The Detection Of Gene Expression In Myocardial Infarction And Establish A Cadiocyte Isolate Platform

Objective:Myocardial infraction will induce heart failure, even worse lead to death. This study is about the express of acute myocardial infarction related gene, and mechanism of action of this progress; we analyses the effective of Tween-80to cadiocyte ICa·L, also discuss how PSEN1relate to cardio-cerebral-vascular develop, those have profound guiding significance for clinic research and therapeutics.Method:1. Express of rat M1related gene(1) Ligature left anterior descending coronary artery to create an acute myocardial infraction model, take rat cardio muscle each0h,2h,4h,12h,24h;(2)solated cadiocyte from group M1and group control, after Fluo-5N/AM stained, use confocal laser scanning microscope determin the calcium cont. of sarcoplasmic reticulum;(3)observe cadiocyte ultrastructure of each group with transmission electron microscope;(4) extract RNA in routine method, real-time PCR detect,quantitate use double standard curve, analyses different gene express as different time;2. Effect of Tween-80to cadiocyte ICa·LGet cadiocyte after enzymolysis, use patch clamp detect how different level (0.05%,0.1%,0.2%) of Tween-80influence cadiocyte ICa·L.3. Effect of PSEN1and building a jimpy mice cadiocyte isolate platform(I) Collect cyema from different gene type mice, use microscope find the different;(2) make heart paraffin section, analyses the effect of PSEN1in histology way;(3) Allomixis PSEN1mice and Myh6-cre/Esr1mice, we can get PSEN1loxp、Myh6-cre/Esrl double-masc. mice. Peritoneal injection Tamoxifen to move PSEN1from cardio gene group, establish a strain of PSEN1gene knock-out mice;(4) based on the demand of cadiocyte isolate,we build a mice cadiocyte isolate system ourselves, and isolate successfully.Result:1.Gene TNF α and d IL6have same express trend in M1, both of them was up-regulation after2h, and reach the peak value12hs later, after24hs return to basis level, this summons that, with the ischemic time pass, the inflammatory reaction occur, and will disappear after24hs; metabolism transcription factor C-Myc up-regulation fast at early MI, it is maintain high level in whole MI progress, this shows after ischemic, cardiac metabolism became abnormal for adapt biology change; ANP will express highly with time pass, and reach the peak at24h, it shows endogenic ANP changing trend after MI, and participate in myocardial preservation; SOD express low at early4hs after ligation, came normal after24h, shows after4h MI, oxidative stress occur; FKBP12.6have no significant change. After MI, with cardiac gene express change, its ultrastructure change too, myoneme dissolve, chondrosome dysfunction; 2.Use patch clamp record every change pre/after medical treatment of each group(pre20s/after150s), results:control group(8.40±0.21and7.4±0.26, n=3);0.05%group(9.35±0.30and8.22±0.48, n=3);0.1%group(5.32±0.37and2.50±0.20,n=3);0.2%group(6.35±0.29and2.54±0.23, n=3), ANOVA analyses shows control group and0.05%group have no significant difference(p>0.05),0.1%group and0.2%group difference is apparently,(P<0.05), it identical low level Tween-80have no effect to cardiac, however high level Tween-80can inhibit ICa·L.Each group show, control group11.2%±5%, n=3;0.05%group12.0%±3.2%, n=3;0.1%group52.7%±6.5%, n=3;0.2%group59.9%±4.5%, n=3; compared each other, control group and0.05%group P>0.05, the other two group have significant different P<0.05, it imply when Tween-80reach to0.1%, electric damp go peak value. With Tween-80go higher, electric damp have no significant change;3. PSEN1gene knock-out mice dysplasia, embryo observe result:anangioplasia, atelencephalia, dysmelia and growth retardation; cardiac histology analyses result: ventricular dilatation, ventricular fold thinner, VSD, double outlet of pulmonary artery. For further research, we built a mice cardiac isolate platform, acquire mice cadiocyte, cell condition well, makes a good fundament of follow work.Conclusion:1. This study found some MI related gene expressed in time dependent, bind with confocal laser scanning microscope and electronmicroscope study the MI physiopathologic change in both molecule and morphology way, provide a theory evidence of MI clinic research and treatment;2. Study the effect of Tween-80to ICa·L at celluar level, low doss Tween-80have weak effect to cardiac, however, when it reach0.1/%, the decline reach peak value, so0.1/%of Tween-80can be seen as a dangerous signal, provide a new standard in biomedicine to use Tween-80.3. This study research the effect of PSEN1to mice cardio-cerebral-vascular develop, we found PSEN1gene knock-out mice have a lot of cacoepy in many system, expend the knowledge of PSEN1gene function. Establish a good fundament of follow work.