鹿瓜多肽联合hBMP-2基因对软骨细胞增殖和Ⅱ型胶原分泌的影响

Proliferation of articular chondrocytes and secretion of collagen type Ⅱ affected by cervus and cucumis polypeptide and BMP-2

目的观察鹿瓜多肽联合hBMP-2基因对软骨细胞的增殖和软骨细胞分泌Ⅱ型胶原是否有协同作用。方法分离培养原代兔软骨细胞,用第3代细胞为模型,实验分为未转染组、腺病毒-绿色荧光蛋白(Ad-GFP)组、Ad-hBMP2-GFP组、鹿瓜多肽+Ad-hBMP2-GFP组,培养48 h后,RT-PCR检测hBMP-2mRNA的表达,流式细胞仪检测软骨细胞的周期。培养48、72 h后,MTT法检测软骨细胞的增殖,W estern印迹检测hBMP-2蛋白和Ⅱ型胶原的表达。结果培养48 h后,Ad-hBMP2-GFP组和鹿瓜多肽+Ad-hBMP2-GFP组hBMP-2 mRNA呈阳性,而未转染组、Ad-GFP组表达呈阴性;与未转染组、Ad-GFP组、Ad-hBMP2-GFP组比较,鹿瓜多肽+hBMP-2基因组能使软骨细胞S-G2/M期细胞比例增加,软骨细胞数量增加(P<0.05),软骨细胞hBMP-2蛋白表达和Ⅱ型胶原分泌增加(P<0.01);培养48与72 h比较,鹿瓜多肽联合hBMP-2基因对软骨细胞增殖和Ⅱ型胶原分泌无显著性差异(P>0.05)。结论鹿瓜多肽能促进hBMP-2基因在软骨细胞中合成hBMP-2蛋白,鹿瓜多肽联合hBMP-2基因对软骨细胞增殖和分泌Ⅱ型胶原的功能有协同作用。

Objective To observe the effect of a combination of cervus and cucumis polypeptide and human bone morphogenetic protein（hBMP）-2 gene for the proliferation of articular chondrocytes and for the secretion of collagen Type Ⅱ.Methods Rabbit＇s articular chondrocytes were primary cultured and separated.The third generation cells were transfected with adenovirus-mediated hBmp-2-green fluorescent protein（Ad-hBMP2-GFP）.Non-transfected group and empty virus group were used as the controls.The groups of Ad-hBMP2-GFP transfection and Ad-hBMP2-GFP combining with cervus and cucumis polypeptide transfection were used as experiment group.hBMP-2 mRNA was determined by RT-PCR,chondrocytes proliferation was determined by MTT,the effect on chondrocytes cycle was detected by flow cytometry,BMP-2 and collagen type Ⅱ protein were detected by Western-blot.Results hBMP-2 mRNA was expressed in Ad-hBMP2-GFP group and Ad-hBMP2-GFP combining with cervus and cucumis polypeptide group,and it was not expressed in non-transfected and Ad-GFP group.The percentage of S-G2/M cells and the number of chondrocytes increased in experiment group（P0.05）.hBMP-2 expression and secretion of collagen type Ⅱ increased in experiment group compared with those of other groups（P0.01）.There were no significant difference in chondrocytes proliferation and collagen type Ⅱ secretion between 48 and 72 h in experiment group（P0.05）.Conclusions A combination of cervus and cucumis polypeptide and hBMP-2 promote coordinately proliferation and secretion of collagen type Ⅱ of chondrocytes.Cervus and cucumis polypeptide promotes hBMP-2 protein synthesis.The research of cervus and cucumis polypeptide combining with hBMP-2 provides experimental evidence for treatment of osteoarthritis.