發(fā)布時間：2018-02-01 02:58

  本文關鍵詞： 骨肉瘤 lncRNA MALAT-1 免疫組化 新輔助化療　出處：《第二軍醫(yī)大學》2017年碩士論文　論文類型：學位論文

【摘要】：如今,惡性腫瘤已成為威脅人類生命健康的重要元兇。而好發(fā)于青少年的骨肉瘤,由于其惡性程度高,早期轉移率高,致死致殘率高,給社會和家庭都造成了沉重的心理壓力和經(jīng)濟負擔。隨著化療方案的不斷改進完善,以及人工關節(jié)材料技術的不斷進步,骨肉瘤的治療手段取得了比較快的發(fā)展,患者的保肢率、5年生存率不斷提高。但是,目前骨肉瘤的治療方案仍存在不少難以克服的局限性。對于存在化療藥物抵抗、出現(xiàn)復發(fā)或轉移等情況的骨肉瘤患者,生存率仍然較低。最近的5~10年,骨肉瘤的治療已遇到了“瓶頸”。尋找腫瘤的靶點,探索分子靶向治療的方向,將成為骨肉瘤治療新的突破口。目前,長鏈非編碼RNA(long non-coding RNA,lncRNA)在腫瘤中的作用正在被逐漸地探索和發(fā)現(xiàn)。許多研究者在不同腫瘤中發(fā)現(xiàn)了許多l(xiāng)ncRNA存在異常表達,并且這些lncRNA的表達程度與腫瘤的分期、遠期預后等密切相關。在腫瘤中改變一些lncRNA的表達,可以改變腫瘤的侵襲性、遷移性和轉移性。這些研究結果提示lncRNA可能成為腫瘤治療的新靶點。MALAT-1(metastasis-associated lung adenocarcinoma transcript 1,肺腺癌轉移相關轉錄本-1)目前已被證實在多種腫瘤的增殖、侵襲、轉移中發(fā)揮作用。但是目前關于MALAT-1在骨肉瘤中表達情況的研究較少,且都存在各自的局限性。本研究旨在進一步探討MALAT-1在骨肉瘤中的表達及其作用,為骨肉瘤的病情評估尋找新指標,以及為將來可能出現(xiàn)的基因治療和分子靶向治療提供依據(jù)和參考。一方面,我們通過裸鼠植瘤試驗,觀察MALAT-1表達對骨肉瘤生長的影響。另一方面,我們收集了科室收治的骨肉瘤患者的腫瘤標本和癌旁正常組織標本。通過對比同一患者的骨肉瘤組織和癌旁正常組織中MALAT-1的表達情況,并與該患者的臨床資料相結合,探索MALAT-1在骨肉瘤中的表達情況及其與臨床特點之間的關系。第一部分:MALAT-1表達對裸鼠體內骨肉瘤生長的影響。在第一部分的研究中,我們使用慢病毒轉染的方法,培養(yǎng)穩(wěn)轉錄的MALAT-1穩(wěn)定低表達的骨肉瘤H2OS細胞系。將12只裸鼠平均分為實驗組和對照組,實驗組裸鼠背部皮下植入MALAT-1穩(wěn)定低表達的骨肉瘤H2OS細胞系,對照組裸鼠背部皮下植入空載慢病毒處理的、MALAT-1表達基本正常的骨肉瘤H2OS細胞系。植瘤后,觀察兩組裸鼠移植瘤的生長情況。處死裸鼠后,比較兩組裸鼠的移植瘤在體積、重量、HE染色、免疫組化之間的差異。結果顯示,實驗組裸鼠皮下的腫瘤生長更慢,相同生長時間內腫瘤的體積更小、重量更輕。HE染色和免疫組化結果顯示,實驗組裸鼠生長的腫瘤中,基質成分增多,細胞密集程度降低,PCNA表達程度降低。這一結果提示MALAT-1表達降低可以抑制骨肉瘤的生長,促進骨肉瘤細胞的凋亡,降低骨肉瘤細胞的侵襲性。從病理學特點方面探討MALAT-1表達對實體骨肉瘤生長影響的研究目前尚無文獻報道。第二部分:MALAT-1表達與骨肉瘤患者臨床特征相關性的研究。在第二部分研究中,我們收集同一患者的骨肉瘤標本和相鄰癌旁正常組織標本,分別測定MALAT-1的表達情況,并將測定結果與患者的年齡、性別、腫瘤部位、堿性磷酸酶、腫瘤分期、取材時機(活檢或根治性手術)、化療情況等相對照,探討MALAT-1表達與患者臨床特點之間的關系。這是目前為止較為詳細的MALAT-1表達情況與臨床特點相關性的研究。結果顯示,MALAT-1表達與骨肉瘤患者的臨床分期、取材前是否經(jīng)歷新輔助化療有關,ⅡB期的骨肉瘤與ⅡA期的骨肉瘤相比,更容易出現(xiàn)MALAT-1表達高于癌旁正常組織的情況。新輔助化療后的骨肉瘤標本中,MALAT-1的表達往往高于癌旁正常組織。未接受化療的骨肉瘤標本中,MALAT-1的表達常常低于癌旁正常組織。這一結果顯示,與其他腫瘤相比,骨肉瘤標本中MALAT-1表達情況可能存在特殊性。由于骨肉瘤特殊的治療過程,臨床特征多變,因此關于MALAT-1用于骨肉瘤的病情評估和分子靶向治療的效果,還需要更大樣本、更深入的研究。
[Abstract]:Now, the malignant tumor has become an important cause of threat to human life and health. And good in adolescents of osteosarcoma, due to its high degree of malignancy, early metastasis rate and high mortality rate is high, to the society and family have caused a heavy psychological pressure and economic burden. With the continuous improvement of chemotherapy, and constantly the progress of artificial joint material, osteosarcoma treatment has achieved relatively fast development, limb salvage rate of patients, 5 year survival rate was increased. However, the treatment of osteosarcoma still exist many limitations difficult to overcome. For the existence of chemotherapy drug resistance occurred in patients with osteosarcoma recurrence or metastasis etc. the survival rate is still low. The recent 5~10 years, the treatment of osteosarcoma has encountered a bottleneck. The target for tumor molecular targeted therapy, explore the direction, will become a new breakthrough in the treatment of osteosarcoma At present, the long chain of non encoding RNA (long non-coding RNA, lncRNA) in tumor cells is gradually explored and found. Many researchers in different tumors found many lncRNA abnormal expression, and the expression level and tumor staging of these lncRNA, the long-term pre is closely related to changes in the expression of some. LncRNA in tumors, invasive tumors can change, migration and metastasis. These results suggest that lncRNA may become a new target for tumor therapy (.MALAT-1 metastasis-associated lung adenocarcinoma transcript 1, lung adenocarcinoma metastasis related transcripts -1) has been confirmed in a variety of tumor proliferation, invasion and metastasis play a role. But at present about the expression of MALAT-1 in osteosarcoma research, and have their own limitations. The purpose of this study is to further explore the MALAT-1 in osteosarcoma table Da and its role, looking for new index for assessing the severity