Dharmavaram R M, Liu G, Tuan R S, Stokes D G, Jiménez S A
Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5541, USA.
Arthritis Rheum. 1999 Jul;42(7):1433-42. doi: 10.1002/1529-0131(199907)42:7<1433::AID-ANR17>3.0.CO;2-G.
To perform stable transfections of human chondrocytes under conditions that allow the maintenance of the chondrocyte-specific phenotype, and to examine the effects of the stable transfection of a mutated type II procollagen gene (COL2A1) on the structure of the cartilaginous extracellular matrix produced in vitro.
A type II procollagen minigene that lacks exons 16-27 was stably transfected into human fetal epiphyseal chondrocytes in vitro. Expression of the minigene was detected by reverse transcriptase-polymerase chain reaction, and the encoded protein was identified by Western blot with a human type II collagen-specific antibody. The cartilaginous extracellular matrix produced by the cultured transfected chondrocytes was characterized using histochemical staining, polarized light microscopy analysis, and transmission electron microscopy.
A shortened type II collagen encoded by the transfected minigene was biosynthesized and produced in the cultures of transfected cells. Histologic analyses demonstrated a more dense, negatively charged cartilaginous matrix in control cultures. In contrast, COL2A1 minigene-transfected cultures were more cellular, were populated with cells of irregular shape and less-chondrocytic appearance, contained abundant intracellular dense granules, and were surrounded by a less-dense matrix. Polarized light microscopy and transmission electron microscopy revealed a well-organized collagen fibrillar matrix in untransfected, control chondrocyte cultures, while the matrix in the transfected cultures was less birefringent and contained numerous truncated collagen fibrils.
The findings illustrate the feasibility of gene transfer into human fetal chondrocytes under conditions that allow the preservation of their specific phenotype, and also shed light on the function of type II collagen in the maintenance of the structural integrity of articular cartilage matrix.
在能够维持软骨细胞特异性表型的条件下对人软骨细胞进行稳定转染，并研究突变的II型前胶原基因（COL2A1）的稳定转染对体外产生的软骨细胞外基质结构的影响。
将缺失外显子16 - 27的II型前胶原小基因体外稳定转染到人胎儿骺软骨细胞中。通过逆转录-聚合酶链反应检测小基因的表达，并用II型胶原特异性抗体通过蛋白质印迹法鉴定编码的蛋白质。使用组织化学染色、偏光显微镜分析和透射电子显微镜对培养的转染软骨细胞产生的软骨细胞外基质进行表征。
转染的小基因编码的缩短型II型胶原在转染细胞培养物中进行生物合成并产生。组织学分析表明，对照培养物中的软骨基质更致密，带负电荷。相比之下，COL2A1小基因转染的培养物细胞更多，细胞形状不规则，软骨细胞样外观较少，含有丰富的细胞内致密颗粒，周围是密度较低的基质。偏光显微镜和透射电子显微镜显示，未转染的对照软骨细胞培养物中有组织良好的胶原纤维基质，而转染培养物中的基质双折射性较低，含有许多截短的胶原纤维。
这些发现说明了在能够保留其特定表型的条件下将基因转移到人胎儿软骨细胞中的可行性，也揭示了II型胶原在维持关节软骨基质结构完整性中的作用。
