X线诱导心肌成纤维细胞促纤维化损伤及黄芪注射液对其干预的实验研究

发布时间：2018-05-13 18:01

本文选题：放射性心脏损伤 + 纤维化　；参考：《兰州大学》2014年博士论文

【摘要】：背景胸腹部肿瘤放射治疗时,位于纵隔的心脏不可避免受到照射而引起的心脏损伤,称为放射性心脏疾病(radiation-induced heart disease, RIHD)。RIHD是胸部肿瘤放疗后最常见的良性致死原因,其发生率呈上升趋势,该病逐渐成为医学研究热点。RIHD的主要病理改变是纤维化,心肌成纤维细胞(cardiac fibroblasts, CFs)是心脏纤维化的主要效应细胞,可合成分泌TGF-β1和胶原蛋白,二者直接参与纤维化病理发展过程,现普遍认为“纤维化”是细胞因子表达失衡的多因素复杂疾病。内质网(endoplasmic reticulum, ER)是细胞内负责蛋白质合成、加工的细胞器,在受到氧化应激等因素刺激时,其内稳机制被破坏引起ER应激(endoplasmic reticulum stress, ERS)反应。越来越多的证据表明ERS参与心脏的纤维化重构,但是否也参与放射性心脏纤维化损伤不清楚。总体来说,RIHD的发病机理不清,临床尚无可靠治疗措施,通常以预防为主,按照循证医学“有证查证用证,无证创证用证”的观点分析,目前RIHD的临床防治处于“无证”或“少证”阶段,这就要求在“查证”基础上进行“创证用证”实践,深入研究放射性心脏纤维化损伤的病理学分子机制,为其临床防治寻找可能的干预靶点,这具有迫切的现实意义。中药黄芪在肝、肾、肺等器官的纤维化防治中疗效明确,在RIHD的临床实践中也已见个别报道,但其改善心脏放射性纤维化的机制不清,有待进一步研究。目的 (1)观察“X线对CFs的促纤维化损伤效应”并构建纤维化损伤的细胞模型；(2)在上述细胞模型上检测分析X线对84个纤维化相关分子表达的影响,筛选出差异表达的基因,从分子水平初步探索放射促纤维化损伤的可能分子机制；(3)由于ER应激可能参与心脏的纤维化重构,故观察X线是否引起CFs中FR应激反应,并通过ER应激抑制剂牛磺熊去氧胆酸钠(tauro ursodesoxy cholic acid, TUDCA)的实验干预论证ER应激与射线促纤维化损伤的关系；(4)观察黄芪对X线促纤维化损伤效应的影响,论证其对受照射CFs是否有保护作用,并从纤维化相关分子表达和ER应激两个方面探讨黄芪保护作用的可能机制,为其临床防治RIHD提供基础理论支持。方法 (1)观察X线的“促纤维化损伤效应”并构建细胞模型：用低剂量X线照射CFs后,通过MTT检测细胞增殖活性,选择实验指标观察时点；通过ELISA观察纤维化指标TGF-β1、I型胶原(Col-1)和Ⅲ型胶原(Col-3)的分泌情况,粗筛造模剂量,最后用RT-PCR和Western Blot技术进一步确定造模剂量并验证“促纤维化损伤”细胞造模是否成功；(2)应用RT-PCR和微阵列相结合的实验新技术"PCR Array"高通量检测84个纤维化相关分子在对照组和模型组之间的差异表达,筛选出两组间表达变化≥2倍,有统计学差异的基因,初步分析并探讨放射促纤维化损伤的可能分子机制；(3)ER应激在射线促纤维化损伤中的作用：首先用透射电镜观察受照射CFs纤维化损伤模型中ER形态变化,然后用RT-PCR和Western Blot比较照射组和未照射组ERS标志分子GRP78、ATF6、p-PERK和CHOP的表达；最后用ERS抑制剂TUDCA预处理受照射CFs,再观察比较纤维化和ERS的标志分子差异表达的情况；(4)黄芪实验干预：通过MTT确定黄芪注射液干预剂量,黄芪注射液预处理受照射CFs,流式细胞术检测黄芪对受照射CFs中ROS的影响；以RT-PCR和Western Blot方法观察未照射组、照射组、高/低剂量黄芪+照射组中纤维化和ERS的标志分子差异表达情况；PCR Array检测84个纤维化相关分子在照射组、黄芪+照射组之间的差异表达。结果 (1)Ⅹ线照射CFs发现Ⅹ线以剂量和时间依赖性方式抑制细胞增殖活性,表现出射线直接照射细胞的损伤效应。低剂量(1、2Gy)X线照射后的前48h CFs增殖活性良好,对比未照射组无差异,之后增殖变慢,和未照射组比有显著差异；同时48h观察到TGF-β1的mRNA表达分别增加了138.64%(1Gy)和88.03%(2Gy)(均P0.D1)；Col-1的mRNA表达分别增加了109.65%(1Gy)和80.62%(2Gy)(均P0.01)；相应的与对照组相比,蛋白TGF-β1的相对表达水平增加到160.05%(1Gy)和98.95%(2Gy),蛋白Col-1的相对表达水平增加到201.37%(1Gy)和129.54%(2Gy)(均P0.01)。4Gy照射后,TGF-β1和Col-1的mRNA和蛋白表达均显著低于对照组。提示1、2Gy对CFs有明显的促纤维化效应。当剂量增大到4Gy,各观察时点细胞增殖抑制均显著,且上述促纤维化效应明显被取消,提示射线剂量过大,细胞活性功能全面下降。1、2Gy X线照射CFs后48h细胞活性良好,同时促纤维化效应最明显,确定为X线促纤维化损伤效应的建模剂量及观察时点。 (2) PCR Array检测84个纤维化相关分子显示模型组(1Gy)和对照组(0Gy)相比有44个基因差异表达,30个基因表达上调,14个基因表达下调；细胞外基质两个基因Col-1A2和Col-3A1表达均上调；重构酶基因中MMP14、MMP3、 MMP8、Plau (uPA)、Serpinala (al-antitrypsin)表达上调,Lox、Plat、Serpinel、 TIMP1表达下调,炎性细胞因子/趋化因子类基因中CXCR4、IL-10, IL-13、 IL-13ra2、IL-1a、IL-1b、TNF表达上调,Ccr2表达下调；TGF-β超家族基因中BMP7、Cavl、Dcn、TGFβ1、Smad2、Smad3、Smad4、TGIF1表达上调,Eng、 Inhbe、Smad7、TGFβr1、Thbs1表达下调；Fasl基因表达也上调了2.1倍。 (3)受照射的CFs中内质网增殖、扩张、排列紊乱。ERS标志分子GRP78和CHOP的mRNA分别较0Gy对照组增加了158.66%(1Gy)、133.12%(2Gy)和106.23%(1Gy)、189.65%(2Gy)(均P0.01),4Gy时两者mRNA表达均下降；类似的对比0Gy组,1和2Gy照射组GRP78蛋白表达分别增加了1.50倍和1.45倍,CHOP蛋白表达分别增加了2.50倍和2.81倍,ATF6蛋白表达分别增加了70.27%和23.74%,p-PERK蛋白表达分别增加了42.9%和28.6%(均P0.01)；以上结果提示ERS在受照射的CFs中被激活。ERS抑制剂TUDCA (0.8mmol/L)明显抑制射线诱导的ERS反应,表现为：1Gy+TUDCA组与1Gy组相比,GRP78和CHOP的mRNA表达分别降低了40.8%和42.46%(均P0.01)；GRP78、 CHOP、ATF6和p-PERK蛋白表达分别降低了54.02%、68.01%、10.83%和22.2%(均P0.01)；同时TUDCA下调纤维化分子的表达,减弱X线促纤维化效应,表现为：对比1Gy组,TUDCA预处理组Col-1的mRNA和蛋白表达分别下降了34.20%和64.01%分别(均P0.01),TGF-β1mRNA的表达下降了52.76%(P0.01),TGF-β1和其下游分子p-Smad2/3的相对蛋白质含量也分别下降了60.86%(P0.01)和55.73%(P001)。 (4)黄芪注射液(AST)抑制受照射CFs内的ROS水平,同时AST(20μg/ml)预处理组对比单纯1Gy照射组,GRP78、CHOP、TGF-β1和Col-1的mRNA表达分别降低了40.8%、42.46%、48.12%和39.22%(均P0.01),蛋白表达分别降低了54.02%、68.01%、48.14%和64.02%,p-Smad2/3蛋白表达降低了38.20%(均P0.01)；AST对纤维化分子的抑制效应类似与对ROS的抑制,表现为剂量依赖性,即：20μg/ml的AST抑制作用强于10μg/ml的AST; PCR Array检测表明20pg/ml的AST预处理受照射CFs后,绝大部分差异表达的纤维化相关基因被逆转,即：射线引起高表达的基因可被AST下调,反之,射线引起表达下调的基因可部分的被AST恢复至基线水平或上调。结论 (1)1、2Gy X线照射CFs后48h细胞活性良好,同时纤维化主要分子TGF-β1和Col-1表达显著增加,促纤维化效应最明显,确定为X线促纤维化损伤的建模剂量及指标观察时点；(2)小剂量X线照射CFs有明确的促纤维化损伤效应,这种效应是多途径多分子共同介导的,主要涉及的分子机制包括：促纤维化和抗纤维化失衡；TGF-β信号级联反应激活,其正性调节被强化而负性调节被抑制；细胞外基质合成增多；促炎因子/趋化因子和基质重构系统等在X线照射早期也被激活。(3)ERS也在受照射的CFs中被激活,ERS抑制剂TUDCA显著抑制X线促纤维化效应,提示ERS参与放射诱导的促纤维化损伤过程。(4)本研究显示中药黄芪可通过其抗氧化特性抑制射线诱导的ERS反应,减弱放射促纤维化损伤效应,逆转射线引起的绝大多数纤维化相关分子的异常表达而有效的保护受照射CFs。体外研究证据支持黄芪应用于放射诱导的纤维化损伤防治,但这仅仅是一个初步探索性实验,其有效性和确切分子机制有待进一步研究。
[Abstract]:background
Radiation-induced heart disease (RIHD).RIHD is the most common cause of benign death after radiotherapy of the chest tumor, which is the most common cause of death after radiotherapy in the chest. The incidence of the disease is increasing, and the disease gradually becomes the focus of medical research,.RIHD. The main pathological changes are fibrosis, cardiac fibroblasts (CFs), the main effect cell of cardiac fibrosis, can synthesize and secrete TGF- beta 1 and collagen, and the two are directly involved in the pathological process of fibrosis, and it is generally believed that "fibrosis" is a complex multifactor disease of cytokine imbalance. Endoplasmic reticulum (endop Lasmic reticulum, ER) is a cell organelle