链脲佐菌素诱导糖尿病大鼠骨髓间充质干细胞成骨分化

山东大学学报(医学版) ›› 2013, Vol. 51 ›› Issue (8): 7-12.

链脲佐菌素诱导糖尿病大鼠骨髓间充质干细胞成骨分化

胡苏1,2，逄曙光2，崔莹2，于春晓1，赵家军1，管庆波1

1.山东大学附属省立医院内分泌科，济南 250021； 2.山东大学附属济南市中心医院内分泌科，济南 250013

收稿日期:2012-12-26 出版日期:2013-08-10 发布日期:2013-08-10
通讯作者: 管庆波，E-mail: guanqingbo317@163.com
基金资助:
国家自然科学基金(81170771)

Osteogenic potential of bone marrow mesenchymal stem cells from streptozotocininduced diabetic rats

HU Su1,2, PANG Shu-guang2, CUI Ying2, YU Chun-xiao1, ZHAO Jia-jun1, GUAN Qing-bo1

1. Department of Endocrinology, Provincial Hospital Affiliated to Shandong University, Jinan 250021, China;
2. Department of Endocrinology, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013, China

Received:2012-12-26 Online:2013-08-10 Published:2013-08-10

摘要/Abstract

摘要：

目的观察糖尿病大鼠模型骨髓间充质干细胞(BMSCs)体外增殖、抗凋亡和成骨分化能力。方法将40只6周龄雌性SD大鼠随机分为2组，每组20只，实验组腹腔注射链脲佐菌素，对照组注射等量的生理盐水。处理10周后，采用贴壁法获得2组大鼠的BMSCs。应用CCK-8检测第2代BMSCs增殖能力，0.3%过氧化氢和血清剥夺诱导的BMSCs凋亡；成骨诱导培养4周后， RT-PCR检测I型胶原蛋白(COL-I)、骨钙素(OCN)和核心结合蛋白因子-2(Runx-2)的mRNA表达水平；应用酶联免疫吸附法定量分析细胞碱性磷酸酶活性；茜素红染色及von Kossa染色分析矿化能力。结果链脲佐菌素成功诱导制备糖尿病大鼠模型。与对照组比较，实验组BMSCs增殖和抗凋亡能力降低，细胞成骨相关基因表达下降，细胞碱性磷酸酶活性显著降低，细胞成骨分化能力减弱(P<0.01)。结论糖尿病大鼠来源的BMSCs体外增殖、抗凋亡作用减弱，成骨分化能力显著下降。

关键词: 糖尿病；骨质疏松症；骨髓间充质干细胞；骨形成；凋亡

Abstract:

Objective To explore the proliferation, anti-apoptosis characteristics and osteogenic differentiation of BMSCs isolated from diabetic rats. Methods Forty 6-week female adult Sprague-Dawley rats were randomly divided into the control group and experimental group, with 20 animals in each group. The rats in the experimental group were administrated with a single intraperitoneal injection of streptozotocin (STZ, 65mg/kg, dissolved in 5mmol/L citrate buffer, pH 4.5) to induce diabetes mellitus. The control group was treated with the same dose of normal saline. After 10-week treatment, BMSCs were isolated from the two groups by using adherent method. The proliferation of secondpassage BMSCs was evaluated by CCK-8 assay. The apoptosis induced by serum deprivation and 0.3% hydrogen peroxide was assessed by flow cytometry. After 4-week osteoinductive culture, the expressions of COL-I, OCN, and Runx-2 mRNA were detected by RT-PCR. Alkaline phosphatase (ALP) activity was determined in cell lysates using enzyme-linked immunosorbent assay (ELISA). Meanwhile, the capacities of mineralization of BMSCs were measured by VonKossa and alizarin red staining. Results Diabetic rat models were successfully induced by intraperitoneal injection of STZ. The proliferative and antiapoptotic abilities of BMSCs derived from diabetic rats were significantly decreased compared with those from normal rats (P<0.01). The mRNA expressions of COL-I, OCN and Runx-2 were down-regulated in experimental group. ALP activity in the experimental group was lower than that in the control group. In addition, the ability of osteogenic differentiation decreased significantly. Conclusion BMSCs derived from diabetic rats show an impaired ability of proliferative, anti-apoptosis and osteogenic differentiation in vitro.

Key words: Diabetes; Osteoporosis; Bone mesenchymal stem cells; Bone formation; Apoptosis

中图分类号:

R587.1

胡苏1,2，逄曙光2，崔莹2，于春晓1，赵家军1，管庆波1. 链脲佐菌素诱导糖尿病大鼠骨髓间充质干细胞成骨分化[J]. 山东大学学报(医学版), 2013, 51(8): 7-12.

HU Su1,2, PANG Shu-guang2, CUI Ying2, YU Chun-xiao1, ZHAO Jia-jun1, GUAN Qing-bo1. Osteogenic potential of bone marrow mesenchymal stem cells from streptozotocininduced diabetic rats[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2013, 51(8): 7-12.

参考文献

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