發(fā)布時間：2018-03-03 02:13

  本文選題：橫紋肌肉瘤　切入點：HDAC　出處：《吉林大學》2017年碩士論文　論文類型：學位論文

【摘要】：橫紋肌肉瘤(rhabdomyosarcoma,RMS)是臨床上兒童常見的軟組織肉瘤,起源于骨骼肌細胞,約占小兒惡性實體瘤的10%左右,占兒童期所有軟組織肉瘤50%~70%。隨著在臨床上綜合治療的應用,其預后已有了明顯提高,5年總生存率達到33%~50%,但仍有超過50%的患者死于腫瘤復發(fā)或遠處轉(zhuǎn)移進展。在我國,兒童橫紋肌肉瘤的患者人口基數(shù)大,總體治療效果雖然有所改善,但是其遠期治愈率并不令人滿意。目前對于RMS的發(fā)病機制有了許多進展,對于其綜合治療方法的改善有一定的幫助。但是對于復發(fā)的患者,目前主要依靠施行化療,但化療的毒副反應則限制了其在兒童患者的使用。因此目前迫切需要治療橫紋肌肉瘤的新型藥物出現(xiàn)。研究報道腫瘤細胞中組蛋白往往呈低乙酰化水平,并且組蛋白去乙�；�(HDAC)活性異常造成的組蛋白乙�；癄顟B(tài)失衡與腫瘤的發(fā)生發(fā)展有密切關系。HDAC抑制劑SAHA能可逆性地逆轉(zhuǎn)組蛋白乙�；癄顟B(tài),從而調(diào)節(jié)基因表達、促進凋亡、細胞周期的阻滯、抗血管生成、DNA的損傷和修復以及減弱腫瘤細胞的侵襲和遷移。因此SAHA有望成為治療橫紋肌肉瘤的一種新型靶向藥物。因此本課題的目的是研究SAHA對人橫紋肌肉瘤RD細胞增殖、細胞周期、凋亡、自噬和遷移能力的影響,并探索其誘導凋亡的機制。方法:不同濃度SAHA作用于RD細胞,MTT法檢測對細胞增殖作用的影響,western blot檢測ERK信號通路蛋白表達;PI染色法分析細胞周期;Annexin V-FITC/PI雙染法檢測細胞凋亡,DAPI染色法驗證細胞凋亡,然后進行以下實驗初步探討誘導凋亡的機制:用JC-1探針檢測線粒體膜電位的變化;western blot檢測凋亡相關蛋白、線粒體途徑相關蛋白的變化;MDC染色法檢測細胞自噬,western blot檢測自噬相關的蛋白的表達;細胞劃痕檢測細胞遷移能力;western blot檢測細胞組蛋白H4的乙�；健＝Y(jié)果:1.saha抑制了rd細胞的增殖,并且具有時間和濃度依賴性。saha作用于rd細胞24h、48h、72h的半數(shù)抑制率ic50值分別為8.503μmol//l、3.965μmol/l和1.921μmol/l;與rd細胞增殖相關erk信號通路的變化是:erk蛋白磷酸化水平下降,而總erk蛋白水平?jīng)]有顯著的變化;2.流式細胞術結(jié)果顯示saha使rd細胞發(fā)生g2/m期阻滯;3.annexinv-fitc/pi雙染法結(jié)果顯示saha能夠誘導rd細胞凋亡;dapi染色法實驗表明,隨著saha濃度的增加,凋亡細胞的數(shù)量隨之增加,jc-1流式細胞術結(jié)果顯示,隨著saha濃度的增高,rd細胞線粒體膜電位水平逐漸降低;westernblot結(jié)果顯示:隨著saha濃度的增高,凋亡相關蛋白caspase-3和parp-1表達量增高,抗凋亡蛋白bcl-2表達量逐漸減低,促凋亡蛋白bax表達量逐漸增加,細胞色素c和caspase-9的表達也均有所增加;4.saha使rd細胞發(fā)生自噬:mdc染色結(jié)果顯示隨著saha濃度的增高,細胞內(nèi)的高亮點狀綠色熒光的斑點數(shù)量逐漸增多,表明細胞發(fā)生自噬。westernblot結(jié)果顯示:隨著saha濃度的增高,p62蛋白表達逐漸減少,beclin1蛋白表達水平逐漸增高,lc3-Ⅱ蛋白的表達也顯著增高。5.saha抑制rd細胞的遷移:細胞劃痕實驗結(jié)果顯示:隨著saha濃度的增高,劃痕愈合率逐漸降低。6.westernblot結(jié)果顯示:隨著saha濃度的增高,組蛋白h4的乙�；街饾u增高。結(jié)論:1.hdac抑制劑saha抑制rd細胞的增殖,使rd細胞發(fā)生g2/m期阻滯,該抑制作用具有時間和濃度依賴性;2.hdac抑制劑saha誘導rd細胞的凋亡,介導凋亡的機制可能為依賴于線粒體途徑和具有caspase依賴性;3.hdac抑制劑saha誘導rd細胞自噬;4.hdac抑制劑saha抑制rd細胞的遷移能力。
[Abstract]:Rhabdomyosarcoma (rhabdomyosarcoma, RMS) is a clinically common soft tissue sarcoma, originated in skeletal muscle cells, accounting for about 10% of pediatric malignant solid tumor in childhood, accounting for all soft tissue sarcoma 50%~70%. with the application of comprehensive treatment in clinic, its prognosis has been improved obviously, 5 year survival rate of 33%~50%. But there are still more than 50% of the patients died of tumor recurrence or distant metastasis. In our country, children with rhabdomyosarcoma of the large population, the treatment effect was improved, but the long-term cure rate is not satisfactory. The pathogenesis of RMS has a lot of progress, for the comprehensive treatment can be improved help. But for patients who relapse, currently rely mainly on the implementation of chemotherapy, but the toxicity of chemotherapy is limited by its use in pediatric patients. So there is an urgent need for treatment The new drug research group reported rhabdomyosarcoma. Tumor cells often showed low protein acetylation, and histone deacetylase (HDAC) activity in the occurrence and development of abnormal histone acetylation imbalance and tumor is closely related to the.HDAC inhibitor SAHA can reversibly reverse histone acetylation, thereby regulating gene the expression of apoptosis, cell cycle arrest, anti angiogenesis, DNA damage and repair and weaken the invasion and migration of tumor cells. Therefore, SAHA is expected to become a new target for the treatment of rhabdomyosarcoma to drugs. So