Effect Of Free Fatty Acid On The Expression Of ENOSmRNA And ET-1mRNA In The Cultured Human Umbilical Vein Endothelial Cells

Objective: About 40~50 percents of 2 type diabetesmellitus (DM) patients have lipometabolism disorders . Somedata has showed that the free fatty acid (FFA) levels areheightened in the blood serum of the insulin resistance (IR)and / or 2 type DM patients .And it has been a independent risnotation of impaired glucose tolerance (IGT). Nowadays mostscholars think that the endothelium disfunction have intimaterelations with the blood vessel lesions in the DM. Someinvestigations have detected that the endothelium-derivedrelaxing(EDVR)is weakened in the DM patients, and this kindof abnormality has played an important role in the blood vessellesions of DM. The dysequilibrium of excretion and regulatingfunction in the endothelium-derived contracting factors (EDCF)and the endothelium-derived relaxing factors (EDRF) isthe chief character of endothelium disfunction and endothelialcellular damage . As we know , endothelins (ET-1) and Nitrogenmonoxide (NO)are the represens of those two type of factorsrespectively. Some studies and our preceding investigations bothshow that FFAs levels rise in 2 type DM patients and FFAs indifferent concentrations have effects on the releasing of NO and 5英 文 摘 要ET-1 in cultured human umbilical vein endothelial cells( hUVECs) . Our study means to observe the effect of FFA onendothelial type nitric oxide synthase ( eNOS ) and endothelinmRNA (ET-1m RNA) in hUVECs . This will help us withour standing of on what regulation level FFAs act on the bloodvessel endothelial cells and the possible mechanism of onset ofgreat vessel deases in DM patients with high FFAs. Thisinvestigation maybe will open up a new thought of earlyrecovery of endothelial cell physiological function and thecardiovascular disease prevention and cure . Methods : We used the hUVECs ECV304 as the objects ,and the culture medium was type M199 which contained 20%cattle foetus blood serum, Benzylpenicillin,estreptomicina,andL-Glutamyl. All the objects were cultured under the condition of370C and 5%CO2 in the incubator . We made the FFAs intodifferent kinds of FFAs/0.1NnaOH solutions , and madethem to dissolve completely by ultraemulsification . Every5ml nutrien was added with different 200uml/ml ＦＦＡsolutions in amount of 2.5ul,5ul,10ul,15ul by turns to makethe FFA concentration in the medium reach to100umol/L,200uml/L,400umol/L,600umol/L one by one . Thecells cultured with medium containing correspongding amountof 0.1NnaOH soution were the control group named group F.The intervention groups were divided into five groups calledＡ,Ｂ,Ｃ,Ｄ and Ｅ according to the different kinds ofFFA,and every group was given different concentrations of 6英 文 摘 要FFA in four levels . And then they were put into theincubator and cultured for 24 hours under the condition givenbefore . We used TRIZOL reagent to extract the whole RNAfrom the cells . After the detect of the integrity and the purity ,the RNA were taken to the next step of RT-PCR. Theamplificating products mixed with the bromophenol blue wereadded into the hole of the 1% sepharose , and were fractionatedthrough the agarose gel electrophoresis. Every DNA strap waswas observed under ultraciolet light and photographed , thenbands on film were scanned by densitometer and quantified oncomputer by the image analysis software . The RT-PCRproducts of GAPDHmRNA were taken as the references in allthe architectures,and were used to calculate the products ofcertain mRNA . The odds of their gray degrees standed for theexpressions of the mRNA we worked on . We drew assistancefrom the SＡS software to analyze the data . Result :When the FFA concentration was higher than200umol/L, the expression of eNOSmRNA was decreased inthe C18:1 group. The same result was observed in the groupC18:2. No notable discrepancy...