【作者】 吴建亮；

【导师】 张海林；

【作者基本信息】 河北医科大学 ， 药理学， 2010， 硕士

【摘要】 心肌肥厚是多种心脏疾病共同的病理过程。其病理变化包括心肌细胞肥大、心肌间质细胞增殖以及心脏细胞外基质改建等多方面的改变,即心肌重构。心肌肥厚时心肌细胞蛋白合成增加、体积增大、直径增宽或长度增加,肌节数量增多并伴有纤维组织增生。心肌肥厚是引起多种心血管疾病的重要危险因素。早期的心肌肥厚可能是心肌细胞对外界的刺激的一种适应性的改变,有一定的代偿意义,有利于维持心输出量,以满足机体的需要,但是长期的心肌肥厚最终会导致心力衰竭,甚至会猝死。在心肌肥厚发生发展过程中,细胞内钙内流都起到了至关重要的作用。当压力负荷作用与心脏或者是某些神经体液因子(AngⅡ、ET-1和PE等)刺激时,可以导致细胞膜上钙库调控的钙通道(SOC)、受体调控的钙通道(ROC)或者电压依赖的钙通道(VDCC)等通道开放,使钙离子内流,细胞内钙会和钙调素(CaM)结合,继而激活钙调磷酸酶(CaN),CaN的活化,又作用于NFAT通路,导致肥大基因的表达升高,进而引起心肌肥厚。TRP通道是存在与细胞膜上一种重要的阳离子通道,与众多的疾病的发生发展有重要的关系,TRP通道几乎存在与机体的各个组织。在心脏中主要是表达的是TRPC通道,也表达部分TRPM和TRPV通道。大多数的TRP通道都可以使钙通过。此外ROC、VDCC和SOC的开放,也可以导致钙内流。而TRPC通道的部分亚型被认为是组成ROC或者SOC的分子基础。ROC和SOC等都与钙内流有关,与心肌肥厚的发生密切相关。因而TRPC通道和心肌肥厚的发生有很重要的关系。但是,目前为止,对于TRP通道和心肌肥厚的关系,TRPC通道的报道较多,其他的TRP通道的亚型报道较少,其在心肌肥厚的发生发展中是否发挥作用尚未知晓,有待于进一步研究。本实验利用Real-Time PCR的方法去研究TRPC、TRPV、TRPM、STIM1和Orai1在正常成年大鼠、新生期大鼠和心肌肥厚模型组大鼠的心肌组织中表达水平。通过与正常成年大鼠心肌组织的表达水平的比较,研究这些通道和心肌肥厚发生发展的关系;另外,通过新生期大鼠和正常成年大鼠和心肌肥厚模型大鼠的比较,也可以了解心脏发育与心肌肥厚的关系。第一部分大鼠心肌肥厚模型的制备和心脏功能和组织学评价目的:制备异丙肾上腺素(Iso)诱导和腹主动脉缩窄(AAC)诱导的心肌肥厚模型。方法:Iso诱导心肌肥厚模型:Iso 5mg/(kg·d),背部皮下注射,连续七天,可以诱导大鼠心肌肥厚模型;AAC诱导的心肌肥厚模型:大鼠常规1%戊巴比妥钠腹腔注射麻醉,左侧卧位,切开皮肤、筋膜和肌肉,游离出一段腹主动脉,然后用4#缝合线将自制的弯曲的8#针头和该段腹主动脉一起结扎,再缓慢的抽出针头,造成腹主动脉在结扎点处内径缩窄,然后依次复位各脏器,缝合腹壁。关笼饲养,常规给予16万U青霉素抗炎一周。饲养十二周成模。结果:血流动力学:与空白对照组比较,Iso模型组和AAC组LVEDP明显升高(P<0.01),而LVSP和±dp/dtmax明显下降(P<0.01)。Iso组和AAC组两组相比,LVEDP、LVSP、±dp/dtmax则无明显差异(P>0.05)。心脏重量指数(HWI):与空白对照组比较,Iso模型组和AAC组心脏重量及HWI明显升高(P<0.01);病理学检查:光镜下可见,空白对照组大鼠心肌纤维排列整齐,横纹明显,胞核结构清晰,无细胞肿胀;Iso组和AAC组心肌细胞显著肥大,核大浓染,肌纤维排列紊乱,呈旋涡状或簇状,肌原纤维走向不一,互相交错排列。结论:经过Iso处理和AAC处理的大鼠均能成功诱导出心肌肥厚,其中Iso造模方法相对简单,成模率高;AAC模型类似临床负荷性心肌肥厚的病理生理学过程。两种方法诱导的心肌肥厚模型成功制备下一步的实验打下基础。第二部分心肌肥厚与TRP离子通道基因表达水平的研究目的:研究正常成年大鼠、新生期大鼠、心肌肥厚模型组大鼠心肌组织中TRP通道、STIM1和Orai1 mRNA的表达水平。方法:严格按照Promega total RNA Isolation Syetem的方法分别提取正常成年大鼠、新生期大鼠和模型组大鼠的心肌组织的总RNA;然后根据Promega Reverse Transcription System的方法合成cDNA的第一链;最后根据Takara SYBR Premix Ex TaqTM荧光试剂盒的说明和cDNA的量,进行Real-Time PCR。数据分析所用的方法为2-ΔCt,其中﹣ΔCt为不同组间PCR扩增进入指数增长期的循环数的差值。结果:1. BNP是心肌肥厚的标记分子之一,与正常成年鼠相比,在Iso诱导的心肌肥厚和AAC诱导的心肌肥厚模型的心肌组织中,BNP的mRNA表达水平分别升高了7.80±0.33倍和8.11±0.37倍(P<0.01),而新生期大鼠的心肌组织中BNP的表达水平升高了8.08±0.39倍(P<0.01)。2. TRPC1、TRPC3和TRPC6通道mRNA在心肌肥厚模型和新生期大鼠的心肌组织中表达水平均升高(P<0.01,数据未列出,详见第二部分Table3和Table4)。上述三个通道在Iso诱导心肌肥厚模型和AAC诱导的心肌肥厚模型的表达水平无差异(P>0.05)。TRPC2、TRPC4和TRPC5通道在三组中的表达水平无明显差异(P>0.05)。TRPC7在新生期大鼠的表达水平要高于其他三组(P<0.01)。正常成年大鼠和模型组大鼠TRPC7的表达水平无明显差异(P>0.05)。3.在正常成年大鼠组、新生期大鼠组和心肌肥厚模型组大鼠的心肌组织中只探测到了TRPM4和TRPM7有表达,其他的TRPM通道均未探测到。其中TRPM4在新生期大鼠组和心肌肥厚模型组的表达水平要高于正常成年大鼠组(P<0.01,详见第二部分Table5和Table6)。在所有组的心肌组织中TRPM7表达水平无明显差异。4.在正常成年大鼠组、新生期大鼠组和心肌肥厚模型组大鼠组的心肌组织中只探测到了TRPV2和TRPV6有表达,其他的TRPV通道均未探测到。TRPV2和TRPV6主要在心肌成纤维细胞上有表达。和正常成年大鼠组相比,心肌肥厚模型组的TRPV2和TRPV6表达水平均升高(P<0.01,详见第二部分Table5和Table6)。正常成年大鼠和新生期的大鼠表达水平则无差异。5.与正常成年大鼠相比,新生期大鼠和心肌肥厚模型组大鼠的心肌组织中STIM1和Orai1的表达水平均升高(P<0.01,详见第二部分Table1和Table2)。结论:在心肌肥厚的发生发展过程中,TRP通道的亚家族的某些亚型可能起到了重要的作用,具体表现在其在心肌组织的表达水平升高。此外,在新生期大鼠的心肌组织TRP通道表达谱有与心肌肥厚模型组心肌组织的TRP通道表达谱有某些相似之处。其中,新生期大鼠和心肌肥厚模型组的心肌组织的BNP、TRPC1、TRPC3、TRPC6、TRPM4、STIM1和Orai1的表达水平比正常成年大鼠高。本实验结果提示TRP通道有作为治疗心肌肥厚药物的作用靶点的潜能。更多还原

