| Part 1Preparation of adenovirus vectors containing thymosin?4 gene sequenceobjective:In this study,the adenovirus vectors were constructed by plasmid with T?4gene,and were packaged and determined.Methods:In this experiment,AdMax as an eukaryotic system was used to package the adenovirus.Firstly,target gene sequence was analyzed and detected.Then,according to the result of gene sequence analysis,we designed and synthesized,enzymed,spliced it.The synthesized gene sequence was carried into pMD-18T vector and transmitted to DH5?cells.After sequencing and amplifying,the target gene sequence was made into the plasmid containing GFP coexpression.Primers for PCR reaction were designed and recombined to the vector of pAD/CMV/V5-DEST with Invitrogen company's systems.The constructed adenovirus expression vector was digested by Pac I and transfected into 293A cells.In this study,the titer of adenovirus was detected with the immunological method.Results:Adenovirus vector recombined plasmid was successfully produced.The titer of adenovirus was 2.12×1010ifu/ml.Conclusion:Adenovirus vector carried T?4 gene sequences can be produced by ways of whole-gene synthesis.Part 2 Study of thymosin ?4 gene modification to endothelial cells in vitro.Objective: To study the effect of T?4 gene modification to endothelial cells with the transfection technique of adenovirus vector.Methods: HUVEC after cultivated,were modified by adenovirus vector with T?4gene.Apoptosis was measured under the conditions of normal oxygen and hypoxia.Cells function was observed in different ways.Cells proliferation was detected by MTT assays.Cells migration was detected by Transwell methods.Expression of proteins in signal pathway was detected by Western Blotting.Results: Under the conditions of normal oxygen and hypoxia,the apoptosis in Ad-T?4 group decreased.Under the condition of hypoxia,the apoptosis rate was significantly reduced in Ad-T?4 group?12.35%?compared with that in Ad-NC group?21.24%,p<0.05?,and in Ad-T?4+Ly294002 group?20.94%,p<0.05?.MTT assays indicated that Cells proliferation significantly increased in Ad-T?4 group?proliferation rate: 60.04%?compared with control group?0%?and Ad-NC group?3.23%,p<0.05?after 72 hours.Transwell indicated that the number of migrating cells significantly increased under the condition of normal oxygen in Ad-T?4 group?54.56±3.09?,compared with Ad-NC group?42.11±2.71?and Ad-T?4+Ly group?14.89±2.80?.Expression of Caspase3 was no different under the condition of normal oxygen in Ad-T?4 group compared with other groups by Western Blotting,but decreased significantly under the condition of hypoxia.Expression of p-Akt increased under hypoxia in Ad-T?4 group,and decreased with Ly294002.Conclusion: T?4 gene can be transfected into HUVEC by adenvirus effectively.The modified endothelial cells have more viability and therapeutic value.Under the condition of hypoxia,T?4 can enhance the expression and enzymolysis activeness of PI3 K,and increase the excitation of Akt.Part 3 Study on the effect of adenovirus with T?4 gene on acute myocardial infarction in pigsObjective: To observe the effects of T?4 gene adenovirus on ischemic injury after acute myocardial infarction in animal experiments.Methods: 12 porcine models of acute myocardial infarction established successfully,were divided into 3 groups?Ad-T?4 group,Ad-NC group and control group?.They were respectively injected into coronary artery with T?4 gene adenovirus,Null vecter adenovirus and normal saline.On the second day and the 30 th day,the cardiac function and myocardial infarction area ratio were measured with 3.0T MRI.The content of T?4 was determined by ELISA method from the homogenate of infarcted myocardial tissue after centrifugation.Results: There were no significant difference in myocardial infarction area ratio and left ventricular ejection fraction between 3 groups on second day after operation.On30 th day,The infarction area ratio?%?significantly decreased in Ad-T?4 group?22.6±3.2?compared with Ad-NC group?28.0±2.7,p<0.05?and Control group?27.1±1.5,p<0.05?.The ejection fraction increased in Ad-T?4 group?48.75±3.77%?compared with other 2 groups.And the difference was significant only compared with control group?41.25±2.99%,p<0.05?.The content of T?4 increased in Ad-T?4 group?9.039±2.531ng/ml?compared with other 2 groups,p<0.05.Conclusion: T?4 gene adenovirus has a beneficial effect on ischemic injury after acute myocardial infarction,and improve the cardiac function,promote angiogenesis.Part 4 The effect of hypersplenism on the level of T?4 in patients with heart failureObjective: To observe the effects of oxidative stress product and the level of thymosin?4 and the endothelial cell function in patients with hypersplenism combined with heart failure.And to reveal the mechanism of hypersplenism to aggravate heart failure.Methods: There were 33 patients with hypersplenism and 35 patients without hypersplenism combined with chronic heart failure?NYHA grade III or IV?.EPCs were isolated by MACS,quantified by PCR.Apoptosis was assessed by TUNEL analysis.Serum concentrations of VCAM-1,ICAM-1,P selectin,E selectin,free plasma hemoglobin and T?4 were detected by ELISA.The serum concentration of free heme was detected by hemosiderin detection.Results: Free plasma hemoglobin?pg/ml?and heme?pg/ul?were significantly increased in CHF patients with hypersplenism compared with in the control group?307.75±130.49 vs.212.46±121.11,P<0.001;and 1.96±0.82 vs.1.26±0.77,P<0.001,respectively?.The expression levels of VCAM-1,ICAM-1,P selectin and E selectin were higher in CHF patients with hypersplenism than in the control group?p<0.001?.After 24 months,compared with the control group,LVEF was significantly decreased in CHF combined with hypersplenism?41.06%±7.77% vs.47.05%±10.62%,P=0.01?.Free plasma hemoglobin and heme levels were significantly correlated with LVEF?r=-0.29,P=0.015;and r=-0.28,P=0.020,respectively?.The proliferation of EPCs in CHF combined with hypersplenism decreased significantly.After 6 hours from ischemic stimulation,TUNEL+ EPCs?%?was significantly increased compared with the control group?36.5±11.8 vs.21.2±7.4,P<0.001?.The level of T?4 in the group of CHF combined with hypersplenism was significantly lower than in the control group?5.80±3.85 ng/ml vs.8.34±4.06 ng/ml,p<0.05?.By linear regression,we found that LVEF as a dependent variable was positively correlated with the level of T?4?R=0.331,P=0.006?.Conclusion: Hypersplenism deteriorates cardiac function by activating oxidative stress response and endothelial dysfunction.Thymosin?4 decreases in hypersplenism and is associated with heart failure. |
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