背景：血管紧张素Ⅱ诱导骨髓间充质干细胞向心肌细胞定向分化的实验研究还较少，目前对于这种新的诱导剂在实验应用中没有一个统一的浓度标准，所以诱导结果有一定的差异。 目的：探讨血管紧张素Ⅱ诱导骨髓间充质干细胞分化为心肌细胞的最佳浓度。 方法：采用密度梯度离心+贴壁法分离培养大鼠骨髓间充质干细胞，将第3代骨髓间充质干细胞分为0.05，0.1，0.2，0.5μmol/L血管紧张素Ⅱ组和对照组，血管紧张素Ⅱ各组接种后24 h加相应的诱导剂诱导24 h后，完全培养液连续培养4周，对照组只用完全培养液培养。采用MTT法检测各组第1，3，5，7天的吸光度值，计算各诱导组的细胞增殖抑制率。不同浓度血管紧张素Ⅱ诱导第1，4周时，免疫荧光细胞化学染色法检测心肌特异蛋白cTnT、β-MHC的表达，RT-PCR检测各组诱导培养4周时心肌早期转录因子GATA-4、Nkx2-5的表达。 结果与结论：各诱导组间的细胞增殖曲线相似，第5天0.05μmol/L血管紧张素Ⅱ组细胞增殖抑制率开始出现负值，与其他组比较差异有显著性意义(P <0.05)。第5，7天0.1μmol/L血管紧张素Ⅱ组细胞增殖抑制率明显低于0.2，0.5μmol/L血管紧张素Ⅱ组(P <0.05)。经4种浓度血管紧张素Ⅱ诱导的骨髓间充质干细胞均表达心肌特异蛋白cTnT、β-MHC，对照组阴性。诱导培养1周，0.5μmol/L血管紧张素Ⅱ组cTnT、β-MHC蛋白阳性表达率明显高于0.05μmol/L组(P <0.05)。诱导培养4周，0.1μmol/L血管紧张素Ⅱ组的cTnT、β-MHC蛋白阳性表达率与0.2μmol/L血管紧张素Ⅱ组差异无显著性意义(P >0.05)。RT-PCR检测各组骨髓间充质干细胞经不同浓度血管紧张素Ⅱ诱导培养4周后心肌早期转录因子GATA-4、Nkx2-5均有表达，对照组阴性。结果表明经血管紧张素Ⅱ诱导的骨髓间充质干细胞表达心肌特异蛋白 cTnT、β-MHC 及心肌特异转录因子GATA-4、Nkx2-5，诱导分化适宜浓度为0.1μmol/L。%BACKGROUND:There are less experimental studies addressing angiotensin II-induced differentiation of bone marrow mesenchymal stem cels into cardiomyocytes, and there is no uniform standard for this new inducer used in the experiments, so the results of the induced experimental studies are certainly different. OBJECTIVE:To discuss the optimal concentration of angiotensin II to induce the differentiation of bone marrow mesenchymal stem cels into cardiomyocytes. METHODS:Bone marrow mesenchymal stem cels from rats were isolated, cultured and purified by the density gradient centrifugation combined with adherence method. Passage 3 cels were divided into four induction groups (0.05, 0.1, 0.2, 0.5 μmol/L angiotensin II) and a control group. In the four induction groups, the corresponding inducer was added 24 hours after cels were seeded, and after 24 hours of induction, the cels were cultured in complete medium for 4 weeks. Cels in the control group were only cultured in the complete medium. MTT assay was used to detect the absorbance values at days 1, 3, 5, 7 to calculate the cel proliferation inhibition rate in each group. Expression of cardiac-specific cTnT and β-MHC in bone marrow mesenchymal stem cels was detected by immunofluorescence staining at 1 and 4 weeks after induction. mRNA expression of cardiac-transcription-factor GATA4 and NKx2.5 in bone marrow mesenchymal stem cels was measured by real-time PCR at 4 weeks after induction. RESULTS AND CONCLUSION: The cel proliferation curves were similar among different groups. The cel proliferation inhibition rate in the 0.05 μmol/L group began to appear negative, which was significantly different from the other groups at the 5th day (P< 0.05). The cel proliferation inhibition rate of 0.1 μmol/L group was significantly lower than that of 0.2 or 0.5 μmol/L groups at the 5th and 7th days. cTnT and β-MHC were detected in the bone marrow mesenchymal stem cels of four induction groups, but the control group was negative for cTnT and β-MHC. After 1 week of induction, the positive rates of cTnT and β-MHC inthe 0.5 μmol/L group was significantly higher than that in the 0.05 μmol/L group (P < 0.05). After 4 weeks of induction, the positive rates of cTnT and β-MHC had no difference between the 0.1 μmol/L and 0.2 μmol/L groups (P > 0.05). The early cardiac transcription factor GATA-4 and NKx2.5 were detected by RT-PCR in the cels of four induction groups after 4 weeks of induction, while their expression was negative in the control group. These findings suggest that after angiotensin II induction, bone marrow mesenchymal stem cels can express the cardiac-specific protein cTnT, β-MHC and transcription factor GATA-4 and NKx2.5. As mentioned above, the suitable concentration of angiotensin II is 0.1 μmol/L.
展开▼
