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 Gene Expression and Its Stimulating Effect on Fibroblast Proliferation and Collagen Synthesis

目的建立稳定表达人血小板衍生生长因子－ＢＢ（ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ－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。

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/10~6 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 synthesis of the fibroblasts, which may have some clinical value in injury repair.