Bit1靶向性小分子RNA对食管鳞癌（细胞）侵袭抑制及治疗作用

发布时间：2018-05-16 01:34

本文选题：Bit1 + 食管鳞癌　；参考：《郑州大学》2015年硕士论文

【摘要】：研究背景食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)是中国常见的恶性肿瘤之一,尤其是在河南最为常见。恶性肿瘤的发生和发展需要经历一系列复杂的过程,可能涉及某些凋亡信号传导通路的激活和与之对应的抗凋亡信号传导通路的失活。Bit1(Bcl-2 inhibitor of transcription 1)是2004年发现的一个蛋白质,其正常的生理功能还不清楚。有研究认为,Bit1是一种线粒体蛋白,在生理状态下定位于线粒体。当细胞受到失黏附信号刺激时,Bit1蛋白转移至细胞胞浆中,转移而来的或者在细胞胞浆中外源表达Bit1蛋白时,其则会通过与胞浆中的AES(amino-terminal enhancer of split)蛋白结合成为复合体,而介导脱粘附发生的失巢凋亡,即非caspase(半胱氨酸蛋白酶)途径依赖的细胞凋亡。亦有研究认为Bit1蛋白定位在高尔基体中,与之前研究理论相悖,具有抗凋亡作用。Bit1与各肿瘤的关系,实验研究及文献报道甚少,且结果各异。至于Bit1与ESCC之间的关系,鲜有报道。为了更进一步探讨Bit1蛋白对食管鳞癌发生发展、侵袭转移的影响,本实验研究拟采用RNA干扰、蛋白免疫印迹(Western Blot)、Transwell侵袭技术、构建裸鼠皮下移植瘤模型并且首次运用干扰质粒(瞬转)与脂质体2000的混合液对瘤体进行干扰治疗,也是本课题的一个创新点,从体内和体外两个层面,更加深入的研究Bit1的表达对对ESCC细胞增殖、侵袭等生物学特性的影响,有助于了解Bit1与食管鳞癌发生、转移的关系,以期能够为食管鳞癌早期诊断靶点和个体化治疗方案提供一个新的思路。方法1.使用Western blot技术,检测6种ESCC细胞系中Bit1蛋白表达水平。2.采用Transwell侵袭技术,检测运用RNAi方法下调Bit1表达水平后EC9706细胞侵袭力的改变。3.构建裸鼠皮下移植瘤并且运用干扰质粒(瞬转)与脂质体2000的混合液对瘤体进行干扰治疗,检测运用RNAi方法持续下调Bit1的表达水平后,观察EC9706细胞成瘤作用的变化。4.采用SPSS17.0软件分析数据,多组定量资料比较采用单因素方差分析ANOVA(其中的两两比较用LSD)法;两样本均数采用t检验而定性资料用秩和。以α=0.05作为检验水准。结果1.Bit1在6种ESCC细胞系中的表达Bit1在4种分化程度较低的ESCC细胞株EC9706、Eca109、TE-13和KYSE70Bit1蛋白的表达量相对较高,而在分化程度较好的2种细胞株TE-1和KYSE450表达相对较低。2.下调Bit1表达水平,EC9706细胞侵袭能力的改变下调Bit1表达水平,转然后72h基因干扰效率达到62%;RNAi组穿膜的细胞个数显著少于阴性对照组(空载体转染组)(P0.01),而在阴性对照组和未转染组之间,则不存在明显的差异(P0.05)。3.裸鼠皮下成瘤实验表明,持续下调Bit1的蛋白表达,可以明显抑制裸鼠食管鳞癌移植瘤的生长。细胞的成瘤率为100%,于接种后第5d,10μg空载体组和10μg RNAi组之间比较,P0.05,差异具有统计学意义,自第9d起至第21d,C组和D组比较,均P0.05,差异均具有统计学意义;5μg RNAi组和5μg空载体组,在各个时间点上差异均无统计学意义(P0.05)。结论1.Bit1蛋白的表达水平可能与ESCC的分化程度有关。2.下调Bit1蛋白的表达能够抑制EC9706细胞的侵袭能力。3.EC9706细胞具有很强的致瘤性,下调Bit1蛋白的表达可以显著抑制裸鼠瘤体的生长。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.1

【参考文献】