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[1]岑芊瑤,劉江源,曾慶賀,等.補(bǔ)腎活血方聯(lián)合人臍帶血間充質(zhì)干細(xì)胞修復(fù)小鼠膝關(guān)節(jié)軟骨缺損的實(shí)驗(yàn)研究[J].中醫(yī)正骨,2023,35(12):5-13，24.
　CEN Qianyao,LIU Jiangyuan,ZENG Qinghe,et al.Bushen Huoxue Fang(補(bǔ)腎活血方)combined with human umbilical cord blood-derived mesenchymal stem cells against knee articular cartilage defects in mice:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2023,35(12):5-13，24.
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補(bǔ)腎活血方聯(lián)合人臍帶血間充質(zhì)干細(xì)胞修復(fù)小鼠膝關(guān)節(jié)軟骨缺損的實(shí)驗(yàn)研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期數(shù):
2023年12期
頁碼:
5-13，24
欄目:
基礎(chǔ)研究
出版日期:
2023-12-20

文章信息/Info

Title:
Bushen Huoxue Fang(補(bǔ)腎活血方)combined with human umbilical cord blood-derived mesenchymal stem cells against knee articular cartilage defects in mice:an experimental study
作者:
岑芊瑤1; 劉江源2; 曾慶賀2; 溫經(jīng)淵2; 吳叢姿2; 王旭2; 吳震3; 金紅婷2; 陳佳麗2
1.浙江中醫(yī)藥大學(xué)第二臨床醫(yī)學(xué)院,浙江 杭州 310053; 2.浙江中醫(yī)藥大學(xué)骨傷研究所,浙江 杭州 310053; 3.浙江省立同德醫(yī)院,浙江 杭州 310012
Author(s):
CEN Qianyao1; LIU Jiangyuan2; ZENG Qinghe2; WEN Jingyuan2; WU Congzi2; WANG Xu2; WU Zhen3; JIN Hongting2; CHEN Jiali2
1.The Second Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 2.Institute of Traumatology and Orthopedics of Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 3.Tongde Hospital of Zhejiang Province,Hangzhou 310012,Zhejiang,China
關(guān)鍵詞:
膝關(guān)節(jié);  軟骨; 關(guān)節(jié);  小鼠;  軟骨缺損;  間質(zhì)干細(xì)胞;  臍帶血;  補(bǔ)腎活血方;  骨微結(jié)構(gòu);  動(dòng)物實(shí)驗(yàn)
Keywords:
knee joint;  cartilage; articular;  mice;  cartilage defects;  mesenchymal stem cells;  umbilical cord blood;  Bushen Huoxue Fang;  bone microstructure;  animal experimentation
摘要:
目的:探討補(bǔ)腎活血方聯(lián)合人臍帶血間充質(zhì)干細(xì)胞(human umbilical cord blood-derived mesenchymal stem cells,hUCB-MSCs)修復(fù)小鼠膝關(guān)節(jié)軟骨缺損的效果及作用機(jī)制。方法:將24只10周齡雌性C57BL/6J小鼠隨機(jī)分為對(duì)照組、模型組、hUCB-MSCs組和補(bǔ)腎活血方聯(lián)合hUCB-MSCs組,每組6只。除對(duì)照組外,其余3組均用針頭在小鼠股骨髁滑車關(guān)節(jié)面上造一個(gè)直徑 0.45 mm、深1 mm的圓柱形缺損。分別于造模后第1周、第2周、第3周,向hUCB-MSCs組、補(bǔ)腎活血方聯(lián)合hUCB-MSCs組小鼠膝關(guān)節(jié)腔內(nèi)注射10 μL的hUCB-MSCs懸液(200個(gè)細(xì)胞·μL-1),向?qū)φ战M和模型組小鼠膝關(guān)節(jié)腔內(nèi)注射10 μL的生理鹽水; 各組小鼠均每周注射1次。造模后第2天,補(bǔ)腎活血方聯(lián)合hUCB-MSCs組小鼠給予補(bǔ)腎活血方濃縮液(20 μL·g-1)灌胃,對(duì)照組、模型組和hUCB-MSCs組小鼠每天給予等量生理鹽水灌胃; 各組小鼠均每天灌胃1次,共4周。灌胃結(jié)束后第2天,脫頸處死各組小鼠,切取小鼠右側(cè)膝關(guān)節(jié),以Micro-CT觀察小鼠膝關(guān)節(jié)軟骨缺損區(qū)骨微結(jié)構(gòu); 以阿爾新藍(lán)-蘇木素染色觀察膝關(guān)節(jié)軟骨缺損區(qū)軟骨退變情況,并采用國際骨關(guān)節(jié)炎研究學(xué)會(huì)(Osteoarthritis Research Society International,OARSI)評(píng)分對(duì)關(guān)節(jié)軟骨退變情況進(jìn)行評(píng)估; 采用免疫組織化學(xué)染色測定小鼠膝關(guān)節(jié)軟骨缺損區(qū)軟骨中Ⅱ型膠原蛋白α1鏈(collagen typeⅡalpha 1 chain,Col2a1)和基質(zhì)金屬蛋白酶13(matrix metalloproteinase 13,MMP13)的表達(dá)量。結(jié)果:①小鼠膝關(guān)節(jié)軟骨缺損區(qū)骨微結(jié)構(gòu)觀察結(jié)果。對(duì)照組、hUCB-MSCs組和補(bǔ)腎活血方聯(lián)合hUCB-MSCs組小鼠膝關(guān)節(jié)軟骨缺損區(qū)骨松質(zhì)的骨密度均高于模型組(LSD-t=18.425,P=0.000; LSD-t=-10.186,P=0.000; LSD-t=-7.487,P=0.000),骨體積分?jǐn)?shù)均高于模型組(LSD-t=7.242,P=0.002; LSD-t=-5.243,P=0.006; LSD-t=-5.441,P=0.006),骨小梁厚度均大于模型組(LSD-t=7.575,P=0.002; LSD-t=-10.005,P=0.002; LSD-t=-5.409,P=0.006),骨小梁數(shù)量均多于模型組(LSD-t=9.166,P=0.000; LSD-t=-10.014,P=0.000; LSD-t=-9.147,P=0.000),骨小梁分離度均小于模型組(LSD-t=-9.120,P=0.000; LSD-t=10.375,P=0.000; LSD-t=7.650,P=0.002); hUCB-MSCs組骨松質(zhì)骨小梁數(shù)量少于對(duì)照組(LSD-t=3.027,P=0.039); 其余各組之間兩兩比較,差異均無統(tǒng)計(jì)學(xué)意義。