G-CSFR在心肌梗死后表达变化及G-CSF干预大鼠亚急性期心肌梗死的实验研究

【作者】 王斌；

【导师】 曾智；

【作者基本信息】 四川大学 ， 心血管内科， 2007， 博士

【摘要】 背景与目的：近期的研究表明，G-CSF对心肌缺血再灌注损伤有一定的心肌保护作用，G-CSF的作用可能由两条途径介导：一是心肌细胞膜上的G-CSFR；二是增加动员骨髓干细胞。G-CSF对亚急性期心肌梗死的干预作用及其机制目前尚无报道。本研究通过探索心肌梗死后大鼠心肌组织G-CSF的表达变化与G-CSFR分布及量的变化，以期寻找在心肌梗死亚急性期进行G-CSF干预的依据。方法：结扎Sprague-Dawley大鼠冠脉前降支，构建心肌梗死模型，通过逆转录聚合酶链式反应（reverse transcription PCR，RT-PCR）检测梗死心肌G-CSF在不同时间点（0h，6h，12h，1d，3d和7d）的mRNA表达变化。同时，采用免疫组织化学技术了解G-CSFR在梗死后心肌组织的定位，并通过免疫印记法检测G-CSFR在不同时间点（1d，3d，5d和7d）的蛋白表达变化规律。结果：心肌梗死后6小时，G-CSF在梗死区域心肌组织内的表达开始增加，在梗死后12小时达峰值，1天后逐渐下降至正常水平。免疫组化检测结果显示G-CSFR在心肌梗死后1天仅定位于非梗死区域，3天后则逐渐在梗死区域出现阳性染色。免疫印记结果显示，随梗死时间延长在梗死区域G-CSFR表达增加，并在梗死后第5天达峰值。结论：G-CSF在梗死后心肌组织表达增加，梗死区域可能通过损伤局部G-CSF的表达变化与骨髓形成信息交流。G-CSFR的表达变化较G-CSF稍滞后，心肌梗死后G-CSFR在梗死区域呈现的表达变化规律，为在心肌梗死亚急性期进行G-CSF干预提供了依据。背景与目的：心肌梗死后，经过心肌坏死，炎性细胞侵入，肉芽组织阶段，最终形成纤维瘢痕。肉芽组织中心肌成纤维母细胞（cardiacmyofibroblasts，CMF）的凋亡在心肌梗死的修复进程中起着关键作用。在第一部分的研究中发现G-CSFR在梗死组织表达增加，本部分将通过体外培养心肌成纤维细胞（cardiac fibroblasts，CF）及CMF，观察CF及CMF是否存在G-CSFR的表达，并了解G-CSF对它们迁移与凋亡的影响；构建大鼠心梗模型，观察肉芽组织的演变过程，间质细胞衍生因子（stromal-cell-derived factor，SDF-1）与CMF的定位关系；进一步通过体外实验，观察G-CSF对CMF中SDF-1表达的影响。方法：建立大鼠心肌梗死模型，通过免疫组化方法观察梗死区不同时间点α-SMA阳性表达情况。通过免疫双荧光方法观察α-SMA与SDF-1在心肌梗死后的表达定位。进行CF及心梗后肉芽组织内CMF的体外培养，用RT-PCR及免疫细胞化学法检测有无G-CSFR的表达，并观察G-CSF对CF细胞迁移、CMF凋亡和SDF-1表达的影响。结果：心肌梗死后α-SMA阳性表达逐渐增多，至7d达高峰。免疫双荧光提示α-SMA⁺与SDF-1⁺共定位于CMF细胞。体外培养发现，CF及CMF细胞均存在G-CSFR表达。此外，G-CSF能有效抑制H₂O₂诱导的CMF细胞凋亡，增加CMF中SDF-1表达，并增加CF的迁移能力。结论：首次发现G-CSFR在CMF细胞表达；G-CSF减少CMF的凋亡，增加CMF中SDF-1的表达，并增加CF的迁移能力；G-CSF可能通过上述作用改善亚急性期心肌梗死的心室重构。背景与目的：在前面的研究中发现，G-CSFR在梗死区域表达增加，在体外，G-CSF能明显抑制CMF凋亡，促进CF迁移，增加CMF分泌SDF-1。本部分我们将在体情况下研究G-CSF干预亚急性期心肌梗死的作用，并进一步探讨G-CSF对亚急性期心肌梗死的潜在作用机制。方法：建立大鼠心肌梗死模型，随机分为干预组（T）和对照组（C），分别在梗死后3天经腹腔注射G-CSF（25μg╱kg／d）或等量生理盐水，连续注射5天。分别在以下三个时间点处死大鼠进行相关检测：1、梗死后八天（Tn=4，Cn=4），免疫印迹观测SDF-1在两组中梗死区域的表达变化；2、梗死后10天（Tn=5，Cn=5），取心脏组织切片，行Tunel及Brdu免疫组织化学检测，观察两组中梗死区域细胞凋亡及增殖情况，并用免疫印迹法检测抗凋亡蛋白Bcl-2的表达变化；3、梗死后4周（Tn=5，Cn=6），分别观察大鼠非梗死区域心肌细胞直径，梗死区细胞α-SMA阳性率，细胞数量，胶原形成及梗死周边的血管生成情况，以评价心室重构情况。结果：梗死后8天，G-CSF干预组梗死区SDF-1表达明显高于对照组；梗死后2周，G-CSF干预组梗死区域细胞凋亡率明显低于对照组，而细胞增殖及Bcl-2表达水平较对照组高；梗死后4周，干预组非梗死区心肌细胞直径、胶原形成较对照组减少，而梗死区细胞数量，梗死周边血管数量及α-SMA阳性区域较对照组增加。结论：G-CSF干预亚急性期心梗可通过减少梗死区细胞凋亡，增加梗死周边区域血管生成改善心肌梗死后心室重构，增加梗死组织SDF-1的表可能是G-CSF促血管生成的机制之一。更多还原

【Abstract】 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:①treatment group （T_n=14）: I.P G-CSF（25μg/kg/d×5d）;②control group （C=15）: I.P N.S（0.1 m1×5d）. The rats were sacrificed at the following time point:①8 days after MI, the hearts were resected, the expression of SDF-1 in infarction area was examined by western blot;②10 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;③4 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:①The expression of SDF-1 in infarction area was significantly stronger among treatment rats than control rats 8 days after MI;② 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;③4 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.更多还原