【摘要】：研究目的： 一些腫瘤細(xì)胞系呈組成性表達(dá)VTCNl。在肺癌、卵巢癌、腎細(xì)胞癌均有VTCN1蛋白的表達(dá)；乳腺癌和子宮內(nèi)膜癌幾乎普遍存在VTCN1的表達(dá),且不依賴腫瘤的分級(jí)和分期,而且這種VTCN1高表達(dá)是明顯增加神經(jīng)膠質(zhì)瘤病例死亡風(fēng)險(xiǎn)的獨(dú)立因素。但VTCN1在神經(jīng)膠質(zhì)瘤腫瘤組織表達(dá)的確切臨床意義還有待進(jìn)一步驗(yàn)證,VTCN1是否可以作為神經(jīng)膠質(zhì)瘤早期診斷和預(yù)后評(píng)價(jià)的指標(biāo)也值得進(jìn)一步深入研究。因此,本部分?jǐn)M探討VTCN1在神經(jīng)膠質(zhì)瘤組織中的表達(dá)及其與臨床病理及預(yù)后的關(guān)系。 研究方法： 以酶聯(lián)免疫反應(yīng)(ELISA)檢測(cè)神經(jīng)膠質(zhì)瘤患者外周血中VTCN1蛋白的含量,以逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)技術(shù)檢測(cè)神經(jīng)膠質(zhì)瘤患者手術(shù)切除的腫瘤組織標(biāo)本中VTCN1基因表達(dá)水平,以免疫組織化學(xué)染色(IHC)檢測(cè)神經(jīng)膠質(zhì)瘤患者手術(shù)切除的腫瘤組織標(biāo)本中VTCN1蛋白質(zhì)表達(dá)水平。 研究結(jié)果： VTCN1蛋白在神經(jīng)膠質(zhì)瘤患者外周血中的含量(44.80±8.20gg/L)明顯高于腦部良性腫瘤患者(35.10±6.33μg/L)和正常人群(32.17±5.96μg/L)(P0.05)。隨后檢測(cè)VTCN1mRNA在30例神經(jīng)膠質(zhì)瘤組織和癌旁組織中均有表達(dá),神經(jīng)膠質(zhì)瘤組織中表達(dá)明顯高于癌旁組織(P0.01),且癌旁組織中的表達(dá)明顯高于腦部良性腫瘤組織(P0.01)。進(jìn)一步檢測(cè)70例神經(jīng)膠質(zhì)瘤組織中VTCN1蛋白的表達(dá),其陽性表達(dá)率68.57%,其中77.08%為高表達(dá),明顯高于腦部良性腫瘤組織(P0.01)。VTCN1蛋白的表達(dá)與神經(jīng)膠質(zhì)瘤組織學(xué)分級(jí)、腫瘤大小及腫瘤復(fù)發(fā)或轉(zhuǎn)移密切相關(guān)(P0.05),而與患者年齡、性別和組織學(xué)類型等無關(guān)(P0.05)。VTCN1高表達(dá)患者生存率明顯低于低表達(dá)患者(P0.05)。 研究結(jié)論： VTCN1在神經(jīng)膠質(zhì)瘤組織表達(dá)狀態(tài)與神經(jīng)膠質(zhì)瘤的惡性程度、進(jìn)展及預(yù)后關(guān)系密切,可以作為反映神經(jīng)膠質(zhì)瘤生物學(xué)行為和判斷預(yù)后的有效指標(biāo)。 研究目的： VTCN1基因家族負(fù)性分子在腫瘤的異常表達(dá)提示其在腫瘤發(fā)生發(fā)展中發(fā)揮了重要作用,這其中涉及的機(jī)制將為干預(yù)腫瘤的發(fā)生發(fā)展提供有效途徑,具有較大的潛在應(yīng)用價(jià)值。越來越多的臨床研究已發(fā)現(xiàn)VTCN1分子與腫瘤免疫調(diào)控密切相關(guān),但在機(jī)體腫瘤免疫應(yīng)答中的作用機(jī)理還有待進(jìn)一步研究。因此本部分通過RNA干擾技術(shù)下調(diào)VTCN1基因在人神經(jīng)膠質(zhì)瘤U251細(xì)胞株中的表達(dá),探討VTCN1基因表達(dá)下調(diào)后對(duì)神經(jīng)膠質(zhì)瘤細(xì)胞生物學(xué)行為的影響。 研究方法： 將針對(duì)VTCNI基因的siRNA序列重組入真核表達(dá)載體中構(gòu)建pU-VTCN1-siRNA,轉(zhuǎn)染至神經(jīng)膠質(zhì)瘤U251細(xì)胞株中,采用逆轉(zhuǎn)錄PCR(RT-PCR)、免疫印跡法檢測(cè)轉(zhuǎn)染pU-VTCN1-siRNA后的U251細(xì)胞中VTCN1基因在基因表達(dá)水平和蛋白質(zhì)表達(dá)水平的變化。采用流式細(xì)胞儀分析轉(zhuǎn)染pU-VTCN1-siRNA后的U251細(xì)胞的細(xì)胞周期變化,應(yīng)用Transwell侵襲小室模型觀察轉(zhuǎn)染pU-VTCN1-siRNA后U251細(xì)胞侵襲力的變化。 研究結(jié)果： 轉(zhuǎn)染pU-VTCN1-siRNA后的U251細(xì)胞中,VTCN1基因的基因表達(dá)水平和蛋白質(zhì)表達(dá)水平顯著下降(P0.05),細(xì)胞周期被阻滯在G1期,細(xì)胞生長(zhǎng)緩慢,體外侵襲能力明顯下降(P0.05)。研究結(jié)論： 通過下調(diào)VTCN1基因的表達(dá),能有效抑制U251細(xì)胞的生長(zhǎng)、增殖和侵襲能力,提示VTCN1基因在神經(jīng)膠質(zhì)瘤的發(fā)生、發(fā)展過程中起重要作用。
[Abstract]:The purpose of the study is:
Some tumor cell lines are composed of VTCNl. in the expression of VTCN1 protein in lung cancer, ovarian cancer and renal cell carcinoma; the expression of VTCN1 is almost common in breast and endometrial cancer, and does not depend on the classification and staging of the tumor. Moreover, this high expression of VTCN1 is an independent factor to increase the risk of death in the cases of glioma. But VTC The exact clinical significance of the expression of N1 in glioma tumor tissue remains to be further verified. Whether VTCN1 can be used as an indicator of early diagnosis and prognostic evaluation of glioma is also worth further study. Therefore, this part is to explore the expression of VTCN1 in glioma tissue and its relationship with clinicopathology and prognosis.
Research methods:
The content of VTCN1 protein in peripheral blood of glioma patients was detected by enzyme linked immunosorbent assay (ELISA), and the expression of VTCN1 gene expression in the tumor tissue specimens of patients with glioma was detected by reverse transcriptase polymerase chain reaction (RT-PCR), and the surgical resection of glioma patients was detected by immunohistochemical staining (IHC). The expression level of VTCN1 protein in tissue specimens.