responsible for protein synthesis and processing within the cell. When stimulated by oxidative stress, its internal stability mechanism is destroyed to cause ER stress (endoplasmic reticulum stress, ERS). More and more evidence shows that ERS is involved in cardiac fibrosis reconstruction, but it is also involved in radioactive cardiac fibrosis. In general, the pathogenesis of RIHD is not clear, and there is no reliable treatment in clinical. It is usually based on the viewpoint of evidence-based medicine, "evidence-based evidence, no proof of evidence". At present, the clinical prevention and control of RIHD is in the stage of "no evidence" or "less evidence", which requires "creation" on the basis of "verification". It has urgent practical significance to study the pathological molecular mechanism of radiological cardiac fibrosis and find possible targets for its clinical prevention and treatment. The curative effect of Astragalus membranaceus in the prevention and treatment of fibrosis in liver, kidney, lung and other organs is clear, and some reports have been seen in the clinical practice of RIHD, but it improves the heart. The mechanism of radiation-induced fibrosis is unclear and needs further study.
objective
(1) observe the effect of X - ray induced fibrosis injury on CFs and construct the cell model of fibrosis injury; (2) the effects of X - ray on the expression of 84 fibrosis related molecules are detected and analyzed on the above cell model, the differentially expressed genes are screened, and the possible molecular mechanism of radiation induced fibrosis damage is preliminarily explored from the molecular level; (3) ER Stress may be involved in the remodeling of cardiac fibrosis, so to observe whether X-ray causes FR stress in CFs, and through the experimental intervention of ER stress inhibitor sodium taurodeoxycholate (Tauro ursodesoxy cholic acid, TUDCA) to demonstrate the relationship between ER stress and radiation induced fibrosis, and (4) observe the effect of Astragalus on the effect of X - ray induced fibrosis To demonstrate whether it has protective effect on the irradiated CFs, and to explore the possible mechanism of the protective effect of Astragalus from the two aspects of the expression of fibrosis related molecules and ER stress, which provides the basic theoretical support for the clinical prevention and treatment of RIHD.
