本文選題：肝癌 + AFP　； 參考：《河北醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:肝癌是全球發(fā)病率第六的惡性腫瘤,每年約有70萬人被診斷為肝癌。在癌癥引起的死亡中30%為肝癌,近年來其病死率呈逐年上升的趨勢,已經(jīng)對人類健康構(gòu)成了極大的威脅。肝癌是涉及多基因、多過程的復(fù)雜疾病,其致病機(jī)理仍不清楚。目前,手術(shù)切除治療仍然是肝癌最有效的治療方法,然而肝癌切除術(shù)或肝移植術(shù)后患者預(yù)后情況往往并不理想,其主要原因之一就是腫瘤的復(fù)發(fā)和轉(zhuǎn)移。因此探討肝癌細(xì)胞轉(zhuǎn)移發(fā)生、發(fā)展的分子生物學(xué)機(jī)制對肝癌的治療具有重要意義,也是肝癌研究的熱點和難點。另外,建立與人類肝癌臨床特征相近的動物模型對于研究肝癌機(jī)理尋找新的治療方法也具有重要的現(xiàn)實意義。甲胎蛋白(alpha—fetoprotein,AFP)是目前已知的與肝癌關(guān)系最密切的基因之一,約70%的肝癌患者高表達(dá)AFP,AFP作為血清標(biāo)記物已廣泛應(yīng)用于肝癌的臨床診斷、治療及預(yù)后判斷。AFP主要在胎兒時期合成,出生后1-2年降至成人水平,健康成年人肝臟失去產(chǎn)生AFP的能力。但是,當(dāng)成年人患肝癌或者肝切除時,肝臟恢復(fù)合成AFP的能力,AFP的合成量與胎兒肝臟或者肝細(xì)胞再生的分裂細(xì)胞數(shù)呈正相關(guān),因此認(rèn)為AFP是分裂增殖的肝細(xì)胞產(chǎn)生,靶向Afp的治療能同時針對所有的肝癌細(xì)胞。近年研究表明,AFP在肝癌細(xì)胞的生長、分化、增殖和遷移中發(fā)揮重要作用,是肝癌進(jìn)展過程中一個重要的調(diào)節(jié)因子。RNA干擾(RNA Interference)通過雙鏈RNA在mRNA水平部分或者完全抑制特異性基因表達(dá)。RNAi在生物體內(nèi)廣泛存在能同時抑制多個基因,并且操作簡單、特異性高、能快速使靶向基因功能抑制,為肝癌的基因治療帶來了希望。本研究采用DEN和NMOR聯(lián)合誘導(dǎo)大鼠產(chǎn)生肝癌,通過組織培養(yǎng)、克隆,篩選出具有高、低轉(zhuǎn)移潛能的大鼠肝癌細(xì)胞各1株。并以得到的高轉(zhuǎn)移潛能大鼠肝癌細(xì)胞為研究對象,構(gòu)建靶向Afp的shRNA并用脂質(zhì)體法轉(zhuǎn)染細(xì)胞,干擾其AFP表達(dá),分析不同AFP表達(dá)水平對細(xì)胞遷移率和克隆形成能力的影響。不同AFP表達(dá)水平的細(xì)胞的獲得可為制備相應(yīng)的大鼠肝癌模型做準(zhǔn)備,從而為肝癌的轉(zhuǎn)移、復(fù)發(fā)機(jī)理研究及新藥治療效果評價等提供與臨床相一致的動物模型。方法:無菌取誘導(dǎo)20周肝癌大鼠肝臟腫瘤進(jìn)行原代培養(yǎng),收取細(xì)胞后經(jīng)Transwell篩選獲得AFP高表達(dá)的高、低轉(zhuǎn)移潛能的肝癌細(xì)胞株Wrh-f2和Wrh-s2,分析細(xì)胞生物學(xué)特性;將靶向AFP的4個shRNA載體及陰性載體轉(zhuǎn)染AFP高表達(dá)的Wrh-f2細(xì)胞,實驗分為空白對照組、陰性對照組、轉(zhuǎn)染AFP-si RNA組,獲得5個穩(wěn)定轉(zhuǎn)染細(xì)胞株。通過Real-time PCR和Western blot方法檢測各組細(xì)胞Afp mRNA和AFP的表達(dá)變化,采用t檢驗和方差分析進(jìn)行比較。選擇干擾效果最好的8號細(xì)胞,檢測細(xì)胞克隆形成率和遷移率。結(jié)果:1 DEN和NMOR聯(lián)合誘導(dǎo)大鼠于20周末獲得誘發(fā)性肝癌模型,肝臟有大的腫瘤塊;腫塊經(jīng)培養(yǎng)、克隆獲得肝癌細(xì)胞。2經(jīng)Transwell篩選獲得高、低轉(zhuǎn)移潛能的肝癌細(xì)胞株Wrh-f2和Wrh-s2,AFP表達(dá)均為陽性。Wrh-f2細(xì)胞具有更高的遷移率和克隆形成率,有多核細(xì)胞,異常核分裂相多見,其特征與病人肝癌組織相同。經(jīng)染色體核型分析發(fā)現(xiàn)Wrh-s2株細(xì)胞為超2倍體,眾數(shù)為55;Wrh-f2細(xì)胞大部分核型為超4倍體,各細(xì)胞內(nèi)部差異較大,f2眾數(shù)為83。且高轉(zhuǎn)移潛能細(xì)胞Wrh-f2易發(fā)生肺部轉(zhuǎn)移,這與人體內(nèi)的肝癌細(xì)胞特性相近。3合成針對Afp的shRNA4個分別為5、6、7、8號,另有陰性對照0號,轉(zhuǎn)染W(wǎng)rh-f2細(xì)胞,獲得穩(wěn)轉(zhuǎn)細(xì)胞。Real-time PCR和Western blot定量結(jié)果顯示,轉(zhuǎn)染8號shRNA的細(xì)胞mRNA及蛋白表達(dá)水平均顯著降低,因此選擇8號細(xì)胞進(jìn)行下面實驗。4經(jīng)檢測Wrh-f2、0號、8號細(xì)胞克隆形成率分別為:(85.7±7.2)%、(59±5.8)%、(5.5±0.7)%,差異極顯著;細(xì)胞遷移率結(jié)果顯示W(wǎng)rh-f2、0號、8號平均穿膜細(xì)胞數(shù)分別為98±12,45±15,30±10,差異顯著。結(jié)論:1經(jīng)Transwell篩選獲得Wrh-f2和Wrh-s2細(xì)胞分別為高、低轉(zhuǎn)移潛能的肝癌細(xì)胞株,AFP表達(dá)均為陽性。Wrh-f2細(xì)胞具有更高的遷移率和克隆形成率,且從形態(tài)學(xué)及染色體分析發(fā)現(xiàn)Wrh-f2分化水平較低,惡化程度較高,且高轉(zhuǎn)移潛能細(xì)胞Wrh-f2易發(fā)生肺部轉(zhuǎn)移,這與人體內(nèi)的肝癌細(xì)胞特性相近。2合成的4組shRNA均可靶向抑制Afp mRNA的表達(dá)量,其中8號可同時顯著抑制蛋白的表達(dá),且干擾效果最好。經(jīng)試驗8號可以有效降低肝癌細(xì)胞的轉(zhuǎn)移能力。
