| ObjectiveObjective1:As we all know,there is difference in some cardiac-celebrovascular diseases between men and women during some particular stages.The study of androgen receptor(AR)on cardial vascular diseases becomes a major important field.The relationships between(CAG)n of AR and cardial vascular diseases have no identical results,and there are no results about the relationships between(CAG)n of AR and celebral vascular diseases.Our study aim is to analysis the CAG polymorphisms of androgen receptor gene and to explore the relationship between CAG polymorphisms of androgen receptor gene and myocardial infarction and acute cerebral infarction in men.Objective2:To set up BM-MSCs culture model in vitro by extraction,isolation,culture and then manage the BM-MSCs by AR knockout and AR overexpression.Then research the effect of AR on BM-MSCs apoptosis in order to offer one method to decrease MSCs apoptosis before BM-MSCs were transplanted.Objective3:The conclution that transplanted BM-MSCs could repair infarction myocardial has been proved by animal and clinical studies.But there are many questions to resolve about the transplantation of BM-MSCs on myocardial infarction.It is not difficult to understand that BM-MSCs have sex difference.Our experiment is to overexpress AR protein of BM-MSCs by exogenous gene transfection skill.Then inject these BM-MSCs into myocardial of the infracted rats.After 1 month,some hemodynamics indexes were measured.The aim of our experiment was to confirm that transplanted BM-MSCs with AR overexpression are likely in favour of recover and improvement of heart function on the rats with infracted hearts.Methods and ResultsMethods1:112 acute myocardial infarct patients of men aged from 31 to 87,who were collected at the Cardiology department of the Second Hospital of Tianjin Medical University,were selected as the acue myocardial infarction group(AMI).73 acute cerebral infarction patients aged from 31 to 87 at the neurology department of the Second Hospital of Tianjin Medical University during the same time were selected as acute celebral infarction group(ACI).107 healthy men aged from 39 to 93,who were collected at the department of Health Care of the Second Hospital of Tianjin Medical University,were as the male control group.All selected objects were collected venous blood 2mL of sodium citrate anticoagulant the next morning.The blood were frozen at-80 ?.The index of lipids,liver function,kidney function,fasting glucose and other clinical and biochemical indicators were recorded for all selected objects.Medical history including gender,age,height,weight,smoking,hypertension,diabetes mellitus(DM),family history of CAD and so on were taken.Whole blood genomic DNA was extracted in accordance with the blood genomic DNA extraction kit.The length of CAG repeats in the peripheral blood AR gene was amplificated through polymerase chain reaction(PCR)and the PCR product was detected by 1.5%agarose gel electrophoresis.Nested PCR reaction products were purified,recovered and sequenced in Beijing Liu he Genomics Institute of Science and Technology.Resultsl:For the male AMI patients,the numbers of CAG repeats ranged from 15-33,with mean values 23.09±3.520.For the male ACI patients,the numbers of CAG repeats ranged from 19-30,with mean values 22.51±3.358.There were 12?31 and 20.30±3.710 for the male controls respectively.There were statistically significant differences among the 3 groups(F=20.528,P<0.01).The patients of AMI and ACI groups have longer CAG repeats than that of the control group(q value was 4.56 and 3.21 respectively,both P<0.05).Men with CAG repeats length of 20 or longer may be in high risk of AMI(odds ratio,2.098,P<0.01),compared to men with CAG repeats length shorter than 20.And in the ACI group men with CAG repeats length of 20 or longer than 20 may be in high risk of ACI(odds ratio,1.353,P<0.01),compared to men with CAG repeats length shorter than 20.Methods2:BM-MSCs were extracted from male rats directedly,and then were isolated by density gradient.The cultured model in vitro were builted by primary and subcultured.BM-MSCs were identificated by cell morphology,immuohistochemistry and flow cytometry.The AR of BM-MSCs were detected by RT-PCR and Western blot.The plasmids of pCMV-AR,pCMV-Vector,pSuperior-ARsiRNA and ARsi-Scramble were transfected into BM-MSCs by LipofectamineTm2000.The AR mRNA expression diffence of aforementioned four models were detected by RT-PCR,and the apoptosis of the models were measured by flow cytometry.Results2:Cultrured BM-MSCs model in vitro were set up succeedly and grew in good state.The surface antigen CD29 and CD44 of BM-MSCs were positive,but the surface antigen CD34 was negative.The positive expression rate of BM-MSCs surface antigen CD90 was 99.63%and that of CD45,CD34 and control cells were 1.06%,0.43%and 0.21%respectively by flow cytometry.BM-MSCs expressed AR by MHC,western blot and RT-PCR,the AR overexpressed and expressed weak after BM-MSCs were transfected by exogenous plasmids.The apoptosis of BM-MSCs with AR overexpression after 36 hours,48 hours and 72 hours from the time when BM-MSCs were transfected were measured by flow cytometry respectively,and that of BM-MSCs with AR knockdown were measured by flow cytometry respectively.The rates of BM-MSCs with AR overexpression after 36 hours,48 hours and 72 hours were 12.02%,14.15%and 17.89%respectively,and that of BM-MSCs with AR knockdown AR were 13.83%,16.31%and 23.24%respectively.Methods3:The infarted male SD rats were individed into experimental group(injected by BM-MSCs with AR overexpression,n=8)and control group(injected by PBS solution,n=7).After 1 month,some hemodynamics index include Left Ventricular End Systolic Pressure(LVESP),Left Ventricular End Diastolic Pressure(LVEDP),Systolic Blood Pressure(SBP),Diastolic BloodPressure(DBP),the maximum rate of rise of left ventricular pressure(LV+dp/dtmax)during isovolumetric systolic stage,the largest decline rate of left ventricular pressure(LV-dp/dtmax)during isovolumetric diastolic stage were measured.Results3:The all index of experimental and control group were respectively LVESP(113.09±28.86 vs 140.62131.50,P=0.086),LVEDP(4.77±20.40 vs-5.42±6.16,P=0.227),SBP(108.42±28.98 vs 141.70±34.57,P=0.063),DBP(82.64±30.79 vs 114.41±22.16,P=0.042),LV+dp/dtmax(7243,00±2206.09 vs 9113.30±1316.87,P=0.073)and LV-dp/dtmax(-5497.00±1800.17 vs-7210.00±1390.48,P=0.062).The DBP of experimental group was more than that of control group.The differences others had no statistical significance between experimental and control groups.ConclutionConclutionl:Androgen receptor gene CAG repeats showed polymorphisms in our study.The androgen receptor gene CAG repeat length of the male AMI and ACI group is greater than that of the male control group.The CAG repeat polymorphism in the androgen receptor is associated with acute myocardial and cerebral infarction.Men with CAG repeats longer may be in high risk of acute myocardial and cerebral infarction.Conclution2:The surface antigens of CD44,CD29 and CD90 of our cells were positive,CD34 and CD45 were negative.BM-MSCs expressed AR antigen by MHC,BM-MSCs expressed AR protein by Western blot and BM-MSCs expressed AR m RNA by RT-PCR,the ARmRNA overexpressed and expressed weak by RT-PCR after BM-MSCs were transfected by exogenous plasmids.The apoptosis of BM-MSCs with AR overexpression after 36 hours,48 hours and 72 hours from the time when BM-MSCs were transfected less than that of BM-MSCs with AR knockdown.Conclution3:The myocardial infarction rat model could be succeeded by the coronary ligation,and the model may be used by treatment of BM-MSCs transplantation.The index(except that DBP of experimental group was more than that of control group)have no statistical significance between the group injected by BM-MSCs with AR overexpression and the control group.There were many regrets in our experiment to be improved later. |
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