Method
(1) observe the "fibrotic injury effect" of X ray and construct the cell model: after irradiating CFs with low dose X-ray, the cell proliferation activity is detected by MTT, and the time points are observed by the experimental index. The secretion of TGF- beta 1, I collagen (Col-1) and type III collagen (Col-3) are observed by ELISA, the dosage of the coarse screening model, and the final use of RT-PCR and W Estern Blot technology further determines the dosage of the model and verifies the success of the "fibrotic injury" cell model. (2) the differential expression of 84 fibrotic related molecules between the control group and the model group is detected by the new technique of RT-PCR and microarray combined with "PCR Array", and the changes of the expression between the two groups are more than 2 times. The genes of statistical difference were preliminarily analyzed and discussed the possible molecular mechanism of radiation induced fibrosis injury; (3) the role of ER stress in the radiation induced fibrosis injury: first, the morphological changes of ER in the irradiated CFs fibrosis model were observed by transmission electron microscopy, and then the ERS markers in the irradiated and unirradiated groups were compared with the RT-PCR and Western Blot. The expression of GRP78, ATF6, p-PERK and CHOP; finally, the ERS inhibitor TUDCA was used to pretreat the irradiated CFs, and then the differential expression of the markers of fibrosis and ERS was observed. (4) Astragalus membranaceus experimental intervention: the dosage of Astragalus membranaceus injection was determined by MTT, the pretreated astragalus injection was irradiated CFs, and the flow cytometry was used to detect Astragalus exposure. The effect of ROS in CFs and RT-PCR and Western Blot were used to observe the differential expression of the markers of fibrosis and ERS in the unirradiated group, the irradiated group, the high / low dose Astragalus + irradiation group and the PCR Array to detect the difference expression between the 84 fibrotic related molecules in the irradiation group and the Astragalus membranaceus + irradiation group.
Result
(1) the line irradiated CFs found that the line inhibited the cell proliferation activity in a dose and time dependent manner, showing the damage effect of direct irradiation of the cells. The proliferation activity of the pre 48h CFs was good after the low dose (1,2Gy) X-ray irradiation, and the proliferation was not significant compared with the unirradiated group, and the proliferation was significantly different from that of the unirradiated group. At the same time, 48h was observed by 48h. The expression of mRNA in TGF- beta 1 increased by 138.64% (1Gy) and 88.03% (2Gy), respectively. The mRNA expression of Col-1 increased by 109.65% (1Gy) and 80.62% (2Gy) (2Gy), respectively, and the relative expression level of protein TGF- beta 1 increased to 160.05% (1Gy) and 98.95%, compared with the control group, and the relative expression level of proteins increased to 201.37%. The mRNA and protein expression of TGF- beta 1 and Col-1 were significantly lower than that of the control group after 129.54% (2Gy) (P0.01).4Gy irradiation. It was suggested that 1,2Gy had obvious fibrotic effect on CFs. When the dose increased to 4Gy, the proliferation inhibition of all the observed time points was significant, and the above fibrotic effect was obviously cancelled, suggesting that the radiation dose was too large and the activity work of the cells was too large. The activity of 48h cells was good and the fibrotic effect was most obvious after.1,2Gy X-ray irradiation of CFs. It was determined to be the dose of modeling and the observation time of the effect of X - ray induced fibrosis.
(2) PCR Array detected 84 fibrotic related molecules in the model group (1Gy) and the control group (0Gy), there were 44 genes differential expression, 30 gene expression up-regulated, 14 gene expression down-regulation, two extracellular matrix Col-1A2 and Col-3A1 expression up-regulated, MMP14, MMP3, MMP8, Plau (uPA), Serpinala (Serpinala) in the reconstructive enzyme. The expression of Lox, Plat, Serpinel, TIMP1 down regulated, CXCR4, IL-10, IL-13, IL-13ra2, IL-1a, IL-1b, IL-1b, IL-1b, TNF expression in the inflammatory cytokines / chemokine genes. The gene expression was also up 2.1 times higher.
