自杀基因ePNP诱导小鼠巨噬细胞凋亡的研究

发布时间：2018-08-30 11:26

【摘要】：研究背景及目的众多研究发现动脉粥样硬化(atherosclerosis,As)晚期巨噬细胞的凋亡造成As斑块形态学改变(尤其是帽/核比值的变小),影响斑块易损性,从而造成斑块不稳定,是导致各种心血管疾病的重要原因。As斑块中的巨噬细胞几乎都变成了泡沫细胞,巨噬细胞/泡沫细胞凋亡导致细胞外脂质核的发生和扩大,在脂质核的非细胞部分可发现凋亡残留体[1-3]。诱导巨噬细胞凋亡,对通过巨噬细胞凋亡建立As易损斑块模型进而了解As易损斑块发生发展的分子机制奠定良好基础。鉴于以往的众多研究已得出的结论,即巨噬细胞凋亡和斑块破裂的关系,从这层关系出发,如果能成功诱导巨噬细胞凋亡,那么就可以采取同样的方法诱导斑块内巨噬细胞凋亡来建立动脉粥样硬化易损斑块模型,从而建立一个体外可控可量化的斑块破裂模型,方法简单易行。因此,我们尝试一种方法来诱导巨噬细胞凋亡,为后期建立类似于人类As病变的易损斑块模型提供依据。大肠杆菌嘌呤核苷酸磷酸化酶(E.coli purine nucleoside phosphorylase,ePNP)基因有较强的细胞杀伤作用,因此一直是基因治疗的热点[1]。ePNP作为一种前药转换酶基因,能将某些腺嘌呤类似物,如fludarabine等转化成高毒性能自由穿透细胞膜的代谢产物,从而对细胞产生毒性作用,引起细胞死亡[4、5]。众多研究已经在肝癌,胰腺癌等肿瘤细胞中证实ePNP自杀基因系统良好的杀伤细胞作用[6-8]。由ePNP杀伤肿瘤细胞的原理,我们推测ePNP基因同样可以诱导巨噬细胞凋亡。因此,本实验拟采用巨噬细胞特异表达的ePNP转基因小鼠,探究自杀基因ePNP联合前体药物fludarabine对巨噬细胞凋亡的诱导作用。实验方法和结果1.自杀基因ePNP诱导转基因小鼠巨噬细胞凋亡(1)实验分ePNP转基因小鼠组和野生型小鼠组,在体外培养的各组小鼠腹腔巨噬细胞中分别加入浓度为0、0.125μg/ml、0.25μg/ml、0.5μg/ml、1.Oμg/ml和2.0μg/ml的前体药物氟达拉滨(fludarabine),采用活细胞计数试剂盒(CCK-8)检测腹腔巨噬细胞加药后的细胞存活率。CCK-8检测显示给药后ePNP小鼠腹腔巨噬细胞存活率明显下降,尤其在浓度为2.0μg/ml,细胞存活率仅为(10.8±0.8)%。(2)给ePNP转基因小鼠和野生型小鼠腹腔注射剂量分别为0、10mg/kg、20mg/kg、60mg/kg的fludarabine,一天两次,给药五天,采用流式细胞术检测加入fludarabine后腹腔巨噬细胞的凋亡情况。流式细胞术结果显示给药后其腹腔巨噬细胞凋亡明显增加,并且存在剂量效应关系。(3)给ePNP转基因小鼠和野生型小鼠腹腔注射剂量分别为0、10mg/kg、20mg/kg、60mg/kg的fludarabine,一天两次,共给药五天,取材(肺、脾),采用原位凋亡检测试剂盒(TUNEL法)检测加入fludarabine后组织巨噬细胞(肺、脾)的凋亡情况。TUNEL结果也显示给药后其肺、脾巨噬细胞凋亡明显增加,并且存在剂量效应关系。CCK-8、流式细胞术和TUNEL法均显示野生型小鼠给药后没有明显巨噬细胞凋亡。结果有统计学意义。2.SR-A1-ePNP 小鼠和 APOE-/-小鼠杂交培养 SR-A1-ePNP/APOE-/-将筛选出的SR-A1-ePNP转基因纯合子小鼠和APOE-/-小鼠杂交,获得的F1代中,经鉴定为ePNP阳性的小鼠,继续杂交,获得的F2代中小鼠,采用普通PCR鉴定ePNP基因和APOE-/-基因。后代中ePNP阳性和APOE基因敲除的小鼠即为目的小鼠。结论自杀基因ePNP能在前体药物fludarabine协同作用下,诱导SR-A1-ePNP转基因小鼠巨噬细胞凋亡。
[Abstract]:BACKGROUND AND OBJECTIVE Numerous studies have found that apoptosis of macrophages in advanced atherosclerosis (As) causes morphological changes of As plaque (especially the decrease of cap/nucleus ratio) and affects plaque vulnerability, resulting in plaque instability, which is an important cause of various cardiovascular diseases. Macrophage/foam cell apoptosis leads to the occurrence and expansion of extracellular lipid nuclei. Apoptosis residues can be found in the non-cellular part of the lipid nucleus [1-3]. Basis. In view of the conclusions of many previous studies, that is, the relationship between macrophage apoptosis and plaque rupture, from this relationship, if macrophage apoptosis can be successfully induced, then the same method can be used to induce apoptosis of intraplaque macrophages to establish Atherosclerotic Vulnerable Plaque model, thereby establishing a body. Therefore, we try to find a way to induce macrophage apoptosis