[1]吴秀丽,阎晓霞,任之强,等.牛膝含药血清对大鼠骨髓间充质干细胞增殖和成骨分化的影响及其作用机制[J].中医正骨,2024,36(10):10-17.
　WU Xiuli,YAN Xiaoxia,REN Zhiqiang,et al.Effects of achyranthes bidentatae radix(TCD)medicated serum on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in rats and its mechanism of action:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2024,36(10):10-17.
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牛膝含药血清对大鼠骨髓间充质干细胞增殖和成骨分化的影响及其作用机制()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期数:
2024年10期
页码:
10-17
栏目:
基础研究
出版日期:
2024-10-20

文章信息/Info

Title:
Effects of achyranthes bidentatae radix(TCD)medicated serum on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in rats and its mechanism of action:an experimental study
作者:
吴秀丽1; 阎晓霞2; 任之强2; 孙楠2; 李金菊2; 谢亚威1; 李龙飞1
1.河南中医药大学骨伤学院,河南 郑州 450046; 2.河南省洛阳正骨医院/河南省骨科医院,河南 郑州 450016
Author(s):
WU Xiuli1; YAN Xiaoxia2; REN Zhiqiang2; SUN Nan2; LI Jinju2; XIE Yawei1; LI Longfei1
1.College of Orthopaedics and Traumatology of Henan University of Chinese Medicine,Zhengzhou 450046,Henan,China 2.Luoyang Orthopedic-Traumatological Hospital,Zhengzhou 450016,Henan,China
关键词:
牛膝(中药);  间质干细胞;  大鼠; Sprague-Dawley;  细胞增殖;  骨生成
Keywords:
achyranthes bidentatae radix(TCD);  mesenchymal stem cells;  rats; Sprague-Dawley;  cell proliferation;  osteogenesis
摘要:
目的:探讨牛膝含药血清对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖、成骨分化的影响及其作用机制。方法:取4周龄雌性SPF级SD大鼠20只,随机分为空白组和牛膝低、中、高剂量组,每组5只。牛膝低、中、高剂量组大鼠分别以相应浓度的牛膝药液灌胃,空白组大鼠以同等剂量生理盐水灌胃,每日1次,共灌胃14 d。最后一次灌胃干预2 h后,取大鼠腹主动脉血,制备空白血清和相应浓度的牛膝含药血清。另取大鼠4只,处死后取出大鼠股骨和胫骨骨髓,进行BMSCs培养。细胞传到第3代时,用流式细胞仪进行细胞表型鉴定。将大鼠BMSCs分为胎牛血清组、空白血清组和牛膝含药血清低、中、高剂量组,分别加入胎牛血清、空白血清和牛膝低、中、高剂量含药血清进行干预,检测各组大鼠BMSCs的增殖活性; 分别加入含相应血清的成骨诱导液进行成骨诱导,采用茜素红染色观察大鼠BMSCs成骨分化情况,采用荧光定量PCR检测大鼠BMSCs中成骨相关因子碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)、Runt相关转录因子2(runt-related transcription factor 2,Runx2)和Osterix的mRNA相对表达量,采用蛋白质印迹法检测BMSCs中Hedgehog信号通路相关蛋白音猬因子(sonic hedgehog,SHH)、Gli2的蛋白相对表达量。结果:①细胞鉴定结果。细胞表型鉴定结果显示,培养的细胞为BMSCs。②大鼠BMSCs增殖活性检测结果。干预24 h、48 h、72 h、96 h后,5组大鼠BMSCs增殖活性组间总体比较,差异均有统计学意义; 干预24 h后,牛膝含药血清高剂量组BMSCs的增殖活性高于胎牛血清组、空白血清组(P=0.006,P=0.008); 干预48 h、72 h、96 h后,牛膝含药血清高剂量组BMSCs的增殖活性均高于胎牛血清组、空白血清组和牛膝含药血清低、中剂量组(P=0.000,P=0.000,P=0.010,P=0.021; P=0.003,P=0.000,P=0.007,P=0.016; P=0.000,P=0.000,P=0.002,P=0.047)。③大鼠BMSCs成骨分化检测结果。茜素红染色显示,各组大鼠BMSCs均出现细胞外矿化结节形成与沉积,其中牛膝含药血清高剂量组阳性染色面积较大,矿化结节明显。牛膝含药血清高剂量组矿化结节面积大于胎牛血清组和空白血清组(P=0.039,P=0.015)。④大鼠BMSCs中成骨相关因子的mRNA相对表达量检测结果。牛膝含药血清低、中、高剂量组大鼠BMSCs中ALP、OCN、Runx2、Osterix的mRNA相对表达量均高于胎牛血清组(P=0.003,P=0.000,P=0.000; P=0.011,P=0.001,P=0.000; P=0.009,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000)和空白血清组(P=0.000,P=0.000,P=0.000; P=0.005,P=0.000,P=0.000; P=0.031,P=0.001,P=0.000; P=0.000,P=0.000,P=0.000)。牛膝含药血清中剂量组大鼠BMSCs中ALP的mRNA相对表达量高于牛膝含药血清低剂量组(P=0.044)。牛膝含药血清高剂量组大鼠BMSCs中ALP、OCN、Runx2、Osterix的mRNA相对表达量均高于牛膝含药血清低剂量组(P=0.002,P=0.006,P=0.002,P=0.008)。牛膝含药血清高剂量组大鼠BMSCs中Runx2的mRNA相对表达量高于牛膝含药血清中剂量组(P=0.047)。⑤大鼠BMSCs中Hedgehog信号通路相关蛋白的蛋白相对表达量检测结果。牛膝含药血清高剂量组大鼠BMSCs中SHH、Gli2高表达。牛膝含药血清低、中、高剂量组SHH、Gli2的蛋白相对表达量均高于胎牛血清组(P=0.000,P=0.000,P=0.000; P=0.026,P=0.016,P=0.000)和空白血清组(P=0.000,P=0.000,P=0.000; P=0.031,P=0.018,P=0.000)。牛膝含药血清中、高剂量组大鼠BMSCs中SHH的蛋白相对表达量均高于牛膝含药血清低剂量组(P=0.000,P=0.000),牛膝含药血清高剂量组大鼠BMSCs中SHH的蛋白相对表达量高于牛膝含药血清中剂量组(P=0.000)。牛膝含药血清高剂量组大鼠BMSCs中Gli2的蛋白相对表达量高于牛膝含药血清低剂量组(P=0.001)。结论:牛膝含药血清可能通过激活Hedgehog信号通路和上调成骨相关因子ALP、OCN、Runx2、Osterix的表达,促进大鼠BMSCs的增殖和成骨分化。
