【作者】 董莉；

【导师】 肖君华；

【作者基本信息】 东华大学 ， 生物化学与分子生物学， 2021， 硕士

【摘要】 本文分成两部分:第一部分是关于Pbx4互作蛋白的筛选和验证;第二部分是利用多重定量PCR(Taqman探针)检测端粒酶活性。将这两部分进行分别描述。第一部分本课题组经过进一步家系分析、全基因组测序及相关的细胞学实验确认了影响小鼠后肢发育的功能基因—Pbx4。Pbx(Pre-B cell leukaemia transcription factors)基因是Hox(Homeobox)蛋白的辅助因子,在哺乳动物中Pbx基因包括Pbx1、Pbx2、Pbx3和Pbx4。Pbxs在小鼠胚胎发育过程中都在表达,作为整合介导的中心信号,参与了多种发育的信号通路,影响胚胎发育的进程。脊椎动物中的Pbx4基因的功能在一定程度上是通过Pbx蛋白与Hox和Meis/Prep家族成员的相互作用介导的。它是Hox蛋白的辅助因子,与Hox形成转录调控复合物来调控下游靶基因的表达,从而影响发育信号的表达。基于上述研究背景,本文通过构建p LL3.7-Pbx4-EGFP载体和15个p LL3.7-Hoxs-HA-EGFP载体,共转染到HEKHEK293T细胞中。外源融合表达Pbx4蛋白以及带有HA标签的15个候选Hox蛋白,通过免疫共沉淀技术和Western Blot技术正向和反向验证与Pbx4蛋白相互结合的Hox蛋白,最终得出Hoxc12与Pbx4基因互作。其中有3个基因——Hoxa13、Hoxd11和Hoxd10由于CDS序列富含高GC,导致合成基因失败,无法验证与Pbx4蛋白的相互作用。其次,为了进一步去研究Hoxc12基因的表达谱,本文取了E10.5、E11.5、E12.5、E13.5和E14.5的小鼠胚胎后肢进行时空表达,发现E13.5的小鼠胚胎中Hoxc12基因表达量最高。最后,取E13.5的小鼠不同组织器官,抽取RNA之后通过SYBR Green实时荧光定量检测Hoxc12基因的相对表达情况,结果发现Hoxc12基因在小鼠后肢中表达量最高,心脏中微量表达而在其他组织器官中几乎不表达。第二部分近年来,随着细胞治疗的快速发展,为了更好的规范化控制细胞质量,端粒酶活性检测可以作为细胞是否具有成瘤性风险评估的标准,从而提高细胞生物安全性。目前的端粒酶活性检测方法大多数较繁琐,一些直接检测法通过提取细胞蛋白以及聚丙烯酰胺凝胶电泳显示相差六个碱基的条带来反映端粒酶活性但由于存在一定的放射性污染且成本高,已经很少使用;一些间接检测方法开始应用市场中,通过SYBR Green法测定端粒酶催化亚基TERT(Telomerase reverse transcriptase)的表达量来间接反映端粒酶活性,但由于TERT基因表达量较低导致Ct值波动性大无法准确定量,结果存在一定的孔间差和非特异性扩增,带来假阳性干扰。因此如何提高反应的特异性和灵敏度,以及对细胞基因进行精确定量检测是一个很大的挑战。针对上述问题,本研究建立一种稳定、高灵敏度的方法来检测端粒酶催化亚基TERT表达量,从而检测端粒酶活性。针对TERT的CDS序列设计特异性逆转录引物、两对定量引物及同一种荧光标记的探针,优化反应体系,与内参GAPDH基因在单管中利用荧光定量PCR(Taqman探针法)进行多重反应。并且选用具有端粒酶活性的肿瘤人源细胞HEKHEK293T为阳性细胞以及无端粒酶活性的人正常分化细胞MRC-5为阴性细胞,通过TERT的扩增曲线以及与GAPDH基因之间的△Ct是否稳定判定有无端粒酶活性。并对方法的特异性、稳定性和可靠性进行验证。结果显示:在以特异性逆转录引物作用下得到的c DNA为模板,将TERT基因两对定量引物探针混合后与GAPDH进行多重定量,显著提高了整个体系的灵敏度和TERT荧光信号量,△Rn增强了3倍。其次,在单管中,阳性细胞的c DNA梯度稀释后,能高效检测出TERT基因表达且GAPDH和TERT之间的△Ct保持稳定;阴性细胞的c DNA梯度稀释后TERT基因Ct值未被检出。组内和组间变异系数均小于1%,证明方法稳定。利用该方法对16例样本进行检测,阳性率44%,符合预期要求。检测结果显示癌细胞与正常细胞的TERT基因相对表达水平有显著差异(p<0.001),证明该体系可靠。此新型的端粒酶活性检测的方案在肿瘤预判和临床诊断中发挥极大重要性。更多还原

