本文選題：TPX2　切入點：宮頸癌　出處：《吉林大學》2015年博士論文

【摘要】：目的：本課題擬設三個部分進行實驗研究。第一部分從細胞水平和組織水平明確TPX2在宮頸不同生理或病理狀態(tài)下表達的差異性，了解從正常宮頸上皮到CIN再到宮頸浸潤癌組織中的TPX2蛋白的表達水平，并結合病例分析，探討其與宮頸癌的臨床分期、病理分化程度和淋巴轉(zhuǎn)移的關系。第二部分探討小干擾RNA靶向沉默TPX2基因?qū)m頸鱗癌SiHa細胞的生物學行為影響。第三部分基于RNAi構建穩(wěn)轉(zhuǎn)染HeLa細胞株，探討RNAi抑制TPX2蛋白表達對細胞生物學行為的影響和激活p53、p21、Rb細胞信號轉(zhuǎn)導通路的可能性。 方法：選取HPV陽性型的人源宮頸鱗癌SiHa細胞株、宮頸腺癌HeLa細胞株和經(jīng)醫(yī)學倫理委員會審查同意后且經(jīng)病理切片診斷證實的97例不同宮頸組織標本作為實驗研究材料。第一部分實驗采用Western-Blot免疫印跡分析、RT-PCR和免疫組化研究宮頸不同生理或病理狀態(tài)下TPX2的表達情況。第二部分實驗利用RNAi對SiHa細胞中的TPX2基因進行特異性抑制，通過流式細胞術、MTT和侵襲試驗檢測TPX2抑制后對細胞生物學行為的影響。第三部分構建重組擔載TPX2-shRNA的真核載體質(zhì)粒，并轉(zhuǎn)染HeLa細胞，構建穩(wěn)轉(zhuǎn)染HeLa細胞株，采用Q-PCR、Western-Blot免疫印跡分析法、MTT、流式細胞術、TUNEL法觀察TPX2-shRNA對HeLa細胞的生物學行為的影響以及E6、TPX2、p53、p21、Rb之間的可能相互作用機理，探討高危型HPV感染后E6、TPX2激活p53、p21、Rb細胞信號和介導細胞信號轉(zhuǎn)導的可能性。 結果：第一部分實驗結果為TPX2蛋白和TPX2mRNA在CIN、宮頸鱗癌組織、宮頸腺癌組織、宮頸鱗癌細胞SiHa和宮頸腺癌細胞HeLa均有不同程度的表達，但是在正常宮頸上皮組織內(nèi)，TPX2幾乎不表達；隨著病情發(fā)展，從CINⅠ到CINⅢ的不同宮頸組織的TPX2蛋白表達逐漸增強，宮頸浸潤癌臨床II期的TPX2表達水平高于I期，淋巴轉(zhuǎn)移陽性的TPX2表達水平高于陰性，病理學低分化的TPX2表達水平高于高分化。第二部分實驗結果為TPX2-siRNA轉(zhuǎn)染細胞后，隨轉(zhuǎn)染時間的延長，細胞凋亡率逐漸升高；在轉(zhuǎn)染后出現(xiàn)細胞增殖減慢，細胞增殖受到抑制，細胞侵襲能力下降，細胞周期受到調(diào)控，主要表現(xiàn)為G1期細胞比例下降，S期和G2期細胞比例升高。第三部分實驗成功構建穩(wěn)定轉(zhuǎn)染重組TPX2-shRNA質(zhì)粒的HeLa細胞株，對細胞內(nèi)TPX2的表達起到了特異性抑制，Western-Blot和Q-PCR實驗說明TPX2-shRNA在下調(diào)TPX2的同時還能夠在轉(zhuǎn)錄和翻譯水平上下調(diào)E6，對p53、p21、Rb也具有下調(diào)作用，證實TPX2-shRNA具有激活p53、p21、Rb細胞信號的能力，RNAi沉默TPX2后介導細胞信號轉(zhuǎn)導的具體作用機制可能是TPX2被靶向沉默后恢復野生型p53的功能活性，促進細胞凋亡。RNAi沉默TPX2后對細胞周期的調(diào)控主要存在兩種途經(jīng)，一方面，p53通過調(diào)控其下游靶基因p21的轉(zhuǎn)錄和翻譯，刺激p21WAF1/CIP1的表達調(diào)控細胞周期，p21WAF1/CIP1能夠通過引起Rb蛋白磷酸化調(diào)控細胞周期；另一方面，從Rb介導的細胞信號轉(zhuǎn)導通路來看，Rb基因通過G1/S期限制點調(diào)控細胞周期，Rb蛋白的磷酸化狀態(tài)決定了細胞增殖能力，非磷酸化Rb可與轉(zhuǎn)錄因子E2F結合，Rb蛋白轉(zhuǎn)變?yōu)榱姿峄筢尫啪哂谢钚缘挠坞xE2F，調(diào)控細胞增殖。E6基因通過其產(chǎn)物E6蛋白使具有抑癌作用的非磷酸化Rb蛋白轉(zhuǎn)化為磷酸化Rb蛋白。野生型p53可以同非磷酸化的Rb作用，通過依賴p53誘導的細胞凋亡途經(jīng)保護Rb的抑癌功能，促使磷酸化Rb蛋白轉(zhuǎn)化為非磷酸化Rb。此外，高危型HPV的E7蛋白可以結合Rb使E2F游離，活化S期轉(zhuǎn)錄。RNAi的效果越好，細胞增殖活性受到抑制的程度越明顯、細胞凋亡數(shù)量越多，停頓于S期和G2/M期的細胞比例越大。 結論：在宮頸鱗狀細胞癌、CIN、宮頸腺癌組織中均存在TPX2過表達，TPX2在正常宮頸組織中幾乎不表達；在宮頸上皮細胞從正常到癌變的發(fā)展過程中，TPX2表達含量逐漸增多；宮頸癌的臨床分期、病理分級和轉(zhuǎn)移均與TPX2過表達水平密切相關，TPX2過表達水平越高，臨床分期越晚、病理分化越差、越容易發(fā)生淋巴結轉(zhuǎn)移。RNAi特異性沉默SiHa、HeLa細胞的TPX2后不僅能夠調(diào)控細胞周期，使細胞周期在S期及G2/M期停頓，而且能夠抑制細胞的增殖活性和侵襲能力，TPX2-shRNA能夠使細胞發(fā)生凋亡。成功構建了穩(wěn)轉(zhuǎn)染TPX2-shRNA重組質(zhì)粒的HeLa細胞株，證實了高危型HPV感染后TPX2在轉(zhuǎn)錄和翻譯水平上均與E6關系密切，TPX2-shRNA能夠激活p53、p21、Rb細胞信號通路，介導細胞信號轉(zhuǎn)導途經(jīng)、調(diào)控細胞周期、誘發(fā)細胞凋亡；E6、TPX2通過抑制p53、p21、Rb的抗癌功能，使細胞周期檢查點功能缺陷，使正常狀態(tài)的細胞發(fā)生癌變。RNAi干擾能夠有效抑制TPX2的過表達，保護p53、p21、Rb的正常功能狀態(tài)，基于RNAi技術抑制TPX2基因的表達可對宮頸癌的發(fā)生和進展起抑制作用，，TPX2基因有望成為一種新的宮頸癌早期診斷和基因治療的候選靶點，為宮頸癌的臨床診斷和治療提供新的思路。