Abstract:
Objective:To observe the effects of achyranthes bidentatae radix(ABR)(TCD)medicated serum on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in rats,and to explore its mechanism of action.Methods:Twenty 4-week-old specific pathogen-free(SPF)-grade female Sprague-Dawley(SD)rats were selected and randomized into blank group,low-dose ABR(L-ABR)group,medium-dose ABR(M-ABR)group,and high-dose ABR(H-ABR)group,5 cases in each group.The rats in L-,M-,and H-ABR group were intervened by intragastric administration with ABR solution in their corresponding concentration,respectively,whereas,the ones in blank group with the same dosage of normal saline,once a day for consecutive 14 days.Two hours after the end of the last intervention,the blood was drawn from the abdominal aorta of rats in each group for making blank serum and ABR medicated serum with the corresponding concentrations.Additionally,another 4 rats were selected and executed,and the bone marrow was harvested from their femurs and tibias for BMSCs culture.The third-generation BMSCs were collected to identify the phenotypes by using flow cytometry.Furthermore,the BMSCs were divided into fetal bovine serum group,blank serum group,L-,M-,and H-ABR medicated serum groups,and were intervened by fetal bovine serum,blank serum,L-,M-,and H-ABR medicated serum,respectively.The proliferation activity of BMSCs in rats was detected,and the osteogenic induction was conducted by adding osteogenic induction medium containing the corresponding serum into the BMSCs.After 21-day induction,the osteogenic differentiation of BMSCs was observed via alizarin red staining(ARS).Besides,the relative mRNA expression levels of osteogenesis-related markers,including alkaline phosphatase(ALP),osteocalcin(OCN),runt-related transcription factor 2(Runx2)and Osterix,in rat BMSCs were detected by fluorescence quantitative PCR,and the relative protein expression le-vels of Hedgehog signaling pathway-related proteins,including sonic hedgehog(SHH)and Gli2,in rat BMSCs were detected by Western blotting.Results:①The results of BMSCs identification.The results of cell phenotype identification indicated that the cultured cells were identified as BMSCs.②The results of detection on the proliferation activity of BMSCs in rats.After 24-,48-,72-,and 96-hour intervention,the difference was statistically significant in proliferation activity of rat BMSCs among the 5 groups in general.The proliferation activity of BMSCs was higher in H-ABR medicated serum group compared to fetal bovine serum group and blank serum group after 24-hour intervention(P=0.006,P=0.008),and was higher in H-ABR medicated serum group compared to fetal bovine serum group,blank serum group,L-,and M-ABR medicated serum groups after 48-,72-,and 96-hour intervention(P=0.000,P=0.000,P=0.010,P=0.021; P=0.003,P=0.000,P=0.007,P=0.016; P=0.000,P=0.000,P=0.002,P=0.047).