MAPK在天花粉蛋白抗HIV-1中的作用以及syncytin基因在白血病细胞中的表达
文献类型：学位论文
| 作者 | 欧阳东云 |
| 学位类别 | 博士 |
| 答辩日期 | 2006-07 |
| 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 | 天花粉蛋白 HIV-1 MAPK Syncytin 白血病 淋巴瘤 |
| 其他题名 | Role of MAPK in the Anti-HIV-1 Activity of Trichosanthin and Overexpression of syncytin, the Envelope Gene of HERV-W, in Leukemia |
| 学位专业 | 动物学 |
| 中文摘要 | 本学位论文主要包括两部分的内容： 一是关于MAPK信号转导在天花粉蛋白（trichosanthin, TCS）抗人类免疫缺陷病毒-1（HIV-1）中的作用的研究。TCS是I型核糖体失活蛋白（RIP），分子量27Kd，可从传统的中期流产和抗绒癌中药栝楼根块茎（天花粉）中提纯获得。该蛋白具有抗HIV-1活性，但其机制尚不清楚。本文用JNK抑制剂CEP-11004，预处理宿主细胞，检测其对TCS抗HIV-1的影响。用以下两种方法检测病毒的复制：一是用ELISA方法检测细胞培养上清中p24抗原的水平，二是检测上清中病毒粒子的逆转录酶（RT）活性。结果显示，TCS剂量依赖性地抑制HIV-1在C8166细胞中的复制。在TCS实验浓度下，HIV-1的复制水平平均为68 ± 4％（p24抗原检测）和52 ± 4％（RT活性检测）。如果用0.4μM CEP-11004对C8166细胞预处理2小时，TCS似乎失去了抗HIV-1活性，HIV-1的复制水平分别恢复为101 ± 4％和101 ± 7％。但无论是p24抗原检测还是RT活性检测，当不含TCS时，CEP-11004预处理病毒宿主细胞，本身并不影响病毒粒子的复制。这说明CEP-11004能够拮抗TCS的抗病毒活性，或者说CEP-11004抑制的信号转导途径的某些信号分子，与TCS的抗HIV-1活性相关。Western Blot方法检测的结果也证明，TCS能够以时间依赖和剂量依赖的方式激活JNK激酶，0.4μM的CEP-11004能有效抑制JNK的磷酸化。因此，TCS与它激活MAPK信号转导途径有关。 二是关于人类内源性病毒HERV-W家族囊膜蛋白基因syncytin在白血病细胞中的表达的研究。该基因在人的胎盘组织中特异性表达，可能与合胞滋养层的形成有关。另外也少量表达于睾丸组织。本论文采用实时定量RT-PCR的方法证明，syncytin能够在白血病细胞系中表达。进一步的检测还表明，syncytin的mRNA也表达于白血病/淋巴瘤患者的外周血细胞，而不表达于作为对照的10名健康志愿者的血细胞。在15名不同类型的白血病/淋巴瘤患者中，有11名有syncytin的表达。细胞系的表达相对稳定，与C8166细胞系的表达量相比较，介于0.5-2.0倍之间；而在白血病患者外周血细胞中的表达则介于0.8-21.7倍不等。上述结果提示，syncytin可能与白血病的形成有关。 |
| 英文摘要 | There are two sections included in this thesis： One studies the role of MAPK signal transduction in the anti-HIV-1 activity of trichosanthin (TCS). TCS is a type I ribosome-inactivating protein with a molecular weight of 27 kilodaltons. It is purified from the root of a traditional mid-term abortive and anti-choriocarcinoma Chinese herb medicine, with a Chinese name Tian-Hua-Fen. This protein possesses broad-spectrum of biolological and pharmaceutical properties, such as immunosupression, mid-term abortion and antiviral activities, in addition to killing leukemia, lymphoma and other tumor potential. 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. But its anti-HIV-1 mechanism is still uncertain. A specific JNK pathway inhibitor, CEP-11004, was used to pretreat host cells and the influence of this inhibitor on the anti-HIV-1 activity of TCS was assayed. The results showed that CEP-11004 could counteract the anti-virus activity of TCS in C8166 cells. CEP-11004 alone had no effect in HIV-1 replication and TCS alone significantly inhibited this process. When CEP-11004 was used together with TCS, the antiviral action of TCS was much reduced. Two methods were used to assess viral replication. (1) By measuring the HIV-1 reverse transcriptase, TCS on the average reduced viral replication to 52 ± 4%. With CEP-11004 pretreatment, TCS appeared to lose the HIV-1 inhibitory activity with viral replication stood at 101 ± 7%. (2) By measuring HIV-1 p24, TCS reduced viral replication to 68 ± 4%. With CEP-11004 pretreatment, TCS again seemed to lose its anti-HIV-1 activity with HIV-1 replication rose back to 101 ± 4%. Both index indicated that CEP-11004 counteracted the antiviral action of TCS. The result demonstrated that some of the components of cell signal tranduction pathway that could be inhibited by CEP-11004 were related to the anti-HIV-1 activity of TCS. And Western Blot assay showed that TCS potentiated JNK activation in a time- and dose-dependent manner, while 0.4 μM CEP-11004 could effectively suppress JNK phosphorylation. The results suggested that MAPK signal transduction was involved in the anti-HIV-1 activity of TCS. The other part of this thesis is on the study of syncytin, the envelope gene of HERV-W. It is specifically expressed in human placenta and might be involved in syncytiotrophoblast morphogenesis, less expressed in testes. Real-time RT-PCR method was recruited in this study and the results showed that this gene was also expressed in leukemia cell lines. Further research demonstrated that syncytin mRNA was expressed in the peripheral blood cells of leukemia patients (11 out of 15 samples), but was not expressed in the blood cells of 10 healthy donors using as control samples. Syncytin expression level in C8166 cell line was set as 1.0. Then relative syncytin expression levels of all tested cell lines were between 0.5 and 2.0, while that in patients’ samples varied from 0.8 to 21.7 folds. Our results suggested that aberrant expression of this gene might be involved in leukemogenesis. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6261]  |
| 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 | 欧阳东云. MAPK在天花粉蛋白抗HIV-1中的作用以及syncytin基因在白血病细胞中的表达[D]. 北京. 中国科学院研究生院. 2006. |
入库方式： OAI收割
来源：昆明动物研究所
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