(3) the proliferation and expansion of endoplasmic reticulum in the irradiated CFs, the mRNA of the disorder.ERS markers, GRP78 and CHOP, were increased by 158.66% (1Gy), 133.12% (2Gy) and 106.23% (1Gy), 189.65% (2Gy) (P0.01), and the expression of the two groups decreased by 1.50 times as compared with those of the 1 and 1 groups. 1.45 times, the expression of CHOP protein increased by 2.50 and 2.81 times, the expression of ATF6 protein increased by 70.27% and 23.74% respectively, and the expression of p-PERK protein increased by 42.9% and 28.6%, respectively, and 28.6% (all P0.01); the above results suggest that ERS is activated by the.ERS inhibitor TUDCA (0.8mmol/L) in the irradiated CFs, which obviously inhibits the ray induced ERS reaction, which is manifested in 1Gy+TU. Compared with group 1Gy, mRNA expression of GRP78 and CHOP decreased by 40.8% and 42.46% (all P0.01), and the expression of GRP78, CHOP, ATF6 and p-PERK proteins decreased by 54.02%, 68.01%, 10.83% and 22.2% (P0.01). The expression of mRNA and protein in 1 decreased by 34.20% and 64.01% respectively (all P0.01), and the expression of TGF- beta 1mRNA decreased by 52.76% (P0.01). The relative protein content of TGF- beta 1 and its downstream molecule p-Smad2/3 also decreased by 60.86% (P0.01) and 55.73% (P001) respectively.
(4) Astragalus Injection (AST) inhibited the ROS level in the irradiated CFs, while the AST (20 g/ml) pretreatment group compared the mRNA 1Gy group with GRP78, CHOP, TGF- beta 1 and Col-1 mRNA expression of 40.8%, 42.46%, 48.12% and 39.22% (P0.01), and the protein expression decreased by 54.02%, 68.01%, 48.14% and 64.02% respectively. The expression of protein decreased by 38.20%. (all P0.01); the inhibitory effect of AST on fibrotic molecules is similar to that of ROS, showing a dose dependence, that is, the AST inhibition of 20 mu g/ml is stronger than the AST of 10 mu g/ml; PCR Array detection shows that after AST pretreatment of 20pg/ml is irradiated, most of the differentially expressed fibrinolytic related genes are reversed, that is, the high expression caused by rays. The genes can be downregulated by AST. Conversely, the genes that cause down-regulation by radiation can partly be restored to baseline or up-regulated by AST.
conclusion
(1) the activity of 48h cells was good after 1,2Gy X ray irradiation for CFs, and the expression of TGF- beta 1 and Col-1 increased significantly, and the fibrotic effect was most obvious. It was determined to be the time point for the modeling dose and index of the X-ray fibrotic injury. (2) the small dose X-ray irradiation of CFs had a definite fibrotic injury effect. This effect was multichannel and multiple. The main molecular mechanisms involved include: fibrosis and anti fibrosis imbalances; TGF- beta signaling cascade activation, positive regulation being enhanced and negatively regulated; extracellular matrix synthesis; proinflammatory factors / chemotactic factors and matrix remodeling systems are also activated in the early X-ray exposure. (3) ERS is also being subjected The irradiated CFs was activated, and the ERS inhibitor TUDCA significantly inhibited the X ray induced fibrosis effect, suggesting that ERS was involved in the process of radiation induced fibrosis injury. (4) the study showed that Astragalus membranaceus could inhibit the radiation induced ERS reaction through its antioxidant properties, weaken the effect of radiation induced fibrosis, and reverse the vast majority of fibrosis induced by radiation. The abnormal expression of the related molecules and effective protection of the irradiated CFs. in vitro evidence supports the application of Astragalus in the prevention and control of radiation induced fibrosis injury, but this is only a preliminary exploratory experiment. Its effectiveness and molecular mechanism need to be further studied.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R965

【参考文献】