Expression of plasma membrane calcium channel, calcium-sensing receptor and adipogenic determination genes during the adipogenesis of porcine bone marrow mesenchymal stem cells
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摘要:目的
研究猪骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)定向分化为脂肪细胞过程中细胞膜上钙离子通道、钙敏感受体(Calcium-sensing receptor, CaSR)基因及成脂定向相关基因的表达。
方法从5~7日龄仔猪骨髓中分离纯化出猪BMSCs，诱导猪BMSCs成脂分化。油红O法和三酰甘油法检测细胞分化聚酯状况。在成脂分化不同时间(0、1、2、5和10 d)收集细胞，利用荧光定量PCR检测锌指蛋白423(Zinc finger protein 423, Zfp423)、脂肪前体细胞因子(Preadipocyte factor 1, Pref-1)、骨形态发生蛋白2(Bone morphogenetic protein 2, BMP2)、骨形态发生蛋白4(Bone morphogenetic protein 4, BMP4)、细胞膜钙离子通道及CaSR基因的mRNA表达变化。
结果油红O染色和三酰甘油检测结果表明，成功诱导猪BMSCs成脂分化；定量PCR结果显示，在猪BMSCs成脂分化第5天，成脂定向标志基因Zfp423、脂肪前体细胞标志基因Pref-1及促进成脂分化基因BMP2、BMP4的mRNA相对表达量显著提高(P < 0.05)，说明第5天是猪BMSCs成脂定向形成脂肪前体细胞的关键时期；同时，细胞膜上的电压门控钙离子通道亚基电压依赖型α/δ亚型1(Voltage-dependentalpha-2/delta subunit 1, CACNA2D1)、钙释放激活钙通道调节分子1 (Calciumr elease-activated calcium channel modulator 1, Orai1)、瞬时受体电位通道传统型1(Transient receptor potential canonical type 1, TRPC1)、瞬时受体电位通道M型7(Transient receptor potential melastatin 7, TRPM7)、瞬时受体电位通道香草素受体亚型1(Transient receptor potential vanilloid receptor1, TRPV1)基因和CaSR基因在诱导成脂第5天mRNA相对表达量也显著提高(P < 0.05)，提示细胞膜钙离子通道及CaSR基因可能参与了猪BMSCs成脂分化过程。
结论揭示了猪BMSCs成脂分化过程中细胞膜钙离子通道、钙敏感受体及成脂定向相关基因的表达模式。
Abstract:ObjectiveTo investigate the mRNA expression of plasma membrane calcium channel, calcium-sensing receptor (CaSR) and adipogenic determination genes during the adipogenesis of porcine bone marrow mesenchymal stem cells (BMSCs).
MethodThe porcine BMSCs, isolated and purified from 5-7 d piglet, were induced to adipogenic differentiation. Oil red O staining and triglyceride assay were used to detect the formation of adipocytes. The cells were collected at various time points (0, 1, 2, 5, and 10 d) during the adipogenic differentiation of porcine BMSCs. The mRNA expression patterns of zinc finger protein (Zfp423), preadipocyte factor 1 (Pref-1), bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 4 (BMP4), plasma membrane calcium channel and CaSR genes were detected by real-time quantitative PCR.
ResultThe results of oil red O staining and triglyceride assay indicated that porcine BMSCs were successfully induced to adipogenic differentiation. The findings of real-time quantitative PCR demonstrated that the mRNA expression of Zfp423, Pref-1, BMP2 and BMP4 significantly increased at day 5(P < 0.05), suggesting that day 5 is the key time for the adipogenic determination of porcine BMSCs. Meanwhile, the mRNA expression of voltage-dependent alpha-2/delta subunit 1 (CACNA2D1), calcium release-activated calcium channel modulator 1 (Orai1), transient receptor potential canonical type 1 (TRPC1), transient receptor potential melastatin 7 (TRPM7), transient receptor potential vanilloid receptor 1 (TRPV1) and CaSR also significantly increased at day 5, implying that calcium channels and CaSR gene might be involved in the adipogenesis of porcine BMSCs.
ConclusionOur data reveal the expression patterns of plasma membrane calcium channel, CaSR and adipogenic determination genes.
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图 1 猪BMSCs成脂分化过程中油红O染色结果
Figure 1. The oil red O staining result of porcine BMSCs during the adipogenic differentiation
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图 2 猪BMSCs成脂分化过程中三酰甘油(TG)质量摩尔浓度的变化
图中数据为平均值±标准误，***表示差异极显著(独立样本t检验分析，P < 0.001)。
Figure 2. Triacylglycerol(TG) assay of porcine BMSCs during the adipogenic differentiation
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图 3 猪BMSCs成脂分化过程中细胞膜钙离子通道(A-E)与CaSR(F)基因的mRNA表达模式
图中数据为平均值±标准误，同一分图中不同诱导时间的柱上凡是有一个相同小写字母者，表示差异不显著(单因素方差分析，P > 0.05)。
Figure 3. The mRNA expression patterns of calcium channel and CaSR genes in plasma membrane during the adipogenic differentiation of porcine BMSCs
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图 4 猪BMSCs成脂分化中成脂定向相关基因的mRNA表达模式
图中数据为平均值±标准误，同一分图中不同诱导时间柱上凡是有一个相同小写字母者，表示差异不显著(单因素方差分析，P > 0.05)。
Figure 4. The mRNA expression patterns of adipogenic determination genes during the adipogenic differentiation of porcine BMSCs
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