干扰TRIP13基因对结直肠癌细胞HCT116增殖的作用

发布时间：2018-05-03 17:45

本文选题：TRIP13 + HCT116　；参考：《吉林大学》2017年硕士论文

【摘要】：背景:从流行病学上来看,结直肠癌(colorectal cancer,CRC)的发病率和死亡率均令人堪忧。在我国所有恶性肿瘤之中,CRC的发病率位居第3-5位。在欧美国家所有恶性肿瘤中,CRC的发病率居第2-3位。在全球癌症相关的死亡原因中,结直肠癌是第四大原因。多数CRC患者就诊时已属中晚期,经手术治疗、放疗、化疗等治疗后发生肿瘤耐药、进展、复发几率较高。基因诊断与治疗为打破CRC传统防治模式提供了一个新的方向,发现与CRC相关的基因并探究其致病机制是迈向这个方向的前提。甲状腺激素受体因子13(Thyroid Hormone Receptor Interactor 13,TRIP13)基因位于5号染色体短臂1区5带,它是一个蛋白编码基因。TRIP13基因参与细胞减数分裂和有丝分裂的相关过程,能通过干扰纺锤体组装检查点进而影响有丝分裂过程,从而导致肿瘤的发生、发展,它被确定为癌基因。TRIP13的过度表达可导致多种人类癌症,目前尚未证实TRIP13基因与CRC相关,TRIP13基因与CRC的关系尚待进一步研究。目的:本实验拟研究TRIP13基因在结直肠癌HCT116细胞中的表达情况,然后用慢病毒感染HCT116细胞的方法构建TRIP13基因过表达和TRIP13基因敲减的HCT116细胞,并检验慢病毒干扰效果。随后,通过进行一系列细胞功能检测(包括细胞周期检测、细胞凋亡检测、克隆形成检测、MTT检测)来研究TRIP13对胃癌细胞生物学特性的影响,探究干扰目的基因TRIP13对HCT116细胞增殖的影响,进一步探讨TRIP13对结直肠癌细胞的生物学作用,为后续结直肠癌基因诊断和治疗提供初步实验依据。方法:(1)采用实时荧光定量检测系统(Real-time Quantitative Polymerase Chain Reaction Detecting System,qPCR)检测HCT116细胞中TRIP13基因的表达,使用TRIP13基因RNA干扰序列的慢病毒感染目的细胞,再用qPCR检测mRNA水平TRIP13基因敲减的效率,若TRIP13基因敲减效率50%,继续进行慢病毒感染;若TRIP13基因敲减效率50%,进行下一步细胞功能检测。(2)在体外细胞功能实验部分,细胞周期检测使用碘化丙啶(Propidium,PI)染色流式细胞仪检测法,来探究TRIP13基因对HCT116细胞生长周期的影响;细胞凋亡检测采用Annexin V-APC单染法,研究TRIP13基因与HCT116细胞凋亡的关系;克隆形成检测使用Giemsa染色法,探究用含有目的基因RNA干扰序列的慢病毒感染后HCT116细胞的成瘤能力;通过MTT检测探究TRIP13基因对HCT116细胞增殖的影响。(3)使用Spss 21.0软件进行统计分析。结果:(1)HCT116细胞中高丰度表达TRIP13基因,慢病毒感染HCT116细胞效率达到80%以上,细胞状态正常,HCT116细胞中TRIP13基因在mRNA水平的表达量下降(p0.05),敲减效率达到85.5%。(2)细胞周期检测结果:相比对照组(shCtrl组),实验组(shTRIP13组)处于S期的细胞减少(P0.05),处于G1期的细胞增加(P0.05),处于G2/M期的细胞无显著变化。(3)细胞凋亡检测结果:与shCtrl组相比,shTRIP13组凋亡细胞数增多(P0.05)。(4)克隆形成检测结果:与shCtrl组相比,shTRIP13组克隆形成数减少(P0.05)。(5)MTT检测结果:相比shCtrl组,shTRIP13组细胞增殖减缓。结论:干扰目的基因TRIP13抑制结直肠癌细胞HCT116的增殖。TRIP13基因为结直肠癌的的防治提供了一个新方向,它可能是CRC治疗的一个潜在靶点,为实现CRC的可靠诊疗具有基础的实验依据。
[Abstract]:Background: the incidence and mortality of colorectal cancer (CRC) are all worrying in epidemiology. Among all the malignant tumors of our country, the incidence of CRC is the No. 3-5. Among all the malignant tumors in Europe and America, the incidence of CRC is the 2-3. The colorectal cancer is fourth among the causes of global cancer related deaths. Most CRC patients are in the middle and late stages of medical treatment. After surgical treatment, radiotherapy, chemotherapy and other treatments, tumor resistance, progression, and recurrence rate are higher. Gene diagnosis and treatment provide a new direction for breaking the traditional CRC prevention and control model, and finding the genes related to CRC and exploring its pathogenesis are the premise of this direction. The thyroid hormone receptor factor 13 (Thyroid Hormone Receptor Interactor 13, TRIP13) gene is located in the 5 band of the short arm 1 of chromosome 5. It is a protein encoding gene.TRIP13 gene involved in cell meiosis and mitosis. It can interfere with the spindle assembly checkpoint and then affect the mitosis process, resulting in swelling. The occurrence and development of the tumor, which is identified as the overexpression of the oncogene.TRIP13, can lead to a variety of human cancers. At present, the TRIP13 gene has not been confirmed to be associated with CRC. The relationship between the TRIP13 gene and CRC remains to be further studied. Objective: To study the expression of the TRIP13 gene in HCT116 cells in colorectal cancer and to infect HCT116 with