The results of the study:
The content of VTCN1 protein in peripheral blood of glioma patients (44.80 + 8.20gg/L) was significantly higher than that of benign tumors in the brain (35.10 + 6.33 g/L) and normal people (32.17 + 5.96 g/L) (P0.05). Then VTCN1mRNA was detected in 30 cases of glioma tissue and para cancerous tissue, and the expression of glioma tissues was significantly higher than that of the para cancerous tissue. The expression of VTCN1 in the tissue (P0.01) was significantly higher than that of the benign tumor tissue (P0.01). The expression of VTCN1 protein in 70 cases of glioma was further detected. The positive expression rate was 68.57%, of which 77.08% was high, which was significantly higher than the expression of.VTCN1 protein in the benign tumor tissue of the brain and the histological grade of glioma. The tumor size and tumor recurrence or metastasis were closely related (P0.05), but the survival rate of patients with high expression of.VTCN1 was significantly lower than that of low expression patients (P0.05), which was not related to age, sex and histology type (P0.05).
The conclusions are as follows:
The expression of VTCN1 in glioma tissue is closely related to the malignancy, progression and prognosis of glioma. It can be used as an effective indicator to reflect the biological behavior of glioma and to judge the prognosis.
The purpose of the study is:
The abnormal expression of negative molecules in the VTCN1 gene family suggests that it plays an important role in the development of tumor. The mechanism involved will provide an effective way to interfere with the development of tumor, and it has great potential application value. More and more clinical studies have shown that VTCN1 molecules are closely related to the immunoregulation of tumor. However, the mechanism of action in the immune response of the body remains to be further studied. Therefore, this part reduces the expression of VTCN1 gene in human glioma U251 cell lines by RNA interference technique, and explores the effect of down regulation of VTCN1 gene expression on the biological behavior of glioma cells.
Research methods:
The siRNA sequence of VTCNI gene was reorganized into the eukaryotic expression vector to construct pU-VTCN1-siRNA and transfected into the glioma U251 cell line. Reverse transcriptase PCR (RT-PCR) was used to detect the change of VTCN1 gene in the gene expression level and protein expression level in U251 cells transfected with pU-VTCN1-siRNA. Flow cytometry was used. The cell cycle changes of U251 cells after transfection of pU-VTCN1-siRNA were analyzed by means of the instrument, and the invasiveness of U251 cells after transfection of pU-VTCN1-siRNA was observed by Transwell invasion model.
The results of the study:
In the U251 cells transfected with pU-VTCN1-siRNA, the gene expression level and protein expression level of VTCN1 gene decreased significantly (P0.05), the cell cycle was blocked in the G1 stage, the cell growth was slow and the invasion ability in vitro decreased significantly (P0.05).
Down regulation of the expression of VTCN1 gene can effectively inhibit the growth, proliferation and invasion of U251 cells, suggesting that the VTCN1 gene plays an important role in the development of glioma.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

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本文編號(hào)：2152160