本文選題：放射性心臟損傷 + 纖維化��； 參考：《蘭州大學》2014年博士論文

【摘要】：背景 胸腹部腫瘤放射治療時,位于縱隔的心臟不可避免受到照射而引起的心臟損傷,稱為放射性心臟疾病(radiation-induced heart disease, RIHD)。RIHD是胸部腫瘤放療后最常見的良性致死原因,其發(fā)生率呈上升趨勢,該病逐漸成為醫(yī)學研究熱點。RIHD的主要病理改變是纖維化,心肌成纖維細胞(cardiac fibroblasts, CFs)是心臟纖維化的主要效應細胞,可合成分泌TGF-β1和膠原蛋白,二者直接參與纖維化病理發(fā)展過程,現(xiàn)普遍認為“纖維化”是細胞因子表達失衡的多因素復雜疾病。內質網(endoplasmic reticulum, ER)是細胞內負責蛋白質合成、加工的細胞器,在受到氧化應激等因素刺激時,其內穩(wěn)機制被破壞引起ER應激(endoplasmic reticulum stress, ERS)反應。越來越多的證據表明ERS參與心臟的纖維化重構,但是否也參與放射性心臟纖維化損傷不清楚�？傮w來說,RIHD的發(fā)病機理不清,臨床尚無可靠治療措施,通常以預防為主,按照循證醫(yī)學“有證查證用證,無證創(chuàng)證用證”的觀點分析,目前RIHD的臨床防治處于“無證”或“少證”階段,這就要求在“查證”基礎上進行“創(chuàng)證用證”實踐,深入研究放射性心臟纖維化損傷的病理學分子機制,為其臨床防治尋找可能的干預靶點,這具有迫切的現(xiàn)實意義。中藥黃芪在肝、腎、肺等器官的纖維化防治中療效明確,在RIHD的臨床實踐中也已見個別報道,但其改善心臟放射性纖維化的機制不清,有待進一步研究。 目的 (1)觀察“X線對CFs的促纖維化損傷效應”并構建纖維化損傷的細胞模型；(2)在上述細胞模型上檢測分析X線對84個纖維化相關分子表達的影響,篩選出差異表達的基因,從分子水平初步探索放射促纖維化損傷的可能分子機制；(3)由于ER應激可能參與心臟的纖維化重構,故觀察X線是否引起CFs中FR應激反應,并通過ER應激抑制劑牛磺熊去氧膽酸鈉(tauro ursodesoxy cholic acid, TUDCA)的實驗干預論證ER應激與射線促纖維化損傷的關系；(4)觀察黃芪對X線促纖維化損傷效應的影響,論證其對受照射CFs是否有保護作用,并從纖維化相關分子表達和ER應激兩個方面探討黃芪保護作用的可能機制,為其臨床防治RIHD提供基礎理論支持。 方法 (1)觀察X線的“促纖維化損傷效應”并構建細胞模型：用低劑量X線照射CFs后,通過MTT檢測細胞增殖活性,選擇實驗指標觀察時點；通過ELISA觀察纖維化指標TGF-β1、I型膠原(Col-1)和Ⅲ型膠原(Col-3)的分泌情況,粗篩造模劑量,最后用RT-PCR和Western Blot技術進一步確定造模劑量并驗證“促纖維化損傷”細胞造模是否成功；(2)應用RT-PCR和微陣列相結合的實驗新技術"PCR Array"高通量檢測84個纖維化相關分子在對照組和模型組之間的差異表達,篩選出兩組間表達變化≥2倍,有統(tǒng)計學差異的基因,初步分析并探討放射促纖維化損傷的可能分子機制；(3)ER應激在射線促纖維化損傷中的作用：首先用透射電鏡觀察受照射CFs纖維化損傷模型中ER形態(tài)變化,然后用RT-PCR和Western Blot比較照射組和未照射組ERS標志分子GRP78、ATF6、p-PERK和CHOP的表達；最后用ERS抑制劑TUDCA預處理受照射CFs,再觀察比較纖維化和ERS的標志分子差異表達的情況；(4)黃芪實驗干預：通過MTT確定黃芪注射液干預劑量,黃芪注射液預處理受照射CFs,流式細胞術檢測黃芪對受照射CFs中ROS的影響；以RT-PCR和Western Blot方法觀察未照射組、照射組、高/低劑量黃芪+照射組中纖維化和ERS的標志分子差異表達情況；PCR Array檢測84個纖維化相關分子在照射組、黃芪+照射組之間的差異表達。 結果 (1)Ⅹ線照射CFs發(fā)現(xiàn)Ⅹ線以劑量和時間依賴性方式抑制細胞增殖活性,表現(xiàn)出射線直接照射細胞的損傷效應。低劑量(1、2Gy)X線照射后的前48h CFs增殖活性良好,對比未照射組無差異,之后增殖變慢,和未照射組比有顯著差異；同時48h觀察到TGF-β1的mRNA表達分別增加了138.64%(1Gy)和88.03%(2Gy)(均P0.D1)；Col-1的mRNA表達分別增加了109.65%(1Gy)和80.62%(2Gy)(均P0.01)；相應的與對照組相比,蛋白TGF-β1的相對表達水平增加到160.05%(1Gy)和98.95%(2Gy),蛋白Col-1的相對表達水平增加到201.37%(1Gy)和129.54%(2Gy)(均P0.01)。4Gy照射后,TGF-β1和Col-1的mRNA和蛋白表達均顯著低于對照組。提示1、2Gy對CFs有明顯的促纖維化效應。當劑量增大到4Gy,各觀察時點細胞增殖抑制均顯著,且上述促纖維化效應明顯被取消,提示射線劑量過大,細胞活性功能全面下降。