TBX18腺病毒使成鼠骨髓间充质干细胞分化为心肌样细胞的研究

TBX18 gene promotes cardiomyogenic differentiation of adult rat bone mesenchymal stem cells

目的研究TBX18腺病毒能否转染成鼠骨髓间充质干细胞(BMSCs),并诱导其向心肌样细胞分化。方法实验于2015年5月—2016年10月在武汉大学心血管病研究所/心血管病湖北省重点实验室进行。全骨髓贴壁法分离培养BMSCs,采用流式细胞仪分析细胞纯度,随机数字表法分为实验组(TBX18组)、空白病毒对照组(GFP组)和空白对照组(B组),用TBX18或GFP重组腺病毒载体分别转染BMSCs,通过实时荧光定量聚合酶链式反应(qRT-PCR)检测α-SMA基因的表达,蛋白质免疫印迹(Western blot)检测α-SMA和cTnI蛋白的表达。结果通过全骨髓贴壁法可分离获得纯度较高的BMSCs(97%～99%)。TBX18组的α-SMA mRNA表达(2. 66±0. 08)明显高于GFP组(1.49±0.09)和B组(1.00±0.00),差异均有统计学意义(t=16. 743、36. 813,P <0. 01); TBX18组α-SMA蛋白的表达(0.66±0.00)明显高于GFP组(0.36±0.08)和B组(0. 17±0.03),差异均有统计学意义(t=6. 751、23.761,P<0.01);cTnI蛋白的表达(0.57±0.12)明显高于GFP组(0.32±0.04)和B组(0.09±0.00),差异均有统计学意义(t=3.298、6.660,P<0.01)。结论 TBX18腺病毒能成功转入骨髓间充质干细胞,并在细胞内稳定表达;TBX18可诱导成鼠骨髓间充质干细胞分化为心肌样细胞。

Objective To observe if TBX18 can transfect adult rat bone mesenchymal stem cells (BMSCs) to differentiate into cardiomyocyte like cells. Methods The TBMSCs were isolated and cultured by whole bone marrow adherence method, then the cells were analyzed by flow cytometry, they were randomly divided into experimental group, empty virus control group and blank control group. The cells were transfected with TBX18 or GFP. Qrt PCR was used to detect the expression of α-SMA mRNA. Western blot was used to detect the protein expressions of α-SMA and cTnI. Results The whole bone marrow adherent method can isolate the higher purity of BMSCs (97%~99%). The expression levels of α-SMA mRNA were higher in TBX18 groups than GFP groups and blank groups(2.66±0.08 vs. 1.49±0.09 vs. 1.00±0.00, t =16.743, t =36.813, P <0.01); the expression levels of α-SMA protein were higher in TBX18 groups than GFP groups and blank groups (0.66±0.00 vs. 0.36±0.08 vs. 0.17±0.03, t =6.751, t =23.761, P <0.01); the expression levels of cTnI protein were higher in TBX18 groups than GFP groups and blank groups (0.57±0.12 vs. 0.32±0.04 vs. 0.09±0.00, t =3.298, t =6.660, P <0.01). Conclusion TBX18 can successfully be transfected into BMSCs and expresses steadily. TBX18 can induce cardiomyogenic differentiation of adult rat bone mesenchymal stem cells.