恒古骨伤愈合剂对大鼠骨髓间充质干细胞成脂分化的影响

发布时间：2018-04-11 17:33

本文选题：恒古骨伤愈合剂 + 骨质疏松症　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:探究恒古骨伤愈合剂调控大鼠BMSCs成脂肪细胞分化的能力及其对防治骨质疏松症的作用机制。方法:以大鼠BMSCs为研究对象,采用全骨髓贴壁培养法,从幼年SD大鼠股骨和胫骨骨髓中分离培养BMSCs;免疫组织细胞化学染色检测BMSCs细胞纯度;恒古骨伤愈合剂分别稀释500倍、1000倍、2000倍、4000倍,相对应分5组(control组、2×10-3组、1×10-3组、0.5×10-3组、0.25×10-3组),其中control组加入溶媒;MTT法检测各浓度恒古骨伤愈合剂对BMSCs细胞毒性;BMSCs在不同浓度恒古骨伤愈合剂干预下分别向脂肪细胞方向诱导分化;在恒古骨伤愈合剂干预后的第21天,Oil Red O染色后显微镜下观察及酶标仪定量检测成脂肪细胞分化情况;在恒古骨伤愈合剂干预后的第7天、14天、21天,Realtime-PCR定量检测成脂肪细胞分化特异性转录因子过氧化物酶体增值物激活受体(PPAR)γ、CCAAT/增强子结合蛋白(C/EBP)α的mRNA相对表达量。结果:?采用细胞贴壁生长法获得大鼠(乳鼠)BMSCs;?免疫组织细胞化学染色后荧光倒置显微镜下观察发现BMSCs特异性表面抗原CD44阳性,阳性率为95.47%,而CD19阴性,提示培养的细胞为骨髓间充质干细胞且纯度高;?MTT法检测结果示:在恒古骨伤愈合剂干预BMSCs后的0.5天、1天、4天、21天,各组与control组比较,差异无统计学意义(P0.05)。提示各浓度恒古骨伤愈合剂不论是早期还是晚期,对BMSCs的增殖及细胞活性均无毒性作用;?在恒古骨伤愈合剂干预BMSCs诱导成脂肪细胞分化的第21天,Oil Red O染色后显微镜下观察发现各浓度药物均对BMSCs成脂肪细胞分化有抑制作用。成浓度依赖性,且浓度越高抑制作用越强。酶标仪定量检测OD值分别为(1.104±0.414、0.785±0.085、0.836±0.138、1.008±0.369、1.000±0.291),2×10-3组、1×10-3组与control组比较,差异有统计学意义(P0.05),与染色观察结果一致;(5)恒古骨伤愈合剂能抑制脂肪细胞生成,下调脂肪细胞特异性转录因子的表达。在恒古骨伤愈合剂干预的第7天、14天、21天,Realtime-PCR定量检测结果提示PPARγ、C/EBPα基因表达量与control组比较下调且差异有统计学意义(P0.05)。结论:恒古骨伤愈合剂可通过下调PPARγ、C/EBPα的表达而抑制大鼠BMSCs成脂肪细胞分化。抑制BMSCs成脂肪细胞分化是恒古骨伤愈合剂防治骨质疏松症的另一作用途径。
[Abstract]:Aim: to investigate the ability of Henggu bone wound healing agent to regulate the differentiation of rat BMSCs adipoblasts and its mechanism of prevention and treatment of osteoporosis.Methods: BMSCs were isolated from femur and tibia bone marrow of young SD rats by whole bone marrow adherent culture, and the purity of BMSCs cells was detected by immunohistochemical staining.Henggu bone wound healing agent diluted 500 times, 1000 times, 2000 times and 4000 times respectively,The differentiation of adipocytes was observed under microscope on the 21st day after the intervention of Henggu bone wound healing agent with oil Red O, and the differentiation of adipocytes was detected quantitatively by enzyme labeling instrument.It turns out to be:?The method of cell adherent growth was used to obtain BMSCs?The positive rate of BMSCs specific surface antigen (CD44) was 95.47%, but CD19 was negative.The results showed that the cultured cells were bone marrow mesenchymal stem cells and the purity was high. The results showed that there was no significant difference between control group and Heng Gu bone wound Healing Agent 0. 5 day, 0. 5 days, 4 days and 21 days, compared with control group, the difference was not statistically significant (P 0. 05).The results suggest that there is no toxic effect on proliferation and cell activity of BMSCs in early or late stage.Oil Red O staining of adipocytes induced by BMSCs on the 21st day after treatment with Henggu bone wound healing agent was observed under microscope. It was found that every concentration of drugs could inhibit the differentiation of BMSCs adipocytes.In a concentration-dependent manner, the higher the concentration, the stronger the inhibitory effect.The quantitative analysis of real time-PCR showed that the expression of PPAR 纬 C / EBP 伪 gene was down-regulated compared with that of control group, and the difference was statistically significant (P 0.05).Conclusion: Henggu bone healing agent can inhibit the differentiation of rat BMSCs adipoblasts by down-regulating the expression of PPAR 纬 -C / EBP 伪.Inhibiting the differentiation of BMSCs adipoblasts is another way to prevent and cure osteoporosis with Henggu bone injury healing agent.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580

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