[Abstract]:Objective: This paper proposed three parts. The first part studied the differences from the cell level and tissue level clear expression of TPX2 in cervical under different physiological or pathological condition, understand the expression of water from normal cervical epithelium to CIN to invasive cervical carcinoma TPX2 protein level, and combined with case analysis, to explore the with the clinical stage of cervical cancer, the relationship between the degree of pathological differentiation and lymph node metastasis. The second part discusses the small interfering RNA targeting TPX2 gene on the biological behavior of cervical squamous cell carcinoma SiHa cells. The effects of the third part of the RNAi construction of stable transfected HeLa cells based on the inhibitory effect of RNAi on expression of TPX2 protein on cell biological behavior and activation of p53 p21, the possibility of signal transduction pathway of Rb cells.
Methods: HPV positive type human cervical squamous cell carcinoma SiHa cell line, cervical cancer cell line HeLa and by the medical ethics committee for examination and approval and 97 cases with different cervical tissue biopsy specimens confirmed the diagnosis as the experimental materials. The first part of the experiment using Western-Blot blot analysis, RT-PCR and immunohistochemical study of cervical the physiological or pathological conditions TPX2 expression. The second part of the experiment using RNAi TPX2 gene on SiHa cells was specifically inhibited by flow cytometry. The effect on cell biological behavior and invasion of MTT TPX2 inhibition test. The third part is to construct the eukaryotic recombinant plasmid carrying TPX2-shRNA, and transfected into HeLa to construct stable cells, transfected HeLa cells, using Q-PCR, Western-Blot blot analysis, MTT, flow cytometry and TUNEL method to observe the effect of TPX2-shRNA on HeLa cells The influence of learning behavior and the possible interaction mechanism between E6, TPX2, p53, p21 and Rb are discussed. The possibility of E6, TPX2 activation p53, p21, Rb cell signal and mediating cell signal transduction after high-risk HPV infection is discussed.
