CAJ | 학술논문

目的构建并鉴定针对LRIG3 基因的小干扰RNA(LRIG3-siRNA)腺病毒表达载体,为膀胱癌基因治疗的研究提供有效的实验工具.方法设计可形成小发夹结构的LRIG3-siRNA模板cDNA 序列,克隆至缺陷型腺病毒质粒pSilencer-U6neo,构建LRIG3-siRNA表达质粒pSilencer-LRIG3.鉴定正确后,将pSilencer-LRIG3转染至HEK293细胞,包装成复制缺陷型腺病毒pAd-LRIG3.大量扩增pAd-LRIG3,氯化铯梯度离心纯化,测病毒滴度.pAd-LRIG3 感染人膀胱癌细胞T24,RT-PCR法检测LRIG3 mRNA的表达.结果酶切分析、测序鉴定表明pSilencer-LRIG3构建成功,PCR分析表明pAd-LRIG3含有LRIG3-siRNA 序列.pAd-LRIG3可抑制 LRIG3 mRNA的表达.结论含有LRIG3-siRNA 的重组腺病毒构建成功,且具有抑制LRIG3基因mRNA 表达的功能.
목적 구건병감정침대LRIG3 기인적소간우RNA(LRIG3-siRNA)선병독표체재체,위방광암기인치료적연구제공유효적실험공구.방법 설계가형성소발협결구적LRIG3-siRNA모판cDNA 서렬,극륭지결함형선병독질립pSilencer-U6neo,구건LRIG3-siRNA표체질립pSilencer-LRIG3.감정정학후,장pSilencer-LRIG3전염지HEK293세포,포장성복제결함형선병독pAd-LRIG3.대량확증pAd-LRIG3,록화색제도리심순화,측병독적도.pAd-LRIG3 감염인방광암세포T24,RT-PCR법검측LRIG3 mRNA적표체.결과매절분석、측서감정표명pSilencer-LRIG3구건성공,PCR분석표명pAd-LRIG3함유LRIG3-siRNA 서렬.pAd-LRIG3가억제 LRIG3 mRNA적표체.결론 함유LRIG3-siRNA 적중조선병독구건성공,차구유억제LRIG3기인mRNA 표체적공능.
Objective To construct and identify adenovirus vector that expresses small interfering RNA(siRNA)against the LRIG3 gene,which will enable development of a gene therapy protocol for the treatment of human bladder carcinoma.Methods A LRIG3-siRNA template DNA sequence,capable of forming a small hairpin structure,was designed.After renaturation,it was cloned into the defective adenoviral expression vector pSilencer-U6neo to construct the LRIG3-siRNA expression vector pSilencer-LRIG3.After verification,the pSilencer-LRIG3 vector was co-transfected into HEK 293 cells where they were packed as the replication-deficient adenovirus pAd-LRIG3.pAd-LRIG3 was abundantly amplified and then virus titer was evaluated.The pAd-LRIG3 was used to infect the human bladder cancer T24 cells.RT-PCR was used to detect the LRIG3 mRNA expression in T24 cells.Results Restriction endonuclease and sequencing analyses showed that pSilencer-LRIG3 was constructed successfully.PCR analysis indicated that the pAd-LRIG3 contained the LRIG3-siRNA sequence.The expression of LRIG3 mRNA was greatly inhibited after transfection in T24 cells.Conclusion The recombinant adenovirus vector containing the LRIG3-siRNA gene was successfully constructed and could specifically inhibit the LRIG3 expression.