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The middle of the last century, it was discovered that, in some cases restore blood flow, tissue and organ ischemia not only fail to reduce the damage to the contrary, lead to further aggravate the injury and organ dysfunction. This ischemic tissue in restoring blood perfusion, ultrastructural, metabolic, functional, and electrophysiological phenomenon further damage known as ischemia / reperfusion injury (ischemia in / reperfusion injury, IRI). In recent years, clinicians have found through shock therapy, cardiopulmonary cerebral resuscitation, angina coronary antispasmodic, cardio-cerebral vascular thrombosis, open heart surgery, percutaneous transluminal coronary angioplasty, organ and limb transplants, burns skin graft and other cases have occurred IRI is an important reason for the patient's condition deteriorated after surgery caused. How to reduce reperfusion injury has become clinically relevant disciplines, especially cardiothoracic surgery research focus. Gene therapy through gene into the body and play a role, can be given to the myocardium against acute myocardial injury, and no secondary surgery brings patients to combat, is bound to bring about a new dawn for the prevention and treatment of acute myocardial injury. The proto-oncogene is a normal cellular gene, a class encoding key regulatory proteins in cell survival, growth, development, differentiation process has important physiological functions, when the proto-oncogene activation may cause overactive cell growth and / or cell death is inhibited. Apoptosis is an acute injury of myocardial cells lost cause myocardial damage. The use of the proto-oncogene effective repair of damaged myocardial tissue and to achieve the purpose of the reversal of heart failure. Have been reported in the literature, myocardial ischemic injury by the original oncogene src, ras, vav tyrosine kinase-mediated pathway able to repair, thereby preventing the occurrence of heart failure. The original the cancer, Ji Yinsi / threonine kinase Pim-3 in structure with Pim-1, Pim-2 belong to Pim kinase family. Each member of the family is highly homologous to the kinase domain, its biological function is also a significant overlap exists. Other wire / threonine protein kinases such as PI3K-Akt, mTOR, Pim kinase family enables the serine / threonine phosphorylation of residues within the protein molecules that play a key role in cell growth pathway, and through different from PI3K-Akt-mTOR another pathway to promote cell survival and reproduction. Has been confirmed by a variety of growth factors, hormones and cell growth pathways such as the JAK / STAT, PI3K, Akt can be mediated Pim expression at different levels. Therefore, Pim kinases play an important role in the regulation of cell growth, and significantly inhibit apoptosis. Experiments show that early high-level expression of Pim-3 gene in the brain tissue around the ischemic injury. Early tissue ischemia, the expression of many genes were significantly inhibited, but Pim-3 gene expression were early induction, suggesting that it may be cell survival and functional recovery is important. Inspired by this project was to investigate in myocardial IRI Pim-3 gene with anti-apoptotic and has a protective effect on the survival of myocardial cells. The overall research ideas: First, using primary cultures of neonatal rat cardiomyocytes, hypoxia / reoxygenation (anoxia / reoxygenation, A / R) injury and hypoxic preconditioning (anoxia preconditioning, APC) protection model based on accepted detection method to confirm acute myocardial injury and the protective effect of a variety of classic and molecular biology detection methods to study the variation of Pim-3 mRNA and protein expression. Then, build Pim-3 expression plasmid, gene transfection technology processing cardiomyocytes. High expression of Pim-3 cells, using a variety of classic, accepted detection method for Pim-3 in the role of acute A / R injury model APC protection model, clarify Pim-3 gene expression in acute myocardial cell injury Significance of the time. Then, based on clear Pim-3 myocardium against A / R injury, we selected an