Results: in the first part of the experimental results of TPX2 protein and TPX2mRNA in CIN, cervical squamous cell carcinoma, adenocarcinoma of the uterine cervix, cervical squamous cell carcinoma SiHa cells and cervical cancer cell line HeLa in varying degrees, but in normal cervical epithelial tissues, there was almost no expression of TPX2; with the disease development, expression increased gradually from different cervical tissues I CIN to CIN III TPX2 protein, the expression level of invasive carcinoma of clinical stage II TPX2 cervical lymph node metastasis was higher than that in stage I, TPX2 positive expression level was higher than the negative, pathological differentiation of low TPX2 expression level was higher in high differentiation. Some experimental results for second TPX2-siRNA after the transfection, the transfection time, cell apoptosis the rate increased gradually; cell proliferation in transfected cell proliferation inhibition and cell invasion ability decreased, cell cycle regulated, mainly for the G1 phase of the cell ratio decreased, S And the proportion of cells in G2 phase of HeLa cells increased. The third part of the experiment successfully constructed stably transfected with recombinant plasmid TPX2-shRNA, the expression of TPX2 in endothelial cells plays a specific inhibition, Western-Blot and Q-PCR experiments indicated that the TPX2-shRNA in the TPX2 can also cut down E6, at the level of transcription and Translation of p53, p21, Rb with a reduced role, has confirmed that the TPX2-shRNA activation of p53, p21, Rb cell signal, the specific mechanism of RNAi silencing TPX2 mediated signal transduction may be TPX2 by targeting functional recovery after wild-type p53, promote apoptosis of.RNAi cells after silencing of TPX2 in the regulation of cell cycle there are two main way on the one hand, through the regulation of p53, p21 and its downstream target gene transcription and translation, cell cycle regulation stimulates p21WAF1/CIP1 expression, p21WAF1/CIP1 can induce Rb protein phosphorylation in cell cycle Period; on the other hand, from the cell signal transduction pathway mediated by Rb, Rb gene restriction point of cell cycle regulation by G1/S, the phosphorylation status of Rb determines the ability of cell proliferation, non phosphorylated Rb can bind to the transcription factor E2F and Rb protein phosphorylation after release into free E2F with activity the regulation of cell proliferation,.E6 gene has tumor suppressor role of non phosphorylated Rb protein into phosphorylated Rb protein by product E6 protein. Rb effect of wild-type p53 with non phosphorylation, cell apoptosis induced by p53 dependent protection via the tumor suppressor function of Rb, the phosphorylation of Rb protein into non phosphorylation of Rb. in high-risk HPV E7 protein can be combined with Rb to make E2F free, the better effect of activation of S phase.RNAi transcription, cell proliferation activity was inhibited more obviously, the number of apoptosis cells more pauses in S phase and G2/M phase cells The greater the proportion.
Conclusion: in cervical squamous cell carcinoma, CIN, overexpression of TPX2 exists in cervical adenocarcinoma, TPX2 almost no expression in normal cervical tissue; in the process of development of cervical epithelial cells from normal to cancer, the expression of TPX2 was increased; the clinical staging of cervical cancer, pathological grading and metastasis with TPX2 over expression of TPX2 is closely related to the higher level of expression, the later clinical stage, pathological differentiation, the more prone to lymph node metastasis of.RNAi silencing of SiHa, HeLa cells after TPX2 can not only regulate the cell cycle, the cell cycle arrest in S phase and G2/M phase, and can inhibit the cell proliferation activity and the invasion, TPX2-shRNA can induce cell apoptosis. HeLa cells was successfully constructed stably transfected with TPX2-shRNA recombinant plasmid, confirmed the high-risk HPV infection after TPX2 at the level of transcription and translation are closely related with E6, T PX2-shRNA can activate p53, p21, Rb cell signaling pathway, mediated via cellular signal transduction, cell cycle regulation, apoptosis; E6, TPX2 p21, Rb by inhibiting p53, anti-cancer function, cell cycle checkpoint function defects, the cancerous.RNAi interference can effectively inhibit the TPX2 expression, p53 protection p21, normal cell, normal functional status of Rb, inhibit the occurrence and development of RNAi technology to inhibit TPX2 gene expression in cervical cancer based on TPX2 gene, is expected to become a new candidate target for early diagnosis of cervical cancer and gene therapy, to provide new ideas for clinical diagnosis and treatment of cervical cancer.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R737.33

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相關期刊論文 前1條

1 張賓;趙海波;種道群;李暢;聶學誠;王家富;;TPX2蛋白在子宮內(nèi)膜樣腺癌中的表達及其臨床意義[J];臨床腫瘤學雜志;2011年04期

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