To study the expression of both Fos and HSP70 proteins in the early stage of acute myocardial ischemia/reperfusion(I/R) in rats with immunohistochemistry in order to search the objective morphologic evidence for the forensic pathological diagnosis.I/R rat model was established by ligation of the anterior branch of the left coronary artery.All animals were divided into 7 groups.Tissues were cut and stained immunohistochemistry with the corresponding anti Fos and anti HSP70 sera as the first antibodies.There was no expression of both Fos and HSP70 proteins in control group.The expression of HSP70 protein began to increase in myocytes 1h after I/R in the ischemic areas and increased gradually wtih the prolongation of ischemia,while it began to express 2h after I/R in the non ischemic areas.Fos protein began to express 0 5h after I/R in the ischemic areas,1h in the non ischemic areas.The expressions of both HSP70 and Fos proteins in the non ischemic areas were weaker than those in the ischemic areas.Meanwhile,the expression of HSP70 and Fos proteins appears firstly in the inner layer of myocardium and extended gradually toward the outer layer.The expression of Fos protein mainly located in the inner layer of myocardium 0 5h after I/R and then extened to the whole layer of myocardium 1h after I/R.The expression of Fos protein in outer layer was stronger than that in inner layer at 4h after I/R.The expression of HSP70 protein located mainly in the inner layer of the myocardium at 1h and 2h after I/R,and then extended to the whole layer of the myocardium at 4h and 6h after I/R.The results indicated that the I/R induced the expression of both HSP70 and Fos proteins in the early stages of myocardial ischemia.The location and the intersity of the immunoreactivity of these two proteins changed at the different stages after I/R.These changes observed in the present study might be useful for the forensic pathological diagnosis of the early myocardial ischemia.

Objective Investigate the postmortem stability of the six immunohistochemical markers of fibronectin(Fn),fibrinogen(Fg),C5 complement(C5), myoglobin(Mb), actin(HHF35)and desmin(Dm)for the postmortem diagnosis of myocardial infarction.Method The areas of depletion ofMb、HHF35 and Dm,and the positive reaction areas of Fn、Fg and C5 in the ischemic myocardium were studied with immunohistochemistry,image analysis technique and statistical system.The postmortem stability of the six immunohistochemical markers was compared.Results The specimens of normal myocardium kept at 4℃ for 1 to 2 days showed homogenous brown reactions for Dm, HHF35 and Mb; depletion of Dm, HHF35 and Mb became evident when these specimens were kept at 4℃ for 3 days postmortem, and the depletion area increased with the lapse of postmortem interval; the depletion area of Dm, HHF35 and Mb in ischemic myocardial tissues also increased with the lapse of postmortem interval;the positive reaction areas of Fg, C5 and Fn in ischemic myocardial tissues decreased with the lapse of postmortem interval.Fg became negative when the ischemic myocardium were kept at 4℃ for 2 weeks postmortem,C5 became negative when kept at 4℃ for 3 weeks postmortem,but Fn remained positive when kept at 4℃ for 4 weeks postmortem.No positive reactions for Fg,C5 and Fn could be found in normal myocardium when kept at 4℃ for different time intervals. The image analysis result showed that the positive reaction areas decreased with the lapse of postmortem interval. Conclusion The Dm and HHF35, Mb showed least postmortem stability, easily influenced by autolysis, only suitable for detection in fresh corpses(1to 2dayspostmortem) ;Fgislittlebitbetter,suitableforcorpsesat4℃ 1weekpostmortem ;C5isbet ter,suitableforcorpsesat 4℃ 2weekspostmortem ;Fnisthebestmarker ,suitableforcorpsesat 4℃ 4 weekspostmortem .

Objective To detect the loss of cardiac troponin T (CTnT) in human myocardial infarction. Methods Loss of cardiac troponin T in human myocardial infarction was detected by immunohistochemical LSAB method. Results Obvious depletion of CTnT was observed in cases died from myocardial infarction,and by the computer image analysis, the mean CTnT-depleting area in myocardial infarction group was significantly different from that in the control group(P

In order to study the morphclogic changes of cardiac conduction system in six cases of acute myocardial infarctions fibronectin (Fn), myoglobin(Mb)and vascular endothelial growth factor (VEGF) were study by immunohistochemical method. It was observed that strong positive Fn staining were present in 3 cases, positive in 1 case, weak positive in 1 case; weak VEGF positive and depletion of Mb in all cases. It is indecated that the Fn staining is sensitive and was stable, and easily observed, and can be used as a good marker for diagnosis of the injury of the cardiac conduction system in acute myocardial infarction.

Experimental acute myocardial ischemia model of dog was established, and the postmortem stability of fibronectin for the diagnosis of mycardial infarction was studied with immunohistochemistry and image analysis technique. The results showed that the positive reaction areas of fibronectin in ischemic myocardial tissues decreased along with the prolongation of postmortem interval, but positive reaction of fibronectin could still be found in ischemic myocardia kept for 4 weeks postmortem. No positive reaction for fibronectin could be found in normal myocardia when kept for different times. So fibronectin is a quite stable marker for the postmortem diagnosis of myocardial infarction, and is of practical value in the forensic practice.

