| Objective: To detect the IgG anti-endothelial cell antibodies(AECA)inserums and discuss the variational trend of AECA between patients with different types of diabetic retinopathy(DR). In the meanwhile, observe the influence of Chinese traditional medicine astragalus on the secretion of TNF—a that had been produced during IgG AECA in serum of DR patients stimulating the endothelial cell(EC)cultivated in vitro.Methods: the study was composed of two parts:1 The research objects were divided into 4 groups: group with diabetes mellitus non-diabetic retinopathy(DM-NDR) , group with background dabetic retinopathy(BDR)and group with proliferative diabetic retinopathy(PDR)according to the examination results of the fluorescein fundus angiography(FFA)and ophthalmoscope, and group with normal control was set up. With the human umbilical vein endothelial cells(HUVEC) cultured in vitro acting as the antigens, the variation of positive rates of IgG AECA in the serums of patients was detected with the indirect immunoflorescence(IIF), and the result was compared with normal control. The datas of the positive rates in groups were analyzed by the R×C chi-square test with the statistics software SPSS12.0. Chi-square decomposition was adopted in comparation of positive rates between groups. Relationship between the course of disease of DM and positive number of IgG AECA was analyzed by bivariate correlate. Comparation of positive rates of IgG AECA in different courses of disease with DM was analyzed by R ×C chi-square test.2 The human HUVEC cultivated in vitro was Stimulated by IgG AECA in serum of diabetic retinopathy patients purified with the ion exchange chromatogram,in the meanwhile, putting astragalus into it. the3-(4, 5-dimethy thioazol-2-yl)-2, 5-diphenyl-tetrazoliumbromide (MTT) was set up to mensurate the injury effect of TNF - a that produced by IgG AECA stimulating HUVEC on cells of WEHI 164.13, and the protective effect of astragalus on HUVEC was observed. The data of experiment was analyzed with one way ANOVA, and LSD multi-comparation was adopted in the comparation between each two groups.Results:1 The positive rates among groups had significant difference By R*C chi-square test(￡2=97.892, P<0.01). By chi-square decomposition, the results between DM-NDR and the normal control had no significant difference (￡2=0.000, P>0.05), these two groups as one group without retinopathy. the results between BDR and the PDR had no significant difference either(/=0.346, P>0.05), these two groups as one group with diabetic retinopathy(DR). the positive rate of the group with DR(82.50%) was higher than that in group without retinopathy (5.00%), there was significant difference between them(￡2=97.625, PO.01). And that, bybivariate correlate, there was positive correlation between course of disese with DM and positive number of IgG AECA(r=0.908, P<0.01). Comparation of positive rates of IgG AECA in groups with different courses of disease with DM had significant difference(/=15.186, PO.01).2 With the increase of concentration in IgG AECA, the output of TNF—a raised, there was significant difference in groups with different concentrations (F=5.093, PO.05). Under different concentrations of antibody, there was significant difference between three concentrations of medication. F=80.451, PO.05 when concentration of antibody was 2.5%. F=30.341, P<0.05 when concentration was 5%. F=\ 14.465, P<0.05 when concentration was 10%. And the killing activity washigh in concentration of no medication, moderate in low concentration, and low in high concentration. Under different concentrations of medication, there was significant difference between three concentrations of antibody. F=30.723, P<0.05 when concentration of medication was high. F=5.187, PO.05 when concentration of medication was low. F=5.093, P<0.05 when concentration of medication was blank, and when concentration of antibody was 10%, the killing activity was the highest, then was 5%, 2.5% in turn. The killing activity was not significant when there was no antibody.Conclusions:1 IIF was fast, economical and could be accurately located, also had good repeatability, which could examine the massive antibodies at the same time.2 The positive rate of IgG AECA in group with DR was obviously higher than that in group without retinopathy. In the relationship between the course of disease with DM and positive rate of IgG AECA, and the comparation of positive rates of IgG AECA in groups with different courses of disease with DM, there was statistics significant, these results showed that IgG AECA had relationship with the onset of DR, with the conversion from normal retina to DR, the positive rates of IgG AECA in serums took on the trend of increasing. While the positive rates between group with BDR and the group with PDR had no significant difference, indicating that the positive DR patients with IgG AECA could develop from BDR into PDR, which had been confirmed in the comparation result of positive rates of IgG AECA in groups with different courses of disease with DM.3 Through MTT, killing activity of TNF-a might be mensurated using the WEHI164.13 cells that were highly sensitive to TNF-a. TNF-a could be secreted by EC that stimulated by IgG AECA, by the examination in killing activity of TNF-a,the damage function of IgG AECA to EC could be appraised. Results of the experiment demonstrated that, TNF- a had remarkable killing effect on WEHI164.13 cells, and showed distinct density dependence. It was understandable that with the increase in concentration of IgG AECA, killing activity of TNF-a secreted by EC that stimulated by IgG AECA raised, which had been clear about the damage function of IgG AECA to EC, namely, higher the concentration of IgG AECA was, higher the damage extent of EC would be, showing there was close relationship between IgG AECA and the vascularpathy of DR.4 Chinese traditional medicine astragalus had certain inhibitory effect on killing activity of TNF—a, indicating that astragalus had protective effect on EC. But further research would be needed to instruct the useness of medicine in clinic.
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