糖尿病大鼠骨髓间充质干细胞成骨和成脂分化的研究

Study of Osteogenesis and Adipogenesis of Bone Marrow Mesenchymal Stem Cells by Diabetic Rats

摘要: 目的：研究糖尿病模型大鼠造模后不同时间段骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells, BMSCs)的成骨及成脂分化能力的改变，探讨糖尿病模型骨质疏松形成与BMSCs成骨及成脂分化能力的相关性。方法：利用链脲佐菌素(Streptozotocin, STZ)加高糖高脂饲料诱导制作大鼠糖尿病模型，正常大鼠作为对照组。分别于注射STZ液4、8、12周后，利用全骨髓贴壁法来扩增和纯化BMSCs，取生长良好的第3代细胞作为本实验的研究对象。用成骨诱导液诱导细胞分化，培养7 d后，检测碱性磷酸酶(Alkaline Phosphatase, ALP)活力，14 d后，进行茜素红染色及检测ALP和骨钙素(Osteocalcin, OC) mRNA含量。用成脂诱导液诱导细胞分化，诱导14 d后，进行油红O染色，计算脂肪细胞转化率；进行油红O染色定量分析；检测脂蛋白脂酶(Lipoprotein Lipase, LPL)和过氧化物酶增殖活化受体(Peroxisome Proliferator Activated Receptor, PPARγ2) mRNA含量。结果：注射STZ液8、12周后，糖尿病组ALP活力明显下降，与对照组比较，P < 0.01。对照组钙化结节数量明显多于糖尿病组。ALP基因相对OD值，糖尿病组(0.26 ± 0.05)低于对照组(0.42 ± 0.09)，P < 0.01。OC基因相对OD值，糖尿病组(0.31 ± 0.09)低于对照组(0.41 ± 0.06)，P < 0.05。注射STZ液4、8、12周后，油红O染色，对照组、糖尿病组镜下观察可见有红色脂滴，对照组脂肪细胞转化率(25.16 ± 5.03)明显低于糖尿病组(66.85 ± 10.01)，P < 0.01。油红O染色定量分析结果(OD值)，对照组(0.016 ± 0.015)油红含量明显低于糖尿病组(0.058 ± 0.016)，P < 0.01。LPL基因相对OD值，糖尿病组(0.89 ± 0.05)高于对照组(0.22 ± 0.05)，P < 0.01。PPARγ2基因相对OD值，糖尿病组(0.38 ± 0.03)高于对照组(0.18 ± 0.01)，P < 0.01。结论：注射STZ液4周以后，糖尿病大鼠BMSCs成脂分化能力增强；8周以后，成骨分化能力明显下降，可能与骨质疏松的形成有一定的关系。

Abstract: Objective: To study osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) by diabetic rats during different time periods, and discuss the correlation of diabetic osteoporosis model formation and BMSCs osteogenesis and adipogenesis. Method: Using Streptozotocin (STZ) with higher sugar high fat feedstuff induced diabetic rats model, normal rats as control group. BMSCs were amplified and purified by whole bone marrow adherent method at 4, 8 and 12 weeks after STZ injection, and the well-grown third-generation cells were selected as the research object of this experiment. Osteogenic induction was used to induce cell differentiation, detecting Alkaline phosphatase (ALP) activity after 7 d; detecting alizarin red staining and ALP and Osteocalcin (Oste-ocalcin, OC) mRNA levels after 14 d. Adipogenic induction was used to induce cell differentiation, and after 14 d detecting quantitative analysis of oil red O staining and Lipoprotein lipase (LPL) and peroxisome proliferator activated receptor (PPARγ2) mRNA levels. Results: The ALP activity of dia-betes group significantly decreased, compared with control group, P < 0.05. Calcified nodules of normal control group are significantly higher than the diabetes groups. The OD relative value of ALP (0.26 ± 0.05) and OC mRNA (0.31 ± 0.09) is lower than the control group (P < 0.01, P < 0.05). After STZ injection fluid 4, 8, 12 weeks, the adipocyte transformation rate and Oil red O dyeing value of control group is lower than the diabetes group. The OD relative value of LPL (0.89 ± 0.05) and PPARγ2 mRNA (0.38 ± 0.03) is lower than the diabetes group (P < 0.01, P < 0.01). Conclusion: Injec-tion of STZ liquid after 4 weeks, the adipogenesis of diabetic rat BMSCs differentiation capacity in-creased obviously; after 8 weeks, the ossification of diabetic rat BMSCs differentiation capacity de-creased obviously, which may have a certain relationship with the formation of osteoporosis.