[1]凌晓宇,周康,姚黎佳,等.血小板裂解液对膝骨关节炎模型大鼠疼痛和软骨损伤的影响及作用机制研究[J].中医正骨,2017,29(11):8-14.
　LING Xiaoyu,ZHOU Kang,YAO Lijia,et al.Effect of platelet lysate on pain and cartilage injury in knee osteoarthritis rat models and its mechanism of action:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(11):8-14.
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血小板裂解液对膝骨关节炎模型大鼠疼痛和软骨损伤的影响及作用机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期数:
2017年11期
页码:
8-14
栏目:
基础研究
出版日期:
2017-11-20

文章信息/Info

Title:
Effect of platelet lysate on pain and cartilage injury in knee osteoarthritis rat models and its mechanism of action:an experimental study
作者:
凌晓宇1; 周康1; 姚黎佳1; 严莉1; 俞光平2; 童培建1; 单乐天1; 肖鲁伟1
1.浙江中医药大学,浙江 杭州 310053; 2.浙江省仙居县中医院,浙江 仙居 317300
Author(s):
LING Xiaoyu1; ZHOU Kang1; YAO Lijia1; YAN Li1; YU Guangping2; TONG Peijian1; SHAN Letian1; XIAO Luwei1
1.Zhejiang University of Traditional Chinese Medicine,Hangzhou 310053,Zhejiang,China 2.Traditional Chinese Medical Hospital of Xianju county,Taizhou 317300,Zhejiang,China
关键词:
骨关节炎; 膝;  血小板裂解液;  疼痛;  软骨;  细胞增殖;  大鼠;  动物实验
Keywords:
Key words osteoarthritis; knee;  platelet lysate;  pain;  cartilage;  cell proliferation;  rats;  animal experimentation
摘要:
目的:探讨血小板裂解液(platelet lysate,PL)对膝骨关节炎(knee osteoarthritis,KOA)模型大鼠疼痛和软骨损伤的影响及可能的作用机制。方法:取4只SD大鼠从其外周血中分离制备PL,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PL。取40只SD大鼠,随机分为空白组、模型组、PL低浓度组、PL中浓度组、PL高浓度组,每组8只。空白组不进行造模处理,其余4组大鼠通过向双侧膝关节腔内注射碘乙酸进行KOA造模。造模后第1天开始进行药物干预,PL低、中、高浓度组大鼠双侧膝关节腔分别注射50 μL低、中、高浓度PL,空白组和模型组大鼠双侧膝关节腔分别注射等量生理盐水,每周1次,共注射4次。分别于造模结束后第2周和第4周测定各组大鼠的压痛阈值和热痛阈值。痛阈测定结束后处死大鼠,取双侧膝关节进行组织病理学观察并评定Mankin's评分。另取5只SD大鼠,从大鼠关节软骨中分离获取软骨细胞进行体外培养,将培养的第3代软骨细胞分为空白组、模型组、PL低浓度组、PL中浓度组和PL高浓度组,空白组以含10%FBS的培养基进行培养,模型组以含10%FBS和碘乙酸的培养基进行培养,PL低、中、高浓度组分别以含10%FBS和低、中、高浓度PL的培养基进行培养,以CCK-8法测定细胞增殖情况。结果:①压痛阈值测定结果。造模结束后第2周时,5组大鼠的压痛阈值比较,差异有统计学意义[(385.04±116.23)g,(179.23±74.75)g,(257.60±70.97)g,(306.79±56.91)g,(352.13±67.03)g,F=8.255,P=0.000]。模型组的压痛阈值低于空白组、PL低浓度组、PL中浓度组和PL高浓度组(P=0.001,P=0.045,P=0.002,P=0.000); PL高浓度组的压痛阈值高于PL低浓度组和PL中浓度组(P=0.000,P=0.001); PL中浓度组的压痛阈值高于PL低浓度组(P=0.001)。造模结束后第4周时,5组大鼠的压痛阈值比较,差异有统计学意义[(540.58±97.70)g,(352.81±54.41)g,(419.17±44.74)g,(460.43±63.73)g,(493.38±62.53)g,F=9.137,P=0.000]。模型组的压痛阈值低于空白组、PL低浓度组、PL中浓度组和PL高浓度组(P=0.000,P=0.018,P=0.003,P=0.000); PL高浓度组的压痛阈值高于PL低浓度组和PL中浓度组(P=0.000,P=0.002); PL中浓度组的压痛阈值高于PL低浓度组(P=0.002)。②热痛阈值测定结果。造模结束后第2周时,5组大鼠的热痛阈值比较,差异有统计学意义[(8.35±2.17)s,(5.90±1.67)s,(6.77±1.08)s,(7.48±0.91)s,(8.24±1.65)s,F=4.248,P=0.007]。模型组的热痛阈值低于空白组、PL中浓度组和PL高浓度组(P=0.013,P=0.014,P=0.007); 模型组与PL低浓度组热痛阈值比较,差异无统计学意义(P=0.118); PL高浓度组的热痛阈值高于PL低浓度组和PL中浓度组(P=0.000,P=0.024); PL中浓度组的热痛阈值高于PL低浓度组(P=0.002)。造模结束后第4周时,5组大鼠的热痛阈值比较,差异有统计学意义[(9.75±2.10)s,(6.78±1.46)s,(7.15±1.58)s,(7.91±1.35)s,(8.67±1.55)s,F=4.310,P=0.006]。模型组的热痛阈值低于空白组、PL中浓度组和PL高浓度组(P=0.005,P=0.009,P=0.002); 模型组与PL低浓度组热痛阈值比较,差异无统计学意义(P=0.634); PL高浓度组的热痛阈值高于PL低浓度组和PL中浓度组(P=0.000,P=0.019); PL中浓度组的热痛阈值高于PL低浓度组(P=0.025)。③膝关节软骨病理学观察结果。5组大鼠膝关节软骨Mankin's评分比较,差异有统计学意义[(1.63 ± 1.11)分,(9.29 ± 1.03)分,(5.14 ± 1.64)分,(3.14 ± 1.73)分,(2.57 ± 1.40)分,F=37.299,P=0.000]。模型组的Mankin's评分高于空白组、PL低浓度组、PL中浓度组、PL高浓度组(P=0.000,P=0.000,P=0.000,P=0.000); PL高浓度组的Mankin's评分低于PL低浓度组(P=0.013); PL中浓度组的Mankin's评分与PL高浓度组、PL低浓度组比较,差异均无统计学意义(P=0.541,P=0.062)。④膝关节软骨细胞增殖测定结果。5组软骨细胞的吸光度比较,差异有统计学意义(0.71 ± 0.06,0.46 ± 0.01,0.58 ± 0.02,0.66 ± 0.11,0.69 ± 0.01,F=25.644,P=0.000)。模型组的吸光度低于空白组、PL低浓度组、PL中浓度组和PL高浓度组(P=0.001,P=0.000,P=0.016,P=0.000); PL高浓度组的吸光度高于PL低浓度组(P=0.000); PL中浓度组的吸光度与PL高浓度组、PL低浓度组比较,差异均无统计学意义(P=0.639,P=0.204)。结论:PL可提高KOA模型大鼠疼痛阈值,修复软骨损伤,且其作用效果与PL的剂量有关,其作用机制可能与PL能促进软骨细胞增殖有关。