For the purpose of establishment of a diagnostic method of mild viral myocarditis,an experimental mild viral myocarditis model was induced in Balb/c murine by coxsackie virus B 3.The cardiac tissue secfion were stained by LSAB-immunohistochemical method with anti fibronectin(Fn) antibody. The results demonstrated that the Fn deposition was found in the myocardium of mice with myocarditis and the mild degeneration of cardiomyocytes could be identified by Fn LSAB immunohistochemical staining.It is suggested that the Fn deposition in the myocardium tissue is one of the reliable marks of myocardium inflammation.

Experimental acute myocardial ischemia model of rat was established, and the changes of C5 complement in the ischemic myocardia were studied with immunohistochemistry and image analysis technique. The results showed that the positive reaction of C5 could be observed in ischemic myocardia at 15 min after ischemia, and the positive reaction area increased along with the prolongation of the ischemic period. It is concluded that the positive reaction of C5 in cardiomyocytes is a quite sensitive marker of early myocardial ischaemia.The immunohistochemical detection of C5 in cardiomyocytes will be a meaningful tool for the postmortem diagnosis of early myocardial ischaemia.

In order to explore the value of fibronectin (Fn) in the postmortem diagnosis of myocardial infarction.The changes of Fn staining in normal, infarcted and other non infarcted myocardial injuries resulted from myocarditis, mechanical asphyxia, electrocution, hemorrhagic shock, cardiac contusion and organophosphate poisoning were studied with immunohistochemistry and image analysis system. The results showed that positive Fn staining could only be observed in groups of myocardial infarction and myocarditis, but could not be found in groups of mechanical asphyxia, electrocution, hemorrhagic shock, cardiac contusion, organophosphate poisoning. It is indicated that positive reaction of Fn could be affected only by myocarditis, so it is quite specific for the diagnosis of myocardial infarction.

In order to explore the significance of cardiomyocyte apoptosis in the early myocardial ischemia,the rat model of acute myocardial ischemia was established,and the apoptotic cells were detected in the early phase of ischemia(within 6h)with TUNEL method.The results showed that scanty apoptotic cells could be observed in the ischemic region 1h after ischemia,and reached the peak 3h after ischemia,then decreased.No apoptotic cells were found in the normal region.In the peri ischemic region,apoptotic cells could also be observed 1h after ischemia,and reached the peak 5h after ischemia.It is indicated that apoptosis is the major form of early ischemic myocardial damage,and the detection of apoptotic cardiomyocyte may provide a new sensitive and objective method for the postmortem diagnosis of early myocardial infarction.

Objective To investigate the effect of glutamine in combination with umbilical cord blood mesenchymal stem cells ( MSCs) transplantation on intestinal ischemia-reperfusion injury in rats.Methods Umbilical cord blood mesenchymal stem cells were isolated, and were labeled with CM-DiI fluorescent dye.Eighty Sprague-Dawley rats were randomly divided into normal control group, ischemia reperfusion injury group, glutamine group, MSCs transplantation group and combined group with 15 rats in each group.The control group received saline enema.The injury group was treated with TNBS ( ethanol dilution) enema.The glutamine group at 1 h after TNBS received intravenous injection of 0.45 g/kg glutamine.The rats of MSCs transplantation group had tail vein injection of 1 ×1010/L umbilical cord blood mesenchymal stem cell suspension, and the combined group received intravenous injection of glutamine 0.45 g/kg and 1 ×1010/L umbilical cord blood mesenchymal stem cell suspension.ELISA was used to detect the midgut fatty acid binding protein (iFABP), interleukin 6 (IL-6), and superoxide dismutase (SOD) content in the rat serum.The water content of intestinal tissue was detected at 1 h and 3 h after reperfusion in each group.The expressions of NF-kB, Bcl-2 and caspase-3 mRNA and proteins in the rat intestinal epithelial cells after treated with glutamine in combination with MSCs were detected by RT-PCR and Western blot assays.Results The fluorescent tracer method revealed that the transplanted MSCs cells were distributed in the intestinal mucosal lymphoid tissues and glandular epithelial cells, indicating that MSCs might be involved in the repair process of intestinal ischemia-reperfusion injury.The content of serum IFABP and IL-6 in the injured group was significantly higher than that in the control group, while significantly reduced in the glutamine group, MSCs transplantation group and combined group, with the most obvious in the combined group.The content of SOD in the injury group was significantly lower than that in the control group, and significantly increased than that in the glutamine group, MSCs transplantation group, with the most striking in the combined group ( P<0.05 for all) .The water content of intestinal tissue in the injury group at 1 and 3 hours after reperfusion was significantly higher than that in the control group, significantly lower in the glutamine group, MSCs transplantation group and the combined group, with the most decreased in the combination group, and there was no significant difference between the glutamine group and MSCs transplantation group (P>0.05).Compared with the control group, the caspase-3 and NF-kB mRNA and protein expressions in the intestinal mucosal epithelial cells of the injury group were significantly increased, and the expressions of Bcl-2 mRNA and protein were significantly reduced ( P <0.05 ) , the expressions of caspase-3 and NF-kB mRNA and protein were significantly reduced in the glutamine group, MSCs transplantation group and combined group.The expressions of Bcl-2 mRNA and protein were significantly increased ( P<0.05) , while no significant difference was shown between the glutamine group and MSCs transplantation group (P>0.05), but there was a significant difference between these two groups and the combined group (P<0.05).Conclusions After treated with glutamine and MSCs transplantation, the degree of intestinal ischemia reperfusion injury is obviously reduced in rats.It may be mediated through inhibiting the expression of caspase-3 and NF-kB and promoting the expression of Bcl-2.