the purpose of this paper is to study the proliferation of SAHA on human rhabdomyosarcoma RD cells cell cycle, apoptosis, autophagy effect and migration, and to explore the mechanism of apoptosis. Methods: different concentrations of SAHA in RD cells. The effect of MTT method to detect cell proliferation, weste Expression of ERK signaling pathway protein RN blot; cell cycle analysis of PI staining method; Annexin V-FITC/PI method to detect cell apoptosis, apoptotic cells verified by DAPI staining, then the following experiments: preliminary study on the mechanism of apoptosis induced by changes in mitochondrial membrane potential detection JC-1 probe; Western blot detection of apoptosis related protein, mitochondrial pathway related changes protein; autophagy was detected by MDC staining, the expression of Western blot was used to detect the protein; cell scratch detection cell migration; acetylation level of histone H4 Western were detected by blot. Results: 1.saha inhibited the proliferation of RD cells, and has a time and concentration dependent effect of.Saha on RD 24h cells. 48h 72h, the half inhibition rate of IC50 was 8.503 mol//l, 3.965 mol/l and 1.921 mol/l; and RD cell proliferation and ERK signal pathway changes: ERK protein phosphorylation The level of decline, but no significant changes in the total protein level of ERK 2.; flow cytometry showed that RD Saha cells g2/m phase arrest; 3.annexinv-fitc/pi staining showed that Saha could induce Rd cell apoptosis; DAPI staining showed that the experiment, with the increase of SAHA concentration, the number of apoptotic cells increased JC-1 flow cytometry showed that with the increase of SAHA concentration, the level of RD cells mitochondrial membrane potential gradually decreased; Westernblot results showed that with the increase of SAHA concentration, apoptosis related protein caspase-3 and PARP-1 expression and anti apoptosis protein bcl-2 expression reduced gradually, the pro apoptotic protein Bax expression increased gradually, the increased expression of cytochrome c and caspase-9 are 4.saha; the autophagy in RD cells: MDC staining results show that with the increase of SAHA concentration, the number of spots of green fluorescent cells within the bright dot Gradually increased, indicating that cell autophagy.Westernblot results showed that with the increase of SAHA concentration, p62 protein expression decreased, Beclin1 protein expression gradually increased, the expression of lc3- protein also increased significantly inhibited Rd cell migration of.5.saha: the experimental results show that the node cell scratch with the increasing of SAHA concentration, the wound healing rate decreased.6.westernblot the results showed that with the increase of SAHA concentration, the acetylation level of histone H4 increased. Conclusion: 1.hdac inhibitor Saha inhibited the proliferation of RD cells, the g2/m phase arrest in RD cells, the inhibition is time and concentration dependent manner; the apoptosis of RD cells induced by 2.hdac inhibitor Saha mediated apoptosis, the possible mechanism for depending on the mitochondrial pathway and dependent on caspase; 3.hdac inhibitor Saha induces autophagy in RD cells; the migration ability of 4.hdac inhibitor Saha inhibited Rd cells.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R738.7
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本文編號：1559090