【Abstract】 This paper is divided into two parts: the first part is about the screening and validation of PBX4 interacting proteins;In the second part,the telomerase activity was detected by multiple quantitative PCR(Taqman probe).Describe the two parts separately.Part AThrough further family analysis,whole genome sequencing and cytological experiments,Pbx4 was identified as the functional gene that affects the development of hind limbs in mice.In mammals,Pbx genes include Pbx1,Pbx2,Pbx3 and Pbx4.PBXs is expressed during the development of mouse embryos.As a central signal mediated by integration,PBXs participates in a variety of developmental signaling pathways and affects the process of embryo development.The function of Pbx4 in vertebrates is mediated,in part,by the interaction of Pbx proteins with members of the Hox and Meis/Prep families.It is a cofactor of Hox protein and forms a transcriptional regulatory complex with Hox to regulate the expression of downstream target genes,thereby affecting the expression of developmental signals.Based on the above research background,we constructed p LL3.7-PBX4-EGFP vector and 15 p LL3.7-HOXS-HA-EGFP vectors,and co-transfected HEK293 T cells with 70% to 80%overgrowth.Pbx4 protein and 15 candidate Hoxs proteins with HA label were fused to express Pbx4 protein.Immunoprecipitation and Western Blot were used to verify the positive and negative Hox protein binding with Pbx4 protein,and finally,Hoxc12 interacted with Pbx4.Among them,three genes--Hoxa13,Hoxd11 and Hoxd10 failed to synthesize genes due to the high GC content of CDS sequences,and the interaction with Pbx4 protein could not be verified.In order to further study the gene expression profile of Hoxc12,the hind limbs of E10.5,E11.5,E12.5,E13.5 and E14.5 mouse embryos were taken for spatio-temporal expression,and it was found that the highest expression level of Hoxc12 gene was found in E13.5 mouse embryos.Finally,RNA was extracted from different tissues and organs of E13.5 mice,and then the relative expression of Hoxc12 gene was detected by SYBR Green real-time fluorescence quantitative analysis.The results showed that Hoxc12 was expressed in the hind limbs of mice with the highest expression level,and the heart was slightly expressed,but almost no expression level in other tissues and organs.Part BIn recent years,with the rapid development of cell therapy,in order to better standardize the control of cell quality,telomerase activity detection can be used as a standard for the risk assessment of tumor-forming cells,so as to improve cell biological safety.Most of the current telomerase activity detection methods are cumbersome.Some direct detection methods can reflect telomerase activity by extracting cell proteins and polyacrylamide gel electrophoresis,but they are rarely used due to certain radioactive pollution and high cost.Some indirect detection methods have been applied in the market.SYBR Green method is used to measure the expression level of telomerase catalyzed subunit TERT to reflect telomerase activity indirectly.However,due to the low expression level of TERT gene,Ct value fluctuation cannot be accurately quantified,resulting in certain pore difference and non-specific amplification,resulting in false positive interference.Therefore,how to improve the specificity and sensitivity of the response,as well as the accurate quantitative detection of cell genes is a great challenge.In view of the above problems,this study established a stable and sensitive method to detect the expression level of telomerase catalytic subunit TERT,thus reflecting telomerase activity.Specific reverse transcription primers,two pairs of quantitative primers and one fluorescent labled probes were designed for the CDS sequence of TERT.The reaction system was optimized and multiple reactions were performed with the reference GAPDH gene in a single tube using fluorescence quantitative PCR(Taqman probe method).In addition,HEK293 T positive cells from tumor-derived human cells with telomerase activity and MRC-5 negative cells from normal human differentiated cells without telomerase activity were selected,and telomerase activity was determined by TERT amplification curve and △Ct stability between GAPDH gene and tumor cells.And the specificity,stability and reliability of the method were verified.Results: Using c DNA obtained under the action of specific reverse transcription primers as template,the two pairs of primer probes of TERT gene were mixed with GAPDH for multiple quantification,which significantly improved the sensitivity and TERT fluorescence signal quantity of the whole system by 3 times.Secondly,in a single tube,TERT gene expression can be detected efficiently and the△Ct between GAPDH and TERT remains stable after gradient dilution of positive cells c DNA.Ct value of TERT gene was not detected after c DNA gradient dilution of negative cells.The intragroup and inter-group coefficient of variation are less than 1%,which proved that the method is stable.Sixteen samples are tested by this method,and the postive rate is 44%,which met the expected requirements.The results show that the relative expression level of TERT gene is significantly different between cancer cells and normal cells(p <0.001),which proved that the system is stable and reliable.This novel telomerase activity detection protocol plays an important role in tumor prediction and clinical diagnosis.更多还原