摘要
目的:观察淫羊藿苷骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)向成骨细胞分化过程中的促进作用及其对Notch信号通路的影响。方法:通过对BMSCs细胞进行体外分离培养,将细胞分为对照组与给药组,给药组加入0.1mg·L-1、0.2 mg·L-1、0.4 mg·L-1、1 mg·L-1、2 mg·L-1、4 mg·L-1、10 mg·L-1、20 mg·L-1淫羊藿苷分别培养3 d、7 d、10 d、14 d、21 d,测定细胞上清液中碱性磷酸酶(alkaline phosphatase,ALP)活性,确定最佳给药剂量。将细胞分为3组,对照组、骨分化诱导组、骨分化诱导+淫羊藿苷处理组(联合组),培育7~21 d后测定ALP阳性染色率,Western blot法检测不同处理后Notch信号通路中Noth1、CBF1、Jagged-1蛋白表达情况,RT-PCR法检测淫羊藿苷对Hes1、Runx2 mRNA的影响。结果:与对照组比较,0.1~1.0 mg·L-1淫羊藿苷处理后ALP活性升高较明显,7~14 d内活性均逐渐升高,14 d时活性达到最大值,其中0.4 mg·L-1处理活性最高。因此,选择淫羊藿苷最佳处理质量浓度为0.4 mg·L-1。对照组、联合组在细胞培养7~14 d间,ALP活性逐渐升高,14~21 d活性逐渐下降,第21天ALP活性维持较低水平;诱导组在培养期间ALP活性持续维持较高水平。第7天、第14天,与对照组比较,诱导组、联合组ALP活性升高,且诱导组升高程度高于联合组,差异有统计学意义(P<0.05)。第21天,联合组ALP活性高于对照组、诱导组,差异有统计学意义(P<0.05)。诱导组、联合组ALP阳性染色率均高于对照组,且联合组高于诱导组,差异有统计学意义(P<0.05)。Western blot结果显示,与对照组比较,联合组、诱导组Noth1、CBF1、Jagged-1蛋白表达量均升高,联合组蛋白表达量明显高于诱导组。RT-PCR结果显示诱导组Hesl、Runx2 mRNA表达量均高于对照组,联合组Hesl、Runx2 mRNA表达量高于诱导组。结论:淫羊藿苷能够促进骨髓间充质干细胞向成骨细胞分化,并通过上调Hesl、Runx2 mRNA表达以及增加Noth1、CBF1、Jagged-1蛋白表达来实现。
Objective： To observe the effects of Icariin promoting the differentiation of bone mesenchymal stem cells（ BMSCs） into osteoblasts and its effects on Notch signaling pathway. Methods： The BMSCs cells were isolated and cultured in vitro,and the cells were divided into the control group and the administration group,icariin with different concentration of 0. 1,0. 2,0. 4,1,10,2,4,20 mg·L-1 were added to the administration group,then cells were cultured 3 d,7 d,10 d,14 d and 21 d respectively,the activityof alkaline phosphatase（ ALP） in the supernatant of cells was measured to determine the optimum dosage. Then the cells were divided into three groups： the control group,the bone differentiation induction group,the bone differentiation induction ＋ icariin treatment group（ the joint treatment group）. The positive staining rate of ALP was tested after 7-21 d cultivation,western blot were used to detect the expression of Noth1,CBF1 and Jagged-1 proteins in Notch signaling pathway after different treatments,RT-PCR was used to detect the effect of Icariin on Hes1 and Runx2 mRNA. Results： Compared with the control group,the activity of ALP increased obviously after icariin treatment with 0. 1-1. 0 mg·L-1,and the activity of ALP gradually increased in 7-14 d,and reached the maximum at 14 d with concentration of 0. 4 mg·L-1,so the best concentration of icariin is 0. 4 mg·L-1. For the control group and the combined group,the activity of ALP increased gradually in cell culture 7-14 d,and decreased gradually in14 d-21 d,and remained lower level at the 21 th d; For the induction group,the activity of ALP remained higher level during culture. Compared with the control group,the activities of ALP in the induced group and the combined group were increased on the7 th d and 14 th d,and the level of induction group was higher than that of the combined group,the difference was statistically significant（ P〈0. 05）. On the 21 th d,the activity of ALP in the combined group was higher than that of the control group and the induced group,the difference was statistically significant（ P〈0. 05）. The positive rate of ALP in the induced group and the combined group was higher than that of the control group,and the combination group was higher than that of the induction group,the difference was statistically significant（ P〈0. 05）. The results of western blot showed that compared with the control group,the expression levels of Noth1,CBF1 and Jagged-1 in the combined group and the induced group were increased,and the expression level of the combined group was significantly higher than that of the induction group. The results of RT-PCR showed that the expressions of Hesl and Runx2 mRNA in the induced group were higher than those of the control group,and the expression of Hesl and Runx2 mRNA in the combined group was higher than that of the induced group. Conclusion： Icariin can promote the differentiation of bone mesenchymal stem cells into osteoblasts by up-regulating the expression of Hesl and Runx2 mRNA and increasing the expression of Noth1,CBF1 and Jagged-1 proteins.
出处
《中医学报》
CAS
2017年第12期2393-2398,2403,共7页
Acta Chinese Medicine
基金
国家自然科学基金项目(81502817)