②小鼠膝關(guān)節(jié)軟骨缺損區(qū)軟骨退變情況觀察結(jié)果。與對(duì)照組相比,模型組小鼠關(guān)節(jié)軟骨退變明顯; 與模型組相比,hUCB-MSCs組和補(bǔ)腎活血方聯(lián)合hUCB-MSCs組小鼠關(guān)節(jié)軟骨退變較輕。對(duì)照組、hUCB-MSCs組和補(bǔ)腎活血方聯(lián)合hUCB-MSCs組小鼠OARSI評(píng)分均低于模型組(LSD-t=-11.762,P=0.000; LSD-t=10.947,P=0.000; LSD-t=14.779,P=0.000),補(bǔ)腎活血方聯(lián)合hUCB-MSCs組和對(duì)照組小鼠OARSI評(píng)分均低于hUCB-MSCs組(LSD-t=-5.635,P=0.005; LSD-t=-3.443,P=0.026),對(duì)照組小鼠OARSI評(píng)分低于補(bǔ)腎活血方聯(lián)合hUCB-MSCs組(LSD-t=-5.914,P=0.004)。③小鼠膝關(guān)節(jié)軟骨缺損區(qū)軟骨Col2a1和MMP13表達(dá)量測定結(jié)果。對(duì)照組、hUCB-MSCs組和補(bǔ)腎活血方聯(lián)合hUCB-MSCs組小鼠膝關(guān)節(jié)軟骨缺損區(qū)軟骨Col2a1陽性表達(dá)面積均大于模型組(LSD-t=9.863,P=0.000; LSD-t=45.990,P=0.000; LSD-t=-17.406,P=0.000),補(bǔ)腎活血方聯(lián)合hUCB-MSCs組小鼠膝關(guān)節(jié)軟骨缺損區(qū)軟骨Col2a1陽性表達(dá)面積大于hUCB-MSCs組(LSD-t=3.623,P=0.022),補(bǔ)腎活血方聯(lián)合hUCB-MSCs組和hUCB-MSCs組小鼠膝關(guān)節(jié)軟骨缺損區(qū)軟骨Col2a1陽性表達(dá)面積與對(duì)照組的差異均無統(tǒng)計(jì)學(xué)意義(LSD-t=-1.643,P=0.176; LSD-t=0.533,P=0.623)。對(duì)照組、hUCB-MSCs組和補(bǔ)腎活血方聯(lián)合hUCB-MSCs組MMP13陽性細(xì)胞數(shù)占比均低于模型組(LSD-t=-14.299,P=0.001; LSD-t=9.688,P=0.001; LSD-t=15.638,P=0.000),補(bǔ)腎活血方聯(lián)合hUCB-MSCs組MMP13陽性細(xì)胞數(shù)占比低于hUCB-MSCs組和對(duì)照組(LSD-t=-11.488,P=0.007; LSD-t=3.578,P=0.023),hUCB-MSCs組MMP13陽性細(xì)胞數(shù)占比高于對(duì)照組(LSD-t=-9.425,P=0.000)。結(jié)論:補(bǔ)腎活血方聯(lián)合hUCB-MSCs能明顯改善小鼠膝關(guān)節(jié)軟骨缺損區(qū)骨微結(jié)構(gòu),修復(fù)軟骨缺損,其作用機(jī)制可能與上調(diào)Col2a1的表達(dá)和抑制MMP13的表達(dá)有關(guān)。
Abstract:
Objective:To explore the effects and mechanism of Bushen Huoxue Fang(補(bǔ)腎活血方,BSHXF)combined with human umbilical cord blood-derived mesenchymal stem cells(hUCB-MSCs)in repairing knee articular cartilage defects in mice.Methods:Twenty-four 10-week-old female C57BL/6J mice were selected and randomized into control group,model group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group,6 ones in each group.A cylindrical defect(0.45 mm in diameter and 1 mm in depth)was created on the articular surface of trochlea of femoral condyle with a needle in mice except for the ones in control group.At week 1,2 and 3 after the mode-ling,the mice in hUCB-MSCs group and BSHXF combined with hUCB-MSCs group were intervened by knee intra-articular injection of hUCB-MSCs suspension(10 μL,200 cells/μL),while the ones in control group and model group with the same dose of normal saline.All mice were intervened once a week.On day 2 after the modeling,the mice in BSHXF combined with hUCB-MSCs group were intragastric administrated with BSHXF concentrate in dosage of 20 μL/g,while the ones in control group,model group,and hUCB-MSCs group with the same dose of normal saline.All mice were intervened once a day for consecutive 4 weeks.On day 2 after the end of intragastric administration,all mice were sacrificed by cervical dislocation and their right knees were harvested.The bone microstructure and cartilage degeneration in the knee articular cartilage defect zone were observed by using Micro-CT and alcian blue-hematoxylin(ABH)staining,respectively,and the knee articular cartilage degeneration was evaluated by using Osteoarthritis Research Society