1,25-维生素-D3体外诱导骨髓间充质干细胞向心肌样细胞分化的最适浓度

刘洋 吕洋 王浩宇 李娇 任君旭 王海萍*

doi:10.16098/j.issn.0529-1356.2019.05.007

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解剖学报 ›› 2019, Vol. 50 ›› Issue (5) : 580-588. DOI: 10.16098/j.issn.0529-1356.2019.05.007

1,25-维生素-D3体外诱导骨髓间充质干细胞向心肌样细胞分化的最适浓度

刘洋吕洋王浩宇李娇任君旭王海萍*

作者信息 +

Optimal concentration of 1,25-vitamin-D₃ to induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro

LIU Yang Lü Yang WANG Hao-yu LI Jiao REN Jun-xu WANG Hai-ping*

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文章历史 +

摘要

目的探讨1，25-维生素D₃（1,25-vitamin-D₃）体外诱导骨髓间充质干细胞（BMSCs）向心肌样细胞分化的最适浓度。方法采用全骨髓贴壁法结合密度梯度离心法分离培养SD大鼠BMSCs，应用3、6、12、24 nmol/L 1,25-vitamin-D₃对第2代BMSCs进行定向诱导，倒置相差显微镜下动态观察细胞生长状况。运用流式细胞仪对BMSCs进行鉴定。应用免疫细胞化学、免疫荧光及Western blotting法检测诱导后4周的BMSCs原肌球蛋白（TPM）、间隙连接蛋白43（Cx43）、心肌肌钙蛋白T（cTnT）的表达情况。运用透射电子显微镜观察分化细胞与心肌细胞类似的超微结构。在诱导后1、2和4周这3个时间点，以Real-time PCR法检测细胞心肌早期转录因子GATA结合蛋白4（GATA4）、Nkx2.5的表达。结果 1.倒置相差显微镜下观察，原代细胞培养72 h后，大部分细胞呈短梭形；培养1周后，细胞呈现出多样化的形态；诱导4周的BMSCs，相邻细胞间联系紧密，排列具有明显的方向性。不同浓度1,25-vitamin-D₃诱导后的BMSCs，其数量及形态存在差异。2.流式细胞术的鉴定结果显示，CD29、CD45、CD90的阳性表达率分别为97.4%、3.3%和91.4%。3.免疫细胞化学、免疫荧光及Western blotting法检测不同浓度1,25-vitamin-D₃诱导4周后的BMSCs均可见TPM、Cx43及cTnT的阳性表达，其中6 nmol/L组的表达均最高，而未诱导组细胞呈弱阳性或阴性表达，差异具有统计学意义（P＜0.05）。4.透射电子显微镜观察，细胞诱导培养至4周，胞质内可见较多平行排列的肌丝及线粒体、核糖体、粗面内质网等细胞器。5.Real-time PCR检测结果显示，GATA4及Nkx2.5基因于诱导后1周表达增强，2周表达减弱，4周表达增强；4个诱导组相比较，6 nmol/L组明显优于其他3组（P＜0.05）。结论 1,25-vitamin-D₃可以诱导BMSCs获得心肌分化表型，其诱导分化最适浓度为6 nmol/L。

Abstract

Objective To explore the optimal concentration of 1,25-vitamin-D₃ for inducing the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocyte-like cells in vitro. Methods BMSCs of SD rats were isolated and cultured by whole bone marrow adherent method combined with density gradient centrifugations. Depending on the final concentration of 1,25-vitamin-D3, the 2nd-generation BMSCs were divided into five groups: 3 nmol/L group, 6 nmol/L group, 12 nmol/L group, 24 nmol/L group and the control group. The adherent cells were observed dynamically under the inverted phase contrast microscope, including their morphology and growth status. The surface antigens, morphological characteristics, protein expression and mRNA expression of the cells in each group were assessed. Results 1. Under the inverted phase contrast microscope, most of the primary cells showed a short spindle shape after 72 hours of culture. After 1 week of culture, the cells showed diversified morphology. The adjacent cells of BMSCs, induced after 4 weeks, were closely connected with each other, and the arrangement had obvious directivity. There were differences in the number and morphology of BMSCs, induced by 1,25-vitamin-D₃ at different concentrations. 2. The result of flow cytometry showed that the positive expression rates of CD29, CD45 and CD90 were 97.4%, 3.3% and 91.4%, respectively, 3. The results of immunofluorescence, immunocytochemistry and Western blotting revealed that the expressions of tropomyosin(TPM), connexin43(Cx43) and cardiac troponin T(cTnT) in 6 nmol/L group were significantly higher than that of the other groups, while that in control group were weak or negative (P<0.05). 4. Transmission electron microscope(TEM) observation showed that the induced cells had a cardiomyocyte-like ultra structure: there were many parallel arranged myofilaments, mitochondria, ribosomes, rough endoplasmic reticulum and other organelles in the cytoplasm. 5. The result of Real-time PCR showed that the induced cells could express GATA binding protein 4(GATA 4) and Nkx2.5 at the 1st week, and then the expression of them decreased at the 2nd week, but then increased at the 4th week. The 6 nmol/L group was superior to the other three groups in gene expression (P<0.05). Conclusion 1,25-vitamin-D₃ can induce BMSCs to obtain myocardial differentiation phenotype, and the optimum concentration of inducing differentiation is 6 nmol/L.

导出引用

刘洋吕洋王浩宇李娇任君旭王海萍. 1,25-维生素-D3体外诱导骨髓间充质干细胞向心肌样细胞分化的最适浓度[J]. 解剖学报. 2019, 50(5): 580-588 https://doi.org/10.16098/j.issn.0529-1356.2019.05.007

LIU Yang Lü Yang WANG Hao-yu LI Jiao REN Jun-xu WANG Hai-ping.

Optimal concentration of 1,25-vitamin-D₃ to induce the differentiation of bone marrow mesenchymal stem cells into cardiomyocyte-like cells in vitro

[J]. Acta Anatomica Sinica. 2019, 50(5): 580-588 https://doi.org/10.16098/j.issn.0529-1356.2019.05.007

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