| Background:As people's living standard has improved,the current incidence rate of diabetic mellitus(DM)is much higher,diabetic nephropathy(DN)is one of the most common and serious complications of diabetes,its incidence is rising year by year.At present,the clinical diagnostic criteria of DN is commonly combining DM history,clinical characteristics of different degree of albuminuria and other complications of DM,such as diabetic retinopathy.Renal biopsy is the golden standard in diagnosis of DN,however,due to its invasive and injurious disadvantages,it is seldom applied to DN patients.Once massive albuminuria occurs in DN patients,the lesions will progress rapidly until end-stage renal disease(ESRD).Therefore,it is of great value to find a relatively early and more specific indicator for diagnosis of DN.As tiny vesicles secreted by cells,exosomes carry a large amount of biological information.Due to its bilayer structure,the relevant biological information in the exosomes could get better protection and a more stable presence.At present,many literatures have reported that exosomes are of great value and significance in the diagnosis and treatment of a variety of diseases.Transforming growth factor ?1(TGF-?1)and connective tissue growth factor(CTGF)are genes associated with fibrosis which play an important role in the process of development in the pathogenesis of DN.A variety of studies have shown that these two factors are connected with podocyte damage,proliferation of mesangial cells and deposition of extracellular matrix(ECM).Currently,by comparing the content of TGF-?1 and CTGF genes in serum or urine of DN patients and healthy subjects,it has been found that the expression of TGF-?1 and CTGF genes in serum or urine of DN patients is increased,suggesting that their expression isV of great significance for the early diagnosis of DN.In this research,exploring the relationship between DN and the expression of TGF-?1 and CTGF in the urinary exosome,which provides a new idea for the early diagnosis of DN.Objective:To investigate the differences in the gene expression of fibrogenic genes TGF-?1and CTGF in urinary exosomes of patients with early DN compared with that of healthy people,so as to find diagnostic markers for early DN.Method:In this study,30 cases in the healthy group and 30 cases in the early DN group were included.Collecting the ordinary materials such as gender,age,smoking history and the clinical data such as hypertension,systolic pressure,diastolic blood pressure,proteinuria,hematuria,glycosuria,urine microalbumin,fasting blood glucose,glutamic-pyruvic transaminase,aspertate aminotransferase,albumin,triglyceride,cholesterol,serum creatinine,blood urea nitrogen,blood uric acid,blood cystine protease inhibitors C,hemoglobin.All the data were collated for statistical analysis.400 ml of morning urine was collected from every candidate and urine exosomes were isolated and extracted by differential and overspeed centrifugation.We use transmission electron microscope(TEM)and western blot(WB)to identify exosome,real-time quantitative PCR(RT-q PCR)is used to detect the relative expression of target genes TGF-?1 and CTGF,and western blot is used to detect the relative expression of proteins.All the data collected are statistically analyzed with SPSS 19.0,p<0.05 is considered statistically significant.Result:1.There were a total of 30 healthy subjects,including 14 males(46.67%)and 16females(53.33%),the ratio of male to female was 0.875:1,the average age was51.57±7.81 years old.There were 30 DN patients,21 males(70.00%)and 9 females(30.00%),the ratio of male to female was 2.33:1,the average age was 54.9±9.15 years old.Compared with healthy subjects,the DN patients had significantly higher levels of hypertension,systolic blood pressure,diastolic blood pressure,proteinuria,diabetes,microalbumin,fasting blood glucose and blood urea nitrogen(p<0.05).2.The urine samples of healthy people and the DN patients obtained by differential ultracentrigation showed a goblet size of about 40-100 nm in diameter under TEM.In the WB experiment,exosome marker proteins CD9 and TSG101 were positive.3.Compared with the healthy group,the m RNA and protein levels of TGF-?1 in urinary exosome of the DN group were significantly increased(p<0.001).4.Compared with the healthy group,the m RNA and protein levels of CTGF in the urinary exosome of patients in the DN group were significantly increased(p<0.001).Conclusion:1.Exosome can be successfully isolated from urine of healthy people and the DN patients by differential ultracentrifugation.2.The m RNA levels and protein contents of TGF-?1 and CTGF genes in the urine exosomes of DN patients were significantly higher than those in the healthy subjects,suggesting that the TGF-?1 and CTGF gene expression levels in the urine exosomes can be used as diagnostic indicators for early DN. |
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