天花粉蛋白诱导HIV-1 慢性感染细胞凋亡及机制研究
文献类型：学位论文
| 作者 | 王媛媛 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郑永唐 |
| 关键词 | HIV-1 TCS 凋亡 线粒体膜电位 caspase-8 |
| 其他题名 | The apoptotic action of TCS on HIV-1 chronically infected cells |
| 学位专业 | 动物学 |
| 中文摘要 | 天花粉蛋白（Trichosanthin,TCS）是一种由247个氨基酸组成的Ⅰ型核糖体失活蛋白（Ribosome Inactivating Protein，RIP），从葫芦科植物栝楼(Trichosanthes Kirilowii)球根中提纯获得。它具有广谱的生物学和药学活性，包括抗肿瘤、免疫抑制、中期引产以及抗病毒活性。上世纪八十年代末，McGrath等发现TCS可以抑制HIV-1在急性感染的T淋巴细胞和慢性感染的巨噬细胞中的复制，从而引起了研究者们极大的兴趣。但迄今为止，其抗HIV-1的作用机制仍不清楚。 我们实验室对TCS的免疫毒理作用和抗HIV-1作用进行了多年的研究，前期工作显示TCS对HIV-1的直接作用并不明显，但对于HIV-1感染细胞却具有很强的毒性作用。提示TCS可能通过作用于宿主细胞来发挥其抗HIV-1活性。在此基础上，我们从细胞方面着手，对TCS选择性杀伤HIV-1感染细胞的作用及机制进行了探讨。首先，通过MTT法检测发现，相同条件下，TCS对于H9/HIV-1IIIB细胞的毒性远远大于其对正常H9细胞的毒性。其次，流式细胞仪检测亚二倍体小峰和琼脂糖凝胶电泳检测片断化DNA的实验证实了TCS对H9/HIV-1IIIB的细胞杀伤作用是通过诱导细胞凋亡实现的。流式细胞仪的结果显示TCS以剂量依赖的方式诱导较多的H9/HIV-1IIIB细胞凋亡，25μg/mlTCS作用24h时，有8.4%的H9细胞凋亡，而H9/HIV-1IIIB细胞的凋亡率则达到24.5%；随着作用浓度的降低，这种差异也在缩小。阳性对照D-Sorbitol对两种细胞的凋亡率没有明显差别，约为25%。琼脂糖凝胶电泳的结果进一步证实了这种推测，相同条件下，TCS诱导H9/HIV-1IIIB细胞出现更多的DNA片断化。 TCS可以选择性的诱导H9/HIV-1IIIB细胞凋亡，为了进行更深入的机制研究，我们建立了另外一株HIV-1慢性感染细胞系，HIV-1慢性感染的Jurkat细胞系（Jurkat/HIV-1ⅢB）来验证TCS的作用。发现相同条件下，TCS可以诱导同等程度的Jurkat/HIV-1ⅢB和Jurkat细胞凋亡，25μg/mlTCS作用24h时，分别有21.08%的Jurkat细胞和27.27%的Jurkat/HIV-1IIIB细胞凋亡。以上的结果说明HIV-1感染H9细胞后增强了感染细胞对TCS的敏感性，而HIV-1感染Jurkat细胞后并不影响其对TCS的敏感性。根据细胞凋亡途径中是否依赖线粒体的参与可以将细胞分成TypeⅠ和TypeⅡ两种类型，H9细胞采取的是线粒体非依赖性的TypeⅠ型凋亡途径，而Jurkat细胞则采取线粒体依赖性的TypeⅡ型凋亡途径。由于Jurkat细胞对TCS诱导的凋亡更加敏感，我们推测HIV-1感染H9细胞后，诱导了细胞凋亡途径由TypeⅠ向TypeⅡ型转变。为此，我们采用流式细胞仪检测了TCS对凋亡细胞内的线粒体膜电位及caspase-8蛋白酶活性的影响，结果显示，H9/HIV-1IIIB、Jurkat和Jurkat/HIV-1IIIB细胞对TCS诱导的凋亡具有相同程度的敏感性，并且伴随着细胞线粒体膜电位的耗散和caspase-8蛋白酶的活化；而相同情况下，TCS对H9细胞的影响则很微弱。由此，进一步证实了我们的推测，即HIV-1感染H9细胞后，通过改变细胞内的某些信号，使H9/HIV-1ⅢB细胞的性状更加倾向于Type Ⅱ型细胞，从而增强其对于TCS的敏感性。 |
| 英文摘要 | TCS is a type I ribosome-inactivating protein with a molecular weight of 27 kilodaltons. It can be purified from the root of tubers of the Chinese medicinal herb Trichosanthes kirilowii Maxim. This protein possesses broad-spectrum of biolological and pharmaceutical properties, such as anti-tumor, immunosupression, mid-term abortion and antiviral activities. At the end of 1980’s, more researchers were attracted to study TCS after McGrath et al found that the plant protein could inhibit HIV-1 replication in the acutely infected T lymphocytes and chronically infected macrophage cells. But the anti-HIV-1 mechanism of TCS is still uncertain. The results of our previous research on anti-HIV-1 activity and the structure-functional relationship of TCS showed that TCS has strong cytotoxicity on