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目的观察晚期糖基化终末产物(AGEs)对SD大鼠骨髓间充质干细胞(BMSCs)成骨分化的影响,探讨AGEs影响BMSCs成骨分化的机制。方法(1)分离提取SD大鼠股骨、胫骨的BMSCs,采用贴壁法培养,传至第三代,用流式细胞仪进行细胞表型鉴定。(2)按处理方法将细胞分为实验组(AGEs组)和对照组(BSA组),分别给予0.2 mg/ml AGEs或牛血清白蛋白(BSA)成骨诱导液干预18 d,然后用茜素红染色,观察比较成骨矿化结节形成情况。(3)将第三代AGEs按处理方法分为正常组、对照组(BSA组)和实验组(AGEs组),正常组细胞不作特殊处理,对照组与实验组分别给予0.2 mg/ml的BSA和0.2 mg/ml的AGEs干预7d,用RNA提取试剂盒提取总RNA,然后用逆转录聚合酶链反应(RT-PCR)技术测定成骨细胞分泌的I型胶原蛋白(Col-Ⅰ)以及Wnt通路相关分子低密度脂蛋白受体相关蛋白5(LRP5)mRNA的表达量。结果(1)细胞从原代传至第三代的过程中,细胞外形由长梭形逐渐变至短棒状,并伸出多个突起,表型鉴定结果:第三代BMSCs特异性表面标志物CD44、CD90的阳性表达率分别为96.07%、99.34%,CD34的阴性表达率为0.19%。(2)与BSA组相比,AGEs组明显减少了矿化结节的形成。(3)Col-ⅠmRNA、LRP5 mRNA表达量在AGEs组和BSA组组间比较差异均有统计学意义(F=70.10,P=0.00;F=47.99,P=0.00)。结论 AGEs抑制BMSCs的增殖,并阻碍BMSCs向成骨细胞分化,此过程可能是通过干扰Wnt/LRP5/β-catenin通路来进行。
Objective To investigate the effects of advanced glycation end products (AGEs) on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in SD rats and to explore the mechanism of AGEs on the osteogenic differentiation of BMSCs. Methods (1) BMSCs from the femur and tibia of SD rats were isolated and cultured. The cells were cultured by adherent method and passed on to the third generation. The phenotypes were identified by flow cytometry. (2) The cells were divided into experimental group (AGEs group) and control group (BSA group) according to the method of treatment, and were respectively given 0.2 mg / ml AGEs or bovine serum albumin (BSA) Su-red staining, observation and comparison of osteogenic mineralization nodule formation. (3) The third generation AGEs were divided into normal group, control group (BSA group) and experimental group (AGEs group) according to the treatment method. The normal group of cells were treated with no special treatment. The control group and the experimental group were given 0.2 mg / ml BSA And 0.2 mg / ml AGEs for 7 days. Total RNA was extracted by RNA extraction kit. The expression of collagen Ⅰ (Col-Ⅰ) secreted by osteoblasts and Wnt Pathway-related molecular low-density lipoprotein receptor-related protein 5 (LRP5) mRNA expression. Results (1) During the process of primary cell passage to the third generation, the cell shape gradually changed from long spindle shape to short bar shape and protruded a number of protrusions. Phenotypic identification results: The third generation of BMSCs specific surface markers The positive rates of CD44 and CD90 were 96.07% and 99.34%, respectively. The positive rate of CD34 was 0.19%. (2) Compared with BSA group, AGEs group significantly reduced the formation of mineralized nodules. (3) The expression of Col-ⅠmRNA and LRP5 mRNA in AGEs group and BSA group were significantly different (F = 70.10, P = 0.00; F = 47.99, P = 0.00). Conclusion AGEs can inhibit the proliferation of BMSCs and hinder the differentiation of BMSCs into osteoblasts. This process may be through interfering with the Wnt / LRP5 / β-catenin pathway.