CAJ | 학술논문

目的：重组慢病毒介导TGF-β3/BMP-2联合基因转染兔骨髓间充质干细胞，诱导其软骨细胞分化，与TGF-β3单基因转染诱导其软骨细胞分化的效率作比较。方法全骨髓贴壁法分离、培养骨髓间充质干细胞，流式细胞术检测细胞表面抗原，培养后通过重组慢病毒分别转染阴性对照（N组）、TGF-β3单基因（T组）、TGF-β3/BMP-2联合基因（TB组），培养14 d后，荧光显微镜下观察各组细胞形态，RT-PCR、Western blot分别检测SOX-9、蛋白多糖、Ⅱ型胶原在基因水平与蛋白水平的表达量。结果流式细胞术检测造血干细胞标志物（CD34，2.07%）、白细胞表面抗原（HLA-DR，1.73%）均为阴性，透明质酸受体（CD44，82.79%）为阳性，荧光显微镜观察诱导后细胞形态多为多角形、方形或者类圆盘状，RT-PCR、Western blot结果均示T组、TB组SOX-9、蛋白多糖、Ⅱ型胶原表达量均比空白对照组（C组）、N组高（P＜0.05），且TB组表达量高于T组（P＜0.01）。结论 TGF-β3/BMP-2联合基因转染骨髓间充质干细胞能诱导其成软骨细胞分化，且联合基因转染效率高于TGF-β3单基因转染。
목적：중조만병독개도TGF-β3/BMP-2연합기인전염토골수간충질간세포，유도기연골세포분화，여TGF-β3단기인전염유도기연골세포분화적효솔작비교。방법전골수첩벽법분리、배양골수간충질간세포，류식세포술검측세포표면항원，배양후통과중조만병독분별전염음성대조（N조）、TGF-β3단기인（T조）、TGF-β3/BMP-2연합기인（TB조），배양14 d후，형광현미경하관찰각조세포형태，RT-PCR、Western blot분별검측SOX-9、단백다당、Ⅱ형효원재기인수평여단백수평적표체량。결과류식세포술검측조혈간세포표지물（CD34，2.07%）、백세포표면항원（HLA-DR，1.73%）균위음성，투명질산수체（CD44，82.79%）위양성，형광현미경관찰유도후세포형태다위다각형、방형혹자류원반상，RT-PCR、Western blot결과균시T조、TB조SOX-9、단백다당、Ⅱ형효원표체량균비공백대조조（C조）、N조고（P＜0.05），차TB조표체량고우T조（P＜0.01）。결론 TGF-β3/BMP-2연합기인전염골수간충질간세포능유도기성연골세포분화，차연합기인전염효솔고우TGF-β3단기인전염。
Objective Combination gene transfection of TGF-β3/BMP-2 mediated by recombinant lentivirus induce chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells(BMSCs), and compared with TGF-β3 single gene transfecting. Methods BMSCs were isolated and cultured using a whole bone marrow adherent method, then detected cells’ surface antigen, respectively transferred negative control (N group), TGF-β3 single gene (T group), TGF-β3/BMP-2 combination gene (TB group), observed cell morphology of each group, the expression of SOX-9, type Ⅱ collagen (COL2), aggrecan (ACAN). Results The BMSCs revealed uniformly negative for the early hematopoietic stem cell marker (CD34, 2.07%) and the leucocytes surface antigen (HLA-DR, 1.73%), positively for the hyaluronan receptor(CD44, 82.79%). The differentiated cells took a shape of polygonal, square or disc-like, the expression of chondrogenic related markers such as sox-9, COL2 and ACAN were upregulated in T group and TB group than in C group and N group (P<0.05), furthermore, those markers were synthetized significantly more in TB group than in T group (P<0.01). Conclusion These findings indicated that BMSCs differentiated into chondrocytes induced by TGF-β3/BMP-2 combination gene, this method was superior to TGF-β3 single gene transfection.