【Abstract】 Cardiac hypertrophy is the common pathology of a variety of cardiovascular diseases. The pathological changes include myocardial hypertrophy,?myocardial interstitial cell proliferation and extracellular matrix modification, namely myocardial remodeling. Characters of cardiac hypertrophy include increase myocardial protein synthesis, increased size of cardiac myocytes with larger dimeter or increased length, accompanied by an increase in fibrous tissue hyperplasia. Cardiac hypertrophy is caused by a variety of risk factors for cardiovascular disease. Although hypertrophy is initially a compensatory mechanism that helps sustain cardiac output, prolonged hypertrophy will inevitably give rise to heart failure and even sudden death. The increase in intracellular Ca2+ ([Ca2+]i) plays an important role in the development of hypertrophy. Hypertrophic stimulation by pressure overload and neurohormonal factors(including AngⅡ, ET-1 and PE et al) lead to the SOC (store-operated calcium channels), ROC (receptor-operated calcium channels) or VDCC (voltage-dependent calcium channels) opening. These Ca2+-entry channels induce a prolonged, low-amplitude rise in Ca2+. This sustained Ca2+ entry dominantly activates the calcineurin/NFAT pathway. The calcineurin/ NFAT pathway produces long-term hypertrophic changes in cardiac myocytes and subsequent cardiac hypertrophy.TRP channels are plasma membrane cation channels. They are expressed in almost every tissue, including the heart and vasculature. In the heart, evidences are accumulating that TRPC channels, and possibly TRPM and TRPV, are present and functional, Most TRP channels are Ca2+ permeable, Moreover, some of the Ca2+ channels such as ROC and SOC may have molecular basis related to some TRPC channel subtypes. ROC and SOC are believed to play important role for Ca2+ influx during cardiac hypertrophy, As thus, TRPC channels may also be important for occurrence of cardiac hypertrophy. Indeed some TRPC channels have been indicated in cardiac hypertrophy. However, little is known about other role of TRP channels subtypes play in cardiac hypertrophy.In this experiment, we use the Real Time PCR method to study expression profiles of the subtypes of TRPC, TRPV snd TRPM channels, as well as STIM1 and Orai1 in myocardial tissue from normal adult rats, neonatal rats and rats with cardiac hypertrophy. Such a comparion between the normal and hypertrophic heart will give us indications which of these TRP channels are possibly involved in cardiac hypertrophy development. And moreover, comparison between neonatal rats and normal adult rat