lentivirus. The cell method constructs the TRIP13 gene overexpression and the TRIP13 gene knockout HCT116 cells, and tests the effect of the lentivirus interference. Subsequently, a series of cell function tests (including cell cycle detection, cell apoptosis detection, clone formation detection, MTT detection) are used to study the effects of TRIP13 on the biological characteristics of gastric cancer cells and explore the interference. The effect of target gene TRIP13 on the proliferation of HCT116 cells, further explore the biological effect of TRIP13 on colorectal cancer cells, and provide a preliminary experimental basis for the follow-up of colorectal cancer gene diagnosis and treatment. Methods: (1) real-time quantitative detection system (Real-time Quantitative Polymerase Chain Reaction Detecting System, qPCR) detection The expression of TRIP13 gene in HCT116 cells was detected by using the TRIP13 gene RNA interference sequence to infect the target cells, and then qPCR was used to detect the efficiency of the mRNA level TRIP13 gene knockout. If TRIP13 gene knockout efficiency was 50%, the slow virus infection continued. If the TRIP13 gene knockout efficiency was 50%, the next step cell function detection. (2) in vitro fines. In cell function experiment part, cell cycle detection using Propidium (PI) staining flow cytometer detection method to explore the effect of TRIP13 gene on the growth cycle of HCT116 cells; cell apoptosis detection using Annexin V-APC single staining method to study the relationship between TRIP13 gene and HCT116 cell apoptosis; clone formation detection using Giemsa staining method, To explore the tumorigenicity of HCT116 cells after lentivirus infection with the target gene RNA interference sequence; explore the effect of TRIP13 gene on the proliferation of HCT116 cells by MTT detection. (3) the use of Spss 21 software for statistical analysis. Results: (1) the high abundance of TRIP13 gene in HCT116 cells, and the efficiency of the lentivirus infection HCT116 cells more than 80% The expression of TRIP13 gene in HCT116 cells decreased (P0.05) in HCT116 cells, and the knockout efficiency reached 85.5%. (2) cell cycle detection results: compared with the control group (shCtrl group), the cells in the experimental group (group shTRIP13) were in S phase (P0.05), the cells in the G1 phase increased (P0.05), and there was no significant change in the cells in the G2/M stage. (3) thin. Apoptosis detection results: compared with the shCtrl group, the number of apoptotic cells in the shTRIP13 group increased (P0.05). (4) the clone formation detection results: compared with the shCtrl group, the number of clones in the shTRIP13 group decreased (P0.05). (5) MTT detection results: compared with the shCtrl group, the cell proliferation slowed down in the shTRIP13 group. Conclusion: the interference target gene TRIP13 inhibits the HCT116 of colorectal cancer cells. The proliferation of.TRIP13 gene provides a new direction for the prevention and control of colorectal cancer. It may be a potential target for the treatment of CRC and is the basis for the reliable diagnosis and treatment of CRC.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34

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