Abstract:
ABSTRACT Objective:To explore the effect of platelet lysate(PL)on pain and cartilage injury in knee osteoarthritis(KOA)rat models and its mechanism of action.Methods:Four SD rats were selected and PL was isolated from their peripheral blood.The PL with 3 different concentration(1×106/mL,1×107/mL and 1×108/mL)were obtained.Another 40 SD rats were selected and were randomly divided into blank group,model group,PL low-concentration group,PL middle-concentration group and PL high-concentration group,8 cases in each group.The KOA models were created in rats of model group,PL low-concentration group,PL middle-concentration group and PL high-concentration group by intra-articular injecting iodoacetic acid into bilateral knees,while the rats in blank group were not given any surgical intervention.At the 1st day after the end of modeling,drug intervention were performed on rats in PL low-concentration group,PL middle-concentration group and PL high-concentration group by injecting low-,middle- and high-concentration PL with dose of 50 μL into bilateral knees respectively,while the rats in the other two groups were administrated with intra-articular injection of the same dose of normal saline into bilateral knees respectively,once a week for consecutive 4 times.The pressure pain threshold(PPT)value and heat pain threshold value were detected in each group at 2 and 4 weeks after the end of modeling.After the end of pain threshold measurement,all rats were executed and their bilateral knee joints were fetched out for histopathological observation and the Mankin's scores were evaluated.Another five SD rats were selected and 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 blank group,model group,PL low-concentration group,PL middle-concentration group and PL high-concentration group.The chondrocytes in blank group and model group were cultured in medium containing 10%FBS and medium containing 10%FBS and iodoacetic acid respectively,and the chondrocytes in PL low-concentration group,PL middle-concentration group and PL high-concentration group were cultured in medium containing 10%FBS and PL with low,middle and high concentration respectively.The cell proliferation were measured by using CCK-8 method.Results:At the 2nd week after the end of modeling,there was statistical difference in PPT values between the 5 groups(385.04+/-116.23,179.23+/-74.75,257.60+/-70.97,306.79+/-56.91,352.13+/-67.03 g,F=8.255,P=0.000).The PPT value was lower in model group compared to blank group,PL low-concentration group,PL middle-concentration group and PL high-concentration group(P=0.001,P=0.045,P=0.002,P=0.000),and was higher in PL high-concentration group compared to PL low-concentration group and PL middle-concentration group(P=0.000,P=0.001),and was higher in PL middle-concentration group compared to PL low-concentration group(P=0.001).At the 4th week after the end of modeling,there was statistical difference in PPT values between the 5 groups(540.58+/-97.70,352.81+/-54.41,419.17+/-44.74,460.43+/-63.73,493.38+/-62.53 g,F=9.137,P=0.000).The PPT value was lower in model group compared to blank group,PL low-concentration group,PL middle-concentration group and PL high-concentration group(P=0.000,P=0.018,P=0.003,P=0.000),and was higher in PL high-concentration group compared to PL low-concentration group and PL