HIV-1 infected cells but little direct effect on the HIV-1. This suggested that the antiviral action of TCS may be related to the interaction with host cells. So we investigated the cytopathic mechanism of TCS on the HIV-1 infected cells. Firstly, cytotoxicity of TCS on H9 T lymphocyte cell line and HIV-1 chronically infected H9 cell line (H9/HIV-1ⅢB) were tested by using MTT colorimetric assay. Apoptotic action of TCS on H9 and H9/HIV-1ⅢB cells were tested by flow cytometry assay and agarose gel electrophoresis. The results showed that TCS could induce more H9/HIV-1ⅢB cells apoptosis in a dose-dependant manner. At TCS concentration of 25μg/ml, 8.4% of normal H9 cells were found to be apoptotic whereas the same concentration induced 24.5% in HIV-1 chronically infected cells in flow cytometry study. DNA fragmentation study also confirmed more laddering in infected cells. Such difference was not found in the control experiments with D-sorbitol treatment. Then the selectively apoptotic action of TCS on H9/HIV-1ⅢB cells was further validated by establishing HIV-1 chronically infected Jurkat(Jurkat/HIV-1ⅢB) cell line. We found that TCS could induce H9/HIV-1ⅢB, Jurkat and Jurkat/HIV-1ⅢB cell lines apoptosis in the same measure. But the effects on H9 cells were unconspicuous.Such confirmed our suggestion that TCS just selectively induced H9/HIV-1ⅢB cells apoptsis and had little selective apoptotic action on other HIV-1 infected cells. Present research suggested that there were two apoptotic pathways according to different cell types. H9 cells were TypeⅠcells which had mitochondria-independent apoptotic pathway.Jurkat cells were TypeⅡcells which had mitochondria-dependent apoptotic pathway. Our results showed that H9/HIV-1ⅢB, Jurkat and Jurkat/HIV-1ⅢB cell lines had similar sensitivity to TCS induced apotosis. But the effects on H9 cells were very inapparent. The loss of mitochondrial membrane potential and activity of caspase-8 in apoptotic cells which measured by flow cytometry assay also proved such above results. So we infered that H9/HIV-1ⅢB cells were more similar to the TypeⅡcells other than TypeⅠcells. And TypeⅡcells have more apoptotic sensitivity to TCS. Such may be responsible for the selective apoptotic action to H9/HIV-1ⅢB cells of TCS. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6105]  |
| 专题 | 昆明动物研究所_分子免疫药理学 |
| 推荐引用方式 GB/T 7714 | 王媛媛. 天花粉蛋白诱导HIV-1 慢性感染细胞凋亡及机制研究[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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