[Abstract]:Objective: liver cancer is a malignant tumor of sixth in the world. About 700 thousand people are diagnosed as liver cancer every year. In the death of cancer, 30% of them are liver cancer. In recent years, the mortality rate is increasing year by year, which has posed a great threat to human health. Liver cancer is a complex disease involving multi cause and multi process. The pathogenesis is still unclear. At present, surgical resection is still the most effective treatment for liver cancer. However, the prognosis of the patients after hepatectomy or liver transplantation is often not ideal. One of the main reasons is the recurrence and metastasis of the tumor. Therefore, it is important to explore the metastasis of hepatoma cells and the development of the sub biological mechanism for the treatment of liver cancer. It is also a hot and difficult point in the study of liver cancer. In addition, it is of great practical significance to establish an animal model similar to the clinical characteristics of human liver cancer for the study of the mechanism of liver cancer. Alpha fetoprotein (AFP) is one of the most closely related genes associated with liver cancer, and about 70% of the liver cancer patients High expression of AFP, AFP as a serum marker has been widely used in the clinical diagnosis of liver cancer. The treatment and prognosis of.AFP are mainly synthesized in the fetal period, 1-2 years after birth to adult level. The liver of healthy adults loses the ability to produce AFP. However, when adults suffer from liver cancer or hepatectomy, the liver can restore the ability to synthesize AFP and the combination of AFP. There is a positive correlation between the number and the number of split cells regenerated from fetal liver or liver cells. Therefore, AFP is considered to be a proliferating hepatocyte, and the targeted Afp therapy can simultaneously target all liver cancer cells. In recent years, the study shows that AFP plays an important role in the growth, differentiation, proliferation and migration of hepatoma cells, and is one of the progress of liver cancer. The important regulatory factor.RNA interference (RNA Interference) can inhibit multiple genes at the same time in the mRNA horizontal part or completely inhibit the specific gene expression of.RNAi in the organism. It has a simple and high specificity, which can quickly inhibit the target gene function and bring hope for the gene therapy of liver cancer. DEN and NMOR were used to induce liver cancer in rats. Through tissue culture and cloning, 1 rat hepatoma cells with high and low metastatic potential were screened, and the high metastatic potential rat hepatoma cells were selected as the research object. The target Afp shRNA was constructed and transfected with liposome method. The expression of AFP was interfered with the expression of AFP, and the expression of different AFP expression was analyzed. The effect of level on cell mobility and clone formation ability. The acquisition of cells with different AFP expression levels can prepare the corresponding rat model of liver cancer, thus providing a consistent animal model for the metastasis of liver cancer, the study of the mechanism of recurrence and the evaluation of the effect of new drug treatment. Method: aseptic induction of liver cancer of 20 weeks in rat liver After