DNA末端定量及其在細胞凋亡檢測中的應用研究
發(fā)布時間：2018-08-19 15:29
【摘要】： 細胞凋亡已成為世界生命科學領域的研究熱點,其重要意義在于有可能闡明多種疾病包括病毒感染、神經(jīng)退化性疾病、免疫缺陷和惡性腫瘤的發(fā)病機理,并為這些疾病治療探索新的方法。最近,有學者重點闡述了細胞凋亡與臨床疾病的關系,細胞凋亡的分子機制和實驗室檢測在細胞凋亡評估過程中的重要作用。由此可知,研究細胞凋亡的實驗室檢測體系非常重要,同時,其檢測方法不斷涌現(xiàn),分析手段日趨完善和成熟。按方法學可分為形態(tài)學、生物化學、免疫化學和分子生物學測定法。也可將其分為以下七類,即(1)形態(tài)學檢測;(2)流式細胞分析;(3)DNA降解分析;(4)凋亡細胞膜改變分析;(5)細胞凋亡相關蛋白分析;(6)細胞凋亡酶學分析;(7)其它細胞凋亡檢測法。 目前檢測DNA降解的方法主要有電泳、流式細胞分析、原位末端標記(TUNEL)和PCR法等。我們欲利用α32磷脫氧三磷酸胞苷(α32P dCTP)與雙脫氧三磷酸胞苷(ddCTP)在外源性終末核苷酸轉(zhuǎn)移酶(TdT)的催化作用下,對同一DNA樣本的3’-羥基末端進行飽和標記,建立定量DNA末端最大標記量(Lmax)的方法(TdT法),并將Lmax與流式細胞分析(FCA)等檢測結(jié)果進行比較。同時,利用DNA末端定量技術(shù)檢測不同年齡高血壓大鼠(SHR)心肌細胞凋亡的動力學關系,并通過其探討雷米普利治療SHR,研究ACE抑制劑對心肌細胞凋亡的影響。 在DNA末端定量應用于細胞凋亡檢測中的研究中,我們以P2z受體的激活劑如三磷酸腺苷(ATP)、苯甲酰苯甲酸ATP(BzATP)等,或抑制劑如氧化型ATP(OxATP)作用于P2z(+)與P2z(-)CLL細胞,采用TdT法以及電鏡、DNA凝膠電泳檢測細胞凋亡,觀察P2z受體介導CLL細胞凋亡中的作用。同時,探討影響P2z受體介導CLL細胞凋亡的各種影響因素,包括各種二價陽離子、EDTA或EGTA、溫度及在膽堿介質(zhì)中的P2z(+)CLL細胞經(jīng)ATP或BzATP誘導凋亡的激活或抑制效應及其可能機理。 本研究共分四部分,包括第一部分DNA末端定量檢測方法的建立;第二部分DNA末端定量在雷米普利抑制自發(fā)性高血壓大鼠細胞凋亡中的應用;第三部分DNA末端定量在P2z受體誘導淋巴細胞白血病細胞凋亡中的應用;第四部分DNA末端定量在P2z受體介導淋巴細胞白血病細胞凋亡機理研究中的應用。
[Abstract]:Apoptosis has become a hot topic in the field of life sciences in the world. Its important significance is that it is possible to elucidate the pathogenesis of many diseases, including viral infection, neurodegenerative diseases, immune deficiency and malignant tumors. And to explore new methods for the treatment of these diseases. Recently, some scholars have focused on the relationship between apoptosis and clinical diseases, the molecular mechanism of apoptosis and the important role of laboratory detection in the evaluation of apoptosis. Therefore, it is very important to study the laboratory detection system of cell apoptosis. At the same time, the detection methods are emerging, and the analytical methods are becoming more and more perfect and mature. According to the methodology can be divided into morphology, biochemistry, immunocytochemistry and molecular biology assay. It can also be divided into the following seven categories: (1) morphological analysis; (2) flow cytometry; (3) DNA degradation analysis; (4) analysis of cell membrane changes; (5) analysis of apoptosis-related proteins; (6) analysis of apoptotic enzymes; and (7) other methods for detecting apoptosis. At present, the main methods for detecting DNA degradation include electrophoresis, flow cytometry, in situ end labeling (TUNEL) and PCR. We want to use 偽 32p dCTP and dideoxycytidine triphosphate (ddCTP) in the presence of exogenous terminal nucleotide transferase (TdT) to label the 3H-terminal of the same DNA. A method of quantifying (Lmax) with maximum end labeling of DNA (TdT method) was established, and the results of Lmax and (FCA) were compared with those of flow cytometry. At the same time, DNA terminal quantitative assay was used to detect the dynamic relationship of apoptosis of (SHR) cardiomyocytes in hypertensive rats of different ages, and to study the effect of ACE inhibitor on myocardial apoptosis. In the study of DNA terminal quantitative application in apoptosis detection, we used P2z receptor activator such as adenosine triphosphate (ATP),) benzoyl benzoic acid ATP (BzATP), or inhibitor such as oxidized ATP (OxATP) to act on P2z () and P2z (-) CLL cells. Apoptosis was detected by TdT assay and electron microscope DNA gel electrophoresis. The role of P2z receptor in CLL cell apoptosis was observed. At the same time, the factors affecting the apoptosis of CLL cells mediated by P2z receptor were discussed, including the effects of divalent cations, temperature, activation or inhibition of P2z () CLL cells induced by ATP or BzATP in choline medium, and the possible mechanism. This study was divided into four parts, including the establishment of DNA terminal quantitative detection method in the first part, the application of DNA terminal quantification in the inhibition of apoptosis in spontaneously hypertensive rats by Ramipril. The third part is the application of DNA terminal quantification in P2z receptor induced apoptosis of lymphoblastic leukemia cells, and the fourth part is the application of DNA terminal quantification in the mechanism of P2z receptor mediated apoptosis in lymphoblastic leukemia cells.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R346
