Effects Of Tertramethylpyrazine And Rat CTGF MiRNA Plasmids On CTGF, TGF-β In High Glucose Stimulated HSC

OBJECITVE:To explore the effects of tetramethylpyrazine（TMP）and rat CTGF miRNA plasmids on the expression of CTGF，TGF-β of rathepatic stellate cells(HSC)，which provide theory foundation to find a newmethod of treatment of liver fibrosis。METHODS: According to the rat CTGF mRNA sequence, threekinds of rat CTGF miRNA plasmids and one kind of negative plasmidswere designed and synthesised, then transfected into rat hepatic stellatecells respectively, Plasmids’ instantaneous transfection rate of hepaticstellate cells were observed by the fluorescence microscope after plasmidswere transfected into rat hepatic stellate cells. CTGF mRNA expressionquantity were detected through the RT–PCR,then chose the highestinterference efficient rat CTGF miRNA plasmids.The chosen CTGFmiRNA plasmids and the negative plasmids were transfected into hepaticstellate cells, through the Blasticidin S HCl’s sustained pressure filter toobtain the rat hepatic stellate cells of stable expression rat CTGF miRNAplasmids and the rat hepatic stellate cells of stable expression negativecontrol plasmids.Rat hepatic stellate cells, rat hepatic stellate cells of stable expression rats CTGF miRNA plasmids, rat hepatic stellate cells of stableexpression negative plasmids were cultured, After TMP and highglucose’s stimulus,extracted total RNA,total protein for the furtherdetection of the expression of CTGF,TGF-beta and collectded the cellsupernatant fluid for the further detection of the expression of collagenⅠ.RESULTS: we filter out the highest interference efficient rat CTGFmiRNA plasmids through the RT-PCR method.We obtain the rat hepaticstellate cells of stable expression rats CTGF miRNA plasmids, rat hepaticstellate cells of stable expression negative plasmids. CTGF mRNA’sexpression quantity and CTGF protein’s expression quantity of the highglucose control group were significantly higher than blank control group（P<0.01）；TGF-beta mRNA’s expression quantity and TGF-beta protein’sexpression quantity of the high glucose group were obviously higher thanblank control group （P<0.01）；collagenⅠ’s expression quantity of thehigh glucose group were significantly higher than blank control group（P<0.01）.Compared to the high glucose group,TMP group significantlyreduced the expression quantity of CTGF mRNA and CTGF protein（P<0.01）; TMP group significantly reduced the expression quantity ofTGF-beta mRNA and TGF-beta protein（P<0.01）; TMP group significantlyreduced the collagenⅠexpression quantity（P<0.01）；rat CTGF miRNAplasmids interference group significantly reduced the expression quantityof CTGF mRNA and CTGF protein（P<0.01）; rat CTGF miRNA plasmids interference group significantly reduced the expression quantity ofTGF-beta mRNA and TGF-beta protein（P<0.01）; rat CTGF miRNAplasmids interference group significantly reduced the collagenⅠexpressionquantity（P<0.01）. TMP group compared with rat CTGF miRNA plasmidsinterference group, CTGF mRNA and protein expression quantity had nostatistical differences (P>0.05); TMP group’s collagenⅠ expressionquantity were lower than rat CTGF miRNA plasmids interference groupand the difference was statistically significant (P <0.01).CONCLUSION: We concluded that:high glucose could increase theexpression quantity of CTGF、 TGF-beta、 collagen Ⅰ,TMP and ratCTGFmiRNA plasmids could reduce the expression quantity of CTGF、TGF-beta、collagenⅠ.TMP was obviously important on the effects of liverfibrosis,and had a strong clinical practical value.