【摘要】：[目的]通過體外人結(jié)直腸癌細(xì)胞同期放化療模型的建立,研究放化療后殘余癌細(xì)胞侵襲轉(zhuǎn)移相關(guān)生物學(xué)行為的變化趨勢(shì),并利用基因芯片篩選與之相關(guān)的關(guān)鍵基因,探索調(diào)變放化療后殘余癌細(xì)胞侵襲轉(zhuǎn)移能力變化的可能。本項(xiàng)目將為進(jìn)一步揭示新輔助放化療后殘余結(jié)直腸癌生物學(xué)特性變化及其機(jī)制打下基礎(chǔ),為提高結(jié)直腸癌的綜合治療療效提供新思路。[方法]1.建立體外同期放化療模型:對(duì)HCT116和HT29細(xì)胞運(yùn)用4Gy 6MVX-線照射和10 umol/L5-Fu藥物同時(shí)處理,重復(fù)多次,得放化療后殘余細(xì)胞株(HCT116CR,HT29CR)。2.采用Transwell遷移實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn)進(jìn)行HCT116CR和HT29CR遷移能力和侵襲能力的測(cè)定。3.利用基因芯片檢測(cè)同期放化療殘余細(xì)胞HCT116CR與其母細(xì)胞HCT116N長(zhǎng)鏈非編碼RNA (long non-coding RNA,1ncRNA)的差異表達(dá)水平,根據(jù)芯片結(jié)果和查閱文獻(xiàn),篩選關(guān)鍵基因。4.運(yùn)用小干擾RNA(siRNA)干預(yù)放化療后殘余細(xì)胞HCT16中關(guān)鍵基因的表達(dá)或(和)功能,并運(yùn)用qRT-PCR進(jìn)行干擾效果的檢測(cè)。5.再次利用Transwell遷移實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn)檢測(cè)siRNA干擾后殘余細(xì)胞siRNA-HCT1 16CR細(xì)胞的遷移能力和侵襲能力。[結(jié)果]1. HCT116和HT29細(xì)胞經(jīng)過4次同期放化療處理后,成功建立體外同期放化療殘余細(xì)胞模型:HCT116CR 和 HT29CR。2. Transwell遷移實(shí)驗(yàn)和Transwell侵襲實(shí)驗(yàn)發(fā)現(xiàn),放化療后殘余腸癌細(xì)胞遷移能力和侵襲能力較母細(xì)胞明顯增強(qiáng)。3.基因芯片檢測(cè)出與放化療后殘余腫瘤細(xì)胞生物學(xué)行為變化的1ncRNA表達(dá)譜,篩選出的三個(gè)關(guān)鍵分子:PVT1,LINC00152和MIR22HG。4. SiRNA技術(shù)干擾HCT116CR細(xì)胞中關(guān)鍵分子的表達(dá)后,siRNA-LINC00152組遷移能力、侵襲能力相比未干擾組明顯減弱。[結(jié)論]1.同期放化療后殘余結(jié)直腸癌細(xì)胞侵襲轉(zhuǎn)移能力增強(qiáng),生物學(xué)行為發(fā)生惡化。2. LINC00152可能是調(diào)控放化療后殘余結(jié)直腸癌細(xì)胞生物學(xué)特性變化的關(guān)鍵的分子之一。
[Abstract]:[objective] to study the changes of biological behavior associated with invasion and metastasis of residual cancer cells after radiotherapy and chemotherapy in vitro, and to screen the key genes by gene chip. To explore the possibility of invasion and metastasis of residual cancer cells after adjuvant radiotherapy and chemotherapy. This project will lay a foundation for further revealing the biological characteristics and mechanism of residual colorectal cancer after neoadjuvant radiotherapy and chemotherapy, and provide a new idea for improving the curative effect of comprehensive therapy for colorectal cancer. [methods] 1. To establish the model of simultaneous radiotherapy and chemotherapy in vitro: HCT116 and HT29 cells were irradiated with 4Gy 6MVX- line and treated with 10 umol/L5-Fu drugs simultaneously and repeated for many times. The residual cell line (HCT116CR-HT29CR). 2 was obtained after radiotherapy and chemotherapy. The migration ability and invasion ability of HCT116CR and HT29CR were measured by Transwell migration test and Transwell invasion test. Gene chip was used to detect the differential expression level of HCT116CR and its mother cell HCT116N. According to the results of microarray and literature review, the key gene was screened. Small interfering RNA (siRNA) was used to interfere with the expression or / or function of key genes in HCT16 of residual cells after radiotherapy and chemotherapy, and qRT-PCR was used to detect the interference effect. Transwell migration assay and Transwell invasion assay were used to detect the migration and invasion ability of siRNA-HCT1 16CR cells after siRNA interference. [result] 1. After four times of simultaneous radiotherapy and chemotherapy, HCT116 and HT29 cells were successfully established in vitro concurrent radiotherapy and chemotherapy residual cell model: HCT116CR and HT29CR.2. Transwell migration assay and Transwell invasion assay showed that the migration and invasion ability of residual intestinal cancer cells after radiotherapy and chemotherapy was significantly higher than that of mother cells. The 1ncRNA expression profiles of biological behavior of residual tumor cells after radiotherapy and chemotherapy were detected by gene chip, and three key molecules: PVT1, LINC00152 and MIR22HG.4were selected. After SiRNA interfered with the expression of key molecules in HCT116CR cells, the migration ability of siRNA-LINC00152 group was significantly decreased than that of non-interference group. [conclusion] 1. After radiotherapy and chemotherapy, the invasion and metastasis of residual colorectal cancer cells increased, and biological behavior deteriorated. 2. 2. LINC00152 may be one of the key molecules to regulate the biological characteristics of residual colorectal cancer cells after radiotherapy and chemotherapy.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34

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本文編號(hào)：2148853