Type 2 Diabetic Pedigrees Serum Proteomics Research

PARTⅠTHE METABOLISM FEATURS AND THE METABOLIC SYNDROME PREVALANCE IN CHONGQING FAMILIES WITH TYPE 2 DIABETES MELLITUSObjective To estimate the metabolic features and the prevalence of metabolic syndrome (MS) and its main components in families with type 2 diabetes mellitus and search the potential genetic marks of diabetes.Methods (1) 1824 subjects (788 male and 1036 female) from 406 Chongqing Han families were enrolled. 1606 subjects of them from T2DM family and 218 subjects of them were their spouses. Anthropometry, blood pressure, lipid levels, high-sensitivity C-reactive protein (hsCRP), non-esterified fatty acid (NEFA) and oral glucose tolerance test(OGTT) were examined.(2) Expect second degree relatives and spouses who had diabetic relatives or who were diabetic, all the subjects were divided into three groups: T2DM ,first degree relatives (FDR) and control group. The clinical and metabolic characteristics and the prevalence of MS and its main components were compared among three groups. (3) They were divided into four groups: nT2DM (diabetic relatives who suffered T2DM less than half a year without medicine therapy), IGT group (FDR who were IGT), FDR-N group (FDR who were normal glucose tolerance) and Control-N group (spouses who had non-diabetic relatives or who were normal glucose tolerance). The clinical and metabolic characteristics were compared among four groups.Results (1) OGTT2h plasma glucose, Homa-IR and triglycerides in FDR group were higher than those in controls (P﹤0.05),and HDL-C lower than that in Controls (P﹤0.05). Although there was lower Dispose Index (DI) than in Control group, there were no statistic difference between two groups (P﹥0.05). IGR, obesity, central obesity, hypertriglyceridemia, decreased blood HDL-C, hypertension and MS were 34.3%, 32.2%, 33.1%, 29.8%, 28.9%, 12.5% and 23.5% respectively. There were higher than control except hypertension. The risk of MS and its main components increased in FDR than in controls : IGR (OR 2.39, P﹤0.01), decreased HDL-C (OR 1.87, P﹤0.01), MS (OR 2.3, P﹤0.05). (2) T2DM had serious metabolic disturbance then the others. The risk of obesity, hypertriglyceridemia, decreased HDL-C and hypertension were approximately 2.5 (OR 2.73,2.53,2.46 and 2.81 respectively,P﹤0.01), the risk of central obesity and MS was 4(OR 3.81 and 7.02 ,P﹤0.01).(3) Increased hsCRP ,TG and Homa-IR (P﹤0.05) and decreased HDL-C(P﹤0.05) were detected in FDR-N group comparing with Control-N group. NEFA was higher than Control-N group, but no statistic difference(P﹥0.05). hsCRP and NEFA were higher in nT2DM and IGT group than FDR-N and Control-N group (P﹤0.05).Conclusion There are more or less metabolic abnormalities in the non-diabetic FDR with T2DM families. The familial aggregation of T2DM was not only hyperglycaemia but also obesity, hypertension, lipid disturbance those related with MS. The changes of TG, HDL-C, Homa-IR and inflammatory factor hsCRP and NEFA probably related to genetic factor. PARTⅡESTATISHIMENT AND OPTIMIZATION OF TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELCETROPHORESIS–MASS PECTROMETRY FOR SERUM PROTEOMICS0bjective To establish and optimize of a Two-dimensional electrophoresis(2-DE)-Mass Spectrometry technique for serum proteomics.Methods Removaling serum album and IgG was achieved by filtering with ProteoExtract Albumin/IgG Removal Kit. 2-DE technique, including isoelectric focusing (IEF) with an immobilized pH gradient (IPG) and SDS-PAGE was used, associated with Mass Spectrometry to evaluate the effectiveness of purification, Sample preparation, IEF, silver staining and other steps were adjusted and optimized.Results Depletion of albumin(ALB) and IgG could improve the resolution of serum 2-DE map and enable some proteins to be identified by Mass Spectrometry due to increase in loading quantity. 