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[1]原曉強,金王東,周云婧,等.純化血小板對大鼠軟骨細胞增殖及膝骨關(guān)節(jié)炎大鼠軟骨修復(fù)的作用研究[J].中醫(yī)正骨,2016,28(12):6-12.
　YUAN Xiaoqiang,JIN Wangdong,ZHOU Yunjing,et al.Effect of purified platelet rich plasma on chondrocyte proliferation in rats and cartilage repair in rats with knee osteoarthritis:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(12):6-12.
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純化血小板對大鼠軟骨細胞增殖及膝骨關(guān)節(jié)炎大鼠軟骨修復(fù)的作用研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第28卷
期數(shù):
2016年12期
頁碼:
6-12
欄目:
基礎(chǔ)研究
出版日期:
2016-12-30

文章信息/Info

Title:
Effect of purified platelet rich plasma on chondrocyte proliferation in rats and cartilage repair in rats with knee osteoarthritis:an experimental study
作者:
原曉強1; 金王東1; 周云婧1; 葉希佳1; 郭佳娜1; 單樂天1; 童培建2; 肖魯偉1
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):
YUAN Xiaoqiang1; JIN Wangdong1; ZHOU Yunjing1; YE Xijia1; GUO Jiana1; SHAN Letian1; TONG Peijian2; XIAO Luwei1
1.Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 2.Zhejiang Provincial Hospital of TCM,Hangzhou 310006,Zhejiang,China
關(guān)鍵詞:
骨關(guān)節(jié)炎;  膝關(guān)節(jié);  軟骨細胞;  富血小板血漿;  細胞增殖;  大鼠;  動物實驗
Keywords:
osteoarthritis;  knee joint;  chondrocytes;  platelet rich plasma;  cell proliferation;  rats;  animal experimentation
摘要:
目的:觀察純化血小板(purified platelet rich plasma,pPRP)對大鼠軟骨細胞增殖及膝骨關(guān)節(jié)炎(knee osteoarthritis,KOA)大鼠軟骨修復(fù)的作用。方法:取5只SD大鼠抽取腹主動脈血,經(jīng)多次離心后獲得pPRP,并制成低(1×106個·mL-1)、中(1×107個·mL-1)、高(1×108個·mL-1)3種濃度的pPRP。5只SD大鼠抽取腹主動脈血后脫頸處死,取膝關(guān)節(jié)軟骨分離軟骨細胞。將培養(yǎng)的第3代軟骨細胞分為生理鹽水組、pPRP低濃度組、pPRP中濃度組和pPRP高濃度組,每組設(shè)5個復(fù)孔。各組細胞以無血清IMDM培養(yǎng)基處理后,生理鹽水組更換為含10%FBS的培養(yǎng)基,pPRP低濃度組、pPRP中濃度組和pPRP高濃度組分別更換為含10%FBS和低、中、高濃度pPRP的培養(yǎng)基。分別于培養(yǎng)24、48、72 h后采用CCK-8法測定細胞增殖情況。另外選取30只SD大鼠隨機分為空白組、KOA模型組和pPRP治療組,每組10只。KOA模型組、pPRP治療組大鼠通過向雙側(cè)膝關(guān)節(jié)腔內(nèi)注射碘乙酸進行KOA 造模,空白組不進行造模。1周后,向pPRP治療組大鼠雙側(cè)后肢膝關(guān)節(jié)腔各注射50 μL高濃度pPRP,向空白組和KOA模型組大鼠膝關(guān)節(jié)腔注射等量生理鹽水,每周1次,共注射4次。藥物干預(yù)結(jié)束后分別對各組大鼠進行步態(tài)分析、膝關(guān)節(jié)X線檢查及膝關(guān)節(jié)病理學(xué)觀察。結(jié)果:①軟骨細胞增殖測定結(jié)果。培養(yǎng)24、48、72 h后4組軟骨細胞活力比較,組間差異均有統(tǒng)計學(xué)意義(0.411±0.014,0.458±0.052,0.473±0.029,0.489±0.011,F=5.860,P=0.007; 0.502±0.003,0.551±0.022,0.568±0.019,0.572±0.029,F=12.196,P=0.000; 0.619±0.008,0.747±0.006,0.754±0.031,0.763±0.018,F=67.065,P=0.000)。培養(yǎng)24 h后,pPRP低濃度組、pPRP中濃度組、pPRP高濃度組細胞活力均高于生理鹽水組(P=0.026; P=0.003; P=0.000); pPRP高濃度組和pPRP中濃度組的細胞活力均高于pPRP低濃度組(P=0.028; P=0.008); pPRP高濃度組的細胞活力高于pPRP中濃度組(P=0.002)。培養(yǎng)48 h后,pPRP低濃度組、pPRP中濃度組、pPRP高濃度組細胞活力均高于生理鹽水組(P=0.002; P=0.008; P=0.006); pPRP高濃度組和pPRP中濃度組的細胞活力均高于pPRP低濃度組(P=0.033; P=0.027); pPRP高濃度組的細胞活力高于pPRP中濃度組(P=0.002)。培養(yǎng)72 h后,pPRP低濃度組、pPRP中濃度組、pPRP高濃度組細胞活力均高于生理鹽水組(P=0.000; P=0.000; P=0.000); pPRP高濃度組和pPRP中濃度組的細胞活力均高于pPRP低濃度組(P=0.016; P=0.033); pPRP高濃度組的細胞活力高于pPRP中濃度組(P=0.029)。②步態(tài)分析結(jié)果。3組大鼠跑步過程中單位時間內(nèi)(1 s)左后肢和右后肢著地面積比較,組間差異均有統(tǒng)計學(xué)意義[(2.36±0.49)cm2,(1.68±0.18)cm2,(1.98±0.26)cm2,F=10.320,P=0.005;(2.82±0.59)cm2,(1.91±0.29)cm2,(2.41±0.31)cm2,F=11.790,P=0.002]。空白組、pPRP治療組大鼠左后肢著地面積均大于KOA模型組(P=0.002; P=0.008); 空白組、pPRP治療組大鼠右后肢著地面積均大于KOA模型組(P=0.001; P=0.002); 空白組與pPRP治療組大鼠左后肢及右后肢著地面積比較,組間差異均無統(tǒng)計學(xué)意義(P=0.050; P=0.068)。