不同年龄大鼠骨髓基质细胞端粒酶活性及端粒长度的变化

Changes of telomerase activities and telomere length with aging in rat bone marrow stromal cells

目的:端粒酶活性表达可能是干细胞维持其自我复制能力的分子基础,然而对于这种酶活性表达在骨髓基质细胞端粒维持和细胞增殖中的作用及其随年龄增长的变化规律还知之甚少。观察不同年龄大鼠骨髓基质细胞端粒酶活性和端粒长度的变化情况。方法:实验于2005-10/2006-08在解放军163医院神经外科实验室进行。①实验材料:清洁级SD大鼠,雌雄不拘,体质量120～150g,购自中南大学湘雅医学院实验动物中心。根据大鼠年龄不同分为1月龄组、3月龄组、6月龄组、1年龄组、2年龄组。②实验方法:采用密度梯度离心法分离培养不同年龄大鼠骨髓基质细胞,观察各组细胞形态学差别,采用MTT法绘制细胞生长曲线。③实验评估:采用流式细胞仪检测细胞周期,PCR-ELISA法检测各组细胞端粒酶活性,Southern印迹法测量各组细胞端粒末端限制性片段长度。结果:①不同年龄各组骨髓基质细胞形态上无明显差别。②各组细胞增殖缓慢,不同年龄组大鼠骨髓基质细胞生长周期显示,G0/G1期细胞多,G2/M期和S期细胞较少。各组间无明显差别。③1月至2年组大鼠骨髓基质细胞端粒酶活性分别为0.651、0.676、0.615、0.815、0.672,各组间差异无显著性意义。④1月至2年组大鼠细胞端粒末端限制性片断长度分别为24.4、22.56、23.56、22.79、22.65kb。除1月组稍长于其他各组外,其余各组间端粒末端限制性片断长度无明显差别。结论:①随着年龄的增长,骨髓基质细胞稳定表达端粒酶活性。②骨髓基质细胞能维持端粒长度,不随年龄增长而缩短,这可能与细胞表达端粒酶活性有关。

AIM： Telomerase activity expression may be the molecular foundation of self replication of stem cells, however, it is still uncertain about the effect of telomerase activity on the maintaining and proliferation of bone marrow stromal cells （BMSCs） and change rules of telomerase activities with age. In this study, the changes of telomerase activities and telomere length of BMSCs in rats of different ages were investigated. METHODS. The experiment was conducted in the Department of Neurosurgery, 163 Hospital of Chinese PLA from October 2005 to August 2006. （1）SD rats of clean grade and 120-150 g, either male or female were purchased from the Experimental Animal Center, Xiangya Medical College of Central South University and divided into 1 month group, 3 months group, 6 months group, 1 year group, and 2 years group according to the age of animals. Density gradient centrifugation was used to isolate BMSCs of different age groups, and the cell morphological differences were observed. The growth curve was drawn by MTT method. （2）Cell cycle was detected by flow cytometry; PCR-ELISA assay was used to detect the telomerase activities, and telomere terminal restriction fragments （TRF） length was measured by Southern blotting. RESULTS： （1）The morphology of BMSCs was similar in different groups. （2）The proliferation of BMSCs was slow in all groups. The cell number of G0/G1 phage was large, while of S phage and G2/M phage was small. There was no obvious difference among groups. （3）From 1 month group to 2 years group, the telomerase activity was 0.651, 0.676, 0.615, 0.815, and 0.672, respectively. There was no significant difference among each group. （4）Telomere TRF was 24.4, 22.56, 23.56, 22.79, and 22.65 kb, respectively from 1-month group to 2-years group. The TRF length of 1-month group was longer than that of other groups, but there was no difference between other age groups. CONCLUSION： （1）Telomerase activity of BMSCs is expressed stably with age. （2）BMSCs could maintain the length of telomere to make it not shorten with age, which is relative to telomerase activity expressed stably in BMSCs.