本文編號：2192080
[Abstract]:Apoptosis has become a hot topic in the field of life sciences in the world. Its important significance is that it is possible to elucidate the pathogenesis of many diseases, including viral infection, neurodegenerative diseases, immune deficiency and malignant tumors. And to explore new methods for the treatment of these diseases. Recently, some scholars have focused on the relationship between apoptosis and clinical diseases, the molecular mechanism of apoptosis and the important role of laboratory detection in the evaluation of apoptosis. Therefore, it is very important to study the laboratory detection system of cell apoptosis. At the same time, the detection methods are emerging, and the analytical methods are becoming more and more perfect and mature. According to the methodology can be divided into morphology, biochemistry, immunocytochemistry and molecular biology assay. It can also be divided into the following seven categories: (1) morphological analysis; (2) flow cytometry; (3) DNA degradation analysis; (4) analysis of cell membrane changes; (5) analysis of apoptosis-related proteins; (6) analysis of apoptotic enzymes; and (7) other methods for detecting apoptosis. At present, the main methods for detecting DNA degradation include electrophoresis, flow cytometry, in situ end labeling (TUNEL) and PCR. We want to use 偽 32p dCTP and dideoxycytidine triphosphate (ddCTP) in the presence of exogenous terminal nucleotide transferase (TdT) to label the 3H-terminal of the same DNA. A method of quantifying (Lmax) with maximum end labeling of DNA (TdT method) was established, and the results of Lmax and (FCA) were compared with those of flow cytometry. At the same time, DNA terminal quantitative assay was used to detect the dynamic relationship of apoptosis of (SHR) cardiomyocytes in hypertensive rats of different ages, and to study the effect of ACE inhibitor on myocardial apoptosis. In the study of DNA terminal quantitative application in apoptosis detection, we used P2z receptor activator such as adenosine triphosphate (ATP),) benzoyl benzoic acid ATP (BzATP), or inhibitor such as oxidized ATP (OxATP) to act on P2z () and P2z (-) CLL cells. Apoptosis was detected by TdT assay and electron microscope DNA gel electrophoresis. The role of P2z receptor in CLL cell apoptosis was observed. At the same time, the factors affecting the apoptosis of CLL cells mediated by P2z receptor were discussed, including the effects of divalent cations, temperature, activation or inhibition of P2z () CLL cells induced by ATP or BzATP in choline medium, and the possible mechanism. This study was divided into four parts, including the establishment of DNA terminal quantitative detection method in the first part, the application of DNA terminal quantification in the inhibition of apoptosis in spontaneously hypertensive rats by Ramipril. The third part is the application of DNA terminal quantification in P2z receptor induced apoptosis of lymphoblastic leukemia cells, and the fourth part is the application of DNA terminal quantification in the mechanism of P2z receptor mediated apoptosis in lymphoblastic leukemia cells.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R346
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