③X線檢查結(jié)果。X線片顯示空白組大鼠膝關(guān)節(jié)軟骨完整,關(guān)節(jié)面平整; KOA模型組關(guān)節(jié)軟骨明顯退變,脛骨平臺與股骨遠端關(guān)節(jié)面均不平整,有骨贅樣組織形成,并有軟骨缺損跡象; 與KOA模型組相比,pPRP治療組大鼠膝關(guān)節(jié)軟骨退變程度較輕微。④病理學(xué)檢查結(jié)果。與空白組相比,KOA模型組大鼠膝關(guān)節(jié)軟骨面明顯缺損,缺損處軟骨細胞丟失,軟骨細胞排列紊亂,軟骨下骨硬化,骨小梁面積明顯減小,骨髓腔細胞增生、排列紊亂,骨小梁中的骨細胞明顯減少; pPRP治療組大鼠膝關(guān)節(jié)軟骨面基本光滑,表面未見明顯缺損或裂隙,部分區(qū)域內(nèi)可見軟骨細胞丟失,軟骨下骨骨小梁排列尚整齊,骨小梁稀疏變窄,部分斷裂。結(jié)論:pPRP可促進大鼠軟骨細胞增殖; 高濃度的pPRP可以在一定程度上修復(fù)KOA大鼠退變的關(guān)節(jié)軟骨,改善運動能力。
Abstract:
Objective:To observe the effect of purified platelet rich plasma(pPRP)on chondrocyte proliferation in rats and cartilage repair in rats with knee osteoarthritis(KOA).Methods:Five SD rats were selected and their blood was drawn from aorta abdominalis.The pPRP with different concentration(1×106/mL,1×107/mL and 1×108/mL)were obtained after repeated centrifugation.Then the five SD rats were executed,and their knee articular cartilages were fetched out for separating chondrocytes.The third-generation chondrocytes of SD rats cultured in vitro were divided into normal saline group,pPRP low-concentration group,pPRP middle-concentration group and pPRP high-concentration group,5 holes in each group.The chondrocytes in each group were cultured in serum-free IMDM medium,and then the serum-free IMDM medium was replaced by 10%FBS-containing medium in normal saline group and medium containing 10%FBS and pPRP with low,middle and high concentration in pPRP low-concentration group,pPRP middle-concentration group and pPRP high-concentration group respectively.The cell proliferation were measured by using CCK-8 method when the chondrocytes were cultured for 24,48 and 72 hours respectively.Another 30 SD rats were selected and were randomly divided into blank group,KOA model group and pPRP treatment group,10 cases in each group.The KOA models were created in KOA model group and pPRP treatment group by intra-articular injecting iodoacetic acid into bilateral knees.One week later,the rats in pPRP treatment group were administrated with intra-articular injection of high-concentration pPRP with dose of 50 μL in bilateral knee joint,while the rats in the other two groups were administrated with intra-articular injection with the same dose of normal saline,once a week for consecutive 4 weeks.After the end of drug intervention,gait analysis,X-ray examination and pathological observation of knee joint were performed on all of the rats.Results:There was statistical difference in chondrocytes activity between the 4 groups after 24-,48- and 72-hour culture respectively(0.411+/-0.014,0.458+/-0.052,0.473+/-0.029,0.489+/-0.011,F=5.860,P=0.007; 0.502+/-0.003,0.551+/-0.022,0.568+/-0.019,0.572+/-0.029,F=12.196,P=0.000; 0.619+/-0.008,0.747+/-0.006,0.754+/-0.031,0.763+/-0.018,F=67.065,P=0.000).After 24-hour culture,the chondrocytes activity were higher in pPRP low-,middle- and high-concentration group compared to normal saline group(P=0.026; P=0.003; P=0.000),and were higher in pPRP high- and middle-concentration group compared to pPRP low-concentration group(P=0.028; P=0.008),and were higher in pPRP high-concentration group compared