to provide a basis for the later establishment of vulnerable plaque model similar to human As lesions. Escherichia coli purine nucleoside phosphorylase (ePNP) gene has stronger cells. As a prodrug converting enzyme gene, ePNP can transform some adenine analogues, such as fludarabine, into metabolites with high toxicity and free penetration through cell membrane, thus producing toxic effects on cells and causing cell death. It was confirmed that ePNP suicide gene system had a good killing effect on tumor cells [6-8]. According to the principle of ePNP killing tumor cells, we speculated that ePNP gene could also induce macrophage apoptosis. Therefore, this study was designed to explore the effect of ePNP suicide gene combined with fludarabine on macrophages by using macrophage-specific expression of ePNP transgenic mice. The suicide gene ePNP induced apoptosis of macrophages in transgenic mice (1) The suicide gene ePNP induced apoptosis of macrophages in transgenic mice and wild type mice (1) The ePNP transgenic mice and wild type mice were divided into two groups. The mice peritoneal macrophages were cultured in vitro with 0,0.125 ug/ml, 0.25 ug/ml, 0.5 ug/ml, 1.O ug/ml and 2.0 ug/ml precursors, respectively. CCK-8 assay showed that the survival rate of peritoneal macrophages in ePNP mice decreased significantly, especially at the concentration of 2.0 ug/ml, and the cell survival rate was only (10.8 0.8)%. (2) The survival rate of ePNP transgenic mice and wild-type mice was small. The apoptosis of peritoneal macrophages was detected by flow cytometry after intraperitoneal injection of fludarabine at doses of 0,10 mg/kg, 20 mg/kg and 60 mg/kg twice a day for five days. Fludarabine of 0,10 mg/kg, 20 mg/kg and 60 mg/kg were injected into abdominal cavity of gene mice and wild type mice twice a day for five days. The apoptosis of macrophages (lung and spleen) was detected by in situ apoptosis assay kit (TUNEL). CCK-8, flow cytometry and TUNEL showed that there was no significant macrophage apoptosis in wild-type mice. The results were statistically significant. E-/-mice were hybridized and identified as ePNP-positive mice in the F1 generation. F2 generation mice were hybridized and identified as ePNP gene and APOE-/-gene by ordinary PCR. The ePNP-positive and APOE-knockout mice in the offspring were the target mice. Conclusion The suicide gene ePNP can induce SR-A1-eP in synergy with the precursor drug fludarabine. Macrophages apoptosis in NP transgenic mice.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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