兩類細胞克隆均有Pax6蛋白表達;僅C組篩選克隆1 CK-12蛋白表達為陽性,其余3組均為陰性。實時熒光定量PCR檢測示,C組篩選克隆1的CK-12 mRNA相對表達量為8.64±0.73,高于篩選克隆2(0.55±0.42)、B組(1.36±0.40)和A組(1.00±0.00)(P0.05);A、B組以及C組篩選克隆2比較差異均無統(tǒng)計學(xué)意義(P0.05)。結(jié)論 Pax6基因轉(zhuǎn)染能夠誘導(dǎo)小鼠ADMSCs分化為角膜上皮樣細胞,同時促進CK-12表達,為組織工程角膜構(gòu)建提供了一種有效的上皮細胞來源。
[Abstract]:Objective to investigate the feasibility of inducing adipose-derived mesenchymal stem cells from adipose-derived mesenchymal stem cells to differentiate into corneal epithelioid cells by Pax6 gene transfection. Methods the bilateral inguinal fat of C57BL / 6 mice was isolated and cultured, and the third passage cells were transfected by gene transfection. After 48 hours of transfection, the blank plasmid group (pcDNA3.1) and the recombinant plasmid pcDNA3.1-Pax6 (group C) were selected for G418 screening. The expression of Pax6 protein and 12(cytokeratin _ (12) CK-12, a specific marker of corneal epithelial cells, was detected by Western blot, and the expression of CK-12 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction (FQ-PCR). Results two kinds of cell clones of different shapes were screened in group C, which were similar to corneal epithelial cells and were named as screening clone 1, while those in group A were reticulated and had 3 ~ 7 cell processes. Western blot analysis showed that the expression of Pax6 protein was negative in both cell clones of group C and group C, but was positive in group C, and negative in group C, while in group C, the expression of Pax6 protein was negative in the other three groups. The relative expression of CK-12 mRNA in group C was 8.64 鹵0.73, which was higher than that in group B (0.55 鹵0.42) and group A (1.00 鹵0.005). There was no significant difference between group A and group C in the screening of clone 2 (P 0.05). The relative expression of CK-12 mRNA in group C was 8.64 鹵0.73, which was higher than that in group B (0.55 鹵0.42) and group A (1.00 鹵0.005). Conclusion Pax6 gene transfection can induce ADMSCs to differentiate into corneal epithelioid cells and promote the expression of CK-12, which provides an effective source of epithelial cells for tissue engineering corneal construction.
【作者單位】： 成都醫(yī)學(xué)院第一附屬醫(yī)院實驗研究中心;成都醫(yī)學(xué)院第一附屬醫(yī)院燒傷整形外科;
【基金】：四川省科技廳應(yīng)用基礎(chǔ)研究資助項目(2011JYZ035) 成都醫(yī)學(xué)院第一附屬醫(yī)院重點學(xué)科建設(shè)資助項目(SSZDXK-001)~~
【分類號】：R329

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