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目的证实人CD59突变抗补体活性下降,促进糖尿病血管并发症的发生发展。方法构建2种HMCD59重组质粒,转染CHO细胞;FCM筛选高表达细胞,荧光免疫测定和固相酶免疫测定加一步检测阳性细胞。糖化培养阳性细胞,BCECF染料释放试验比较糖化人突变CD59和糖化人正常CD59抗补体活性。ELISA测定30例糖尿病无血管病变组和30例Ⅱ型糖尿病血管病组血清PDGF、bFGF含量。结果筛选细胞株表达率分别为59.8%、53.0%、54.5%;BCECF染料释放试验显示糖化后人突变CD59细胞比较糖化前染料释放率高(P<0.01);而正常CD59细胞较糖化后细胞染料释放率差异不明显(P>0.05)。Ⅱ型糖尿病有血管病变组与糖尿病无血管病变组相比,血清PDGF、bFGF明显增高(P<0.01)。结论证实人CD59突变后易糖化抗补体活性下降加速糖尿病血管病的发生发展。
Objective To confirm that human CD59 mutation has decreased anti-complement activity and promoted the occurrence and development of diabetic vascular complications. Methods Two HMCD59 recombinant plasmids were constructed and transfected into CHO cells. Highly expressed FCM cells were screened by FCM. The positive cells were detected by fluorescence immunoassay and solid phase enzyme immunoassay. Positive cells were saccharified and the BCECF dye release assay compared the glycated human mutant CD59 and the glycated human normal CD59 anti-complement activity. Serum levels of PDGF and bFGF were measured by ELISA in 30 cases of diabetic neovascular disease and 30 cases of type 2 diabetic vascular disease. Results The results of BCECF dye release assay showed that the rate of CD59 cells after glycation was significantly higher than that of the pre-saccharification (P <0.01), while the percentage of normal CD59 cells was higher than that of the glycated cells The difference of release rate was not obvious (P> 0.05). The serum levels of PDGF and bFGF were significantly higher in patients with type Ⅱ diabetes mellitus than those without diabetes (P <0.01). Conclusions It is confirmed that the decline of the activity of easy glycosylated anti-complement after human CD59 mutation accelerates the occurrence and development of diabetic vascular disease.