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[1]吳剛,童培建.補(bǔ)腎活血湯含藥血清干預(yù)體外培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞成軟骨分化及補(bǔ)腎活血湯聯(lián)合骨髓間充質(zhì)干細(xì)胞治療大鼠膝骨關(guān)節(jié)炎的實(shí)驗(yàn)研究[J].中醫(yī)正骨,2018,30(01):6-11.
　WU Gang,TONG Peijian.Impact of Bushen Huoxue Tang(補(bǔ)腎活血湯)medicated serum on chondrogenic differentiation?of rat’s bone marrow derived mesenchymal stem cells cultured in vitro[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(01):6-11.
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補(bǔ)腎活血湯含藥血清干預(yù)體外培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞成軟骨分化及補(bǔ)腎活血湯聯(lián)合骨髓間充質(zhì)干細(xì)胞治療大鼠膝骨關(guān)節(jié)炎的實(shí)驗(yàn)研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第30卷
期數(shù):
2018年01期
頁(yè)碼:
6-11
欄目:
基礎(chǔ)研究
出版日期:
2018-01-20

文章信息/Info

Title:
Impact of Bushen Huoxue Tang(補(bǔ)腎活血湯)medicated serum on chondrogenic differentiation?of rat’s bone marrow derived mesenchymal stem cells cultured in vitro
作者:
吳剛1; 童培建2
1.浙江中醫(yī)藥大學(xué),浙江 杭州 310053; 2.浙江省中醫(yī)院,浙江 杭州 310006
Author(s):
WU Gang1; TONG Peijian2
1.Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China2.Zhejiang Provincial Hospital of Traditional Chinese Medicine,Hangzhou 310006,Zhejiang,China
關(guān)鍵詞:
骨關(guān)節(jié)炎;  骨髓;  間質(zhì)干細(xì)胞;  軟骨; 關(guān)節(jié);  補(bǔ)腎活血湯;  大鼠;  動(dòng)物實(shí)驗(yàn)
Keywords:
Keywords osteoarthritis;  bone marrow;  mesenchymal stem cells;  cartilage; articular;  Bushen Huoxue Tang;  rats;  animal experimentation
摘要:
目的:觀察補(bǔ)腎活血湯含藥血清對(duì)大鼠骨髓間充質(zhì)干細(xì)胞(bone marrow stem cell,BMSC)成軟骨分化的作用,及補(bǔ)腎活血湯聯(lián)合BMSC治療大鼠膝骨關(guān)節(jié)炎(knee osteoarthritis,KOA)的作用。方法:從4周齡SD大鼠股骨中分離培養(yǎng)BMSC,以8周齡SD大鼠制備補(bǔ)腎活血湯含藥血清和空白血清。選擇體外培養(yǎng)的第3代BMSC,分為3組,分別加入含10%空白血清的DMEM培養(yǎng)液(空白組)、含10%空白血清的DMEM培養(yǎng)液及基礎(chǔ)誘導(dǎo)液(誘導(dǎo)組)、含10%補(bǔ)腎活血湯含藥血清的DMEM培養(yǎng)液及基礎(chǔ)誘導(dǎo)液(含藥血清組),干預(yù)后分別進(jìn)行甲苯胺藍(lán)染色、Ⅱ型膠原免疫組化染色,并以Real-time PCR法檢測(cè)各組細(xì)胞中Ⅱ型膠原mRNA、聚集蛋白聚糖mRNA的水平。取18只8周齡SD大鼠,隨機(jī)分為模型組、BMSC組及聯(lián)合組,每組6只。通過(guò)切斷右前交叉韌帶對(duì)各組大鼠進(jìn)行KOA造模。造模術(shù)后1周,向BMSC組與聯(lián)合組大鼠右側(cè)膝關(guān)節(jié)腔內(nèi)注射5×106個(gè)·mL-1BMSC-PBS溶液(每次0.1 mL,每周1次),向模型組大鼠右側(cè)膝關(guān)節(jié)腔注射等量PBS溶液; 聯(lián)合組在關(guān)節(jié)腔注射的基礎(chǔ)上每天以補(bǔ)腎活血湯灌胃(每次4 mL,每天1次)。