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[1]凌曉宇,周康,姚黎佳,等.血小板裂解液對膝骨關(guān)節(jié)炎模型大鼠疼痛和軟骨損傷的影響及作用機制研究[J].中醫(yī)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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血小板裂解液對膝骨關(guān)節(jié)炎模型大鼠疼痛和軟骨損傷的影響及作用機制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期數(shù):
2017年11期
頁碼:
8-14
欄目:
基礎(chǔ)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:
凌曉宇1; 周康1; 姚黎佳1; 嚴莉1; 俞光平2; 童培建1; 單樂天1; 肖魯偉1
1.浙江中醫(yī)藥大學(xué),浙江 杭州 310053; 2.浙江省仙居縣中醫(yī)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
關(guān)鍵詞:
骨關(guān)節(jié)炎; 膝;  血小板裂解液;  疼痛;  軟骨;  細胞增殖;  大鼠;  動物實驗
Keywords:
Key words osteoarthritis; knee;  platelet lysate;  pain;  cartilage;  cell proliferation;  rats;  animal experimentation
摘要:
目的:探討血小板裂解液(platelet lysate,PL)對膝骨關(guān)節(jié)炎(knee osteoarthritis,KOA)模型大鼠疼痛和軟骨損傷的影響及可能的作用機制。方法:取4只SD大鼠從其外周血中分離制備PL,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PL。取40只SD大鼠,隨機分為空白組、模型組、PL低濃度組、PL中濃度組、PL高濃度組,每組8只。空白組不進行造模處理,其余4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。造模后第1天開始進行藥物干預(yù),PL低、中、高濃度組大鼠雙側(cè)膝關(guān)節(jié)腔分別注射50 μL低、中、高濃度PL,空白組和模型組大鼠雙側(cè)膝關(guān)節(jié)腔分別注射等量生理鹽水,每周1次,共注射4次。分別于造模結(jié)束后第2周和第4周測定各組大鼠的壓痛閾值和熱痛閾值。痛閾測定結(jié)束后處死大鼠,取雙側(cè)膝關(guān)節(jié)進行組織病理學(xué)觀察并評定Mankin's評分。另取5只SD大鼠,從大鼠關(guān)節(jié)軟骨中分離獲取軟骨細胞進行體外培養(yǎng),將培養(yǎng)的第3代軟骨細胞分為空白組、模型組、PL低濃度組、PL中濃度組和PL高濃度組,空白組以含10%FBS的培養(yǎng)基進行培養(yǎng),模型組以含10%FBS和碘乙酸的培養(yǎng)基進行培養(yǎng),PL低、中、高濃度組分別以含10%FBS和低、中、高濃度PL的培養(yǎng)基進行培養(yǎng),以CCK-8法測定細胞增殖情況。結(jié)果:①壓痛閾值測定結(jié)果。造模結(jié)束后第2周時,5組大鼠的壓痛閾值比較,差異有統(tǒng)計學(xué)意義[(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]。模型組的壓痛閾值低于空白組、PL低濃度組、PL中濃度組和PL高濃度組(P=0.001,P=0.045,P=0.002,P=0.000); PL高濃度組的壓痛閾值高于PL低濃度組和PL中濃度組(P=0.000,P=0.001); PL中濃度組的壓痛閾值高于PL低濃度組(P=0.001)。造模結(jié)束后第4周時,5組大鼠的壓痛閾值比較,差異有統(tǒng)計學(xué)意義[(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]。模型組的壓痛閾值低于空白組、PL低濃度組、PL中濃度組和PL高濃度組(P=0.000,P=0.018,P=0.003,P=0.000); PL高濃度組的壓痛閾值高于PL低濃度組和PL中濃度組(P=0.000,P=0.002); PL中濃度組的壓痛閾值高于PL低濃度組(P=0.002)。②熱痛閾值測定結(jié)果。造模結(jié)束后第2周時,5組大鼠的熱痛閾值比較,差異有統(tǒng)計學(xué)意義[(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]。模型組的熱痛閾值低于空白組、PL中濃度組和PL高濃度組(P=0.013,P=0.014,P=0.007); 模型組與PL低濃度組熱痛閾值比較,差異無統(tǒng)計學(xué)意義(P=0.118); PL高濃度組的熱痛閾值高于PL低濃度組和PL中濃度組(P=0.000,P=0.024); PL中濃度組的熱痛閾值高于PL低濃度組(P=0.002)。造模結(jié)束后第4周時,5組大鼠的熱痛閾值比較,差異有統(tǒng)計學(xué)意義[(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]。模型組的熱痛閾值低于空白組、PL中濃度組和PL高濃度組(P=0.005,P=0.009,P=0.002); 模型組與PL低濃度組熱痛閾值比較,差異無統(tǒng)計學(xué)意義(P=0.634); PL高濃度組的熱痛閾值高于PL低濃度組和PL中濃度組(P=0.000,P=0.019); PL中濃度組的熱痛閾值高于PL低濃度組(P=0.025)。③膝關(guān)節(jié)軟骨病理學(xué)觀察結(jié)果。5組大鼠膝關(guān)節(jié)軟骨Mankin's評分比較,差異有統(tǒng)計學(xué)意義[(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]。模型組的Mankin's評分高于空白組、PL低濃度組、PL中濃度組、PL高濃度組(P=0.000,P=0.000,P=0.000,P=0.000); PL高濃度組的Mankin's評分低于PL低濃度組(P=0.013); PL中濃度組的Mankin's評分與PL高濃度組、PL低濃度組比較,差異均無統(tǒng)計學(xué)意義(P=0.541,P=0.062)。④膝關(guān)節(jié)軟骨細胞增殖測定結(jié)果。5組軟骨細胞的吸光度比較,差異有統(tǒng)計學(xué)意義(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)。模型組的吸光度低于空白組、PL低濃度組、PL中濃度組和PL高濃度組(P=0.001,P=0.000,P=0.016,P=0.000); PL高濃度組的吸光度高于PL低濃度組(P=0.000); PL中濃度組的吸光度與PL高濃度組、PL低濃度組比較,差異均無統(tǒng)計學(xué)意義(P=0.639,P=0.204)。結(jié)論:PL可提高KOA模型大鼠疼痛閾值,修復(fù)軟骨損傷,且其作用效果與PL的劑量有關(guān),其作用機制可能與PL能促進軟骨細胞增殖有關(guān)。
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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備注/Memo

備注/Memo:
基金項目:國家衛(wèi)生和計劃生育委員會科學(xué)研究基金-浙江省醫(yī)藥衛(wèi)生重大科技計劃省部共建項目(201487674); 浙江省科技廳重大科技專項社會發(fā)展項目(2014C03035); 浙江省醫(yī)藥衛(wèi)生科技計劃項目(2016155990) 通訊作者:童培建 E-mail:[email protected]
更新日期/Last Update: 2018-04-02