of osteosarcoma, and for gene therapy and molecular targeted therapy may arise in the future and provide the basis for reference. On the one hand, we transplanted nude mice through the experiment, we observed the expression of MALAT-1 effect on the growth of osteosarcoma. On the other hand, we collected osteosarcoma patients admitted to department the tumor specimens and adjacent normal tissues. The expression of MALAT-1 in osteosarcoma tissues compared to the same patients and normal breast tissues, and combined with the clinical data of the patients, to explore the relationship between MALAT-1 in osteosarcoma expression and its clinical characteristics. The first part: the expression of MALAT-1 effect on the growth of osteosarcoma in nude mice. In the first part of the study, we use the method of lentiviral transfection, cultured osteosarcoma cell line H2OS. The transcription of MALAT-1 stable low expression. 12 Nude mice were divided into experimental group and control group, the H2OS osteosarcoma cell line of nude mice subcutaneously implanted MALAT-1 stable low expression, control group of nude mice by subcutaneous implantation of empty lentivirus treatment, the expression of MALAT-1 in osteosarcoma cell line H2OS and normal basic plants. After the tumor growth was observed in the two groups of nude mice. Mice were killed after the comparison of two groups of nude mice transplantation tumor volume, weight, HE staining, immunohistochemical differences. The results showed that the experimental group of nude mice subcutaneous tumor growth is slower, smaller within the same growth time of tumor, lighter weight.HE staining and immunohistochemistry showed that the experimental group of nude mice the growth of tumors, matrix increased, cell density decreased, PCNA expression decreased. These results suggest that the decreased expression of MALAT-1 can inhibit the growth of osteosarcoma, promote the apoptosis of osteosarcoma cells, reduce osteosarcoma fine The invasion of cells. To explore the expression of MALAT-1 impact on the real growth of osteosarcoma is reported from pathological characteristics. The second part: Study on the clinical features of patients with osteosarcoma and the relationship between the expression of MALAT-1. In the second part of this study, we collected the same patients with osteosarcoma specimens and adjacent normal tissues specimens. Determination of the expression of MALAT-1 respectively, and the measurement results were related with age, gender, tumor location, tumor staging, alkaline phosphatase, sampling time (biopsy or radical surgery, chemotherapy) such as contrast, to explore the relationship between the MALAT-1 expression and clinical characteristics of patients. This is by far the more detailed study of the MALAT-1 expression the correlation with clinical characteristics. The results showed that the expression of MALAT-1 and the clinical stage of patients with osteosarcoma were taken before neoadjuvant chemotherapy experience, stage B osteosarcoma Compared with stage A osteosarcoma, are more likely to have higher expression of MALAT-1 in normal bladder tissues. Specimens of osteosarcoma after neoadjuvant chemotherapy, the expression of MALAT-1 is higher than that in adjacent normal tissues. Osteosarcoma specimens did not receive chemotherapy, the expression of MALAT-1 is often lower than in adjacent normal tissues. This result show that compared with other tumors, the expression of MALAT-1 in osteosarcoma specimens may have particularity. Because the treatment process of osteosarcoma special clinical features, changeable, so MALAT-1 used on osteosarcoma patient assessment and molecular targeted treatment effects, but also need more samples and more in-depth research.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R738.1

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