2-DE-MS technique for serum proteomics which was better reproducibility and sharpness of separation was established stably.Conclusion Establishment and optimization of 2-DE-MS technique for serum proteomics may be foundation in searching serology marker of diseases. PARTⅢSCREENING OF SERUM PROTEINS IN CHONGQING FAMILIES WITH TYPE 2 DIABETES BY TWO-DIMENSIONAL ELECTROPHORESIS - MASS SPECTROMETRY TECHNIQUEObjective To illuminate the relationship between the disparation proteins and the genesis and development of type 2 diabetes, to find the biomarkers related type 2 diabetes, according the results of family survey with type 2 diabetes, and Two-dimensional electrophoresis(2-DE)- Mass Spectrometry(MS)technique was applied to find the difference of serum proteins expression, also Peptide Mass Finger printing (PMF) and western blot were adopted to identify the differential proteins.Methods (1) There were enrolled from Chongqing Han families with type 2 diabetes, been randomized into four groups: nT2DM(diabetic relatives who suffered T2DM less than half a year without medicine therapy, male 30/female 30), IGT(male 15/female 15), first degree relatives (FDR-N) who had normal glucose tolerance(male 30/female 30), and Control-N was enrolled the subjects who had normal glucose tolerance and had not DM family history(male 23/female 27). Mixing samples of one group albumin and immunoglobulin depletion and desalting, then four samples were selected to check the reproducibility of 2-DE, PDQuest7.3.1 was applied to compare the 2-DE maps of each group, finally the disparation proteins spots were achieved. (2) Disparation proteins spots were extracted and been enzymolysis and progressing mass spectrogram identification, then according the results of Peptide Mass Finger printing (PMF) to find the disparation proteins by searching internet using Mascot and MS-Fit. (3) 10 serum samples of each group nT2DM, group IGT, FDR-N and Control-N were enrolled to identify disparation proteins by Western blot.Results (1) Four groups of 2-DE maps with high resolution and reproducibility were obtained. (2) 32 differential expression proteins were found by comparing the four groups using PDQuest. 13 proteins were over expressed in group nT2DM, group IGT and group FDR-N, 11 proteins were over expressed only in group nT2DM, group IGT, two proteins were under expressed in group nT2DM, group IGT, three proteins were over expressed only in group nT2DM, one protein were expressed only in group nT2DM, one protein were over expressed in group nT2DM and under expressed in group IGT, and one protein were deletion expressed in group cnotrol-N. (3) All differential proteins were identified by Mass Spectrometry, among the total, 27 spots were belonged to 9 proteins, CRP, Complement C3, Complement C4b binding protein, Heptoglobin, TTR, RBP4, ApoM, ApoAⅣand ApoE, only 5 spots were not identified. (4) ApoM were identified by Western blot, and there was no significant change in all group.Conclusions (1) In this investigation we first time applied Two-dimensional electrophoresis(2-DE)- Mass Spectrometry(MS)technique to find differential protein expression in family survey with type 2 diabetes, and 9 proteins were identified, namely CRP, Complement C3, Complement C4b binding protein, Heptoglobin, TTR, RBP4, ApoM, ApoAⅣand ApoE. (2) In this study proteomics and Western blot just were first step, Apo M and other proteins changing in family survey with type 2 diabetes need to be further investigation. (3) CRP, Complement C3, Complement C4b binding protein, Heptoglobin, TTR, RBP4, ApoM, ApoAⅣand ApoE were over expressed in Chongqing Han families with type 2 diabetes, it was speculated that it related closely to the inheritance of families with type 2 diabetes, and these proteins could be the biomarker of families with type 2 diabetes. These changes also found in obesity, hypertension, hyperlipoidemia, and coronary heart disease, so further investigation should be carry out.