富血小板血浆对硝普钠诱导的软骨细胞Wnt/β-catenin信号通路的影响

Effect of platelet-rich plasma on Wnt/β-catenin signaling pathway in chondrocytes induced by nitroprusside

目的:观察富血小板血浆(platelet-rich plasma,PRP)对硝普钠诱导的软骨细胞Wnt/β-catenin信号通路的影响。方法:取5月龄新西兰大白兔耳静脉血制成富血小板血浆。取兔双膝关节软骨,分离、培养软骨细胞。取培养的第2代软骨细胞,分为空白对照组、硝普钠对照组、PRP组和Wnt蛋白生成抑制剂2(the inhibitor of Wnt production-2,IWP-2)组,空白对照组与硝普钠对照组加入生理盐水、PRP组加入PRP、IWP-2组加入IWP-2干预1 h后,除空白对照组外,其余3组再加入硝普钠干预24 h。采用实时荧光定量PCR法检测各组软骨细胞Wnt1、β-catenin和糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)mRNA的表达情况,采用Western-blot法检测各组软骨细胞Wnt1、β-catenin和GSK-3β蛋白表达情况。结果:经甲苯胺蓝染色及Ⅱ型胶原免疫组化染色,鉴定所培养细胞为软骨细胞。干预后,4组软骨细胞Wnt1、β-catenin和GSK-3βmRNA相对表达量组间总体比较,差异均有统计学意义(0.429±0.015,0.968±0.058,0.693±0.019,0.228±0.012,F=4.673,P=0.013;0.434±0.021,0.948±0.067,0.702±0.024,0.191±0.011,F=3.918,P=0.008;0.816±0.039,0.225±0.017,0.459±0.018,0.929±0.118,F=3.015,P=0.005)。PRP组软骨细胞Wnt1、β-catenin mRNA相对表达量均较空白对照组和IWP-2组高(P=0.036,P=0.000;P=0.026,P=0.000),但均较硝普钠对照组低(P=0.017;P=0.013)。PRP组软骨细胞GSK-3βmRNA相对表达量较空白对照组和IWP-2组低(P=0.001,P=0.000),但较硝普钠对照组高(P=0.016)。干预后,4组软骨细胞Wnt1、β-catenin和GSK-3β蛋白相对表达量组间总体比较,差异均有统计学意义(0.565±0.017,0.941±0.134,0.725±0.031,0.251±0.013,F=5.062,P=0.017;0.526±0.024,0.872±0.016,0.653±0.016,0.262±0.018,F=3.592,P=0.006;0.753±0.014,0.262±0.015,0.579±0.024,0.823±0.025,F=2.384,P=0.002)。PRP组软骨细胞Wnt1和β-catenin蛋白相对表达量较空白对照组和IWP-2组高(P=0.031,P=0.000;P=0.041,P=0.002),但低于硝普钠对照组(P=0.032,P=0.025);PRP组软骨细胞中GSK-3β蛋白相对表达量较空白对照组和IWP2组低(P=0.035,P=0.011),但高于硝普钠对照组(P=0.010)。结论:PRP可抑制兔膝关节软骨细胞中Wnt1、β-catenin的表达,促进GSK-3β的表达。PRP可抑制硝普钠诱导的软骨细胞Wnt/β-catenin通路活性;但与Wnt蛋白生成抑制剂–2相比,PRP对软骨细胞Wnt/β-catenin通路活性的抑制作用较弱。

Objective： To observe the effect of platelet - rich plasma （ PRP） on Wnt /β- catenin signaling pathway in chondrocytes in-duced by nitroprusside. Methods ： Five - month - old New Zealand rabbits were selected and their blood was drawn from auricular veins for making PRP. Then the rabbits were executed and their articular cartilages were fetched out from both knees for separating and culturing chondrocytes. The second - generation chondrocytes of New Zealand rabbits cultured in vitro were divided into blank control group, nitro- prusside control group,PRP group and the inhibitor of Wnt production -2（ IWP - 2） group. The chondrocytes in blank control group,nitro- prusside control group, PRP group and IWP - 2 group were intervened with normal salin （ NS ） , NS, PRP and IWP - 2 respectively for 1 hour,moreover,the chondrocytes in nitroprusside control group, PRP group and IWP -2 group were intervened with nitroprusside for 24 hours. After intervention, the mRNA and protein expression levels of Wntl, β- catenin and glycogen synthase kinase - 3 p （ GSK - 3 p ） in chondrocytes of each group were detected by using Real - time fluorescent quantitative PCR and Western - blot assays respectively. Results ： The cultured cells were identified as chondrocytes through toluidine blue staining and type II collagen immunohistochemical stai-ning. After intervention,there was statistical difference in relative expression levels of Wntl mRNA, β- catenin mRNA and GSK - 3p mR-NA in chondrocytes between the 4 groups in general（0.429 ＋/- 0. 015,0. 968 ＋/-0. 058 ,0. 693 ＋/-0. 019,0. 228 ＋/- 0. 012 = 4. 673 ,P=0.013; 0.434 V-0.021,0.948 ＋/-0. 067,0. 702 ＋/-0. 024,0.191 ＋/-0. Oi l ,F = 3. 918,P =0.008; 0. 816 ＋ / -0 . 039,0. 225 ＋ / - 0. 017,0. 459 ＋/-0. 018,0. 929 ＋/- 0. 118,F = 3. 015,P =0. 005） . The relative expression levels of Wntl mRNA and β- catenin mRNA in chondrocytes were higher in PRP group compared to blank control group and IWP -2 group（P =0. 036,P =0. 000;P =0. 026,P = 0.000） and were lower in PRP group compared to nitroprusside control group （P =0. 017 ;P =0. 013）. The relative expression level of GSK - 3p mRNA in chondrocytes was lower in PRP group compared to blank control group and IWP - 2 group（P = 0. 001 ,P = 0. 000） and was higher in PRP group compared to nitroprusside control group （ P = 0. 016）. After intervention, there was statistical difference in the rela-tive expression levels of Wntl protein, β- catenin protein and GSK - 3p protein in chondrocytes between the 4 groups in general （0.565 ＋/-0.017,0.941 ＋/-0.134,0.725 ＋/-0. 031,0. 251 ＋/-0. 013, F = 5. 062,P = 0. 017; 0. 526 ＋ / -0 .0 2 4 ,0 . 872 ＋ / -0 .0 1 6 , 0.653 ＋/-0.016,0. 262 ＋/-0.018 =3. 592,P =0.006; 0. 753 ＋ / -0 . 014,0. 262 ＋ / -0 . 015,0. 579 ＋ / -0 . 024,0. 823 ＋ / -0 . 02 5,F =2. 384 = 0. 002）. The relative expression levels of Wntl protein and β- catenin protein in chondrocytes were higher in PRP group com-pared to blank control group and IWP -2 group（P =0. 031 ,P =0. 000;P =0. 041 ,P =0. 002）and were lower in PRP group compared to nitroprusside control group（P =0. 032,P =0.025）. The relative expression level of GSK -3p protein in chondrocytes was lower in PRP group compared to blank control group and IWP -2 group（P =0. 035,P =0.011）and was higher in PRP group compared to nitroprusside control group（P = 0. 010） . Conclusion：PRP can inhibit the expression of Wntl and p - catenin and promote the expression of GSK - 3p in chondrocytes of rabbit knees. Meanwhile, PRP can inhibit the activity of Wnt/β- catenin pathway in chondrocytes induced by nitroprus- side. However,PRP is inferior to IWP -2 in inhibiting the activity of Wnt/β- catenin pathway in chondrocytes.