G-csfr Was Expression Changes And Experimental Study Of G-csf On Rats Sub-acute Phase Of Myocardial Infarction After Myocardial Infarction

PART IExpression of G-CSF and G-CSFR in myocardial infarction area in ratsBackground and objective: G-CSF has been reported to protect ischemia -reperfusion injury of heart through binding with the G-CSFR on the myo-cardiocytes or play cardio-protective role by increasing the mobilization of stem cells. However, the effect of G-CSF on sub-acute myocardial infarction and the mechanism underlying it still haven't been reported. To search evidence for the treatment of G-CSF in sub-acute myocardial infarction, the time course of G-CSF and G-CSFR expression after myocardial infarction in rats will be investigated.Methods: Myocardial infarction rat model was induced by permanent left anterior descending branch ligation. The expression of G-CSF mRNA was measured by RT-PCR in different time points (0h, 6h, 12h, 1d, 3d and 7d). The location of G-CSFR was detected by immunohistochemistry and the expression change of G-CSFR was determined by western blot at 12h, 1d, 3d, 5d and 7d.Results: G-CSF mRNA was significantly increased at 6h and peaked at 12h post operation in infarction area, then attenuated gradually within 24h. But in the non-infarcted area G-CSF mRNA were similar at various time points at a low level. 1 day after MI, G-CSFR located merely in non-infarcted area, but G-CSFR positive-staining was found in infarcted area 3 days after MI. It was shown by western blot assay that G-CSFR increased gradually and peaked in5 days, then decreased continuously.Conclusions: G-CSF mRNA increased after myocardial infarction, and thelocal increase of G-CSF in infarction area may be one of the messengersconnecting the injured heart with bone marrow. The augment of G-CSFR ininfarction area is following that of G-CSF. The change pattern of G-CSFR ininfarcted area suggests that proper administration of G-CSF would beadvantageous to myocardial infarction. PART IIEffect of G-CSF on apoptosis and expression of SDF-1 of CMF and migration of CFBackground and objective: Myocardial infarction (MI) progresses from the acute death of myocytes and the infiltration of inflammatory cells into granulation tissue, followed by scar. The apoptosis of cardiac myofibroblast (CMF) in granulation tissue plays a key role in the healing precession. In previous study, we have found that the expression of G-CSFR was increased significantly after MI, which indicated that G-CSF/G-CSFR axis may has some function in the injured heart. In present study, the expression of G-CSFR will be examined in primary culture cardiac fibroblast (CF) and CMF, then the effects of G-CSF on the apoptosis and SDF-1 expression of CMF and the migration of CF will be studied.Methods: After induction of myocardial infarction,α-SMA positive cell had been observed at different time point, double immunofluorescence (IF) of SDF-1 andα-SMA was also examined 3 days after MI. To test whether G-CSFR is expressed on primary culture CF and CMF, we performed RT-PCR and immunocytochemistry (ICC) using specific primers and antibody for rats G-CSFR. Annexin-V apoptosis assay and scratch assay were performed separately to test the effects of G-CSF on the apoptosis and migration of CMF or CF. Then, RT-PCR was used to measure the expression of SDF-1 of CMF after G-CSF intervention in vitro. Results:α-SMA positive cell was increased gradually during 7 days after MI.The co-location ofα-SMA and SDF-1 was verified in infarction area usingdouble IF. G-CSFR is expressed in both CF and CMF in vitro. G-CSF candecrease the apoptosis of CMF induced by H₂O₂ dramatically, and increasethe expression of SDF-1 in CMF and the migration of CF.Conclusions: G-CSFR is expressed in CMF. After exposed to G-CSF,apoptosis of CMF was reduced, and expression of SDF-1 in CMF andmigration of CF was increase, all of which indicated G-CSF may improvethe remodeling of ventricular after MI. PART IIIEffects of G-CSF on sub-acute stage of myocardial infarction in ratsBackground and objective: In the previous study, we have found that the expression of G-CSFR was increased after MI, and then we verified that G-CSF can prevent CMF from apoptosis by binding with G-CSFR. Additionally, G-CSF can promote the migration of CF and SDF-1 expression of CMF in vitro. Furthermore, we will examine whether G-CSF confers its benefits on infarcted heart in vivo as it did in vitro, and elucidate what's mechanism underlying that.Methods: Rat myocardial infarction models were induced, and randomized into two groups:â‘ treatment group (T_n=14): I.P G-CSF(25μg/kg/d×5d);â‘¡control group (C=15): I.P N.S(0.1 m1×5d). The rats were sacrificed at the following time point:â‘ 8 days after MI, the hearts were resected, the expression of SDF-1 in infarction area was examined by western blot;â‘¡10 days after MI, the slices of heart were prepared for the Tunel apoptosis and Brdu proliferation assay, and expression of Bcl-2 was semi-quantified by western blot;â‘¢4 weeks after MI, quantitative assessments of myocardial cell size, cell population, vessel population, ratio of area ofα-SMA-positive cells and fibrotic area were preformed using histological analysis methods.Results:â‘ The expression of SDF-1 in infarction area was significantly stronger among treatment rats than control rats 8 days after MI;â‘¡ Comparing with control rats, the incidence of Tunel-positive was much less, and Brdu-positive cells were more marked, the expression of Bcl-2 was increased significantly 10 days after MI;â‘¢4 weeks after MI, the cell size and fibrotic area was smaller in treatment rats,α-SMA-positive cells, the cell population, vessel population were marked increased in G-CSF treatment rats.Conclusions: At the sub-acute stage of myocardial infarction, G-CSF administration can prevent the apoptosis of cell in the infarction area, promote angiogenesis in the peri-infarction domain, and inhibit the ventricular remodeling. The pro-angiogenesis function of G-CSF may partially contribute to augment of SDF-1 expression.