【摘要】：目的：大腸癌是常見的消化道惡性腫瘤,占腫瘤相關(guān)死因的第二位。近年來(lái)盡管在提高患者生存率的化學(xué)療法上有了很大的進(jìn)展,但是大腸癌晚期患者的預(yù)后仍很差,總的死亡率在40%左右。闡明大腸癌分子機(jī)制是治療大腸癌的基礎(chǔ)和前提。研究提示大腸癌干細(xì)胞參與了大腸癌的發(fā)生、浸潤(rùn)、轉(zhuǎn)移和藥物抵抗。Wnt通路在大腸癌干細(xì)胞的自我更新中起了重要作用,然而Wnt通路作用于大腸癌干細(xì)胞的具體機(jī)制仍不明。本研究通過篩選和鑒定大腸癌干細(xì)胞中Wnt通路激活相關(guān)的蛋白,發(fā)現(xiàn)核受體相互作用蛋白2(Nuclear receptor-interacting protein2, Nrip2)在大腸癌干細(xì)胞中表達(dá)增加,并檢測(cè)Nrip2在大腸癌干細(xì)胞自我更新中的作用,鑒定其關(guān)鍵作用靶蛋白,尋找Nrip2作用于大腸癌干細(xì)胞的潛在靶點(diǎn),從而明確Nrip2對(duì)大腸癌生物學(xué)行為的影響。 方法：通過構(gòu)建SW620克隆球cDNA逆轉(zhuǎn)錄病毒文庫(kù),篩選出Wnt通路激活相關(guān)的蛋白Nrip2表達(dá)增加。為探討Nrip2在大腸癌中的表達(dá)情況,采用免疫組化和mRNA熒光原位雜交驗(yàn)證大腸癌病理組織切片中Nrip2蛋白的表達(dá),并采用Real-time qPCR檢測(cè)大腸癌細(xì)胞和臨床樣本富集的克隆球中Nrip2的表達(dá)情況。為評(píng)價(jià)Nrip2在大腸癌干細(xì)胞自我更新中的作用,構(gòu)建穩(wěn)定表達(dá)Nrip2的大腸癌細(xì)胞系,觀察過表達(dá)Nrip2對(duì)大腸癌細(xì)胞克隆球成球能力、iPS和EMT相關(guān)因子的表達(dá)和體內(nèi)致瘤能力的影響。為研究Nrip2對(duì)大腸癌干細(xì)胞自我更新的作用機(jī)制,質(zhì)譜篩選Nrip2的靶蛋白Frizzled1(Fzd1),構(gòu)建各種結(jié)構(gòu)域突變體分析Nrip2與Fzdl蛋白的結(jié)合位點(diǎn),以及Nrip2和下游靶分子的結(jié)合情況。通過免疫共沉淀(co-immunoprecipitation)和Western blot篩選和鑒定了Nrip2非經(jīng)典Wnt通路的靶蛋白維甲酸相關(guān)孤兒受體β (Retinoic acid-related orphan receptor beta, Rorp),觀察了過表達(dá)Rorp對(duì)大腸癌細(xì)胞克隆球形成能力、EMT相關(guān)因子的表達(dá)和體內(nèi)致瘤能力的影響。通過基因芯片篩選出Rorp的下游靶基因HMG盒轉(zhuǎn)錄因子1(High Mobility Group-box transcription factor1, HBP1)。并通過PepstatinA處理過表達(dá)Nrip2的大腸癌細(xì)胞觀察Nrip2的酶活性對(duì)其調(diào)控大腸癌干細(xì)胞自我更新的影響。 結(jié)果：構(gòu)建SW620克隆球cDNA逆轉(zhuǎn)錄病毒文庫(kù),篩選出Wnt通路激活相關(guān)蛋白Nrip2表達(dá)增加。Nrip2表達(dá)于正常大腸組織和大腸癌組織細(xì)胞中,在大腸癌干細(xì)胞中表達(dá)明顯升高。過表達(dá)Nrip2促進(jìn)了大腸癌干細(xì)胞的自我更新。我們發(fā)現(xiàn)Nrip2能夠與Fzdl結(jié)合,但不與β-catenin結(jié)合。免疫共沉淀篩選出Nrip2與Rorβ結(jié)合,其酶活性能夠降解Rorβ,阻止Rorβ入核。Rorβ通過抑制Wnt通路活性抑制大腸癌干細(xì)胞的自我更新�；蛐酒Y選發(fā)現(xiàn)Rorβ的下游靶基因是HBP1, HBP1通過競(jìng)爭(zhēng)性結(jié)合TCF/LEF4抑制Wnt通路下游靶分子的轉(zhuǎn)錄。Nrip2的酶活性的滅活阻斷了Nrip2對(duì)大腸癌干細(xì)胞的自我更新作用。 結(jié)論：首次發(fā)現(xiàn)了Nrip2和Rorp表達(dá)于正常大腸組織和大腸癌組織細(xì)胞中。Nrip2在大腸癌干細(xì)胞中表達(dá)增加,與Rorβ結(jié)合呈酶活性依賴型,通過降解Rorβ,抑制HBP1的轉(zhuǎn)錄,啟動(dòng)Wnt通路下游靶蛋白的表達(dá),促進(jìn)了大腸癌干細(xì)胞的自我更新,從而介導(dǎo)了一條新的非經(jīng)典Wnt通路Nrip2/Rorβ/HBP1通路。Nrip2的酶活性是干預(yù)阻斷大腸癌干細(xì)胞自我更新的潛在治療靶點(diǎn)。
文內(nèi)圖片：
圖片說明：富集大腸癌細(xì)胞克隆球大腸癌細(xì)胞系HT29SW620和原代大腸癌細(xì)胞P1在無(wú)血清克隆球培養(yǎng)基培養(yǎng)7-
[Abstract]:Objective: Colorectal cancer is a common malignant tumor of the digestive tract. In recent years, although much progress has been made in chemical therapy to improve the survival rate of patients, the prognosis of patients with advanced colorectal cancer is still poor and the total mortality is around 40%. The mechanism of molecular mechanism of colorectal cancer is the basis and premise of the treatment of large intestine cancer. The study suggests that colorectal cancer stem cells are involved in the occurrence, infiltration, metastasis and drug resistance of colorectal cancer. Wnt pathway plays an important role in the self-renewal of colorectal cancer stem cells. In this study, by screening and identifying the protein involved in Wnt pathway activation in colorectal cancer stem cells, the expression of nuclear receptor-interacting protein 2 (NIP2) in colorectal cancer stem cells was increased, and the role of NIP2 in the self-renewal of colorectal cancer stem cells was detected, and the key target protein was identified. The potential target of NIP2 on colorectal cancer stem cells was found, and the effect of NIP2 on the biological behavior of colorectal cancer was confirmed. Methods: Wnt pathway activation related protein Ntri2 expression was selected by constructing SW620 clone ball cDNA reverse transcription virus library. In order to study the