温肾阳方及其拆方通过TGF-β信号通路促进小鼠肢芽干细胞增殖与分化  

Effect of Wenshenyang Formula and Its Disassembled Formula Containing Serum on Proliferation and Differentiation to Mouse Limb Bud-derived Cells through TGF-β Signaling

作　　者：薛纯纯[1] 王利波[2] 李晓锋[2] 施杞[2,3,4] 王开强[1] 王拥军[2,3,4] 舒冰[2,3,4] XUE Chun-chun;WANG Li-bo;LI Xiao-feng;SHI Qi;WANG Kai-qiang;WANG Yona-iun;SHU Bina(Pain Department,Shanghai Municipal Hospital of Traditional Chinese Medicine,Shanghai 200071;Orthopedics Department,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032;Spine Institute,Shanghai Academy of Traditional Chinese Medicine,Shanghai 200032;KeyLaboratory of Theory and Therapy of Muscles and Bones,Ministry of Education of China,Shanghai 200032)

机构地区：[1]上海中医药大学附属市中医医院疼痛科,上海200071 [2]上海中医药大学附属龙华医院骨科,上海200023 [3]上海中医药大学脊柱病研究所,上海200023 [4]教育部筋骨理论与治法重点实验室,上海200032

出　　处：《中国中西医结合杂志》2021年第9期1100-1106,共7页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家重点研发计划项目(No.2018YFC1704300);国家自然科学基金重点项目(No.81730107);国家自然科学基金项目(No.81804122,No.81973881,No.81973876);上海市青年科技英才扬帆计划(No.17YF1417800);上海市“医苑新星”青年医学人才培养资助计划[No.沪卫计人事(2019)72];上海市中医药三年行动计划(2018-2020)项目[No.ZY(2018-2020)-CCCX-3003]。

摘　　要：目的探讨温肾阳方及方中单味中药骨碎补、淫羊藿是否通过TGF-β信号通路促进小鼠肢芽干细胞增殖与分化。方法分别采用温肾阳方(0.42 g生药/mL)、骨碎补(0.09 g生药/mL)及淫羊藿(0.09 g生药/mL)煎剂和等体积生理盐水给大鼠灌胃,制备含药血清,分离孕12.5天小鼠肢芽干细胞进行微团培养,含药血清干预后,CCK-8法检测肢芽干细胞增殖能力,阿尔新蓝染色检测软骨分化能力,IPP软件统计软骨球个数及面积,Real-time PCR法检测Col2a1及Aggrecan表达水平。采用293T细胞,脂质体转染TGF-β信号通路报告基因4×SBE,荧光素酶实验分析各组对TGF-β信号通路的调节作用。采用TGF-β信号通路抑制剂SB431542抑制肢芽干细胞中TGF-β信号通路,进行阿尔新蓝染色及软骨细胞外基质检测。结果与空白组比较,温肾阳方、淫羊藿、骨碎补组干预48 h和72 h后小鼠肢芽干细胞增殖均增强(P<0.01,P<0.05)。阿尔新蓝染色显示,温肾阳方、淫羊藿、骨碎补组软骨球个数及面积增加(P<0.05),软骨细胞基质Col2a1及Aggrecan的表达增强(P<0.01)。荧光素酶实验发现,与空白组比较,各中药组TGF-β信号通路活性上调(P<0.01,P<0.05)。加入TGF-β信号通路抑制剂SB431542后,与空白组比较,抑制剂组软骨球个数及面积均明显减少(P<0.01);而温肾阳方、淫羊藿、骨碎补组软骨生成抑制显著缓解(P<0.05),但与空白组比较,软骨球个数及面积仍明显减少(P<0.01,P<0.05),Col2a1及Aggrecan水平也有相似的变化趋势(P<0.01)。结论温肾阳方及温肾阳药骨碎补、淫羊藿可以部分通过TGF-β信号通路促进小鼠肢芽干细胞增殖与分化。Objective To explore whether Wenshenyang Formula and single Chinese herb(Rhizoma drynariae and Epimedium) can promote the proliferation and differentiation of mouse limb bud-derived stem cells through TGF-β signal pathway.Methods The decoction of Wenshenyang Formula(0.42 g/mL),Rhizoma drynariae(0.09 g/mL) and Epimedium(0.09 g/mL) and an equal volume of saline were administered to rats by gavage to prepare formula containing,then 12.5 days embryonic limb bud-derived cells with micromass culture were prepared in medicated serum.Cell proliferation was measured by CCK-8,alcian blue staining was observed to investigate chondrogenesis with the number and area of cartilaginous nodules using IPP software.The expression of cartilage extracellular matrix markers(Col2a1,Aggrecan) were analyzed by Real-time PCR.The regulatory effect of different groups on TGF-β signal pathway were analyzed by detecting luciferase activity with 4 x SBE promoter transfection in 293 T cells.Limb bud-derived stem cells with SB431542(TGF-β signaling pathway inhibitor)were prepared to observe the cartilage formation with alcian blue staining and Real-time PCR for Col2a1,Aggrecan.Results Compared with the control group,the proliferation of limb mesenchyme cells were significantly promoted in Wenshenyang Formula and Rhizome drynaria,Epimedium groups after 48 h and 72 h intervention(P<0.05,P<0.01).The alcian blue staining showed that the number and area of cartilaginous nodules in the intervention groups increased(P<0.05),and the mRNA expressions of Col2a1 and Aggrecan were upregulated(P<0.01).Meanwhile,the luciferase experiment found that,compared with the blank group,the activity of TGF-β signal pathway in each Chinese medicine group was up-regulated(P<0.01,P<0.05).After adding TGF-β signaling pathway inhibitor SB431542,compared with the control group,the number and area of cartilaginous nodules in the inhibitor group were significantly reduced(P<0.01),while the inhibition of chondrogenesis in Wenshenyang Formula,Rhizome drynaria and Epimedium

关 键 词：温肾阳 骨碎补 淫羊藿 软骨生成 转化生长因子-Β 

分 类 号：R285.5[医药卫生—中药学]