International(OARSI)score.Furthermore,the expression levels of collagen typeⅡalpha 1 chain(Col2a1)and matrix metalloproteinase 13(MMP13)in the cartilage of knee articular cartilage defect zone were detected by using immunohistochemical staining.Results:①In the knee articular cartilage defect zone,the control group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group exhibited higher bone mineral density and bone volume fraction,thicker trabeculae,more trabeculae,and lower trabecular separation in cancellous bone compared with that of model group(LSD-t=18.425,P=0.000; LSD-t=-10.186,P=0.000; LSD-t=-7.487,P=0.000; LSD-t=7.242,P=0.002; LSD-t=-5.243,P=0.006; LSD-t=-5.441,P=0.006; LSD-t=7.575,P=0.002; LSD-t=-10.005,P=0.002; LSD-t=-5.409,P=0.006; LSD-t=9.166,P=0.000; LSD-t=-10.014,P=0.000; LSD-t=-9.147,P=0.000; LSD-t=-9.120,P=0.000; LSD-t=10.375,P=0.000; LSD-t=7.650,P=0.002).The hUCB-MSCs group displayed less trabeculae compared to control group(LSD-t=3.027,P=0.039).Further pairwise comparison among other groups showed no statistical significance.②Compared to control group,the knee articular cartilage degenerated more obviously in mice of model group; while,compared to model group,the mice in hUCB-MSCs group and BSHXF combined with hUCB-MSCs group exhibited slight knee articular cartilage degeneration.The OARSI score was lower in control group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group compared to model group(LSD-t=-11.762,P=0.000; LSD-t=10.947,P=0.000; LSD-t=14.779,P=0.000),and was lower in BSHXF combined with hUCB-MSCs group and control group compared to hUCB-MSCs group(LSD-t=-5.635,P=0.005; LSD-t=-3.443,P=0.026),and was lowest in control group(LSD-t=-5.914,P=0.004).③The positive expression area of Col2a1 in cartilage of knee articular cartilage defect zone was larger in mice of control group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group compared to model group(LSD-t=9.863,P=0.000; LSD-t=45.990,P=0.000; LSD-t=-17.406,P=0.000),and was larger in mice of BSHXF combined with hUCB-MSCs group compared to hUCB-MSCs group(LSD-t=3.623,P=0.022); while,the BSHXF combined with hUCB-MSCs group and hUCB-MSCs group didn't differ from control group in the positive expression area of Col2a1 in cartilage of knee articular cartilage defect zone in mice(LSD-t=-1.643,P=0.176; LSD-t=0.533,P=0.623).The proportion of MMP13-positive cells was lower in control group,hUCB-MSCs group and BSHXF combined with hUCB-MSCs group compared to model group(LSD-t=-14.299,P=0.001; LSD-t=9.688,P=0.001; LSD-t=15.638,P=0.000),and was lower in BSHXF combined with hUCB-MSCs group and control group compared to hUCB-MSCs group,and was lowest in BSHXF combined with hUCB-MSCs group(LSD-t=-11.488,P=0.007; LSD-t=3.578,P=0.023; LSD-t=-9.425,P=0.000).Conclusion:BSHXF combined with hUCB-MSCs can significantly improve the bone microstructure,and repair the cartilage defects in knee articular cartilage defect zone of mice.It may exert the effects by up-regulating the expression of Col2a1 and inhibiting the expression of MMP13.

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備注/Memo

備注/Memo:
基金項(xiàng)目:浙江省中醫(yī)藥科技計(jì)劃項(xiàng)目(2023ZL037); 浙江省基礎(chǔ)公益研究計(jì)劃項(xiàng)目(LGF20H270005)
通訊作者:陳佳麗 E-mail:[email protected]
更新日期/Last Update: 1900-01-01