Gene Cloning Of Hypoxia-inducible Factor-1α And Expression Research Of HIF-1α-pcDNA3.1

Coronary Heart Disease (CHD) is a kind of heart diseasecharacterized by ischemia and anoxia of cardiac muscle. CHD seriouslydamages our health due to its high morbidity and mortality. In China, themorbidity and mortality of CHD is increasing year by year. It has beenone of the widespread diseases that threaten people's health. Nowadays,the treatments of CHD mainly focus on drugs, operation, intervention andso on. Myocardial cell is a kind of cell that differentiation at the end stageand can't regenerate. After myocardial infarction, it can only be repairedthrough the hyperplasia of fibrous tissue and the formation of scar tissue.So the treatments mentioned above are unable to enhance the regenerateof infracted myocardial cell and improve the long-term prognosis ofmyocardial infarction patients significantly. Therefore it is urgent inrecently clinical study to find a novel and better treatment that can repairand regenerate the necrotic myocardial cell, prevent or slow thereconstruction of ventricle and heart failure.As a brand-new treatment, gene therapy (therapeutic angiogenesis)and cellular therapy (cellular cardiomyoplasty) have been an interestingfield in cardio-vascular area.Hypoxia-inducible factor-1(HIF-1) is a neucleoprotein withtranscription activity. HIF-1 can not only promote the transcription andexpression of many VGF and its receptor, increase the regeneration ofvesscle, raise the filling of blood flow, but also enhance the adaption ofischemic myocardial cell in anoxia condition, intensify the process ofzymolysis, and supply myocardial cell with more energy. So it is possiblethat the therapeutic effect of HIF-1 would be significantly better than thatof VEGF or FGF simply.Cellular treatment is a method that transplants self or variant cellswith differentiative potency into human tissue which can proliferate into tissue cells needed in certain condition, then play a role of repairing tissueand recovering organ. It might cooperate and promote the function ofgene or cellular treatment mutually if we could transplant HIF-1 gene intotarget cells to gain effective expression and then transplant cells withexogenous HIF-1 gene into infarctive area. This combinative effectshould be superior to the single effect.This experiment is divided into two parts. PartⅠ: Gene cloning ofHIF-1αand construction of the HIF-1α-pcDNA3.1 expression plasmid.PartⅡ: transfection research of Hela cells with the HIF-1α-pcDNA3.1vector.PartⅠ: Gene cloning of HIF-1αand construction of the HIF-1α-pcDNA3.1 expression plasmid.Objective: Construct HIF-1αgene into pcDNA3.1 vector, thensequence and confirm it.Method: mRNA extracted from myocardial cell and was used as thetemplate to obtain cDNA through reverse transcription. Then to amplifytarget gene HIF-1αby PCR, construct it to pGEM-T vector, followed bysequencing on ABI3100, then HIF-1αfragment was double enzymedigestion and transfer to pcDNA3.1 vector.Result: agarose gel electrophoresis showed the intrest fragmentamplified by RT-PCR was about 2480bp. the sequencing result iscoincident with GenBank'report, which means HIF-1α-pcDNA3.1 wasconstructed successfully.Conclusion: HIF-1αwas constructed into the pcDNA3.1 vectorsuccessfully. There are no mutations by sequencing confirmationPartⅡ: Transfection research of Hela cells with HIF-1α-pcDNA3.1construct.Objective: Expression research of the constructed HIF-1α-pcDNA3.1in uterine cervix cancer cells(Hela).Method: To transfect HIF construct into Hela cells by using positiveiron liposome method, and then by fluorescent staining to confirm theefficiency of transfection. Followed by Western Blot confirm of theexpression of HIF-1α, then selected the stable expression cell line withG418. Result: 24 hours after the transfection of the HIF-1α-pcDNA3, theslides of fluorescent staining were observed under fluorescencemicroscope. The transfection efficiency calculated is about 30%.Conclusion: The transfection efficiency of the HIF-1α-pcDNA3 intoHela cells is about 30%, and a Strain of HIF-1α-pcDNA3.1 with stableexpression was screened, which provided the base for the further study.