CAJ | 학술논문

背景：端粒酶反转录酶(telomerase reverse transcriptase，TERT)对端粒酶活化起重要作用，但利用构建针对其基因的慢病毒抑制其在脊髓星形胶质细胞中的表达却少有报道。 　　目的：构建用于RNA干扰的靶向大鼠脊髓源星形胶质细胞端粒酶反转录酶基因的慢病毒载体，并观察其对端粒酶反转录酶表达的抑制作用。 　　方法：通过设计合成出shRNA-TERT序列，经聚合酶链反应扩增后定向连接到pLentilox3.7U6载体上构建重组质粒，经转染DH5α细胞筛选阳性菌落后行测序鉴定。将pLentilox3.7U6-TERT重组质粒转染293T细胞，包装产生重组慢病毒Le-TERT并测定其滴度，再用Le-TERT感染大鼠脊髓星形胶质细胞，实时定量-聚合酶链反应检测各细胞株TERT基因表达水平，并利用Western blot和免疫荧光分别检测TERT蛋白的表达。 　　结果与结论：基因测序鉴定证明，pLentilox3.7U6-TERT 重组质粒构建成功，并成功包装慢病毒。实时定量PCR、Western blot和免疫荧光检测结果表明，Le-TERT转染星形胶质细胞4 d后，对TERT mRNA的干扰效率可达(63.98±2.6)%，Le-TERT在转然后的星形胶质细胞中呈低表达。结果证实，实验构建的重组表达载体pLentilox3.7U6-TERT能够产生有效滴度的慢病毒，感染大鼠脊髓星形胶质细胞后，该载体可以有效抑制TERT的表达。
배경：단립매반전록매(telomerase reverse transcriptase，TERT)대단립매활화기중요작용，단이용구건침대기기인적만병독억제기재척수성형효질세포중적표체각소유보도。 　　목적：구건용우RNA간우적파향대서척수원성형효질세포단립매반전록매기인적만병독재체，병관찰기대단립매반전록매표체적억제작용。 　　방법：통과설계합성출shRNA-TERT서렬，경취합매련반응확증후정향련접도pLentilox3.7U6재체상구건중조질립，경전염DH5α세포사선양성균락후행측서감정。장pLentilox3.7U6-TERT중조질립전염293T세포，포장산생중조만병독Le-TERT병측정기적도，재용Le-TERT감염대서척수성형효질세포，실시정량-취합매련반응검측각세포주TERT기인표체수평，병이용Western blot화면역형광분별검측TERT단백적표체。 　　결과여결론：기인측서감정증명，pLentilox3.7U6-TERT 중조질립구건성공，병성공포장만병독。실시정량PCR、Western blot화면역형광검측결과표명，Le-TERT전염성형효질세포4 d후，대TERT mRNA적간우효솔가체(63.98±2.6)%，Le-TERT재전연후적성형효질세포중정저표체。결과증실，실험구건적중조표체재체pLentilox3.7U6-TERT능구산생유효적도적만병독，감염대서척수성형효질세포후，해재체가이유효억제TERT적표체。
BACKGROUND:Telomerase reverse transcriptase (TERT) plays an important role in telomerase activation, however there is rare report addressing the construction of the lentivirus targeted its genes to inhibit its expression in the spinal cord astrocytes. OBJECTIVE:To construct recombinant lentivirus vector expressing smal interfering RNA against TERT gene and to evaluate its potential for inhibiting the TERT expression. METHODS:After shRNA-TERT sequence was designed and synthesized, the sequence was amplified by PCR and then connected to plasmid pLentilox3.7U6-hTERT to construct recombinant plasmid. The recombinant plasmid was then transfected to DH5αcel s to screen positive colony, and the sequence was identified. The recombinant plasmid pLentilox3.7U6-TERT was transfected in 293T cel s, generating recombinant lentivirus Le-TERT. The titer of recombinant lentivirus was determined and Le-TERT was transfected into the rat spinal cord astrocytes. The expression of TERT in astrocytes was detected by RT-PCR, western blot and immunofluorescence assay. RESULTS AND CONCLUSION:The gene sequencing analysis confirmed that, recombinant plasmid pLentilox3.7U6-TERT was successful y constructed. The real-time quantitative PCR, western blot analysis and immunofluorescence assay indicated that, after Le-TERT was transfected in the astrocytes for 4 days, the inhibition rate of TERT mRNA was (63.98±2.6)%, and Le-TERT was lowly expressed in the transfected astrocytes. Recombinant expression vector pLentilox3.7U6-TERT can produce the lentivirus at high titer and effectively inhibit TERT expression in the transfected astrocytes.