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目的:观察龟鹿二仙胶汤对骨髓基质干细胞(BMSCs)增殖的影响,确定影响BMSCs增殖的最佳中药血清浓度。方法:经前期体外分离、纯化、培养获得兔BMSCs,运用不同浓度的龟鹿二仙胶汤中药含药血清对获得的兔BMSCs进行药物干预24、48、72 h后,采用MTT、免疫组化、流式细胞仪等方法,研究龟鹿二仙胶汤对BMSCs增殖的影响。结果:MTT比色实验显示,龟鹿二仙胶各浓度组均能不同程度地促进BMSCs的能量代谢,促进BMSCs增殖,与空白对照组相比有显著差异(P<0.01),而且以15倍药物浓度组为最佳。免疫组化结果证实,龟鹿二仙胶汤含药血清可通过促进PCNA表达来控制DNA复制,以15倍龟鹿二仙胶汤药物浓度血清培养时BMSCs表达致密的棕褐色颗粒最多,其他浓度组及TGF-β_3组较多,空白对照组则最少。FCM结果显示,二仙胶汤药物血清能显著提高BMSCs增殖指数,且以15倍药物浓度龟鹿血清培养时最明显。结论:龟鹿二仙胶汤刺激BMSCs增值与药物浓度有关,低浓度龟鹿二仙胶汤就能显著刺激BMSCs增殖,无论是48 h、72 h测验均为15倍药物浓度时具有显著的增值作用,而更高浓度龟鹿二仙胶汤则表现为促进BMSCs增殖作用减弱。
Objective: To observe the effect of Guilu Erxianjiao decoction on the proliferation of bone marrow stromal cells (BMSCs), and to determine the best Chinese herbal serum concentration that affects the proliferation of BMSCs. METHODS: Rabbit BMSCs were isolated, purified and cultured in vitro. Rabbit BMSCs were treated with different concentrations of Guilu Erxian gum decoction for 24, 48, and 72 h after intervention with MTT and immunohistochemistry. Methods such as flow cytometry were used to study the effects of Guilu Erxian Decoction on the proliferation of BMSCs. RESULTS: MTT colorimetric experiments showed that each concentration group of Guilu Erxian gum could promote the energy metabolism of BMSCs to different degrees, promote the proliferation of BMSCs, and there was a significant difference compared with the blank control group (P<0.01), and 15 times. The drug concentration group is the best. The results of immunohistochemistry confirmed that the serum containing Guilu Erxian Decoction could control DNA replication by promoting the expression of PCNA. When cultured with serum of 15 times of Guilu Erxian Decoction, BMSCs expressed dense brown particles, and other concentrations The group and TGF-β_3 group were more, while the blank control group was the least. The results of FCM showed that the serum of Erxianjia Decoction could significantly increase the proliferation index of BMSCs, and it was the most obvious when cultured with Guizhi deer serum at 15 times the drug concentration. Conclusion: The effect of Guilu Erxian Decoction on the proliferation of BMSCs is related to the drug concentration. Low concentrations of Guilu Erxianjiao Decoction can significantly stimulate the proliferation of BMSCs, which has significant value-added when the 48-hour and 72-hour tests are all 15 times drug concentration. The effect of higher concentrations of Guilu Erxian Decoction was to promote the weakening of BMSCs proliferation.