1、2Gy X線照射CFs后48h細胞活性良好,同時促纖維化效應最明顯,確定為X線促纖維化損傷效應的建模劑量及觀察時點。 (2) PCR Array檢測84個纖維化相關分子顯示模型組(1Gy)和對照組(0Gy)相比有44個基因差異表達,30個基因表達上調,14個基因表達下調；細胞外基質兩個基因Col-1A2和Col-3A1表達均上調；重構酶基因中MMP14、MMP3、 MMP8、Plau (uPA)、Serpinala (al-antitrypsin)表達上調,Lox、Plat、Serpinel、 TIMP1表達下調,炎性細胞因子/趨化因子類基因中CXCR4、IL-10, IL-13、 IL-13ra2、IL-1a、IL-1b、TNF表達上調,Ccr2表達下調；TGF-β超家族基因中BMP7、Cavl、Dcn、TGFβ1、Smad2、Smad3、Smad4、TGIF1表達上調,Eng、 Inhbe、Smad7、TGFβr1、Thbs1表達下調；Fasl基因表達也上調了2.1倍。 (3)受照射的CFs中內質網增殖、擴張、排列紊亂。ERS標志分子GRP78和CHOP的mRNA分別較0Gy對照組增加了158.66%(1Gy)、133.12%(2Gy)和106.23%(1Gy)、189.65%(2Gy)(均P0.01),4Gy時兩者mRNA表達均下降；類似的對比0Gy組,1和2Gy照射組GRP78蛋白表達分別增加了1.50倍和1.45倍,CHOP蛋白表達分別增加了2.50倍和2.81倍,ATF6蛋白表達分別增加了70.27%和23.74%,p-PERK蛋白表達分別增加了42.9%和28.6%(均P0.01)；以上結果提示ERS在受照射的CFs中被激活。ERS抑制劑TUDCA (0.8mmol/L)明顯抑制射線誘導的ERS反應,表現(xiàn)為：1Gy+TUDCA組與1Gy組相比,GRP78和CHOP的mRNA表達分別降低了40.8%和42.46%(均P0.01)；GRP78、 CHOP、ATF6和p-PERK蛋白表達分別降低了54.02%、68.01%、10.83%和22.2%(均P0.01)；同時TUDCA下調纖維化分子的表達,減弱X線促纖維化效應,表現(xiàn)為：對比1Gy組,TUDCA預處理組Col-1的mRNA和蛋白表達分別下降了34.20%和64.01%分別(均P0.01),TGF-β1mRNA的表達下降了52.76%(P0.01),TGF-β1和其下游分子p-Smad2/3的相對蛋白質含量也分別下降了60.86%(P0.01)和55.73%(P001)。 (4)黃芪注射液(AST)抑制受照射CFs內的ROS水平,同時AST(20μg/ml)預處理組對比單純1Gy照射組,GRP78、CHOP、TGF-β1和Col-1的mRNA表達分別降低了40.8%、42.46%、48.12%和39.22%(均P0.01),蛋白表達分別降低了54.02%、68.01%、48.14%和64.02%,p-Smad2/3蛋白表達降低了38.20%(均P0.01)；AST對纖維化分子的抑制效應類似與對ROS的抑制,表現(xiàn)為劑量依賴性,即：20μg/ml的AST抑制作用強于10μg/ml的AST; PCR Array檢測表明20pg/ml的AST預處理受照射CFs后,絕大部分差異表達的纖維化相關基因被逆轉,即：射線引起高表達的基因可被AST下調,反之,射線引起表達下調的基因可部分的被AST恢復至基線水平或上調。 結論 (1)1、2Gy X線照射CFs后48h細胞活性良好,同時纖維化主要分子TGF-β1和Col-1表達顯著增加,促纖維化效應最明顯,確定為X線促纖維化損傷的建模劑量及指標觀察時點；(2)小劑量X線照射CFs有明確的促纖維化損傷效應,這種效應是多途徑多分子共同介導的,主要涉及的分子機制包括：促纖維化和抗纖維化失衡；TGF-β信號級聯(lián)反應激活,其正性調節(jié)被強化而負性調節(jié)被抑制；細胞外基質合成增多；促炎因子/趨化因子和基質重構系統(tǒng)等在X線照射早期也被激活。(3)ERS也在受照射的CFs中被激活,ERS抑制劑TUDCA顯著抑制X線促纖維化效應,提示ERS參與放射誘導的促纖維化損傷過程。(4)本研究顯示中藥黃芪可通過其抗氧化特性抑制射線誘導的ERS反應,減弱放射促纖維化損傷效應,逆轉射線引起的絕大多數纖維化相關分子的異常表達而有效的保護受照射CFs。體外研究證據支持黃芪應用于放射誘導的纖維化損傷防治,但這僅僅是一個初步探索性實驗,其有效性和確切分子機制有待進一步研究。
[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.

【學位授予單位】：蘭州大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R965

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