CAJ | 학술논문

目的观察转染骨形态发生蛋白7(BMP7)基因的骨髓间充质细胞(BMSCs)所表达的BMP7蛋白在体外诱导该细胞向软骨细胞方向分化的作用.方法分别取诱导液诱导的BMSCs(诱导组)、转染BMP7基因的BMSCs(转染组)以及未处理的BMSCs(对照组),在6孔板中细胞爬片,经HE、阿尔新蓝染色后,在倒置显微镜下观察形态.3组BMSCs培养7、14、21 d后,以半乳糖醛酸为对照品绘制标准曲线法测定培养液中糖胺多糖(GAG),ELISA法测定胶原蛋白Ⅱ.结果 HE、阿尔新蓝染色后,转染组和诱导组BMSCs分别呈红色和蓝色,具有软骨细胞特性.BMP7诱导后的细胞团表达了GAG和胶原蛋白Ⅱ.第21天,转染组GAG为(39.5±5.4)mg/L,胶原蛋白Ⅱ为(152.8±14.5)μg/L;诱导组GAG为(40.8±6.1)mg/L,胶原蛋白Ⅱ为(155.5±19.3)μg/L;对照组GAG为(17.1±3.4)mg/L,胶原蛋白Ⅱ为(89.7±14.3)μg/L;与对照组比较,转染组与诱导组GAG、胶原蛋白Ⅱ水平明显增高(P均<0.05),转染组与诱导组比较,则未见明显改变(P>0.05).结论转染BMP7基因的BMSCs表达的活性BMP7能够诱导BMSCs向软骨细胞方向分化,且其诱导水平达到了诱导液的诱导水平.
목적 관찰전염골형태발생단백7(BMP7)기인적골수간충질세포(BMSCs)소표체적BMP7단백재체외유도해세포향연골세포방향분화적작용.방법 분별취유도액유도적BMSCs(유도조)、전염BMP7기인적BMSCs(전염조)이급미처리적BMSCs(대조조),재6공판중세포파편,경HE、아이신람염색후,재도치현미경하관찰형태.3조BMSCs배양7、14、21 d후,이반유당철산위대조품회제표준곡선법측정배양액중당알다당(GAG),ELISA법측정효원단백Ⅱ.결과 HE、아이신람염색후,전염조화유도조BMSCs분별정홍색화람색,구유연골세포특성.BMP7유도후적세포단표체료GAG화효원단백Ⅱ.제21천,전염조GAG위(39.5±5.4)mg/L,효원단백Ⅱ위(152.8±14.5)μg/L;유도조GAG위(40.8±6.1)mg/L,효원단백Ⅱ위(155.5±19.3)μg/L;대조조GAG위(17.1±3.4)mg/L,효원단백Ⅱ위(89.7±14.3)μg/L;여대조조비교,전염조여유도조GAG、효원단백Ⅱ수평명현증고(P균<0.05),전염조여유도조비교,칙미견명현개변(P>0.05).결론 전염BMP7기인적BMSCs표체적활성BMP7능구유도BMSCs향연골세포방향분화,차기유도수평체도료유도액적유도수평.
Objective To examine the induction effects of bone marrow mesenchymal stem cells(BMSCs) transfected with bone morphogenetic protein 7 (BMP7) gene differentiating into chondrocytes. Methods We observed the phenotype of cells which were stained with alcian blue and HE climbing to the six pore plate with invert microscope. The glycosaminoglycan (GAG) value in culture medium was detected in control group,BMP7 transfect and culture medium induced groups after 7,14 and 21 days using standard curve method. Standard curve was described using galacturonic-acid as reference substance. The content of collagen Ⅱ was detected by ELISA method. Results HE and Alcian blue staining showed that BMP7 gene transfection group and the group induced by fluid possess the characteristics of chondrocyte. BMP7 induced BMSCs differentiation to chondrocyte which secrete specific protein called collagen Ⅱ and GAG. Content of GAG were (17.1±3.4),(39.5±5.4),(40.8±6.1)mg/L in control group,BMP7 gene transfected group and induced group,collagen Ⅱ were (89.7±14.3),(152.8±14.5),(155.5± 19.3)μg/L in these three groups separately. Comparing with control group,GAG and collagen Ⅱ of BMP7 gene transfected group and culture medium induced group increased obviously(all P < 0.05),but there was no significant difference between BMP7 gene transfeeted group and culture medium induced group (P > 0.05). Conclusion This active protein induces BMSCs differentiating into chondrocyte,in a level similar to that of inducing medium.