[1]原晓强,金王东,周云婧,等.纯化血小板对大鼠软骨细胞增殖及膝骨关节炎大鼠软骨修复的作用研究[J].中医正骨,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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纯化血小板对大鼠软骨细胞增殖及膝骨关节炎大鼠软骨修复的作用研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第28卷
期数:
2016年12期
页码:
6-12
栏目:
基础研究
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:
原晓强1; 金王东1; 周云婧1; 叶希佳1; 郭佳娜1; 单乐天1; 童培建2; 肖鲁伟1
1.浙江中医药大学,浙江 杭州 310053;
2.浙江省中医院,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
关键词:
骨关节炎;  膝关节;  软骨细胞;  富血小板血浆;  细胞增殖;  大鼠;  动物实验
Keywords:
osteoarthritis;  knee joint;  chondrocytes;  platelet rich plasma;  cell proliferation;  rats;  animal experimentation
摘要:
目的:观察纯化血小板(purified platelet rich plasma,pPRP)对大鼠软骨细胞增殖及膝骨关节炎(knee osteoarthritis,KOA)大鼠软骨修复的作用。方法:取5只SD大鼠抽取腹主动脉血,经多次离心后获得pPRP,并制成低(1×106个·mL-1)、中(1×107个·mL-1)、高(1×108个·mL-1)3种浓度的pPRP。5只SD大鼠抽取腹主动脉血后脱颈处死,取膝关节软骨分离软骨细胞。将培养的第3代软骨细胞分为生理盐水组、pPRP低浓度组、pPRP中浓度组和pPRP高浓度组,每组设5个复孔。各组细胞以无血清IMDM培养基处理后,生理盐水组更换为含10%FBS的培养基,pPRP低浓度组、pPRP中浓度组和pPRP高浓度组分别更换为含10%FBS和低、中、高浓度pPRP的培养基。分别于培养24、48、72 h后采用CCK-8法测定细胞增殖情况。另外选取30只SD大鼠随机分为空白组、KOA模型组和pPRP治疗组,每组10只。KOA模型组、pPRP治疗组大鼠通过向双侧膝关节腔内注射碘乙酸进行KOA 造模,空白组不进行造模。1周后,向pPRP治疗组大鼠双侧后肢膝关节腔各注射50 μL高浓度pPRP,向空白组和KOA模型组大鼠膝关节腔注射等量生理盐水,每周1次,共注射4次。药物干预结束后分别对各组大鼠进行步态分析、膝关节X线检查及膝关节病理学观察。结果:①软骨细胞增殖测定结果。培养24、48、72 h后4组软骨细胞活力比较,组间差异均有统计学意义(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)。培养24 h后,pPRP低浓度组、pPRP中浓度组、pPRP高浓度组细胞活力均高于生理盐水组(P=0.026; P=0.003; P=0.000); pPRP高浓度组和pPRP中浓度组的细胞活力均高于pPRP低浓度组(P=0.028; P=0.008); pPRP高浓度组的细胞活力高于pPRP中浓度组(P=0.002)。培养48 h后,pPRP低浓度组、pPRP中浓度组、pPRP高浓度组细胞活力均高于生理盐水组(P=0.002; P=0.008; P=0.006); pPRP高浓度组和pPRP中浓度组的细胞活力均高于pPRP低浓度组(P=0.033; P=0.027); pPRP高浓度组的细胞活力高于pPRP中浓度组(P=0.002)。培养72 h后,pPRP低浓度组、pPRP中浓度组、pPRP高浓度组细胞活力均高于生理盐水组(P=0.000; P=0.000; P=0.000); pPRP高浓度组和pPRP中浓度组的细胞活力均高于pPRP低浓度组(P=0.016; P=0.033); pPRP高浓度组的细胞活力高于pPRP中浓度组(P=0.029)。②步态分析结果。3组大鼠跑步过程中单位时间内(1 s)左后肢和右后肢着地面积比较,组间差异均有统计学意义[(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]。空白组、pPRP治疗组大鼠左后肢着地面积均大于KOA模型组(P=0.002; P=0.008); 空白组、pPRP治疗组大鼠右后肢着地面积均大于KOA模型组(P=0.001; P=0.002); 空白组与pPRP治疗组大鼠左后肢及右后肢着地面积比较,组间差异均无统计学意义(P=0.050; P=0.068)。③X线检查结果。X线片显示空白组大鼠膝关节软骨完整,关节面平整; KOA模型组关节软骨明显退变,胫骨平台与股骨远端关节面均不平整,有骨赘样组织形成,并有软骨缺损迹象; 与KOA模型组相比,pPRP治疗组大鼠膝关节软骨退变程度较轻微。④病理学检查结果。与空白组相比,KOA模型组大鼠膝关节软骨面明显缺损,缺损处软骨细胞丢失,软骨细胞排列紊乱,软骨下骨硬化,骨小梁面积明显减小,骨髓腔细胞增生、排列紊乱,骨小梁中的骨细胞明显减少; pPRP治疗组大鼠膝关节软骨面基本光滑,表面未见明显缺损或裂隙,部分区域内可见软骨细胞丢失,软骨下骨骨小梁排列尚整齐,骨小梁稀疏变窄,部分断裂。结论:pPRP可促进大鼠软骨细胞增殖; 高浓度的pPRP可以在一定程度上修复KOA大鼠退变的关节软骨,改善运动能力。
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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备注/Memo

备注/Memo:
基金项目:国家自然科学基金青年项目(81302989); 浙江省重点科技创新团队自主项目(2011R50022-02); 高校博士点基金青年教师项目(20133322120006); 浙江省中医药科技计划项目(2013ZQ007)
通讯作者:单乐天 E-mail:letian.shan@foxmail.com
更新日期/Last Update: 2016-12-30