| Objectives Glucose and lipid metabolism disorders related diabetes and cardiovascular disease has become the main cause of harm to people's health,often characterized by insulin resistance and dyslipidemia in patients with deposits,including islet beta cells and vascular endothelial cell function injury is the key to its development,but the onset of molecular mechanism is not fully clear.Recently a lot of studies have shown that tiny RNA(micro RNA,mi RNA)sugar and lipid metabolism disorders in type 2 diabetes(type 2 diabetes,T2DM),vascular endothelial cell injury plays an important role.Physi CAL's previous research has shown that people there are rich in peripheral blood circulation,stable expression of micrornas,is a kind of new Type of disease biomarkers,but glucose and lipid metabolism disorders related to T2 DM microvascular complications(Type 2 diabetes microvascular complications,T2DMC)crowd circulating mi RNA research reported less,circulating micrornas as glucolipid metabolic abnormalities crowd new biomarkers value,physiological functions,and participate in the glucolipid metabolic abnormalities related disease molecular mechanism is unclear.So this thesis research mainly has the following objectives: 1)To determine the serum levelsof mi RNA in the serum of highglucose patients,highlipid patients and highglucoselipid patients,analyzes its glucolipid metabolic abnormalities in patients with disease development potential risk prediction potential molecular indicators.2)detecting serum insulin and endothelial function in patients with T2 DM and T2 DMC injury related mi RNA levels and expression characteristics,discusses specific changes of micrornas existence forms and clinical value of assessment as biomarkers in patients with T2 DM and T2 DMC new auxiliary potential.3)Building a high sugar and high fat in vitro induced endothelial cell damage model,detect changes in patients with T2 DM and T2 DMC specific mi RNA expression changes in cells,prediction of micro RNAs endothelial cell function of related target genes,discusses specific micrornas through regulating the target gene expression in high sugar and high fat molecules induced endothelial cell damage mechanism..Methods 1)Collected highglucose patients,highlipid patients,highglucoselipid patients and healthy controls serum samples each 30 cases,based on the lock nucleotides(Locked Nucleic Acid,LNA)technology Exiqon micro RNAs chip technology early screening serum micro RNAs expression spectrum,highglucose patients,highlipid patients and highglucoselipid patients early screening and controls in the chip significant changes of mi RNAs and combined with some literature reports of mi RNAs,using real-time fluorescent quantitative PCR(q RT-PCR)at the beginning of the above samples to sieve the mi RNA level after screening and validation,screening of high sugar and high fat in patients with significant changes in serum mi RNA,and through the receiver-operating characteristic curve(ROC curve),logistic regression analysis methods to analyze it as a disease development potential risk prediction of molecular index value.2)Collect serum specimens of 131 cases with T2 DM ?131 cases with T2 DMC and 129 cases of healthy controls,using q RT-PCR to detect a variety of mi RNAs level which related to islet?cells?vascular endothelial cells and functions in patients with T2 DM,T2DMC and healthy controls serum;Biochemical analyzer to determine patients and controls related biochemical indicators,further changes to a specific serum mi RNAs,explore its existing form,finally through the ROC curve,logistic regression analysis the auxiliary diagnostic value of serum mi RNAs in T2 DM and T2 DMC.3)Building high sugar and high fat induced endothelial cell damage model,then observe cell count,shape change;QRT-PCR detection of T2 DM and the change of serum T2 DMC patient's mi RNA expression level changes in the cells,and in view of the significant changes in specific mi RNAs,using bioinformatics method to predict the potential target genes,the luciferase reports experimental verification of micrornas with target gene targeting regulation relationship,Western blot verify its target gene expression levels in the cell model.Results 1)According to the results of he mi RNAs chip,the difference of serum mi RNAs expression between highglucose patients?highlipid and highglucoselipid patients and contrast was obvious;6 kinds of serum mi RNAs(including mi R-25-5P,mi R-4658,mi R-4773,mi R-4132,mi R-5771-5P and mi R-3165)in a significant rise in high sugar and high fat people,two kinds of mi RNAs(mi R-130 and mi R-494-5P)decreased significantly(P < 0.05);Combined with the literature reported results and further q RT-PCR test results showing that mi R-33 a is higher in highglucose patients(8.85± 4.2)× 10-3? highlipid patients(25.14± 4.9)× 10-3 and highglucoselipid(21.18± 5.4)×10-3than controls(1.95±0.58)×10-3,the marked increase in ROC curve analysis showed that the area under the curve of mi R-33 a as highglucose patients?highlipid and highglucoselipid patients' auxiliary diagnostic indicatorswere 0.702(95% CI = 0.535 ~ 0.535),0.975(95% CI = 0.00 ~ 1.00)and 0.00(95% CI = 0.802 ~ 1.00);Logistic regression analysis shows that it can be used as independent risk factors of glucoselipid metabolism disorders [6.33(0.67 ~ 60.16),P = 0.67;171(14.24 ~ 2.05),P < 0.001)and 76(7.7 ~ 750.5),P < 0.001].2)Determination of serum levels of mi RNAs in patients with T2 DM and T2 DMC using q RT-PCR technology,according to the results of two groups of patients with horizontal {T2DM [mi R-16 =(23.74 + 2.70)×10-5,mi R-126 =(25.01 + 4.13)×10-5,mi R-221 =(84.76 + 11.79)×10-5 and mi R-7 =(173.8 + 33.77)×10-5] and T2 DMC [mi R-16 =(43.74 + 9.61)× 10-5,mi R-126 =(17.66 + 2.20)× 10-5,mi R-221 =(82.52 + 12.48)×10-5 and mi R-7 =(176.2 + 49.27)×10-5]} significant rise than healthy controls [mi R-16 =(14.35 + 1.00)×10-5,mi R-126 =(11.75 + 1.47)×10-5 and mi R-221 =(32.26 + 3.98)×10-5 and mi R-7 =(67.47 + 8.44)×10-5],can be used as auxiliary diagnosis of patients with T2 DM and T2 DMC potential indicator;Further to patients with specific mi R-7 form change,according to a study of serum mi R-7 mainly free rather than outside secrete body(exosome)parcel form,and serum free mi R-7 diagnostic value in patients with T2 DMC than serum mi R-7.3)Under the stimulus of high sugar,high fat,vascular endothelial cells decreased and change significantly;Hhigh sugar and high fat can stimulate mi R-661 levels increased significantly,bioinformatics prediction and luciferase experimental results show that peroxidase body growth activated receptor gamma(peroxisome proliferators-activated receptors,PPARs)auxiliary activation factor alpha 1(PGC-1?)is a potential target genes of mi R-661,Westernblot results show that the high sugar and high fat stimulus to PGC-1?significantly reduced.Conclusion 1)Serum mi RNAs expression spectrum of highglucose patients,highlipid and highglucoselipid patientswere obviously different with control group,and the significantly increased expression level of serum mi R-33 a can be used as a disease development potential risk prediction of molecular target in patients with glucoselipid metabolic abnormalities.2)Serum mi R-7,mi R-16,mi R-126 and mi R-221 expression level were significantly increased in patients with T2 DM and T2 DMC,are independent risk factors and potential and auxiliary diagnosis of non-invasive markers in patients with T2 DM and T2 DMC,significant changes in serum mi R-7 mainly exists in the form of free,and free mi R-7 diagnostic value in patients with T2 DMC was higher than serum mi R-7.3)High sugar,high fat can induce obvious changes in vascular endothelial cells,and increase intracellular mi R-661 levels inhibit the expression of PGC-1?. |
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