important signal transduction pathway MAPKs pathways in cardiac cells, MAPKs, including three major subfamilies: ERK1 / 2, JNK and p38 MAPK to study exactly which or how many MAPKs subfamily mediated Pim-3 expression in the myocardial cell acute injury. Finally, to explore the mechanism of Pim-3 A / R injury against acute myocardial mitochondrial apoptosis signaling pathways start to clarify the mechanism of Pim-3 against acute myocardial injury, play a protective role. The first part: Pim-3 expression in acute myocardial cells A / R injury and the significance of the purpose of the establishment of the neonatal rat primary cultured myocardial cell acute A / R injury and APC protection model, using a variety of classic, the recognized physiological, biochemical such as detection indicators confirm that the model has been successful on the basis of using RT-PCR, Western Blotting detection methods such as molecular biology to explore Pim-3 expression and variation; build pcDNA3.1/Pim-3 gene expression plasmid, transfected into myocardial cells, and to investigate the protective effect proto-oncogene, Pim-3 on myocardial cell acute hypoxia / reoxygenation (A / R) injury. The experiment is divided into five groups, each repeated six times. ① Control group; ② A / R group; ③ APC A / R group; ④ empty plasmid pcDNA3.1 A / R group; ⑤ pcDNA3.1/Pim-3 the A / R group. Pim-3 mRNA and protein expression levels (RT-PCR, Western-blotting method) changes measured after the end of the experiment, the simultaneous detection of lactate dehydrogenase (LDH) in the culture medium activity, tetrazolium salt (MTT) colorimetric test to determine the cell survival, TUNEL assay of apoptosis. Results: 1. Myocardial cells Pim-3 mRNA expression in A / R group Pim-3 mRNA expression was significantly higher (0.37 ± 0.052) compared to the Control (control) group (0.05 ± 0.008) (p lt; 0.01), APC A / R group (0.75 ± 0.094) further increased (p lt; 0.01) similar pcDNA3.1 the A / R group Pim-3 mRNA expression with the A / R group, no significant difference (p gt; 0.05), pcDNA3 .1/Pim-3 A / R group Pim-3 mRNA expression (0.82 ± 0.11) compared with the A / R group is further increased (p lt; 0.01). Myocardial cells Pim-3 protein expression of the A / R group Pim-3 protein expression (1.07 ± 0.24) compared to the Control (control) group (0.3 ± 0.05) was significantly higher (p lt; 0.01), the APC the A / R group further increased (2.31 ± 0.37) (p lt; 0.01), pcDNA3.1 the A / R group Pim-3 protein expression of the A / R group similar (p gt; 0.05), pcDNA3.1/Pim-3. A / Pim-3 expression R group (2.57 ± 0.41) is further increased (p lt; 0.01). 3. Pim-3 gene on cell survival after myocardial cells in A / R injury, of LDH the impact of the A / R group cell viability was 36.7 ± 6.7% of LDH value of 27.1 ± 5.0, and Control (control) group cells significantly lower survival rate, LDH increased (p lt; 0.01); cell survival rate of the APC the A / R group was 80.3 ± 7.8% of LDH value of 8.4 ± 1.2, pcDNA3.1/Pim-3 A / R group and the APC A / cell viability and LDH values ??similar to the R group, no significant difference, but the two groups of cell viability compared with the A / R group were increased, LDH decreased (p lt; 0.01). Prompt myocardial cells transfected with Pim-3 against A / R injury-induced decline in cell viability and elevated LDH levels significantly. 30.4 ± 4.5% 4. Pim-3 gene on the A / R injury of myocardial cells apoptosis A / R group myocardial cells apoptosis Control group of few apoptotic cells (p lt; 0.01) ; APC A / R group, the apoptosis of myocardial cells pcDNA3.1/Pim-3 A / R group significantly reduced apoptotic index was 14.7 ± 2.7% and 17.0 ± 3.2%, respectively, with the A / R group difference significant (p lt; 0.01). Prompted myocardial cells transfected with Pim-3 can significantly reduce apoptosis induced by A / R injury. Discussion and conclusion cardiomyocyte apoptosis is an important feature of the A / R injury, APC is currently the most powerful known methods against A / R injury, the study confirmed that the APC against A / R injury mechanism is upregulated within endogenous protective proteins, these proteins can inhibit apoptosis of myocardial