本文選題：Bit1 + 食管鱗癌　； 參考：《鄭州大學》2015年碩士論文

【摘要】：研究背景食管鱗狀細胞癌(esophageal squamous cell carcinoma,ESCC)是中國常見的惡性腫瘤之一,尤其是在河南最為常見。惡性腫瘤的發(fā)生和發(fā)展需要經(jīng)歷一系列復雜的過程,可能涉及某些凋亡信號傳導通路的激活和與之對應的抗凋亡信號傳導通路的失活。Bit1(Bcl-2 inhibitor of transcription 1)是2004年發(fā)現(xiàn)的一個蛋白質(zhì),其正常的生理功能還不清楚。有研究認為,Bit1是一種線粒體蛋白,在生理狀態(tài)下定位于線粒體。當細胞受到失黏附信號刺激時,Bit1蛋白轉(zhuǎn)移至細胞胞漿中,轉(zhuǎn)移而來的或者在細胞胞漿中外源表達Bit1蛋白時,其則會通過與胞漿中的AES(amino-terminal enhancer of split)蛋白結(jié)合成為復合體,而介導脫粘附發(fā)生的失巢凋亡,即非caspase(半胱氨酸蛋白酶)途徑依賴的細胞凋亡。亦有研究認為Bit1蛋白定位在高爾基體中,與之前研究理論相悖,具有抗凋亡作用。Bit1與各腫瘤的關(guān)系,實驗研究及文獻報道甚少,且結(jié)果各異。至于Bit1與ESCC之間的關(guān)系,鮮有報道。為了更進一步探討B(tài)it1蛋白對食管鱗癌發(fā)生發(fā)展、侵襲轉(zhuǎn)移的影響,本實驗研究擬采用RNA干擾、蛋白免疫印跡(Western Blot)、Transwell侵襲技術(shù)、構(gòu)建裸鼠皮下移植瘤模型并且首次運用干擾質(zhì)粒(瞬轉(zhuǎn))與脂質(zhì)體2000的混合液對瘤體進行干擾治療,也是本課題的一個創(chuàng)新點,從體內(nèi)和體外兩個層面,更加深入的研究Bit1的表達對對ESCC細胞增殖、侵襲等生物學特性的影響,有助于了解Bit1與食管鱗癌發(fā)生、轉(zhuǎn)移的關(guān)系,以期能夠為食管鱗癌早期診斷靶點和個體化治療方案提供一個新的思路。方法1.使用Western blot技術(shù),檢測6種ESCC細胞系中Bit1蛋白表達水平。2.采用Transwell侵襲技術(shù),檢測運用RNAi方法下調(diào)Bit1表達水平后EC9706細胞侵襲力的改變。3.構(gòu)建裸鼠皮下移植瘤并且運用干擾質(zhì)粒(瞬轉(zhuǎn))與脂質(zhì)體2000的混合液對瘤體進行干擾治療,檢測運用RNAi方法持續(xù)下調(diào)Bit1的表達水平后,觀察EC9706細胞成瘤作用的變化。4.采用SPSS17.0軟件分析數(shù)據(jù),多組定量資料比較采用單因素方差分析ANOVA(其中的兩兩比較用LSD)法;兩樣本均數(shù)采用t檢驗而定性資料用秩和。以α=0.05作為檢驗水準。結(jié)果1.Bit1在6種ESCC細胞系中的表達Bit1在4種分化程度較低的ESCC細胞株EC9706、Eca109、TE-13和KYSE70Bit1蛋白的表達量相對較高,而在分化程度較好的2種細胞株TE-1和KYSE450表達相對較低。2.下調(diào)Bit1表達水平,EC9706細胞侵襲能力的改變下調(diào)Bit1表達水平,轉(zhuǎn)然后72h基因干擾效率達到62%;RNAi組穿膜的細胞個數(shù)顯著少于陰性對照組(空載體轉(zhuǎn)染組)(P0.01),而在陰性對照組和未轉(zhuǎn)染組之間,則不存在明顯的差異(P0.05)。3.裸鼠皮下成瘤實驗表明,持續(xù)下調(diào)Bit1的蛋白表達,可以明顯抑制裸鼠食管鱗癌移植瘤的生長。細胞的成瘤率為100%,于接種后第5d,10μg空載體組和10μg RNAi組之間比較,P0.05,差異具有統(tǒng)計學意義,自第9d起至第21d,C組和D組比較,均P0.05,差異均具有統(tǒng)計學意義;5μg RNAi組和5μg空載體組,在各個時間點上差異均無統(tǒng)計學意義(P0.05)。結(jié)論1.Bit1蛋白的表達水平可能與ESCC的分化程度有關(guān)。2.下調(diào)Bit1蛋白的表達能夠抑制EC9706細胞的侵襲能力。3.EC9706細胞具有很強的致瘤性,下調(diào)Bit1蛋白的表達可以顯著抑制裸鼠瘤體的生長。
[Abstract]:Background esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors in China, especially in Henan. The occurrence and development of malignant tumors require a series of complicated processes, which may involve the activation of some apoptotic signaling pathways and the corresponding anti apoptotic signal transduction. The inactivation of.Bit1 (Bcl-2 inhibitor of transcription 1) is a protein found in 2004 and its normal physiological function is not clear. It is considered that Bit1 is a mitochondrial protein that is located in the mitochondria in physiological state. When the cell is stimulated by the lost signal, the Bit1 protein is transferred to the cytoplasm and transferred to the cytoplasm. Or when Bit1 protein is expressed in the cytoplasm, it will be combined with the AES (amino-terminal enhancer of split) protein in the cytoplasm, which mediates the apoptosis of the deadhesion, that is, the cell apoptosis dependent on the non caspase (cysteine protease) pathway. There is also a study that Bit1 protein is located in Golgi. In vivo, it is contrary to previous research theory, with the relationship between anti apoptotic effect of.Bit1 and various tumors. Experimental research and literature report are very few, and the results are different. As to the relationship between Bit1 and ESCC, there are few reports. In order to further explore the effect of Bit1 protein on the development and invasion of esophageal squamous cell carcinoma, this experimental study intends to use RNA