middle-concentration group(P=0.000,P=0.002),and was higher in PL middle-concentration group compared to PL low-concentration group(P=0.002).At the 2nd week after the end of modeling,there was statistical difference in the heat pain threshold values between the 5 groups(8.35+/-2.17,5.90+/-1.67,6.77+/-1.08,7.48+/-0.91,8.24+/-1.65 s,F=4.248,P=0.007).The heat pain threshold value was lower in model group compared to blank group,PL middle-concentration group and PL high-concentration group(P=0.013,P=0.014,P=0.007).There was no statistical difference in the heat pain threshold value between model group and PL low-concentration group(P=0.118).The heat pain threshold value was higher in PL high-concentration group compared to PL low-concentration group and PL middle-concentration group(P=0.000,P=0.024),and was higher in PL middle-concentration group compared to PL low-concentration group(P=0.002).At the 4th week after the end of modeling,there was statistical difference in the heat pain threshold values between the 5 groups(9.75+/-2.10,6.78+/-1.46,7.15+/-1.58,7.91+/-1.35,8.67+/-1.55 s,F=4.310,P=0.006).The heat pain threshold value was lower in model group compared to blank group,PL middle-concentration group and PL high-concentration group(P=0.005,P=0.009,P=0.002).There was no statistical difference in the heat pain threshold value between model group and PL low-concentration group(P=0.634).The heat pain threshold value was higher in PL high-concentration group compared to PL low-concentration group and PL middle-concentration group(P=0.000,P=0.019),and was higher in PL middle-concentration group compared to PL low-concentration group(P=0.025).There was statistical difference in Mankin's scores of knee articular cartilage between the 5 groups(1.63+/-1.11,9.29+/-1.03,5.14+/-1.64,3.14+/-1.73,2.57+/-1.40 points,F=37.299,P=0.000).The Mankin's score was higher in model group compared to blank group,PL low-concentration group,PL middle-concentration group and PL high-concentration group(P=0.000,P=0.000,P=0.000,P=0.000),and was lower in PL high-concentration group compared to PL low-concentration group(P=0.013).There was no statistical difference in Mankin's score between PL middle-concentration group and PL high-concentration group and between PL middle-concentration group and PL low-concentration group(P=0.541,P=0.062).There was statistical difference in the absorbance of chondrocytes between the 5 groups(0.71+/-0.06,0.46+/-0.01,0.58+/-0.02,0.66+/-0.11,0.69+/-0.01,F=25.644,P=0.000).The absorbance was lower in model group compared to blank group,PL low-concentration group,PL middle-concentration group and PL high-concentration group(P=0.001,P=0.000,P=0.016,P=0.000),and was higher in PL high-concentration group compared to PL low-concentration group(P=0.000).There was no statistical difference in the absorbance between PL middle-concentration group and PL high-concentration group and between PL middle-concentration group and PL low-concentration group(P=0.639,P=0.204).Conclusion:PL can increase pain threshold value and repair injuried articular cartilage dose-dependently in KOA rat models.The mechanism of action may be related to the promotion of chondrocyte proliferation.

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备注/Memo

备注/Memo:
基金项目:国家卫生和计划生育委员会科学研究基金-浙江省医药卫生重大科技计划省部共建项目(201487674); 浙江省科技厅重大科技专项社会发展项目(2014C03035); 浙江省医药卫生科技计划项目(2016155990) 通讯作者:童培建 E-mail:tongpeijian@163.com
更新日期/Last Update: 2018-04-02