拉伸應力刺激對骨髓間充質干細胞成骨分化過程的影響研究
發(fā)布時間：2018-08-24 19:09
【摘要】：實驗目的:通過對小鼠BMSCs施加一定的拉伸應力刺激,研究力學因素對小鼠BMSCs成骨分化過程的影響,進一步探討成骨分化過程中細胞的生物力學-生物化學偶聯機制。實驗方法:無菌條件下提取昆明小鼠BMSCs,體外傳代培養(yǎng)至第3代后,進行細胞表型的鑒定。將細胞接種于底部具有彈性膜的拉伸六孔板上。根據細胞不同的培養(yǎng)條件,實驗共分為3組。實驗組(A組):成骨誘導培養(yǎng)基培養(yǎng)+拉伸刺激;單純誘導組(B組):成骨誘導培養(yǎng)基培養(yǎng);空白對照組(C組):普通培養(yǎng)基培養(yǎng)。采用Flex-cell5000細胞力學加載系統(tǒng)給予實驗組6%形變幅度的正弦波刺激,頻率0.5Hz,4h/d。各組細胞均2天換液1次,共培養(yǎng)1周。實驗結束后RT-PCR法檢測各組細胞BMP-2、Runx2和ALP m RNA的表達情況,ELISA法檢測培養(yǎng)基中Col1的含量,并對各組細胞進行茜素紅染色觀察鈣結節(jié)形成情況。實驗結果:1.提取的原代細胞呈圓形或類圓形,所含雜質較多,24h后首次換液,去除漂浮死細胞,顯微鏡下觀察可見有部分細胞貼壁,呈短梭形或多角形。繼續(xù)培養(yǎng)48h后以1:2進行細胞傳代,顯微鏡下觀察傳代后細胞生長加速,呈長梭形、漩渦狀生長。流式細胞儀細胞表型鑒定結果:96.4%的細胞表達CD29,97.2%的細胞表達CD90,1.7%的細胞表達CD34,1.3%的細胞表達CD45。2.RT-PCR檢測:細胞培養(yǎng)1周后,與C組相比,A、B兩組BMP-2、Runx2、ALP的mRNA表達量均明顯升高,差異具有統(tǒng)計學差異(P0.05)。與B組相比,A組BMP-2、Runx2、ALP mRNA表達量增高,差異有統(tǒng)計學差異(P0.05)。3.ELISA檢測Col1的含量:A、B兩組各時間段培養(yǎng)基中Col1的含量均高于C組,且隨著誘導時間的延長逐漸增加。A組培養(yǎng)基中Col1終濃度高于B組,誘導效果最明顯。4.茜素紅染色:倒置顯微鏡下觀察A、B兩組均出現明顯深染鈣結節(jié),證明兩組誘導成骨分化均顯著,且A組視野中鈣結節(jié)多于B組,C組并無出現深染鈣結節(jié)。實驗結論:拉伸應力刺激在小鼠BMSCs向成骨細胞分化的過程中具有重要作用。其可能通過促進BMSCs成骨因子BMP-2的分泌,從而調控下游靶基因Runx2的表達,進而促進BMSCs向成骨細胞分化。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R580
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