拉伸应力刺激对骨髓间充质干细胞成骨分化过程的影响研究
发布时间：2018-08-24 19:09
【摘要】：实验目的:通过对小鼠BMSCs施加一定的拉伸应力刺激,研究力学因素对小鼠BMSCs成骨分化过程的影响,进一步探讨成骨分化过程中细胞的生物力学-生物化学偶联机制。实验方法:无菌条件下提取昆明小鼠BMSCs,体外传代培养至第3代后,进行细胞表型的鉴定。将细胞接种于底部具有弹性膜的拉伸六孔板上。根据细胞不同的培养条件,实验共分为3组。实验组(A组):成骨诱导培养基培养+拉伸刺激;单纯诱导组(B组):成骨诱导培养基培养;空白对照组(C组):普通培养基培养。采用Flex-cell5000细胞力学加载系统给予实验组6%形变幅度的正弦波刺激,频率0.5Hz,4h/d。各组细胞均2天换液1次,共培养1周。实验结束后RT-PCR法检测各组细胞BMP-2、Runx2和ALP m RNA的表达情况,ELISA法检测培养基中Col1的含量,并对各组细胞进行茜素红染色观察钙结节形成情况。实验结果:1.提取的原代细胞呈圆形或类圆形,所含杂质较多,24h后首次换液,去除漂浮死细胞,显微镜下观察可见有部分细胞贴壁,呈短梭形或多角形。继续培养48h后以1:2进行细胞传代,显微镜下观察传代后细胞生长加速,呈长梭形、漩涡状生长。流式细胞仪细胞表型鉴定结果:96.4%的细胞表达CD29,97.2%的细胞表达CD90,1.7%的细胞表达CD34,1.3%的细胞表达CD45。2.RT-PCR检测:细胞培养1周后,与C组相比,A、B两组BMP-2、Runx2、ALP的mRNA表达量均明显升高,差异具有统计学差异(P0.05)。与B组相比,A组BMP-2、Runx2、ALP mRNA表达量增高,差异有统计学差异(P0.05)。3.ELISA检测Col1的含量:A、B两组各时间段培养基中Col1的含量均高于C组,且随着诱导时间的延长逐渐增加。A组培养基中Col1终浓度高于B组,诱导效果最明显。4.茜素红染色:倒置显微镜下观察A、B两组均出现明显深染钙结节,证明两组诱导成骨分化均显著,且A组视野中钙结节多于B组,C组并无出现深染钙结节。实验结论:拉伸应力刺激在小鼠BMSCs向成骨细胞分化的过程中具有重要作用。其可能通过促进BMSCs成骨因子BMP-2的分泌,从而调控下游靶基因Runx2的表达,进而促进BMSCs向成骨细胞分化。
[Abstract]:Objective: to investigate the effects of mechanical factors on the osteogenic differentiation of BMSCs in mice and to explore the biomechanic-biomechanical coupling mechanism of the cells during osteogenic differentiation. Methods: BMSCs, extracted from Kunming mice was subcultured to the third passage in vitro, and the phenotype was identified. The cells are seeded on a stretch six-hole plate with an elastic membrane at the bottom. According to different culture conditions, the experiment was divided into three groups. Experimental group (group A): osteoblast induction medium culture and tensile stimulation; simple induction group (group B): osteoblast induction medium culture; blank control group (C group): ordinary medium culture. Flex-cell5000 cell mechanical loading system was used to stimulate the experimental group with 6% deformation amplitude of sine wave at a frequency of 0.5 Hz / d. The cells in each group were changed once for 2 days and cultured for 1 week. After the experiment, the expression of BMP-2,Runx2 and ALP m RNA was detected by RT-PCR method and the content of Col1 in culture medium was detected by Elisa, and the formation of calcium nodules was observed by alizarin red staining. The result of the experiment was 1: 1. The extracted primary cells were round or round. After 24 hours with more impurities, the cells were changed for the first time to remove the dead floating cells. Some of the cells were observed to be adherent to the wall under microscope, showing short fusiform or polygonal shape. After 48 hours of culture, the cells were subcultured at 1:2. The growth of the cells was observed under microscope, which was spindle-shaped and whirlpool. The results of flow cytometry showed that the expression of CD90,1.7% was detected in 96. 4% of the cells expressing CD29,97.2%. The expression of CD45.2.RT-PCR in CD34,1.3% was detected. After 1 week of cell culture, the mRNA expression of BMP-2,Runx2,ALP in both groups was significantly higher than that in group C, and the expression of BMP-2,Runx2,ALP in both groups was significantly higher than that in group C. The difference was statistically significant (P0.05). Compared with group B, the expression of BMP-2,Runx2,ALP mRNA in group A was significantly higher than that in group B (P0.05). 3. The content of Col1 in culture medium of group B was higher than that of group C (P0.05). The final concentration of Col1 in group A was higher than that in group B with the increase of induction time, and the induction effect was the most obvious. 4. Alizarin red staining: under inverted microscope, there were obvious deep stained calcium nodules in both groups, which proved that the osteogenic differentiation was significant in both groups, and there were no deep calcium nodules in the visual field of group A than those in group B and C. Conclusion: tensile stress stimulation plays an important role in the differentiation of mouse BMSCs into osteoblasts. It may regulate the expression of downstream target gene Runx2 and promote the differentiation of BMSCs into osteoblasts by promoting the secretion of BMSCs osteogenic factor BMP-2.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580
