【摘要】 目的:探讨胰岛素样生长因子I基因转染人软骨细胞的可能性及其在软骨细胞中的表达,并初步判断转基因细胞的生物学功能变化。方法:实验于2002-07/2003-06在中山大学附属第二医院医学研究中心完成。①构建pcDNA3.1-胰岛素样生长因子I/GS质粒,将构建质粒在大肠杆菌DH10B中大量扩增后,抽提纯化质粒,并对质粒进行测序鉴定。②将鉴定的质粒用阳离子脂质体转染人关节软骨细胞,通过逆转录-聚合酶链反应、Westren-blot等方法检测胰岛素样生长因子I基因在人关节软骨细胞中的表达。③分别将同代软骨细胞与转基因细胞的培养液、软骨细胞与转基因细胞裂解液和二甲基亚甲蓝共孵育后,检测各混合液的吸光度,以此判断同代软骨细胞、转基因细胞、软骨细胞培养液、转基因细胞培养液中的蛋白多糖含量。结果:①质粒鉴定结果:质粒抽提纯化后电泳可见3条DNA条带,符合质粒的电泳图谱;质粒的测序结果和胰岛素样生长因子I基因序列完全吻合。②胰岛素样生长因子I基因在软骨细胞中的表达:质粒转染软骨细胞后,反转录-聚合酶链反应能见到298bp的胰岛素样生长因子ImRNA片断的表达;Western-blot可见7.6ku的胰岛素样生长因子I蛋白表达条带。③转胰岛素样生长因子I基因对软骨细胞分泌蛋白多糖的影响:转基因软骨细胞裂解液和同代的软骨细胞裂解液的蛋白多糖含量分别是(5.23±0.62)mg/L和(2.47±0.31)mg/L,两组比较,差异有显著性意义(t=2.88,P<0.05);转基因软骨细胞培养液和同代非转染软骨细胞培养液中的蛋白多糖含量分别为(9.92±1.04)mg/L和(4.56±0.51)mg/L,两组比较,差异有显著性意义(t=6.47,P<0.05)。结论:胰岛素样生长因子I基因转染人关节软骨细胞后能在软骨细胞中表达,并能促进软骨细胞分泌软骨基质蛋白多糖,具有促DNA合成和维持软骨细胞表型稳定的能力。转胰岛素样生长因子I基因软骨细胞的研究为体外软骨组织工程和关节软骨病的基因治疗提供了直接的实验依据。更多还原

【Abstract】 AIM: To explore the possibility of transferring insulin-like growth factor-I (IGF-I) gene to human articular chondrocytes and its expression in chondrocytes, and primarily judge the biological functional changes of the transgeneic cells.METHODS: The experiment was finished in the Medical Research Center of the Second Affiliated Hospital of Sun Yat-sen University between July 2002 and June 2003. ① Plasmid pcDNA3.1-IGF-I was constructed and it was multiplied in Escherichia coli DH10B. After it was extracted and purified from Escherichia coli DH10B, pcDNA3.1-IGF-I was identified through sequence analyzing. ② With the help of lipidosome, the cultured articular chondrocytes were transfected with pcDNA3.1-IGF-I. The evidence of successful transfection in chondrocytes could be obtained by revere transcription-polymerase chain reaction (RT-PCR) and western-blot. ③ Culture media of chondrocytes and transferring IGF-I gene chondrocytes, pyrolysis fluid of chondrocytes and transferring IGF-I gene chondrocytes were incubated with dimethylmethylene blue. Contents of proteoglycan were determined by the absorbance in ultraviolet spectrophoto-meter.RESULTS: ①Identified results of plasmid: After the purification, the electrophoresis of plasmid showed that there were 3 DNA straps, which was accorded with the electrophoretogram of plasmid. Sequence distribution of object gene was concordant with IGF-I gene. ② Expression of IGF-I in chondrocytes: The expression of 298 bp IGF-I mRNA segment could be observed by RT-PCR after it was transferred with IGF-I gene. The transferred chondrocytes could express IGF-I protein that presented a 7.6 ku protein with the western-blot method. ③ Influence of IGF-I gene on the chondrocytes secreted proteoglycan contents: The contents of proteoglycan in chondrocytes and transferred chondrocytes were (5.23±0.62) mg/L and (2.47±0.31) mg/L respectively, and there were significant differences (t =2.88,P < 0.05).The contents of proteoglycan in medium from chondrocytes and transferred chondrocytes were (9.92±1.04) mg/L and (4.56±0.51) mg/L, which were significantly different (t=6.47, P < 0.05). CONCLUSION: IGF-I gene could be transferred to chondrocytes and it could prompt chondrocytes express IGF-I protein temporarily and secrete proteoglycan, which has provided derect experimental evidence for the in vitro cartilage tissue engineering and the gene therapy of articular cartilage diseases.更多还原