to pPRP middle-concentration group(P=0.002).After 48-hour culture,the chondrocytes activity were higher in pPRP low-,middle- and high-concentration group compared to normal saline group(P=0.002; P=0.008; P=0.006),and were higher in pPRP high- and middle-concentration group compared to pPRP low-concentration group(P=0.033; P=0.027),and were higher in pPRP high-concentration group compared to pPRP middle-concentration group(P=0.002).After 72-hour culture,the chondrocytes activity were higher in pPRP low-,middle- and high-concentration group compared to normal saline group(P=0.000; P=0.000; P=0.000),and were higher in pPRP high- and middle-concentration group compared to pPRP low-concentration group(P=0.016; P=0.033),and were higher in pPRP high-concentration group compared to pPRP middle-concentration group(P=0.029).The results of gait analysis showed that there was statistical difference in the touching ground areas of left and right hindlimbs within unit time during running between the 3 groups(2.36+/-0.49,1.68+/-0.18,1.98+/-0.26 cm(2),F=10.320,P=0.005; 2.82+/-0.59,1.91+/-0.29,2.41+/-0.31 cm(2),F=11.790,P=0.002).The touching ground areas of left hindlimbs were larger in blank group and pPRP treatment group compared to KOA model group(P=0.002; P=0.008).The touching ground areas of right hindlimbs were larger in blank group and pPRP treatment group compared to KOA model group(P=0.001; P=0.002).There was no statistical difference in the touching ground areas of left and right hindlimbs between blank group and pPRP treatment group(P=0.050; P=0.068).The X-ray films showed that the knee articular cartilages were complete and the knee articular surfaces were smooth in rats of blank group; while obvious knee articular cartilage degeneration,rough tibial plateau and distal femoral articular surfaces,osteophytes and articular cartilage defects were found in rats of KOA model group.The degrees of knee articular cartilage degeneration were slighter in pPRP treatment group compared to KOA model group.The results of pathological examination showed,obvious knee articular cartilage surface defects,chondrocytes loss at defect site,disorganized chondrocytes,sclerosis of subchondral bone,significant decrease in trabecular bone areas,disorganized hyperplastic cells in marrow cavity and significant decrease in number of trabecular bone cells in rats of KOA model group.The knee articular cartilage surfaces were basically smooth and no obvious defects and gaps were found in knee articular cartilage surfaces in rats of pPRP treatment group.Chondrocytes loss were found in some regions and sparse and narrow bone trabeculas arranged in order in subchondral bone.Conclusion:The pPRP can effectively promote the proliferation of chondrocytes in rats,and high-concentration pPRP can repair the degenerative articular cartilages and improve exercise abilities of rats with KOA to some extent.

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備注/Memo

備注/Memo:
基金項目:國家自然科學(xué)基金青年項目(81302989); 浙江省重點科技創(chuàng)新團隊自主項目(2011R50022-02); 高校博士點基金青年教師項目(20133322120006); 浙江省中醫(yī)藥科技計劃項目(2013ZQ007)
通訊作者:單樂天 E-mail:[email protected]
更新日期/Last Update: 2016-12-30