Preliminary Study On The Mechanism Of Calcification In Diabetic Rabbits With Syphilis Renal

Objective:To compare the renal calcification of experimental group and screen the genes expressed differently in renal tissue by gene chip,and to make preliminary study on the mechanism of renal calcification in diabetic rabbits with syphilis through detection the gene and protein expression level related renal calcification.Methods:1.Animal grouping and model construction: New Zealad male rabbits were randomly divided into normal group,Diabetes mellitus group(group DM),Treponema pallidum group(group TP)and Diabetes combined syphilis group(group H).2.Hematoxylin-eosin staining: The kidneys of each group were fixed,dehydrated and transplanted.Then embeded in wax,sliced,dehydrated and dehydrated.Hematoxylin and ethanol were separated finally.Sealed with neutral gum,and analyzed the renal calcification in the rabbit of each experimental group.3.Gene chip detection: Taked the renal of each experimental group rabbit to screen the differently expressed genes related to calcification by gene chips.4.The Real-time quantitative m RNA PCR detection of differently expressed genes related to calcification: Real-time quantitative PCR was used to detect the relative content of calcification-related gene in renal tissues of each experimental group.5.Western blot(WB)detection of differently expressed protein related to calcification: Total protein of renal tissue were extracted and determined the concentration of the protein.After the operation,the samples were run with glue,transferred to membrane,blocked with milk,and incubated with the first antibody.Then washed with the TBST solution and incubated with the second antibody for 1 hour in the incubator.The expression of differently expressed proteins related to calcification in renal tissues was detected.6.Immuno-histochemistry: Taked the paraffin sections of kidney tissue,dewaxing and hydration after baking slices,incubated with 3% hydrogen peroxide(H2O2)in deionized water,washed with PBS,and then blocked with 5%-10% goat serum.Then incubated with the second antibody,then counterstained,dehydrated,and seeded.Results:1.Animal model of group DM,group TP and group H were succes-sfully Constructed.2.HE staining showed that there was no obvious abnormal change in group C.But the obvious glomerular capillary dilation and proximal tubule swelling were observed in glomeruli of group TP,group DM and group H.And the protein tube type,interstitial mucus-like change,blister-like change were also found in renal medulla district of group DM.Calcium deposition,Glomerular capillary dilatation and proximal tubule swelling is more serious in group H compared with other groups.3.The results of gene chip showed that the ADRA1 A,CACNA1E and CCKAR genes were significantly different in each experimental group compared with group C.The expression of ADRA1 A,CACNA1E and CCKAR genes in group TP,group DM and H were all up-regulated,group H were up-regulated most obviously.4.The Real-time quantitative PCR results of ADRA1 A,CACNA1E and CCKAR m RNA showed that,the expression of ADRA1 A,CACNA-1E genes were up-regulated in group TP,group DM and group H.Compared with group C,group DM and group H had higher expression level than group TP.and the group H had the highest expression level.Which was Consistent with the results of the gene chip.The expression of CCKAR genes were up-regulated in group TP,group DM and group H.Compared with normal group,group DM and group H had higher expression level than group TP.and there are no difference between the group H and group DM.The real-time quantitative m RNA PCR detection of TLR-4 and MAPK1 gene showed that the expression of these two genes was consist-ent with those of ADRA1 A,CACNA1E.5.WB detection: Protein expression level of ADRA1 A,CACNA1E,CCKAR,TLR-4 and MAPK1 was consistent with those of real-time quantitative PCR results.6.The results of immuno-histochemistry showed that the positive cells of ADRA1 A,CACNA1E,CCKAR,TLR-4 and MAPK1 were increased in group TP,group DM,group H.Compared with group C,There were more positive cells in group DM than group TP,but less than group H.Conclusion:1.Compared with the group DM and group TP,Inflammatory response and renal calcification is more obvious in group H.2.Compared with the group DM and group TP,renal calcification in group H may be related to the increase of calcium influx by the up-regulation of ADRA1 A and CACNA1 E.and may be irrelevant to the regulation of CCKAR.3.Compared with the group DM and group TP,Renal calcification in group H may be related to the more severe inflammatory response by the up-regulation of TLR-4 and MAPK1.