【作者】 张志远；

【导师】 钱桂生；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 肺动脉平滑肌细胞(PASMC)的异常增殖与凋亡是低氧肺动脉高压肺血管重建发病的关键环节。在此病理过程中,多种细胞因子、生长因子通过细胞内信号传导途径将细胞外增殖信号传至核内,在核内调节基因表达,启动细胞应激反应。然而在此过程中PASMC的信号转导机制尚未完全阐明。Hedgehog(Hh)信号通路是与发育相关的重要信号转导途径。在脊椎动物Hedgehog主要有三个成员,包括Shh(Sonic hedgehog)、Ihh(Indian hedgehog)和Dhh(Desert hedgehog)。Patched (Ptc)和Smoothened (Smo)共同介导Hedgehog信号向胞内的传递,在无Hedgehog情况下,Ptc抑制Smo,当Hedgehog与Ptc结合后,则解除了其抑制作用,继而Smo激活转录因子Gli,从而增强或抑制一系列Hh信号靶基因的转录,产生一系列细胞效应。Hh信号通路在胚胎发育中发挥重要作用,对于胚胎细胞的增殖、分化起着极为关键的调节作用,如果此通路发生突变,会导致多种严重的先天发育缺陷。近年来发现,Hh信号通路在成熟机体的多种生理病理过程中也发挥重要调节作用。Shh缺陷的小鼠肺组织血管结构存在障碍,而Shh过度表达会导致血管形成过度。最近报道,生物机械应力可降低大鼠血管平滑肌Hh信号通路主要分子的表达,对动脉血管重建具有显著影响。因此,本研究使用人原代PASMC培养模型,观察Hh信号通路主要分子在正常及低氧时的表达和活性变化,并通过特异性拮抗剂及激活剂调节此通路的活化水平,观察其对PASMC增殖、凋亡及细胞因子分泌等病理变化的影响,以阐明Hh信号通路在低氧肺血管重建中的作用及机制,从而为低氧肺动脉高压的防治提供理论依据和新的治疗靶点。方法:1.购买并培养HPASMC细胞株,RT-PCR检测正常HPASMC中Shh、Ptc-1和Gli-1基因mRNA表达;Western blot法检测以上蛋白表达,免疫荧光法检测Ptc-1与Gli-1蛋白在静息状态下HPASMC的定位。Shh刺激实验观察Hh信号通路的功能。2. Real-time RT-PCR和Western blot检测低氧条件下HPASMC中Shh与Ptc-1 mRNA与蛋白表达变化,免疫荧光法检测Gli-1细胞定位的变化。3.不同时相点低氧处理HPASMC, Alamar Blue法和流式细胞仪检测低氧条件下阻断Hh信号通路对HPASMC细胞增殖、凋亡与细胞周期分布的变化。4. ELISA法检测cyclopamine阻断Hh信号通路对低氧诱导的HPASMC分泌MCP-1及IL-6活性的影响。5. Alamar Blue法检测维生素D3对低氧诱导的HPASMC增殖的影响。Real-time RT-PCR检测维生素D3对低氧诱导的HPASMC中Gli-1基因mRNA的表达影响,免疫荧光法检测Gli-1蛋白细胞定位的变化。结果:1.RT-PCR显示HPASMC有Shh、Ptc-1与Gli-1 mRNA表达,免疫荧光检测显示Ptc-1表达在HPASMC细胞膜,而Gli-1在静息HPASMC的胞浆而非胞核表达;Western blot法均检测到以上蛋白表达。Shh刺激可明显促进HPASMC增殖,并可被Hh通路特异性抑制剂cyclopamine阻断。2.实时定量RT-PCR与Western blot均显示Shh与Ptc-1的mRNA和蛋白表达在低氧4h后升高,8h达到高峰,12h下降。低氧可诱导Gli-1蛋白的胞核聚集,并可被cyclopamine所阻断。3.HPASMC经24h静息处理后进行常氧与低氧培养,低氧显著促进细胞增殖,其增殖效应主要发生在低氧4h、8h与12h。cyclopamine可明显抑制此低氧诱导的增殖效应,并可明显增加G0/G1期、减少S期与G2/M期细胞周期分布。Annexin -V染色法流式细胞仪检测显示,与常氧对照比较,低氧刺激后HPASMC凋亡明显减少,而cyclopamine对凋亡具有明显促进作用。4.常氧对照HPASMC培养基上清中可检测到IL-6活性,但无MCP-1分泌,低氧8h处理后二者活性均明显增高,cyclopamine处理可明显抑制二者分泌。5.维生素D3可明显抑制低氧诱导的HPASMC Gli-1mRNA的表达和蛋白的核内聚集,同时对低氧诱导的HPASMC的增殖具有明显抑制作用。结论:1.正常HPASMC有Hh信号通路主要分子的表达,并对细胞生长具有调节功能。2.低氧可明显活化HPASMC中Hh信号通路的表达,并经由此通路促进HPASMC增殖、抑制凋亡及调节致炎性细胞因子的分泌,提示Hh通路可能是低氧肺血管重建病变进展的重要分子基础,抑制此通路的活化可能是低氧肺动脉高压等疾病治疗新的靶点。3.维生素D3可通过抑制Hh信号通路活化对低氧肺血管重建起到治疗作用,具有潜在的临床应用价值。更多还原

【Abstract】 Abnormal changes of proliferation and apoptosis in pulmonary artery smooth muscle cells (PASMCs) play a key role on vascular remodeling that occurs in chronic hypoxic pulmonary hypertension (HPH). Different growth factors, cytokine and proinflammatory mediators convey extracelluar signal to the nucleus by interacting with receptors on the cell surface, then regulate expression of target genes and contribute to cell stress reaction. However, the cellular signal mechanism of PASMC involved in hypoxic pulmonary vascular remodeling are not fully understood.The Hedgehog(Hh) signaling pathway has been well investigated in the studies of development. To date, three members of the hedgehog family have been identified in vertebrates: Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). The stimuli of secreted hedgehog family peptides are mediated by a receptor system on the cell surface composed with two proteins: Patched (Ptc) and Smoothened (Smo). The hedgehog receptor Ptc represses the 7-transmembrane protein Smo, inhibiting the pathway in the absence of hedgehog ligands. Upon hedgehog ligand binding, Ptc is inactivated, and then Smo is de-repressed from the suppression of Ptc and is able to transduce the intracellular signals to transcriptor Gli. Gli transfers the signals into the nucleus and then activates and represses many target gene transcription in response to Hh signaling.The Hedgehog signaling pathway plays a fundamental role in the development of various embryonic tissues development. The pathway has been shown to have effects on cell proliferation, cell survival and cell fate determination in embryonic development. Mutations in the pathway often result in gross defects across species. Its regulatory roles in signal transduction in mature/adult cells and their implications in disease are now well-recognized recent years. Several observations highlight the involvement of Hh in the development of embryonic vascular tissues, including hypervascularization