| Background:Diabetes mellitus(DM)is a metabolic disorder caused by a variety of causes,which is characterized by chronic high blood glucose levels,insufficiencies or dysfunction of insulin secretion.In recent years,with the improvement of people's living quality in China,the incidence of diabetes mellitus is increasingly high,according to the statistics of the international diabetes federation(IDF)in 2017,the number of diabetes mellitus in China ranks the first in the world,among which about25% ~ 40% can develop into diabetic nephropathy(DN).For the pathogenesis of DN,a large number of literature studies have reported that the DN is the result of a combination of multiple complex factors,among which the increase of inflammatory cytokine levels plays an important role in the evolution of DN.A study on type 2diabetes found that patients with microalbuminuria had higher MCP-1 values than healthy control groups and those with normal proteinuria in DN,suggesting that MCP-1 was involved in the inflammatory response of DN,leading to the occurrence and development of DN.The main function of MCP-1 is to activate monocytes into macrophages in the blood circulation and migrate to the inflammatory site to play a regulatory role.Therefore,studying the changes of MCP-1 levels has certain guiding significance for the early diagnosis and treatment of DN.Since the exosome was discovered in the 1980 s,there have been more and more studies on exosomes,which were defined as the material generated by the release of confluent muitivesi cular bodies with plasma membrane in lumen.Analysis of exosomal contents found that it contains a variety of proteins,nucleic acids and lipids,which can be secreted by most cells and released into the microenvironment,as a medium involved in intercellularcommunication,protein and RNA transmission.In this study,the change of MCP-1gene content in urine exosomes was extracted and detected in order to find a marker for early diagnosis of DN.Objective:This study intends to demonstrate that exosomes could be extracted from the urine of healthy people and DN patients by differential and overspeed centrifugation,and to evaluate the differences in gene and protein expression levels of cytokines MCP-1 in the urine of healthy people and early DN patients,we expected to clarify the change laws of MCP-1 in early DN,and to explore a new method for early diagnosis of DN.Method:According to the screening criteria,30 patients each with early stage DN and 30 healthy normal subjects were included,which were divided into the experimental group and the control group.Collecting their ordinary materials such as gender,age,smoking history and the clinical data such as proteinuria,hematuria,hypertension,systolic pressure,diastolic blood pressure,urine microalbumin,glutamic-pyruvic transaminase,aspertate aminotransferase,albumin,fasting blood glucose,triglyceride,cholesterol,blood creatinine,blood urea nitrogen,blood uric acid,hemoglobin.After sorting all the above clinical data,we conducted analysis of differences between groups by T-test or Mann Whitney U test according to whether the normal distribution was obeyed.400 ml of fresh morning urine was collected from each healthy person and patient,and the exosomes of each sample urine were extracted by differential and overspeed centrifugation.Western blotting and transmission electron microscope(TEM)were used to identify exosomes,the mRNA of urinary exosomes was extracted by kit method,and the mRNA expression of MCP-1 in exosomes was detected by real-time quantitative PCR(RT-q PCR),the protein in urinary exosomes in each group was extracted,and the level of MCP-1 protein was detected by Western blotting.We also applied the statistical method T-test to analyze the difference between groups,p<0.05 is considered statistically significant.Result:1.This study involved 60 candidates,including 30 healthy people as the normal control group,of which 11 were male and 19 were female,with an average age of55.00±13.19,and 30 patients with diabetic nephropathy as the diabetic nephropathy group,of which 15 were male and 15 were female,with an average age of56.37±11.89.There was no significant difference between the two groups in gender,age,smoking history or hypertension history.The levels of microalbumin,fastin-g blood glucose,urea nitrogen and albuminuria in uremic nephropathy group were significantly higher than those in normal control group(P<0.05).2.The urine samples of normal control group and diabetic nephropathy group were extracted by differential ultracentrifugation,and the obtained components of exosomes were showed quasicircular cup-shaped small bodies under TEM.In the Western Blot experiment,exosome marker proteins CD9 and TSG101 were positive.3.Compared with the normal control group,the mRNA and protein levels of MCP-1 in urinary exosome of the diabetic nephropathy group were significantly increased(p<0.005).Conclusion:1.Exosome can be successfully isolated from urine of healthy people and the DN patients by differential ultracentrifugation.2.The level of MCP-1 gene expression in urinary exosomes of diabetic nephropathy group was significantly higher than that of normal control group,indicating that the level of MCP-1 gene expression in urinary exosomes could be used as a marker of early clinical diagnosis of early diabetic nephropathy. |
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