③The results of detection on osteogenic differentiation of BMSCs in rats.The ARS result showed that the extracellular mineralized nodules formed and deposited in BMSCs of rats in each group,with H-ABR medicated serum group displaying a larger positive staining area and more prominent mineralized nodules,and the area of minera-lized nodules was larger in H-ABR medicated serum group compared to fetal bovine serum group and blank serum group(P=0.039,P=0.015).④The results of detection on the relative mRNA expression levels of osteogenesis-related markers in rat BMSCs.The relative mRNA expression levels of ALP,OCN,Runx2 and Osterix in rat BMSCs were higher in L-,M-,and H-ABR medicated serum groups compared to fetal bovine serum group and blank serum group(P=0.003,P=0.000,P=0.000; P=0.011,P=0.001,P=0.000; P=0.009,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000; P=0.005,P=0.000,P=0.000; P=0.031,P=0.001,P=0.000; P=0.000,P=0.000,P=0.000),and were higher in H-ABR medicated serum group compared to L-ABR medicated serum group(P=0.002,P=0.006,P=0.002,P=0.008).The relative mRNA expression level of ALP in rat BMSCs was higher in M-ABR medicated serum group compared to L-ABR medicated serum group(P=0.044); while,the relative mRNA expression level of Runx2 was higher in H-ABR medicated serum group compared to M-ABR medicated serum group(P=0.047).⑤The results of detection on the relative protein expression levels of Hedgehog signaling pathway-related proteins in rat BMSCs.The SHH and Gli2 were highly expressed in rat BMSCs in H-ABR medicated serum group.The relative protein expression levels of SHH and Gli2 in rat BMSCs were higher in L-,M-,and H-ABR medicated serum groups compared to fetal bovine serum group and blank serum group(P=0.000,P=0.000,P=0.000; P=0.026,P=0.016,P=0.000; P=0.000,P=0.000,P=0.000; P=0.031,P=0.018,P=0.000).The relative protein expression level of SHH in rat BMSCs was higher in M-,and H-ABR medicated serum groups compared to L-ABR medicated serum group(P=0.000,P=0.000),and was highest in H-ABR medicated serum group(P=0.000).The relative protein expression level of Gli2 in rat BMSCs was higher in H-ABR medicated serum group compared to L-ABR medicated serum group(P=0.001).Conclusion:ABR(TCD)medicated serum may promote the proliferation and osteogenic differentiation of BMSCs by activating Hedgehog signaling pathway and up-regulating the expression of ALP,OCN,Runx2 and Osterix in rats.

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备注/Memo

备注/Memo:
基金项目:国家中医药管理局青年岐黄学者培养项目(国中医药人教函〔2022〕256号); 河南省中医药科学研究专项课题(2022ZYZD14,2024ZY2115,2024ZY2108); 河南省科技发展计划项目(232102310423)
通讯作者:阎晓霞 E-mail:641594851@qq.com
更新日期/Last Update: 1900-01-01