cells, which play a myocardial protection. The original the cancer, Ji Yinsi / threonine kinase Pim-3 in structure with Pim-1, Pim-2 belong to Pim kinase family. A large number of experimental studies have demonstrated that a clear correlation exists, the Pim kinase upregulation of apoptosis inhibition. Pim-3 gene in ischemic brain tissue damage around a high level of expression of the early, early tissue ischemia, the expression of many genes is significantly suppressed, Pim-3 gene expression but was induced upregulation tips may cell survival and functional recovery is important. Therefore, this study used primary cultured neonatal rat cardiomyocytes, the establishment of the A / R injury, APC protection model, results showed that hypoxic preconditioning significantly increased Pim-3 expression construct pcDNA3.1 / Pim-3 gene expression plasmid transfected into myocardial cells, results showed that exogenous Pim-3 gene import against A / R-induced acute injury, to play a similar protective effect of hypoxic preconditioning. The second part: p38 MAPK pathway mediated Pim-3 expression in the myocardial cell acute injury when the purpose of the establishment of the neonatal rat primary cultured myocardial cell acute A / R injury and APC protection model, with a variety of classic and recognized physiological detect indicators of biochemical and ultrastructural changes in the confirmation has been generated against acute damage to the A / R on the basis of a protective effect, were given specific inhibitor U0126, SP600125, and SB203850 blocking MAPKs pathway of ERK1 / 2, JNK, p38 MAPK three subfamilies. Western Blotting detection methods of molecular biology, detection of MAPKs pathway of ERK1 / 2, JNK, p38 MAPK three subfamilies phosphorylated protein expression levels of Pim-3 protein expression levels, research MAPKs pathway mediated original oncogene Pim -3 confrontation acute myocardial injury. The experiment is divided into six groups, each repeated six times. ① Control group; ② A / R group; ③ APC A / R group; ④ U0126 APC A / R group; ⑤, SP600125, APC A / R group; ⑥ SB203850 APC A / R group. Before hypoxia preconditioning with a final concentration of 10 μM SB203850, U0126, SP600125, and cells were incubated for 30 min, and then the line of APC and A / R processing. The MAPKs pathway of ERK1 / 2, JNK, protein levels of p38 MAPK phosphorylation and Pim-3 protein expression levels were measured after the end of the experiment, simultaneous detection of the activity of lactate dehydrogenase (LDH) in the culture medium, the tetrazolium salt (MTT) than color test determination of cell viability, TUNEL assay of apoptosis. Results of MAPKs pathway Pim-3 expression using specific phosphorylation of MAPKs antibodies were used to detect phospho-ERK1 / 2, phospho-p38 MAPK and phospho-JNK protein expression. Compared with the Control group, A / R group of phospho-ERK1 / 2, phospho-p38 MAPK protein levels were increased by 2.3 and 3.2-fold (p lt; 0.01), and, APC Group further increased by 2.6 and 3.2 times (p lt; 0.01). phospho-JNK in the A / R group than the Control group increased 1.4-fold (p lt; 0.05), but did not, APC expression of phospho-JNK increased further. Blockers were given three pathways, elevated phosphorylation induced by APC or A / R level is to cancel all prove U0126, SP600125, and SB203580 can specifically block of ERK1 / 2, JNK, and p38 MAPK activation. APC induced Pim-3 expression increased only in the p38 MAPK pathway is blocked significantly down-regulated (p lt; 0.01), that Pim-3 expression and activation of p38 MAPK, and ERK1 / 2, JNK, nothing to do. Therefore, following the results of the main study p38 MAPK pathway mediated Pim-3 expression on myocardial protection. SB203850 on cell viability, LDH A / R injury of myocardial cells A / R group and Control group cell viability decreased significantly, LDH increased (p lt; 0.01); the APC the A / R group and A / R group compared to cell survival rates were higher, LDH decreased (p lt; 0.01). Give p38 MAPK pathway specificity blockers, the protective effect of APC cancel 3. SB203850 on cardiac myocytes A / R injury in myocardial cell apoptosis in the A / R group 32.4 ± 