of neuroectoderm following overexpression of Shh ,and poor vascularization of the developing lung in Shh-deficient mice. Recently a study showed that Hh signaling is present in adult vascular smooth muscle cells and can be diminished by biomechanical stimulation in vitro and in vivo and thus may play a fundamental role in arterial remodeling. According to these, we hypothesized that Hh pathway may potentially have regulatory effects on VSMC’s response in hypoxic vascular remolding .This study was therefore undertaken to observe the expression of Hh signaling pathway and its role on human PASMCs under hypoxia conditions.Methods1. After HPASMC were purchased and cultured, expressions of Shh, Ptc-1 and Gli-1 mRNA and protein were detected by RT-PCR and Western blot, locations of Ptc-1 and Gli-1 in static HPASMC were detected by immunofluorescence. Shh was used to investigate the role of Hh signal pathway on HPASMC growth.2. The quantitative assessment of Shh and Ptc-1 mRNA expression under hypoxia by Real-time RT-PCR and the protein expression by Western blot analysis. Gli-1 nuclear accumulation induced by hypoxia was detected by immunofluorescence.3. After various hours hypoxia treatment, HPASMC proliferation was detected by Alamar Blue assay, cell cycle distribution and apoptosis was measured by flow cytometry analysis.4. After hypoxia treatment, with or without cyclopamine block, the activities of both cytokine The activities of IL-6 and MCP-1 in HPASMC cell supernatant was detected by ELISA.5. HPASMC was cultured in hypoxia with or without Vitamin D3 treatment, the proliferation was measured by Alamar Blue assay,the expression of Gli-1 mRNA was measured by real-time RT-PCR, and the location of Gli-1 protein was detected by immunofluorescence。Results1.RT-PCR revealed that Shh, Ptc-1, and Gli-1 mRNA are expressed in HPASMC. Immunofluorescence showed that Ptc-1 protein was present on the plasma membranes of HPASMC, and Gli-1 protein was present on the cytoplasm but not nucleus of static HPASMC. Western blot analysis showed that Shh, Ptc-1,and Gli-1 protein were expressed in HPASMC. Shh enhanced proliferation of HPASMC in a dose-dependent manner. The enhancement can be inhibited by cyclopamine(a Hh signaling inhibitor).2.The expressions of Shh and Ptc-1 mRNA level in HPASMC measured by real-time RT-PCR were promoted after 4h hypoxic exposure, peaked after 8h and decreased after 12h. And the protein expression level of Shh, Ptc-1 measured by Western blot changed as the same mode, respectively. Gli-1 nuclear accumulation induced by hypoxia and can be blocked by cyclopamine.3. HPASMC were serum starved at least 24h and then incubated for various periods in normoxic or hypoxic conditions. Hypoxia significantly promoted HPASMC proliferation at the phase of 4h, 8h, and 12h. Cyclopamine inhibited the proliferation, increased G0/G1 phrase, decreased S and G2/M phrase cell cycles significantly. Effect of Hh signaling pathway on HPASMC apoptosis in 8h hypoxic exposure was assessed by annexin V staining. Compared with normoxia control, the rate of HPASMC apoptosis in hypoxia group was lower, which can be increased by cyclopamine significantly.4.The IL-6 activity in normoxia HPASMC cell supernatant was detectable by ELISA, but MCP-1 not. After hypoxia exposure, the activities of IL-6 and MCP-1 both increased significantly, and can be inhibited remarkably by cyclopamine.5.After hypoxic treatment, vitamin D3 can inhibit the Gli-1mRNA expression level and nuclear accumulation of HPASMC and decrease the proliferation induced by hypoxia.Conclusions1. Hh signal pathway main components are expressed in normal adult HPASMC and involve in the growth regulation.2. Hh signal pathway can be activated by hypoxia, which involes in the abnormal changes of proliferation, apoptosis, and proinflammation cytokine secretion of HPASMC induced by hypoxia.3. Vitamin D3 can inhibit the activation of Hh signal pathway and decrease HPASMC proliferation induced by hypoxia, which indicate its potential value for the treatment of hypoxic pulmonary vascular remodeling.更多还原