primary culture, after collecting cells, the tumor cells were screened by Transwell to obtain high, low metastatic potential liver cancer cell lines, Wrh-f2 and Wrh-s2, to analyze the cell biological characteristics, and transfect AFP highly expressed Wrh-f2 cells with 4 shRNA carriers and negative vectors targeting AFP, which were divided into blank control group, negative control group and AFP-si transfected AFP-si. In group RNA, 5 stable transfected cell lines were obtained. The expression of Afp mRNA and AFP in each cell was detected by Real-time PCR and Western blot. T test and variance analysis were used to compare. Select the 8 cells with the best interference effect and detect the cell clone formation rate and mobility. Fruit: 1 DEN and NMOR combined rats were obtained at the end of the 20 week. The liver has a large tumor block, the liver has a large tumor block, and the tumor is cloned to obtain the liver cancer cell.2, which is screened by Transwell to obtain high, low metastatic potential liver cancer cell lines Wrh-f2 and Wrh-s2. The expression of AFP is the positive.Wrh-f2 cells with higher mobility and clone formation rate. The liver cancer tissues of the patients were the same. By karyotype analysis, Wrh-s2 cells were found to be ultra 2 fold and 55, and most of the karyotypes of Wrh-f2 cells were super 4 ploidy, the internal difference between each cell was large, the number of F2 was 83. and the high metastatic potential cell Wrh-f2 was easily transferred to the lung, which was similar to the.3 synthesis of Afp shRNA4 Wrh-f2 cells were transfected with.Real-time PCR and Western blot, respectively. The transfection of.Real-time PCR and Western blot showed that the expression of mRNA and protein in the cells transfected to shRNA was significantly reduced, so 8 cells were selected to test the Wrh-f2,0 number of.4, and the clone formation rate of No. 8 cells was respectively: (85.7) 7.2%, (59 + 5.8)%, (5.5 + 0.7)%, and the difference was very significant. The cell migration rate showed that the number of Wrh-f2,0 and 8 average membrane cells was 98 + 12,45 + 15,30 + 10, and the difference was significant. Conclusion: 1 was screened by Transwell to obtain Wrh-f2 and Wrh-s2 cells with high and low metastatic potential respectively. The expression of AFP was more than that of positive.Wrh-f2 cells. The high mobility and clone formation rate, and from the morphological and chromosome analysis, the Wrh-f2 differentiation level is low, the deterioration degree is high, and the high metastatic potential cell Wrh-f2 is prone to pulmonary metastasis, which can target the expression of Afp mRNA in the 4 groups of shRNA, which are similar to the human hepatoma cells in the human body, and the number of Afp mRNA can be inhibited at the same time. The expression of protein is the best and the interference effect is the best. Test 8 can effectively reduce the metastatic ability of liver cancer cells.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.7

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