Transfecting Retroviral Vector Expressing Livin And Its Effects On Cardiac Ischemia-reperfusion Injury In Rats

Partâ… Construction and production of retroviral vectorexpressing LivinObjectives: Construction a retroviral vector carrying Livin cDNA. To explore the effect of Livin over-expression on cardiac ischemia reperfusion.Methods: Livin gene from mice MA782 cell was amplificated by reverse-transcription polymerase chain reaction (RT-PCR) and first cloned into pGEM-T vector. Then digested by Xhoâ… /Hindâ…¢and identified by PCR.And then subcloned into the retroviral vector pLNCX₂. pLNCX₂-Livin was transferred into packaging cell PT67 after Xhoâ… /Hindâ…¢digestion and identified by PCR. PT67/pLNCX2-Livin cells were selected. A pure packaging cell line/pLNCX2-Livin cells expressing Livin was cultured. The virus titer was assayed.Results:816bp Livin cDNA was obtained by RT-PCR amplification. The recombinant vector pLNCX2-Livin containing Livin gene was confirmed. The virus titer of the pure packaging cell line/ PT 67/ pLLNCX2-Livin was 3.9×10⁷ IU/ml.Conclusion:A kind of recombinant retroviral vector pLNCX2-Livin as well as a pure packaging cell line producing high virus titer are constructed successfully. PART 2 Effects of Livin on Myocardial Ischemiareperfusion Injury in RatsObjectives: To observe the livin protein expression in rat myocardial after the transfection of retroviral vectors and its effects on ischemia-reperfusion (I/R) injury.Methods: (1)90 healthy SD rats were divided into Contrl group, I/R group and Livin group. Rats were subjected to 30 min of left coronary artery occlusion followed by 120 min of reperfusion with (Livin group) or without (IR group) treating the rats with retrovirus vector expressing Livin protein by intramyocardial injection 24h before left coronary artery occlusion. Rats in Control group underwent thoracotomy without coronary ligation and injection of vector.(2) No-reflow area was detected by thioflavin S and the myocardial infarction size was evaluated by TTC dyeing method.(3)Caspase-3 and Livin expression detected with S? immunohistochemical staining.(4)Cardiomyocytes apoptosis was determined with terminal deoxynucleotidyl transferase-mediated Dutp-fluorescein nick end labeling( TUNEL) method .Results:1.The expression of Livin was detected in myocardial cells by immunohisto- chemistry. The level of Livin was increased by Ischemia-reperfusion injury. There is no statistical significance between IR group (3.10±0.52) and Control group (2.35±0.47).2.The level of Caspase-3 increased significantly during ischemia-reperfusion; There was significant diference between IR group(103.39±8.24) and Control group (91.39±4.82). The expression of Caspase-3 was decreased after livin injection, there was significant difference between Livin group (97.43±11.15) and IR group.3. The infarct size (12.80±1.19 vs 20.82±2.82) and the no-reflow area (19.39±6.25 vs 25.93±6.45) were also decreased in Livin groups compared with the IR hearts.4. Using TUNEL assays, we examined the presence of apoptosis during I/R, which was significantly higher in IR group compared with the control hearts (10.35±3.34 vs 1.1±0.42). Apoptosis rate was significantly reduced after Livin rejection (1.7±1.57) versus IR, but had no difference with control group. Conclusion1.There is Livin expression in normal rat myocardial.2.Retrovirus vector expressing Livin protein could express successfully in rat myocardial.3.Livin could inhibit caspase-3 expression in myocardial during I/R.4.Livin inhibits apoptosis, reduces no-reflow area and infartion size in myocardial, and protects the heart against ischemia/reperfusion injury.PART 3 Caspase-3, Livin mRNA detection by real time polymerase chain reactionObjectives: To observe the livin and Caspase-3 mRNA expression in rat myocardial with and without retroviral vectors transfection and its effects on ischemia-reperfusion (I/R) injury.Methods: Both Caspase-3 and Livin mRNA expression were detected by real time polymerase chain reaction (PCR).Results: The livin mRNA expression was positive in normal rats detected by real time quantification PCR. The livin mRNA expression was (8.41±1.39)×10^-3 in control group, (7. 45±2. 51)×10^-3 in vector without livin gene group, (7.82±3.22)×10^-3 in IR group, (145±89)×10^-3 in Livin group. Compared with the Control and IR groups, Livin vector transfection significantly increased Livin mRNA expression.The Caspase-3 mRNA expression was (5.12±2.11)×10^-3 in control group, (6.88±2.31)×10^-3 in vector without livin gene group. Compared with the control group, I/R significantly increased Caspase-3 mRNA expression [(92.1±34.6)×10^-3, I/R group , P<0.01], which indicated that reperfusion could increase Caspase-3 mRNA expression. The Caspase-3 mRNA expression was (56.2±21.1)×10^-3 in Livin group, which was significantly decreased compared to IR group (P<0. 05) . These suggest that Livin overexpression could depress Caspase-3 expression in myocardial during I/R. Conclusions:1.There is Livin expression in normal rat myocardial.2.Retroviral vector expressing Livin protein could express successfully in rat myocardial.3.Livin could depress caspase-3 expression in myocardial during I/R.