黄芪诱导大鼠骨髓间充质干细胞分化早期胞内钙调蛋白mRNA的转录水平

Transcriptional level of calmodulin mRNA during the earlier differentiation of rat bone marrow mesenchymal stem cells induced by astragalus mongholicus

背景:黄芪可促进骨髓间充质干细胞向神经元方向分化,但对于诱导机制的报道较少,目前普遍认为诱导作用可能与其具有抗氧化功能有关。目的:观察黄芪注射液诱导大鼠骨髓间充质干细胞分化早期进程中细胞内钙调蛋白mRNA转录水平的变化。设计、时间及地点:细胞学体外对照观察,于2007-10/12在河北北方学院实验中心完成。材料:清洁级6周龄雄性大鼠1只,购自中国医科院实验动物中心。黄芪注射液为大理药业有限公司产品,批号060105。方法:体外分离培养大鼠骨髓间充质干细胞,传至第4代按4×105L-1密度接种于12孔板,加入含200g/L黄芪注射液、体积分数为15%胎牛血清的DMEM培养基进行诱导分化,制备的细胞爬片行免疫细胞化学染色。传至第5代细胞平均分配到4个离心管内,1管为对照组,另外3管加入上述诱导培养基分别作用30,60,120min,消化获取的细胞采用RT-PCR技术检测钙调蛋白mRNA的转录水平。主要观察指标:黄芪对骨髓间充质干细胞的诱导作用,诱导后钙调蛋白mRNA的转录水平。结果:黄芪诱导5h后,可见少量细胞形态发生改变,胞体变圆,自胞体伸出细长突起,免疫细胞化学染色后可见巢蛋白阳性细胞和神经元特异性烯醇化酶阳性细胞,少量细胞呈微管相关蛋白2阳性,而神经胶质纤维酸性蛋白呈弱阳性。RT-PCR实验显示,在黄芪诱导骨髓间充质干细胞分化的早期,钙调蛋白基因进行了转录,随着诱导时间的延长,钙调蛋白mRNA的相对含量逐渐增加,诱导30,60,120min组与对照组钙调蛋白mRNA的相对含量比较差异有显著性意义(F=153.315,P=0.000)。结论:黄芪诱导骨髓间充质干细胞向神经元方向分化初期细胞内钙调蛋白转录水平上调,提示其诱导作用可能与钙调蛋白介导的信号转导途径有关。

BACKGROUND： Astragalus mongholicus can promote differentiation of bone marrow mesenchymal stem cells （BMSCs） into neurons, but induction mechanism was few. At present, the induction may be associated with the ability of anti-oxidation. OBJECTIVE： To observe the changes in the transcriptional level of calmodulin mRNA during the earlier differentiation period of rat BMSCs induced by astragalus mongholicus injection. DESIGN, TIME AND SETTING： The cytological in vitro controlled study was performed at the Experimental Center of Hebei North University from October to December 2007. MATERIALS： One clean male rat aged 6 weeks were purchased from Experimental Animal Center, Chinese Academy of Medical Sciences. Astragalus mongholicus injection （lot number 060105） was purchased from Dali, China. METHODS： After isolation, culture and passage, four-passage BMSCs at 4×10^5/L were incubated in a 12-well plate in DMEM containing 200 g/L astragalus mongholicus injection and 15% fetal bovine serum. BMSCs underwent immunocytochemical staining. At fifth passage, BMSCs were equally assigned into 4 centrifuge tubes. Tube 1 served as control group. BMSCs in other three tubes were treated with above-mentioned medium for 30, 60 and 120 minutes. The harvesting cells were treated with RT-PCR to measure the transcriptional level of camodulin mRNA. MAIN OUTCOME MEASURES： Induction of astragalus mongholicus on BMSCs, and transcriptional level of camodulin mRNA after induction were measured. RESULTS： After BMSCs are induced by astragalus mongholicus for 5 hours, the morphology of a small number of cells was changed. The cell body was round and the long and thin prominences stretched out from the body. The results of immunocytochemical staining showed that there were some positive staining cells for nestin and neurone specific enolase, a small number of cells presented positive staining for microtubule-associated protein-2, but the degrees of the positive staining was weak comparatively in glial fibrillary acidic protein positive cells. The results of RT-PCR indicated that the gene of calmodulin had been transcribed at the earlier period of BMSC differentiation induced by astragalus mongholicus and the transcriptional level was upregulated gradually with prolonged induction time. Significant differences in relative amount of calmodulin mRNA between the 30, 60, 120 min groups and control groups （F=153.315, P=0.000）. CONCLUSION： Astragalus mongholicus can promote the differentiation of BMSCs into neurons. During early induction period, the transcriptional level is upregulated. It is indicated that the induction effect may be associated with calmodulin-mediated signal transduction.