Construction Of Eukaryotic Expression Vector Containing Gene Cystatin C And The Effect Of Cystatin C On Human Vascular Smooth Muscle Cells Apoptosis Induced By Ox-LDL

OBJECTIVECystatin C,a strongly unglycosylated cationic low-molecular weight protein,is almost freely filtered across the glomerular membrane.The filtered cystatin C is reabsorbed and catabolized almost completely in proximal renal tubular cells. Consequently,cystatin C has been extensively studied as an potential endogenous serum marker of glomerular filtration rate.Furthermore,the recent studies showed that cystatin C correlates closely with the development of atherosclerosis,aneurysm and restenosis after percutaneous transluminal coronary angioplasty.To clone human cystatin C gene and construct its eukaryotic expression vector and obtain its actived protein are very important for approaching further bionomics,searching for potential signal pathways and gene intervention and therapy.Vascular smooth muscle cells(VSMC)play an influential role during the course of development of atherosclerosis,aneurysm and restenosis after percutaneous transluminal coronary angioplasty.The VSMC's apoptosis is an important physiological regulation style for the quantity of vessel wall cells.To study the factors that affect the VSMC's apoptosis is very significant,which may help finding new intervened targets to restrain the apoptosis of VSMC,strengthening the stability of atherosclerosis advanced stage plaque,preventing and delaying the growth of aneurysm and regulating the vessel remodeling during the course of restenosis.As an important protease residing in intracellular and extracellular,Cystatin C deserves further study in the aspect of regulation to VSMC. Therefore,to culture VSMC with high lipoids in vitro,to construct the apoptosis model of VSMC induced by oxidized low density lipoprotein(ox- LDL),to transfect cystatin C to VSMC and to detect the apoptosis items are very significant for researching the peculiarity of cystatin C in cardiovascular disease and doing the groundwork for animal studies.METHODS1 To culture the human umbilical vein endothelial cells cloned line(HUV-EC -C)2 To culture and identify the human VSMCHuman arteriae aorta was obtained from elective abortions.Human VSMC were isolated and cultured from original cells to passage cells,and assessed byα-actin immunochemistry staining method and inverted phase contrast microscope.3 To extract the total RNA and RT-PCRThe total RNA was extracted from HUV-EC-C based on the direction of TRIzol kit.To make use of PCR primer,cystatin C gene was obtained including BamH and Xbaâ… restriction enzyme situs.4 To construct eukaryotic expression plasmid pCDNA3.1-Cystatin CCystatin C obtained through gelatum connected with pMD-18-simpleT vector, and then transformed to competence colibacillus by heat shock.To extract the recombinant plasmid and to obtain cystatin C gene by restriction enzyme,and then pCDNA3.1 expression vector was transformed to competence colibacillus by heat shock.Through gelatum the vector frag was obtained by restriction enzyme including BamH and Xbaâ… situs.Cystatin C connected with the vector frag by T₄-joiningenzyme was transformed to competence colibacillus by heat shock and the competence colibacillus was cultured.After identified by PCR and restriction enzyme including BamH and Xbaâ… situs,the most masculine plasmid was extracted for transfection.5 To construct the model of human VSMC expressing cystatin CUsing liposome-mediated transfection,the eukaryotic expression vector pCDNA3.1-Cystatin C was transfected into human VSMC and cultured the human VSMC for 12 hours,24 hours and 48 hours respectively.The expression of cystatin C in the human VSMC was proved by RT-PCR and Westem blot.6 To construct the apoptosis model of human VSMC induced by ox-LDLThe human VSMC were induced by ox-LDL and the cells' survival rate was detected by MTT.Afterthat,the best concentration and time points of inducing by ox-LDL were determined.The human VSMC in exponential phase of growth were randomly divided into normal control group,pCDNA3.1-Cystatin C-treaded group, ox-LDL-induced group and ox-LDL-induced+ pCDNA3.1-Cystatin C-treaded group.7 To detect apoptosis related items of every group cellsThe quantities of lactate dehydrogenase(LDH),reactive oxygen species(ROS), adenosine triphosphate(ATP)and activity of Caspase-3 were examined in VSMC induced by ox-LDL for 24 hours.The expression of Bax and Bcl-2 were examined by reverse transcription polymerase chain reaction(RT-PCR)and Western-blot.RESULTS1 The human VSMC were cultured successfully.2 The human cystatin C gene was cloned successfully,and the eukaryotic expression vector pCDNA3.1-Cystatin C was constructed correctly and was expressed steadily in human VSMC.3 Compared with normal control group,the expression ofLDH,ROS,ATP, Caspase-3,Bax and Bcl-2 in pCDNA3.1-Cystatin C-treaded group had no significant difference.Compared with ox-LDL- induced group,the quantities of LDH,ROS and the activity of Caspase-3 in ox-LDL-induced +pCDNA3.1-Cystatin C-treaded group were decrease significantly(P＜0.01),while the quantity of ATP was notably increase (P＜0.01).Compared with ox-LDL-induced group,the expression of Bcl-2 by RT-PCR and Western- blot analysis were significantly increased in ox-LDL-induced + pCDNA3.1- Cystatin C-treaded group,while the expression of Bcl-2 was markedly decreased(P＜0.05).CONCLUSIONS1 By PCR and T-A cloning technique,the human cystatin C gene was cloned successfully,and the eukaryotic expression vector pCDNA3.1-Cystatin C was constructed correctly and was expressed steadily in human VSMC.2 Cystatin C could inhibitthe apoptosis of VSMC induced by ox-LDL,which may correlated with improving the stability of cellular membrane and the function of chondriosome,and up regulation of Bcl-2 expression and down regulation of Bax expression.INNOVATION AND LIMITATION1 InnovationPrevious studies focused on the relationship between serum concentration of Cystatin C and human diseases.This study emphasized on the genie investigation of Cystatin C.By cloning human Cystatin C gene and constructing its eukaryotic expression vector and transfecting it to human VSMC and detecting the influence of Cystatin C on VSMC apoptosis induced by ox-LDL,the basic effect mechanism of Cystatin C on VSMC apoptosis was observed, which may be helpful for the study of mechanism of Cystatin C during the course of artherosclerosis development and will be useful to studying the gene therapy and intervention related to Cystatin C.2 LimitationBeacuse time and fund for scientific research were limited,the cells' detailed signal pathways of effect of Cystatin C on VSMC apoptosis induced by ox-LDL were not detected at the same time.Furthermore,the interaction between Cystatin C and other extracellular proteases deserves being studied.