ABSTRACT
OBJECTIVE: To construct a recombinant vector that expresses 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem and its effect in vitro METHODS: Ventricular myocytes were collected from newborn Wistar rats and cultured. Five sites targeting on the rat Kir2.1 gene were selected. Accordingly 5 pairs of oligonucleotide fragments were designed, synthesized, and annealed to obtain double-stranded DNAs. The 5 pairs of oligonucleotide were then cloned into the vector pGenesil-1 by repeated excision and ligation successively. The tandem recombinant vector pEGFP6-1Kir2.1 was thus constructed and transfected into the cultured rat myocytes. RT-PCR and Western blotting were used to detect the mRNA and protein expression of Kir2.1 in the myocytes. Sequence not related to Kir2.1 sequence with mismatched bases was designed and used as control. RESULTS: A recombinant vector that expresses 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem was constructed. 96 hours after the transfection RT-PCR showed that the Kir2.1 mRNA transcription was suppressed by 83, 6%, and Western blotting showed that the Kir2.1 protein transcription was suppressed by 68.1% in comparison with the control. CONCLUSION: The vector that expresses the 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem is able to suppress the expression of Kir2.1 in rat ventricular myocytes. Application of such vector may be a new method to produce a new type of heart biological pacemaker.
Subject(s)
Gene Silencing , Genetic Vectors , Myocytes, Cardiac/metabolism , Potassium Channels, Inwardly Rectifying/genetics , RNA, Small Interfering/genetics , Animals , Animals, Newborn , Cells, Cultured , Myocytes, Cardiac/cytology , Potassium Channels, Inwardly Rectifying/biosynthesis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Wistar , Tandem Repeat Sequences , TransfectionABSTRACT
OBJECTIVE: To review the surgical treatment of supracardiac type of total anomalous pulmonary venous connection (TAPVC). METHODS: Twenty-four patients with supracardiac total anomalous pulmonary venous connection underwent surgical correction from July 1995 to June 2004. There were 11 males, 13 females. The patients aged from 40 days to 35 years (mean 4.5 years). Twenty-three patients were under 6 years old. The weight was from 3.8 to 54.0 kg (mean 17.5 kg). Ten patients were through right atrial incision, 8 through double-atrium incision to anastomose the left atria and the common pulmonary veins. Two used large patches separating the portal of the common pulmonary veins and the enlarged atrial septal defects to the left atria. The left atrium and the common pulmonary veins were anastomosed in one case using a large patch separating the portal of the common pulmonary vein and the enlarged atrial septal defect to the left atrium. Three anastomosed the top of the left atria and the common pulmonary veins. RESULTS: One patient died postoperatively of low cardiac output syndrome. One with cardiac tamponade after operation was cured by reoperation. Six with arrhythia were all cured. One with acute pulmonary edema was cured by reopen of the vertical vein and closed later. The postoperative follow-up period ranged from 3 months to 7 years (mean 2.5 years). One complicated severe supracardiac arrhythia 2 weeks later. One with late pulmonary occlusion was cured by reoperation. There was no long term complication of the others. CONCLUSIONS: The operative methods of TAPVC depend on the cardiac deformation. Proper surgery timely treatment of the postoperative complications may achieve a satisfactory result. The method of only use patch in the right atrium should select proper repair material. Anastomosing the top of the left atrium and the common pulmonary vein is a good method. It is easy for exposure and operating, meanwhile it has low occurrence of arrhythmias and should be recommended.
