DNA末端定量及其在细胞凋亡检测中的应用研究
发布时间：2018-08-19 15:29
【摘要】： 细胞凋亡已成为世界生命科学领域的研究热点,其重要意义在于有可能阐明多种疾病包括病毒感染、神经退化性疾病、免疫缺陷和恶性肿瘤的发病机理,并为这些疾病治疗探索新的方法。最近,有学者重点阐述了细胞凋亡与临床疾病的关系,细胞凋亡的分子机制和实验室检测在细胞凋亡评估过程中的重要作用。由此可知,研究细胞凋亡的实验室检测体系非常重要,同时,其检测方法不断涌现,分析手段日趋完善和成熟。按方法学可分为形态学、生物化学、免疫化学和分子生物学测定法。也可将其分为以下七类,即(1)形态学检测;(2)流式细胞分析;(3)DNA降解分析;(4)凋亡细胞膜改变分析;(5)细胞凋亡相关蛋白分析;(6)细胞凋亡酶学分析;(7)其它细胞凋亡检测法。 目前检测DNA降解的方法主要有电泳、流式细胞分析、原位末端标记(TUNEL)和PCR法等。我们欲利用α32磷脱氧三磷酸胞苷(α32P dCTP)与双脱氧三磷酸胞苷(ddCTP)在外源性终末核苷酸转移酶(TdT)的催化作用下,对同一DNA样本的3’-羟基末端进行饱和标记,建立定量DNA末端最大标记量(Lmax)的方法(TdT法),并将Lmax与流式细胞分析(FCA)等检测结果进行比较。同时,利用DNA末端定量技术检测不同年龄高血压大鼠(SHR)心肌细胞凋亡的动力学关系,并通过其探讨雷米普利治疗SHR,研究ACE抑制剂对心肌细胞凋亡的影响。 在DNA末端定量应用于细胞凋亡检测中的研究中,我们以P2z受体的激活剂如三磷酸腺苷(ATP)、苯甲酰苯甲酸ATP(BzATP)等,或抑制剂如氧化型ATP(OxATP)作用于P2z(+)与P2z(-)CLL细胞,采用TdT法以及电镜、DNA凝胶电泳检测细胞凋亡,观察P2z受体介导CLL细胞凋亡中的作用。同时,探讨影响P2z受体介导CLL细胞凋亡的各种影响因素,包括各种二价阳离子、EDTA或EGTA、温度及在胆碱介质中的P2z(+)CLL细胞经ATP或BzATP诱导凋亡的激活或抑制效应及其可能机理。 本研究共分四部分,包括第一部分DNA末端定量检测方法的建立;第二部分DNA末端定量在雷米普利抑制自发性高血压大鼠细胞凋亡中的应用;第三部分DNA末端定量在P2z受体诱导淋巴细胞白血病细胞凋亡中的应用;第四部分DNA末端定量在P2z受体介导淋巴细胞白血病细胞凋亡机理研究中的应用。
[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 伪 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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346
本文编号：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 伪 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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346
【参考文献】
相关期刊论文 前7条
1 张芹,彭黎明;P2z/P2x7嘌呤能受体的研究进展[J];细胞生物学杂志;2001年02期
2 彭黎明;吞噬细胞对凋亡细胞的识别与吞噬[J];中华病理学杂志;1999年04期
3 彭黎明;六种细胞凋亡检测方法的比较[J];中华病理学杂志;1999年01期
4 彭黎明,ＭｉｋｅＢｒｉｓｃｏ,ＰａｍＳｙｋｅｓ,ＡｌｅｃＭｏｒｌｅｙ;用〔α~(32)P〕dCTP标记断裂DNA检测DNA降解程度[J];中华核医学杂志;1997年01期
5 彭黎明;细胞凋亡检测的研究进展[J];中华医学检验杂志;1996年06期
6 彭黎明;用Annexin法定量分析淋巴瘤细胞凋亡与坏死[J];中华医学检验杂志;1999年01期
7 彭黎明;嘌呤能P2z受体介导白血病细胞凋亡的研究[J];中华医学杂志;1998年07期
，本文编号：2192080
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2192080.html
最近更新
教材专著