4.5% apoptosis, Control group of apoptotic cells rarely (p lt; 0.01), the APC the A / R group apoptotic index is 13.8 ± 2.6%, and the difference between the A / R group was significantly (p lt; 0.01). P38 MAPK pathway and give specific blocker, the apoptotic index was 26.7 ± 4.1%, compared with APC A / R group differences significant (p lt; 0.01). Discussion and Conclusion In early experiments confirmed that hypoxic preconditioning significantly increased the expression of Pim-3, thus combating the A / R injury of myocardial cells, in this section we further explore hypoxia preconditioning upregulated Pim-3 involved myocardial cell signaling pathways. A number of studies have shown that, MAPKs ischemia, hypoxia, stretch, hormones, growth factors and cytokines and other extracellular stimuli-induced gene expression, cell proliferation, nuclear reaction common pathway or aggregation of point, and a variety of extracellular stimuli ( including ischemia, stress, hormones, cytokines, growth factors, etc.), whether it is through the activation of G-protein or protein tyrosine kinase can activate Raf → MAPK kinase (MAPK kinase) → MAPK phosphorylation chain reaction After a MAPK nuclear translocation bit, causing the reaction of the transcription factor phosphorylation, regulating the expression of proto-oncogenes and stress protein gene to promote increased protein synthesis concerned, the completion of extracellular stimuli. MAPKs include three main subfamilies: ERK1 / 2, JNK and of p38 MAPK. Caused by the activation of ERK1 / 2 such as growth factor stimulation of mitosis, leading to cell proliferation, differentiation, nuclear reactions; of JNK and of p38 MAPK in ischemia and hypoxia, is activated under stress, UV, cytokines and other stimuli, thus affecting different pathological under the conditions of the biological behavior of the cells and tissues and organs function. But the exact role of these three pathways in cells is not well understood. In the experiment, three paths were given specific blockers block the activation induced by APC, had no significant effect on the expression of Pim-3 was found to block the activation of ERK1 / 2 and JNK, whereas blocking The activation of p38 MAPK, Pim-3 expression is significantly decreased, suggesting that elevated Pim-3 expression induced by APC and p38 MAPK pathway. Part III: the role of the purpose of the establishment of the mitochondrial apoptosis signaling pathway in Pim-3 anti-myocardial acute injury neonatal rat primary cultured myocardial cell acute A / R injury models, with a variety of classic recognized physiological, biochemical on the basis of indicators, such as testing, confirm that acute A / R injury will build good pcDNA3.1/Pim-3 to expression plasmids were introduced into myocardial cells to explore the role myocardium against acute injury of the proto-oncogene Pim-3 with mitochondrial signal transduction pathways mediated by cytochrome C (Cyt C) release. Methods The experiment is divided into four groups, each repeated six times. ① Control group; ② A / R group; ③ pcDNA3.1 A / R group; ④ pcDNA3.1/Pim-3 the A / R group. Will have to build good plasmids pcDNA3.1 and pcDNA3.1/Pim-3, import myocardial cells, lactate dehydrogenase (LDH) activity detected in the culture after the end of the experiment, tetrazolium salt (MTT) colorimetric test to determine the cell survival by TUNEL method to detect cell apoptosis, spectrophotometric detection of myocardial mitochondrial permeability transition, Western blot detection Pim-3 protein expression levels, the dynamic changes of mitochondria, cytoplasmic Cyt C and caspase-3 activity. Results. Myocardial cells Pim-3 protein expression in A / R group Pim-3 protein expression (1.31 ± 0.22) compared with the Control group (0.25 ± 0.04) elevated the the pcDNA3.1 the A / R group protein A / R group no significant difference (p gt; 0.05) pcDNA3.1/Pim-3 A / R group Pim-3 expression is further increased (2.89 ± 0.48) (p lt; 0.01). 