PDGF-B基因表达及其对成纤维细胞增生和胶原合成的促进作用.docVIP

PDGF-B基因表达及其对成纤维细胞增生和胶原合成的促进作用 PDGF-B基因表达及其对成纤维细胞增生和胶原合成的促进作用 更新日期：2010-09-08 王忠彪　孙逊　李勇　俞璎　戴金凤　陈常庆　范维琥 　　【摘要】　目的　建立稳定表达人血小板衍生生长因子-BB(platelet-derived growth factor-BB, PDGF-BB)的细胞株,并考察该重组蛋白与损伤修复有关的生物学作用。方法　将含PDGF-B基因的真核表达载体pcDNA3转染小鼠成纤维细胞NIH3T3中,在G418选择压力下得到抗性克隆,收集扩增的抗性克隆培养液并制备细胞膜上PDGF-BB蛋白质,3H-TdR掺入法表达产物的生物学活性,并以3H-Pro掺入测胶原合成率。结果　免疫荧光染色法证实抗性细胞中PDGF-BB的表达,膜上PDGF-BB显著促进NIH3T3细胞DNA合成,活性可达约31.2U/106细胞,而培养液的致丝裂活性低于可检测水平。抗性细胞的胶原合成率显著增高。结论　PDGF-BB的真核表达系统已得以成功构建,该重组蛋白可促进成纤维细胞增生和胶原合成,提示其在损伤修复中有潜在应用价值。 　　【关键词】　血小板衍生生长因子　基因　表达　NIH3T3细胞 PDGF-B Gene Expression and Its Stimulating Effect on Fibroblast Proliferation and Collagen Synthesis　　WANG Zhong-biao*, SUN Xun, LI Yong, et al. *Dept. of Cardiology, Huashan Hospital, Shanghai Medical University, Shanghai 200040 　　【Abstract】　Aim　To study the biological effect of human platelet-derived growth factor (PDGF)-BB on injury healing. Methods　A recombinant eukaryotic vector containing human PDGF-B gene was transduced into mouse fibroblasts NIH3T3. The positive clones were obtained under the selection of G418-media. The media and the membrane fractions of the drug resistance cells were well respectively prepared for assaying their biological activity by means of 3 H-TdR incorpration. The effect of PDGF-BB on the collagen synthesis of the fibroblasts was estimated with 3 H-Proline incorpration. Results　The recommbinant protein of the drug resistance cell membrane significantly stimulated the DNA synthesis of NTH3T3 cells with the mitogenic activity about 31.2U/106 cells while the activity in the media was below the detectable level. The PDGF-BB antigen in drug resistance cells was positive in immunofluorescent staining while that in the control group was negative, and the drug resistance cells had significantly higher 3 H-proline incorpration than that of the control cells. conclusion　A cell strain expressing biologically active PDGF-BB was successfully established. The recombinant PDGF-BB stimulated the mitogenesis and collagen sy