and hypertrophyic rats would also shed light on relationship between cardiac development and hypertrophy.Part 1 Establishment of rat cardiac hypertrophy model and evaluation of cardiac function and histologyObject: To establish rat cardiac hypertrophy models by means of isoproterenol injection and abdominal aorta coarctation. Method: Isoproterenol (Iso)-induced cardiac hypertrophy model: Iso 5mg/(kg·d), back subcutaneous injection for consecutive seven days, was used to induce cardiac hypertrophy; Abdominal aorta coarctation (AAC)-induced cardiac hypertrophy model: intraperitoneal injection of 1% sodium pentobarbital was used to anesthetize rats. The anetsthetized rats were laid in a left-lateral position. Abdominal was cut open to expose abdominal aorta. A 4# suture was used to tight the abdominal aorta together with a self-made 8# cured injection needle, causing abdominal aorta coarctation. After this operation, abdominal fascia and skin were sutured. Intraperitoneal injection of benzylpenicillin potassium (16U, a week) was applied to prevent infection. After 12 weeks rat cardiac hypertrophy were established.Results: Hemodynamic parameters: compare with the control group, LVEDP was significantly higher (P<0.01), whereas LVSP and±dp/dtmax were significantly decreased in model groups (P<0.01). However, LVSP, LVEDP and±dp/dtmax were not significantly different within two model groups (P>0.05). Heart weght index (HWI): compare with control group, HWI was significantly higher in model groups (P<0.01). Histopathological examination: the myocardial structure in the normal rat group was clearly evident; myofibrils are the main components of cytoplasm, and they appeared orderly, with bright and dark areas clearly evident; In Iso group and the AAC group, myocardial cells appeared hypertrophy, with an increase in cell size and deposition of fibrous tissue.Conclusion: Isoproterenel injection and abdominal aorta coarctation successfully induced rat cardiac hypertrophy. The Isoproteronel method is relatively simple and had high successful rate. AAC method mimics better the pathophysiology of clinical cardiac hypertrophy with overload. Both models can be used for further study.Part 2 Study of the relationship between cardiac hypertrophy and TRP channel mRNA expression levels.Object: To Study the mRNA expression levels of the TRP channels, STIM1 and Orai1 in rat myocardium of normal adult rats, neonatal rat and cardiac hypertrophy model rats.Method: Using Promega total RNA Isolation Syetem method to isolate total RNA from myocardial tissues of normal adult rats, neonatal rats and model rats. Using Promega Reverse Transcription System to synthesize single-strandea cDNA from total RNA. Using Takara SYBR Premix Ex TaqTM to perform Real-Time PCR from synthesized cDNA. The