interference, Protein immunoblotting (Western Blot), Transwell invasion technique, construction of subcutaneous transplanted tumor model in nude mice and interference plasmids (transient) and liposome 2000 mixture for the first time to interfere with the tumor, is also an innovative point in this subject. From two levels in vivo and in vitro, the expression of Bit1 to ESCC cells is further studied. The effects of biological characteristics such as proliferation and invasion can help to understand the relationship between Bit1 and the occurrence and metastasis of squamous cell carcinoma of the esophagus, in order to provide a new idea for the early diagnosis of target and individualized treatment of esophageal squamous cell carcinoma. Method 1. Western blot technique was used to detect the Bit1 protein expression level of.2. in the 6 ESCC cell lines using Transwell invasion. The technique was to detect the changes of EC9706 cell invasiveness by using the RNAi method to reduce the Bit1 expression level..3. constructed the subcutaneous transplanted tumor of nude mice and interfered with the mixed solution of the plasmid (transient) and liposome 2000. The changes of the tumorigenesis of the EC9706 cells were observed after the RNAi method was kept down to the level of Bit1. 4. SPSS17.0 software was used to analyze the data, and the multiple quantitative data were compared with single factor analysis of variance ANOVA (22 of them using LSD); two samples were tested with t test and the qualitative data were used as a test level. The results of 1.Bit1 in the 6 ESCC cell lines were Bit1 at 4 ESCC cell lines with lower differentiation degree, EC97. 06, the expression of Eca109, TE-13 and KYSE70Bit1 protein was relatively high, while the 2 cell lines with better differentiation, TE-1 and KYSE450 expressed relatively low.2., down regulated the Bit1 expression level. The invasion ability of EC9706 cells decreased the Bit1 expression level, and the interference efficiency of the 72h gene reached 62%, and the number of cells in the RNAi group was significantly less than that of the negative. In the control group (P0.01), there was no significant difference between the negative control group and the untransfected group (P0.05). The subcutaneous tumor formation test in.3. nude mice showed that the continuous downregulation of the protein expression of Bit1 could obviously inhibit the growth of the xenoesophageal squamous cell carcinoma in nude mice. The cell formation rate was 100%, and the 5D, 10 u g empty carrier group after inoculation, and the group of nude mice after inoculation. The difference between the 10 g RNAi groups was statistically significant. The difference was statistically significant from 9D to 21d, C and D groups, and the difference was statistically significant. There was no significant difference between the 5 g RNAi group and the 5 mu g space carrier group (P0.05). Down regulation of the expression of Bit1 protein can inhibit the invasion of EC9706 cells and.3.EC9706 cells have a strong tumorigenicity. Down regulation of the expression of Bit1 protein can significantly inhibit the growth of nude mice.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.1

【參考文獻】

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1 楊歡;車甌;陳姍;孫靚;季愛民;;聚乙烯亞胺介導siRNA分子體內(nèi)外基因沉默VEGFR2表達[J];藥學學報;2010年05期

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