ABSTRACT
Objective To explore the relationship between selenium and diabetic nephropathy (DN)by determining the selenium content in the plasma and whole blood of DN patients and the changes of selenoprotein expressions in human mesangial cells stimulated by high glucose.Methods We collected the samples and clinical indicators of DN patients and healthy controls to determine selenium content by atomic absorption spectrometry method.The mRNA in human mesangial cells was isolated after stimulation with 30 mmol/L of glucose for 24 hours by Trizol method to detect the changes of selenoprotein expressions by Real-time PCR.We analyzed the differences between DN patients and healthy controls and the relationship between clinical indicators by unpaired t-test and Pearson correlation analysis,respectively.Results The selenium contents in the plasma (P＜0.0001)and whole blood (P＜0.001)were significantly lower in DN patients than in the healthy controls.In addition,plasma selenium contents had a significant negative correlation with renal function and a positive correlation with glycosylated hemoglobin in DN patients.Among the 2 1 human selenoproteins,the mRNAs of Gpx1 ,TrxR2 ,TrxR3 ,Dio2 , Dio3 ,SelK,SelN,SelP,SelR,SelT,SelW and SPS2 were significantly lower in the high-glucose group than in the normal-glucose group which produced by human mesangial cells after high glucose stimulation for 24 hours. Conclusion There was obvious selenium deficiency in DN patients.Human mesangial cells have a significantly low expression of some selenoproteins in the high glucose environment.These results provide clinical and experimental evidence for illuminating the role of low selenium-sensitive selenoproteins in the occurrence and development of DN.
ABSTRACT
Objective To construct the recombinant plasmids of normal and truncated selenoprotein S genes so as to observe their biological function in vitro or in vivo.Methods We constructed the recombinant plasmids of normal and truncated selenoprotein genes by gene recombinant technology.The gene of truncated selenoprotein was coding domain sequence(CDS)fragment of mRNA;the gene of normal selenoprotein was CDS and 3'untranslated region(including Sec insertion sequence)fragment of mRNA.We confirmed the sequence of recombinant genes by sending them to a company for comparison.The recombinant plasmids of normal and truncated genes of SelS were transfected into cells by Lipofectamine 2000.After 24 hours,the expression of green fluorescent protein was observed and transfection efficiency was detected by FACS analysis.We collected the cells to isolate the total RNA by TRIzol method,and then cDNA was obtained by mRNA reverse transcription and amplified by PCR.Results The sequencing results showed that the recombinant genes were completely the same as the target genes,indicating that we constructed the plasmids successfully.The expression of green fluorescent protein could be observed and transfection efficiency was detected up to 40% by FACS analysis.PCR results showed that the target selenoprotein gene was highly expressed in the experimental group than in control group.Conclusion The truncated and normal selenoprotein S genes were successfully constructed and transfected into cells where they were highly expressed.It lays foundation for observing the biological effect of truncated and normal selenoprotein in cell line or animal body.