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[1]吳秀麗,閻曉霞,任之強(qiáng),等.牛膝含藥血清對(duì)大鼠骨髓間充質(zhì)干細(xì)胞增殖和成骨分化的影響及其作用機(jī)制[J].中醫(yī)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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牛膝含藥血清對(duì)大鼠骨髓間充質(zhì)干細(xì)胞增殖和成骨分化的影響及其作用機(jī)制()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第36卷
期數(shù):
2024年10期
頁(yè)碼:
10-17
欄目:
基礎(chǔ)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:
吳秀麗1; 閻曉霞2; 任之強(qiáng)2; 孫楠2; 李金菊2; 謝亞威1; 李龍飛1
1.河南中醫(yī)藥大學(xué)骨傷學(xué)院,河南 鄭州 450046; 2.河南省洛陽(yáng)正骨醫(yī)院/河南省骨科醫(yī)院,河南 鄭州 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
關(guān)鍵詞:
牛膝(中藥);  間質(zhì)干細(xì)胞;  大鼠; Sprague-Dawley;  細(xì)胞增殖;  骨生成
Keywords:
achyranthes bidentatae radix(TCD);  mesenchymal stem cells;  rats; Sprague-Dawley;  cell proliferation;  osteogenesis
摘要:
目的:探討牛膝含藥血清對(duì)大鼠骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)增殖、成骨分化的影響及其作用機(jī)制。方法:取4周齡雌性SPF級(jí)SD大鼠20只,隨機(jī)分為空白組和牛膝低、中、高劑量組,每組5只。牛膝低、中、高劑量組大鼠分別以相應(yīng)濃度的牛膝藥液灌胃,空白組大鼠以同等劑量生理鹽水灌胃,每日1次,共灌胃14 d。最后一次灌胃干預(yù)2 h后,取大鼠腹主動(dòng)脈血,制備空白血清和相應(yīng)濃度的牛膝含藥血清。另取大鼠4只,處死后取出大鼠股骨和脛骨骨髓,進(jìn)行BMSCs培養(yǎng)。細(xì)胞傳到第3代時(shí),用流式細(xì)胞儀進(jìn)行細(xì)胞表型鑒定。將大鼠BMSCs分為胎牛血清組、空白血清組和牛膝含藥血清低、中、高劑量組,分別加入胎牛血清、空白血清和牛膝低、中、高劑量含藥血清進(jìn)行干預(yù),檢測(cè)各組大鼠BMSCs的增殖活性; 分別加入含相應(yīng)血清的成骨誘導(dǎo)液進(jìn)行成骨誘導(dǎo),采用茜素紅染色觀察大鼠BMSCs成骨分化情況,采用熒光定量PCR檢測(cè)大鼠BMSCs中成骨相關(guān)因子堿性磷酸酶(alkaline phosphatase,ALP)、骨鈣素(osteocalcin,OCN)、Runt相關(guān)轉(zhuǎn)錄因子2(runt-related transcription factor 2,Runx2)和Osterix的mRNA相對(duì)表達(dá)量,采用蛋白質(zhì)印跡法檢測(cè)BMSCs中Hedgehog信號(hào)通路相關(guān)蛋白音猬因子(sonic hedgehog,SHH)、Gli2的蛋白相對(duì)表達(dá)量。結(jié)果:①細(xì)胞鑒定結(jié)果。細(xì)胞表型鑒定結(jié)果顯示,培養(yǎng)的細(xì)胞為BMSCs。②大鼠BMSCs增殖活性檢測(cè)結(jié)果。干預(yù)24 h、48 h、72 h、96 h后,5組大鼠BMSCs增殖活性組間總體比較,差異均有統(tǒng)計(jì)學(xué)意義; 干預(yù)24 h后,牛膝含藥血清高劑量組BMSCs的增殖活性高于胎牛血清組、空白血清組(P=0.006,P=0.008); 干預(yù)48 h、72 h、96 h后,牛膝含藥血清高劑量組BMSCs的增殖活性均高于胎牛血清組、空白血清組和牛膝含藥血清低、中劑量組(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成骨分化檢測(cè)結(jié)果。茜素紅染色顯示,各組大鼠BMSCs均出現(xiàn)細(xì)胞外礦化結(jié)節(jié)形成與沉積,其中牛膝含藥血清高劑量組陽(yáng)性染色面積較大,礦化結(jié)節(jié)明顯。牛膝含藥血清高劑量組礦化結(jié)節(jié)面積大于胎牛血清組和空白血清組(P=0.039,P=0.015)。④大鼠BMSCs中成骨相關(guān)因子的mRNA相對(duì)表達(dá)量檢測(cè)結(jié)果。牛膝含藥血清低、中、高劑量組大鼠BMSCs中ALP、OCN、Runx2、Osterix的mRNA相對(duì)表達(dá)量均高于胎牛血清組(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)和空白血清組(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)。牛膝含藥血清中劑量組大鼠BMSCs中ALP的mRNA相對(duì)表達(dá)量高于牛膝含藥血清低劑量組(P=0.044)。牛膝含藥血清高劑量組大鼠BMSCs中ALP、OCN、Runx2、Osterix的mRNA相對(duì)表達(dá)量均高于牛膝含藥血清低劑量組(P=0.002,P=0.006,P=0.002,P=0.008)。牛膝含藥血清高劑量組大鼠BMSCs中Runx2的mRNA相對(duì)表達(dá)量高于牛膝含藥血清中劑量組(P=0.047)。⑤大鼠BMSCs中Hedgehog信號(hào)通路相關(guān)蛋白的蛋白相對(duì)表達(dá)量檢測(cè)結(jié)果。牛膝含藥血清高劑量組大鼠BMSCs中SHH、Gli2高表達(dá)。牛膝含藥血清低、中、高劑量組SHH、Gli2的蛋白相對(duì)表達(dá)量均高于胎牛血清組(P=0.000,P=0.000,P=0.000; P=0.026,P=0.016,P=0.000)和空白血清組(P=0.000,P=0.000,P=0.000; P=0.031,P=0.018,P=0.000)。牛膝含藥血清中、高劑量組大鼠BMSCs中SHH的蛋白相對(duì)表達(dá)量均高于牛膝含藥血清低劑量組(P=0.000,P=0.000),牛膝含藥血清高劑量組大鼠BMSCs中SHH的蛋白相對(duì)表達(dá)量高于牛膝含藥血清中劑量組(P=0.000)。牛膝含藥血清高劑量組大鼠BMSCs中Gli2的蛋白相對(duì)表達(dá)量高于牛膝含藥血清低劑量組(P=0.001)。結(jié)論:牛膝含藥血清可能通過激活Hedgehog信號(hào)通路和上調(diào)成骨相關(guān)因子ALP、OCN、Runx2、Osterix的表達(dá),促進(jìn)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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基金項(xiàng)目:國(guó)家中醫(yī)藥管理局青年岐黃學(xué)者培養(yǎng)項(xiàng)目(國(guó)中醫(yī)藥人教函〔2022〕256號(hào)); 河南省中醫(yī)藥科學(xué)研究專項(xiàng)課題(2022ZYZD14,2024ZY2115,2024ZY2108); 河南省科技發(fā)展計(jì)劃項(xiàng)目(232102310423)
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更新日期/Last Update: 1900-01-01