Telomerase Reverse Transcriptase Gene Expression In The Myocardium And Bone Marrow Stromal Cell Culture Solution On Myocardial Protection Mechanism

PART 1. Effects of hypoxia on promoter of Telomerase reverse transcriptase and cell cycle distribution in neonatal rat cardiac myocytesObjective Currently, researchers have attached importance to the role of telomerase in cardiac muscle cell growth and survival. The chief aim of the present study is to investigate the effects of hypoxia on cell cycle distribution and expression of Telomerase reverse transcriptase (TERT) in neonatal rat cardiac myocytes ( CMs) and roughly understand how hypoxia affects activity of TERT promoter, and elucidate the implication of self-protective mechanisms to repair damaged myocardium before the remodeling or apoptosis process of the pathologic heartMethods Cultured neonatal rat CMs were divided into two groups, namely normal control and hypoxia groups cultured in an incubator 95%N₂ and 5%CO₂ for 6 and 12 hours. CMs viability under hypoxia was measured by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide colorimetry (MTT). and the percentage of cells in G1, S and G2 /M phase by flow cytometer (FCM). The expression of TERT was quantified by RT-PCR. Green fluorescent protein (GFP) was used as reporter gene. The plasmids (pEGFP) carrying TERT promoter were constructed and transfected into CMs. After hypoxia treatment. GFP activity of cells was detected by laser confocal microscopy.Results Compared with normal controls(11.28 ± 3.00), the percentage of cells in S phage increased in 6h group(21.27 ± 5.70,P>0.05),decrease in 12h group(7.98 ± 0.98,P<0.01). TERT was expressed in fetal and neonatal rat CMs. The expression of TERT was significantly decreased after 5 days, and was higher with hypoxia for 6 hours than that without hypoxia. A trend of increasing GFP activity of cells with hypoxia was noted.Conclusions The percentage of cells in S phage and the expression of TERT was increased in 6h group, CMs experience a process of proliferation prior to the death and the apoptosis due to hypoxia .With roughly understanding, hypoxia may play a role in the activation of promoter of TERT. This finding reveals that CMs, to a certain extent, have capability of proliferation.PART 2.1 Expression of CyclinDl and CDK4 in neonatal rat cardiac myocytes under hypoxiaKeywords Cardiac myocytes; Hypoxia; Cell cycle distribution; CyclinDl; CDK4Objective To investigate the the effects of hypoxia on cell cycle distribution and protein expression of CyclinDl and CDK4 in neonatal rat cardiac myocytes under hypoxiaMethods Cultured neonatal rat CMs were divided into two groups, namely normal control and hypoxia groups cultured in an incubator 95%N2 and 5%CO2 for 6 hours and 12 hours. The percentage of cells in G1, S and G2 /M phase was measured by flow cytometer (FCM), and theexpression of CyclinDl and CDK4 by immuno-cytochemistry and image analysis.Results Compared with normal controls, the percentage of cells in S phage was increased in 6h group(P<0.01),decrease in 12h group(P<0.05). The expression of CyclinDl and CDK4 was significantly increased under hypoxia for 6 hours than that without hypoxia.Conclusions The percentage of cells in S phage and the expression of CyclinDl and CDK4 was increased in 6h group, CMs experience a process of proliferation prior to the death and the apoptosis due to hypoxia . This finding reveals that CMs, to a certain extent, have capability of proliferation.PART 2.2 Expression of TERT and proto oncogenes in myocardium of rat with myocardial infarction and its significance Objective To investigate the changes of expression of TERT, c-myc , c-fos and c-jun to evaluate the self-protection and proliferation potency in ischemic myocardium after acute myocardial infarction. To probe into the correlation between TERT and proto oncogenes.Methods Infarcted rats were divided into 9 groups, 2 hours, 6 hours , 1 days, 3 days, 5 days, 7days, 9days, 12days, 15 days. Tracheotomy was performed and an intubation cannula was connected to a rodent ventilator. The left thoracotomy was performed at thefifth intercostal space. The pericardium was mcised and the heart w...