摘要
第一部分TKTL1靶向RNA干扰重组体的构建及序列分析
目的:本研究利用RNA干扰技术,以TKTL1为靶基因,设计构建重组体,并进行序列分析,为下一步探索肿瘤基因治疗的新途径打好基础。
方法:设计有小发夹结构的两条DNA序列,经退火形成互补双链,再克隆至载体pEGFP-C1-U6中构建重组体,转化DH5а菌株,提取质粒行酶切鉴定后,进行测序分析。
结果:将合成的DNA序列退火后克隆到载体上,经测序鉴定确实为所需序列。
结论: TKTL1靶向RNA干扰重组体的成功构建和序列分析,可进一步利用克隆所得的小RNA序列干扰TKTL1的mRNA转录,从而达到治疗肿瘤的目的。
第二部分TKTL1基因对人类结肠癌细胞株生长增殖的影响
目的:探讨转酮酶样基因TKTL1在体外培养的人类结肠癌细胞株(LoVo)生长增殖中的作用。
方法:将携带针对TKTL1的小干扰RNA质粒转染人类结肠癌LoVo细胞,实时定量PCR检测转染前后LoVo细胞转酮酶基因家族(TKT、TKTL1、TKTL2)mRNA表达水平的变化,连续监测法检测总转酮酶活性的变化,通过流式细胞术和MTT实验检测转染前后LoVo细胞增殖和细胞周期的改变。
结果:LoVo细胞实验组(携带有pEGFP-C1-U6/TKTL1)、质粒对照组(携带有(pEGFP-C1-U6/UC)和空白对照组(未经转染细胞)中TKT和TKTL2 mRNA的表达水平无显著差异。然而,实验组细胞中TKTL1的表达水平与对照组相比明显下调,且实验组总转酮酶活性明显下降,细胞增殖明显被抑制,被阻滞在G0/G1期。
结论:TKTL1在人类结肠癌细胞(LoVo细胞系)的生长增殖中起重要作用,TKTL1可作为肿瘤治疗研究的新靶点。
Cloning and sequence analyzing of the recombinant plasmid affecting gene TKTL1 translation by RNA interfering
Objective: To clone the recombinant plasmid affecting gene TKTL1 translation by RNA interfering and analyze the nucleic acid sequence of the recombinant for further searching new gene therapy method of tumor.
Methods: Two DNA sequences containing small hairpin structure were designed and synthesized. The complement form was obtained by annealing and inserted into vector pEGFP-C1-U6, and the recombinant plasmid was transformed into DH5аstrain. Finally the plasmid identified by restriction enzyme was used for sequence analysis.
Results: The recombinant was cloned and the aim sequence was obtained.
Conclusion: Successful cloning of the recombinant helps to search new gene therapy method of tumor.
The effect of TKTL1 gene on proliferation of human colon cancer cell line
Objective: The aim of this study was to investigate the effect of TKTL1 on proliferation of LoVo cell in vitro.
Methods: A plasmid, which carries the DNA to be transcribed into a siRNA against TKTL1 mRNA, was transfected human colon cancer cell. Real-time PCR were used to determine the mRNA expression of TKT gene family in the human colon cell line. Continuous monitoring assay were used to determine total transketolase activity. Flow cytometry and MTT were used for detecting the effect of anti-TKTL1 siRNA on cell proliferation and cell cycle in the LoVo cell.
Results: There was no significant difference in the expression level of TKT and TKTL2 gene between the LoVo cells transfected with siRNA TKTL1 construct and the cells transfected with control vector or untransfected LoVo cells (P>0.05). However, the expression level of TKTL1 gene was significantly down regulated in the LoVo cells transfected with siRNA TKTL1 construct compare with the cell transfected with control vector. The total transketolase activity significant decrease, cancer cells proliferation was significantly inhibit, and cancer cells were arrested in G0/G1 phase cyclein the LoVo cells transfected with siRNA TKTL1 construct.
Conclusion: TKTL1 plays an important role in the total transketolase activity and cell proliferation of human colon cancer. It also indicates that TKTL1 can be seen as a potential target for novel anti-transketolase cancer therapies.
引文
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