Restoration Of Impaired Myocardial Sarcoplasmic Reticulum Calcium Uptake By Overexpressing Sarcoplasmic Reticulum Calcium ATPase And Knocking Down Phospholamban Through Recombinant Adenovirus And Adeno-associated Virus

[Background] Congestive heart failure (CHF) is an important field in modern medical research. The calcium regulation not only influences the constriction and heart rate, but also plays important role in the process of hypertrophy and remodeling.In the calcium handling proteins, the ratio of PLN/SERCA2a reflexes the heart inotropic function. During CHF, the calcium pump function is decreased, subsequently calcium uptake is down-regulated, meanwhile the expression level of SERCA2a is lowered, and PLN expression is upregulation with abnormal phosphorylation, so the ratio of PLN/SERCA2a increased. Some biotechniques like transgenic technique (eg. SERCA2a, mutant PLN gene), antisense technique etc can enhance the constractility and the dynamics condition of CHF animal model can improve.RNA interference (RNAi) is a form of posttranscriptional control in which the double-stranded RNA (dsRNA) leads to specific degradation of mRNAs with complementary sequence. The evidence that RNAi occurs in cultured human cells suggests that this procedure could be developed as a powerful tool for gene therapy in humans.We make SERCA2a overexpressed in cardiac myocytes through virus vector, meanwhile knock down PLN expression through RNAi vector, in order to decrease the PLN/SERCA ratio, to improve SR calcium transport activity, to decrease the calcium concentration in the plasma in diastolic period, and to promote inotropic function. We constructed a rAAV containing 2-adrenal receptor gene as well.[Methods] After inserted SERCA2a (-18 - +3486) cDNA into the down stream of CMV promoter in shuttle vector pAdTrack-CMV plsmid of Adeasy adenovirus system and recombinated in E.coli BJ5183, and then delivered into 293 cells, we harvested the recombinant adenovirus rAd-trSERCA2a. Southern Blot analysis revealed that the SERCA2a gene was integrated into the virus vector. Hereditary stability in different generation rAV-SERCA2a was determined by PCR.Cardiac myocytes of newborn rats were isolated by collagenase and trypsin digestion and were cultured in DMEM. Determined the purity of cardiac myocytes by calculating the percent of beating cell or actin positive cell by immunohistochemistry.Three days after infected the cultured cardiac myocytes with rAd-trSERCA2a, the cardiac myocytes were collected. RT-PCR and Western blot were performed to semi-quantity the expression level of SERCAla.The BHK-21 cell was transfected by pSNAV-SERCA2a plsmid, which inserted SERCA2a cDNA into the down stream of CMV promoter. Selected by G418, the cell line BHK/SERCA2a containing SERCA2a expression box was got. To rescue the recombinant virus rAAV-SERCA2a, the BHK/SERCA2a cell line was infected by HSV1-RC/AUL2 after cultured to large quantity.rAAV was labelled with FITC to observe the dynamic infection process of rAAV in cardiac myocytes by confocal microscope. The GFP expression ratio in cardiac myocytes after transfected by rAAV-GFP was measured by flow cytometry and fluorescent microscope. RT-PCR was performed to analysis SERCA2a expression in BHK-21 cell infected with rAAV-SERCA2a.U6 snRNA and H1RNP promoter was cloned by semi-nested PCR. The target sequence was chosen in PLN mRNA (NM₀22707), Ril(59-78nt), Ri2(73-93nt) and Ri3(103-123nt). Constructed the plasmid pSNAV-phRil by adaptor method, and the plasmid pSNAV-phRi2,3 by PCR method. Their accuracy is determined by sequencing.The recombinant virus rAAV-phRil was packed and produced. RT-PCR and Western blot were performed to semi-quantity PLN expression in cardiac myocyte transfected by three pSNAV-phRi plasmids or infected by rAAV-phRil.The long hairpin duplexes were constructed by linkage two segments (corresponding to -108⁺552 and +8⁺552)cloned from phospholamban mRNA with 5'-5" head-to-head mode, which transcriptional RNA would form to a 541bp stem and a 116nt loop structure. Then the duplexes were inserted into the pTract-CMV plasimd under CMV promoter of the AdEasy system to product recombination adenovirus rAd-TCPLN-lhRNAi. The conformity and hereditary stability...