干預(yù)8周后處死大鼠,取右側(cè)膝關(guān)節(jié)軟骨及軟骨下骨組織,HE染色后在光學(xué)顯微鏡下觀察。結(jié)果:干預(yù)7 d后,含藥血清組細(xì)胞甲苯胺藍(lán)染色呈陽(yáng)性,細(xì)胞質(zhì)呈藍(lán)紫色; 空白組和誘導(dǎo)組染色均為陰性。干預(yù)14 d 后,含藥血清組染色進(jìn)一步加深,染色面積增大,呈明顯的紫藍(lán)色; 空白組染色為陰性,誘導(dǎo)組可見(jiàn)少量藍(lán)紫色染色。干預(yù)7 d、14 d后,Ⅱ型膠原免疫組化染色結(jié)果顯示,空白組細(xì)胞均未見(jiàn)明顯陽(yáng)性染色,誘導(dǎo)組及含藥血清組細(xì)胞胞漿和細(xì)胞間質(zhì)內(nèi)呈棕黃色或棕褐色染色,其中干預(yù)14 d后含藥血清組細(xì)胞內(nèi)染色深于誘導(dǎo)組。干預(yù)7 d后,3組大鼠BMSC中Ⅱ型膠原mRNA水平、聚集蛋白聚糖mRNA水平比較,組間差異均有統(tǒng)計(jì)學(xué)意義(1.02±0.23,1.33±0.11,2.11±0.23,F=47.181,P=0.000; 1.03±0.32,1.36±0.16,1.93±0.10,F=26.508,P=0.000)。含藥血清組的Ⅱ型膠原mRNA水平、聚集蛋白聚糖mRNA水平均高于空白組和誘導(dǎo)組(P=0.000,P=0.000; P=0.000,P=0.000),誘導(dǎo)組的Ⅱ型膠原mRNA水平、聚集蛋白聚糖mRNA水平均高于空白組(P=0.017; P=0.029)。藥物干預(yù)8周后,模型組大鼠膝關(guān)節(jié)軟骨表面粗糙,破壞嚴(yán)重,可見(jiàn)明顯纖維增生; BMSC組軟骨可見(jiàn)部分修復(fù),表面仍較粗糙,細(xì)胞纖維化不明顯; 聯(lián)合組關(guān)節(jié)軟骨面光滑,軟骨表面磨損不明顯,無(wú)軟骨表面纖維化。結(jié)論:補(bǔ)腎活血湯含藥血清能有效促進(jìn)體外培養(yǎng)的大鼠BMSC向軟骨細(xì)胞分化; 補(bǔ)腎活血湯灌胃聯(lián)合BMSC膝關(guān)節(jié)腔內(nèi)注射能有效防治KOA大鼠膝關(guān)節(jié)軟骨破壞。
Abstract:
ABSTRACT Objective:To observe the effect of Bushen Huoxue Tang(補(bǔ)腎活血湯,BSHXT)medicated serum on chondrogenic differentiation of rat’s bone marrow derived mesenchymal stem cells(BMSCs)and the curative effect of BSHXT and BMSCs on knee osteoarthritis(KOA)in rats.Methods:The BMSCs were isolated from the femurs of 4-week-old SD rats and were cultured,and BSHXT medicated serum and blank serum were prepared by using 8-week-old SD rats.The third-generation BMSCs cultured in vitro were selected and randomly divided into 3 groups,and they were placed into the DMEM culture fluid added with 10% blank serum(blank group),DMEM culture fluid added with 10% blank serum and basic induced fluid(induced group)and DMEM culture fluid added with 10% BSHXT medicated serum and basic induced fluid(medicated serum group)respectively.After the intervention,the BMSCs were received toluidine blue staining and typeⅡcollagen immunohistochemical staining respectively,and the expression level of typeⅡcollagen mRNA and aggrecan mRNA were detected by using Real-time PCR method.Eighteen eight-week-old SD rats were selected and randomly divided into model group,

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通訊作者:吳剛 E-mail:[email protected]
更新日期/Last Update: 2018-06-02