[Abstract]:Objective: to investigate the effects of mechanical factors on the osteogenic differentiation of BMSCs in mice and to explore the biomechanic-biomechanical coupling mechanism of the cells during osteogenic differentiation. Methods: BMSCs, extracted from Kunming mice was subcultured to the third passage in vitro, and the phenotype was identified. The cells are seeded on a stretch six-hole plate with an elastic membrane at the bottom. According to different culture conditions, the experiment was divided into three groups. Experimental group (group A): osteoblast induction medium culture and tensile stimulation; simple induction group (group B): osteoblast induction medium culture; blank control group (C group): ordinary medium culture. Flex-cell5000 cell mechanical loading system was used to stimulate the experimental group with 6% deformation amplitude of sine wave at a frequency of 0.5 Hz / d. The cells in each group were changed once for 2 days and cultured for 1 week. After the experiment, the expression of BMP-2,Runx2 and ALP m RNA was detected by RT-PCR method and the content of Col1 in culture medium was detected by Elisa, and the formation of calcium nodules was observed by alizarin red staining. The result of the experiment was 1: 1. The extracted primary cells were round or round. After 24 hours with more impurities, the cells were changed for the first time to remove the dead floating cells. Some of the cells were observed to be adherent to the wall under microscope, showing short fusiform or polygonal shape. After 48 hours of culture, the cells were subcultured at 1:2. The growth of the cells was observed under microscope, which was spindle-shaped and whirlpool. The results of flow cytometry showed that the expression of CD90,1.7% was detected in 96. 4% of the cells expressing CD29,97.2%. The expression of CD45.2.RT-PCR in CD34,1.3% was detected. After 1 week of cell culture, the mRNA expression of BMP-2,Runx2,ALP in both groups was significantly higher than that in group C, and the expression of BMP-2,Runx2,ALP in both groups was significantly higher than that in group C. The difference was statistically significant (P0.05). Compared with group B, the expression of BMP-2,Runx2,ALP mRNA in group A was significantly higher than that in group B (P0.05). 3. The content of Col1 in culture medium of group B was higher than that of group C (P0.05). The final concentration of Col1 in group A was higher than that in group B with the increase of induction time, and the induction effect was the most obvious. 4. Alizarin red staining: under inverted microscope, there were obvious deep stained calcium nodules in both groups, which proved that the osteogenic differentiation was significant in both groups, and there were no deep calcium nodules in the visual field of group A than those in group B and C. Conclusion: tensile stress stimulation plays an important role in the differentiation of mouse BMSCs into osteoblasts. It may regulate the expression of downstream target gene Runx2 and promote the differentiation of BMSCs into osteoblasts by promoting the secretion of BMSCs osteogenic factor BMP-2.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580
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