Pax6基因转染对脂肪来源间充质干细胞的诱导分化研究

发布时间：2018-06-12 23:35

本文选题：组织工程角膜 + 脂肪来源间充质干细胞　；参考：《中国修复重建外科杂志》2014年08期

【摘要】：目的探讨应用Pax6基因转染诱导脂肪来源间充质干细胞(adipose-derived mesenchymal stem cells,ADMSCs)分化为角膜上皮样细胞的可行性。方法取健康5~6周龄C57BL/6小鼠双侧腹股沟脂肪,分离、培养ADMSCs,取第3代细胞进行基因转染。设未转染组(A组)、pcDNA3.1空质粒组(B组)和pcDNA3.1-Pax6重组质粒组(C组),转染48 h后取B、C组进行G418筛选,倒置显微镜下观察细胞形态学改变;Western blot检测各组Pax6蛋白以及角膜上皮细胞特异性标志分子——细胞角蛋白12(cytokeratin 12,CK-12)表达;实时荧光定量PCR检测各组CK-12mRNA表达。结果 A、B组细胞形态无改变;C组筛选出两类不同形状的细胞克隆,一类呈"铺路石"状,形态类似角膜上皮细胞,命名为筛选克隆1;另一类呈网状,有3~7个细胞突起,命名为筛选克隆2。Western blot检测示,A、B组Pax6蛋白表达均为阴性,C组两类细胞克隆均有Pax6蛋白表达;仅C组筛选克隆1 CK-12蛋白表达为阳性,其余3组均为阴性。实时荧光定量PCR检测示,C组筛选克隆1的CK-12 mRNA相对表达量为8.64±0.73,高于筛选克隆2(0.55±0.42)、B组(1.36±0.40)和A组(1.00±0.00)(P0.05);A、B组以及C组筛选克隆2比较差异均无统计学意义(P0.05)。结论 Pax6基因转染能够诱导小鼠ADMSCs分化为角膜上皮样细胞,同时促进CK-12表达,为组织工程角膜构建提供了一种有效的上皮细胞来源。
[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 卤0.73, which was higher than that in group B (0.55 卤0.42) and group A (1.00 卤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 卤0.73, which was higher than that in group B (0.55 卤0.42) and group A (1.00 卤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.
【作者单位】：成都医学院第一附属医院实验研究中心;成都医学院第一附属医院烧伤整形外科;
【基金】：四川省科技厅应用基础研究资助项目(2011JYZ035) 成都医学院第一附属医院重点学科建设资助项目(SSZDXK-001)~~
【分类号】：R329

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