2. Pim-3 gene transfected cell viability after myocardial cells in A / R injury, the LDH impact A / R group and Control group significantly reduced comparing cell viability, LDH increased (p lt; 0.01), pcDNA3.1 A / R group cell viability, LDH values ??were 40.1 ± 6.7% and 30.2 ± 5.3, compared with the A / R group, no significant difference pcDNA3.1/Pim-3 the A / R group and A / R group than increased cell viability, LDH reduce, the difference was significant (p lt; 0.01). 3. Pim-3 gene transfection myocardial cells apoptosis after myocardial cells in A / R injury affect the A / R group 31.2 ± 5.2% apoptosis, apoptotic cells rarely Control group (p lt; 0.01 ); pcDNA3.1/Pim-3 A / R group apoptosis of myocardial cells significantly reduced apoptotic index was 18.7 ± 3.2%, the difference between the A / R group was significantly (p lt; 0.01). 4. Pim-3 gene transfection of mitochondrial permeability transition in myocardial cells in A / R injury in the A / R group, mPTP open, ODA540 decline in absolute value (ΔODA540) Control group significantly increased, the difference was statistically significance (p lt; 0.01); the pcDNA3.1 the A / R group ΔODA540 with the A / R group showed no significant difference (p gt; 0.05); the mPTP opening up of pcDNA3.1/Pim-3 A / R group compared with A / R group was significantly suppress, ΔODA540 with the A / R group compared to significantly reduce (p lt; 0.01) (see Fig.13). 5. Pim-3 gene transfection on the A / R injury of myocardial cells Cyt C release from the mitochondria to the cytoplasm in the Control group Cyt C present in the mitochondria, cytoplasmic small amounts, in the A / R group, Cyt C from mitochondria massive release to the cytoplasm pcDNA3.1/Pim-3 A / R group significantly inhibited Cyt C release from mitochondria to the cytoplasm, compared with the A / R group differences was significant (p lt; 0.01 ). 6. Pim-3 gene transfection on the level of caspase-3 in the A / R injury of myocardial cells in the A / R group caspase-3 (1.47 ± 0.16) compared with the Control group (0.52 ± 0.09) was significantly higher (p lt; 0.01), pcDNA3.1 the A / R group, the expression of caspase-3 (1.65 ± 0.17) compared with the A / R group, no significant difference (p gt; 0.05), caspase-3 pcDNA3.1/Pim-3 A / R group The level was significantly decreased (p lt; 0.01). Discussion and Conclusion This study confirmed Pim-3 gene was transfected into myocardial cells, the reversal of the A / R-induced acute myocardial injury Pim-3 gene produces myocardial protection mechanism to explore. Apoptosis is a cardiac myocytes A / R injury of myocardial cells lost cause myocardial damage, Pim-3 as a proto-oncogene against apoptosis, we hypothesized that Pim-3 against acute myocardial injury by inhibition of apoptosis which functions. Molecular biology of apoptosis mechanism is quite complex, in recent years, the mitochondrial signaling pathways mediated apoptosis mechanism much concern that the apoptotic process in mitochondria may play a central role in mPTP excessive openness by making resulting in mitochondrial swelling, outer membrane rupture, release of Cyt C to apoptosis occurred in the cytoplasm. The results of this study indicate that myocardial cells suffer damage to the A / R increased mitochondrial permeability transition, Cyt C release from mitochondria to the cytoplasm of a large number of Pim-3 transfected group was significantly inhibited mitochondrial swelling, thereby inhibiting the release of Cyt C from mitochondria to the cytoplasm but also improve the indicators of myocardial cells played a myocardial protective effect. These data demonstrate that the opening of the mPTP, cytochrome C release from mitochondria to the cytoplasm is triggered apoptosis key steps. When the after apoptotic stimuli factor induced cytochrome C release into the cytosol from the mitochondrial membrane gap, the the initiator caspase cascade reaction, the whole process of a positive feedback, the activation of caspases to its substrate specificity cutting, DNA fragmentation, cells apoptosis. Caspase network caspase-3 apoptosis execution, known as the \The experiment found that the A / R injury associated with caspase-3 activation, and Pim-3 transfected group inhibited the activation of caspase-3, prove Pim-3 anti-apoptosis through the mitochondrial pathway inhibition of caspase network and .
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