difference in amount of original cDNA from different experimental rat groups were calculated using expression of 2-ΔCt; -ΔCt is the difference of PCR circle number in threshold between different groups.Results:1. Compared with normal adult rats, the BNP mRNA expression levels in the Iso-induced cardiac hypertrophy and AAC-induced cardiac hypertrophy models were increased by 7.80±0.33-fold and 8.11±0.37-fold, respectively (P<0.01), and the neonatal rat mRNA expression level was increased by 8.08±0.39-fold (P<0.01).2. Compared with normal adult rats, TRPC1, TRPC3 and TRPC6 channel mRNA expression levels in the cardiac hypertrophy model rats and neonatal rats were higher (P<0.01, For detailed data, see the Part 2 Table3 and Table4). However, there was no significant difference in mRNA expression levels (TRPC1, TRPC3 and TRPC6) between model rats and neonatal rats. The mRNA expression levels of TRPC2, TRPC4 and TRPC5 in all groups were not significantly different (P>0.05). TRPC7 mRNA expression level in neonatal rats was higher than other groups (P<0.01), and the normal adult rats groups and model groups were not significantly different (P>0.05).3. Among all TRPM family members, only TRPM4 and TRPM7 expression were detected. The mRNA expression level of TRPM4 in neonatal rats group and model rats group was higher than normal rats group (P<0.01, for detailed data, see Part 2 Table5 and Table6). The TRPM7 channel mRNA expression level was not significant different in all groups.4. Among all TRPV family members, only TRPV2 and TRPV6 expression were detected. The mRNA expression levels of TRPV2 and TRPV6 in cardiac hypertrophy model rats group were higher than normal rats group and neonatal rats groups (P<0.01, for detailed data, see Part 2 Table5 and Table6). There was no significant difference between hypertrophy model groups.5. Compared with normal adult rats, mRNA expression levels of STIM1 and Orai1 in neonatal rats group and cardiac hypertrophy model rats group were significantly higher (P<0.01, for detailed data, see part 2 Table1 and Table2).Conclusion: Expression levels of some TRP channels subtypes were selectively increased in cardiac hypertrophy of rats. These data suggest that these TRP channels may play an important role in the occurrence and development of cardiac hypertrophy. Furthermore, general expression profile of TRP channels in myocardial tissue from neonatal rats is similar to that from cardiac hypertrophy models rats. Thus, compared with normal rats groups, the expression levels of BNP, TRPC1, TRPC3, TRPC6, TRPM4, STIM1 and Orai1 in neonatal rats and cardiac hypertrophy model rats were increased. The results of this study indicate that TRP channels may represent an important therapeutic target for the treatment of cardiac hypertrophy.更多还原