expression of Ntri2 in colorectal cancer, the expression of the Ntri2 protein in the pathological tissue sections of the large intestine was verified by immunohistochemistry and mRNA fluorescence in situ hybridization, and the expression of the Ntri2 in the clone balls enriched by the colorectal cancer cells and the clinical samples was detected by Real-time qPCR. In order to evaluate the role of NIP2 in the self-renewal of colorectal cancer stem cells, a stable expression of NIP2-expressing colorectal cancer cell line was constructed, and the expression of the expression of the expression of NIP2 on the cloning of the colorectal cancer cells, the expression of iPS and EMT-related factors and the in vivo tumorigenicity were observed. In order to study the mechanism of Ntri2 on the self-renewal of colorectal cancer stem cells, the target protein Frizzled 1 (Fzd1) of Trip2 was screened by mass spectrometry, and the binding sites of the Ntri2 and Fzdl proteins were analyzed by various domain mutants, as well as the binding of the Ntri2 and the downstream target molecules. In this paper, the target protein, Retinoic acid-related receptor beta, Rorp, of the NIP2 non-classical Wnt pathway was screened and identified by co-immunopreservation and Western blot, and the expression of the EMT-related factors and the shadow of the in vivo tumorigenicity were observed. In response, the downstream target gene HMG-box transcription factor 1 (HBP1) of the Rorp was screened by the gene chip ). The human colorectal cancer cells overexpressing Ntri2 were treated with PepstatinA to observe the activity of NIP2 to regulate the self-renewal of colorectal cancer stem cells. In response to that result, a reverse transcription virus library of SW620 clone was construct, and the Wnt pathway activation related protein Ntri2 was screen. Up-to-date. Ntri2 is expressed in normal large intestine tissue and large intestine cancer cells, and is expressed in colorectal cancer stem cells. Significant increases. Overexpression of NIP2 promotes the self-adaptation of colorectal cancer stem cells. I'm updating. We've found that NIP2 can be combined with Fzdl, but not with I-cati. N-binding. The combination of NIP2 with Ror is screened by the immunoprecipitation, and the activity of the enzyme can degrade the Ror antigen and prevent the Ror. The inhibition of the activity of Wnt pathway in colorectal cancer stem cells by inhibiting Wnt pathway I update. The gene chip screen found that the downstream target gene of the Ror gene is HBP1 and HBP1 inhibits the downstream target molecule of the Wnt pathway by competitive binding of TCF/ LEF4 The inactivation of the enzyme activity of NIP2 blocks the self-improvement of the NIP2 to the colorectal cancer stem cells. Conclusion: Ntri2 and Rorp were found to be expressed in normal large intestine and colorectal cancer for the first time. In the tissue cells, the expression of NIP2 in the stem cells of the colorectal cancer is increased, and the expression of the target protein downstream of the Wnt pathway is inhibited by degrading the Ror antigen, inhibiting the transcription of the HBP1, starting the expression of the downstream target protein of the Wnt pathway, and promoting the stem cell of the colorectal cancer. Self-update to mediate a new non-classical Wnt pathway, Trip2/ Ror I/ H BP1 pathway. The enzyme activity of Trip2 is the potential for intervention to block the self-renewal of colorectal cancer stem cells
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.34

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