CAJ | 학술논문

目的：构建hsa‐miR‐424基因慢病毒表达载体，并鉴定miR‐424在细胞内表达水平，研究hsa‐miR‐424对宫颈癌 He‐la 细胞增殖的影响。方法以人基因组 DNA 为模板，设计合成 miR‐424的上下游引物，PCR 扩增目的片段，回收产物将其连入pMD18T 载体中，进行测序。再以 pMD18T‐miR424为模板进行 PCR 扩增，将其中表达 pMD18T‐miR424的结构经酶切后插入pLentis‐CMV‐GFP‐MCS‐PGK‐PURO 载体，构建成 pLentis‐CMV‐GFP‐miR424‐PGK‐PURO ，在293T 细胞中与 pSPAX2、pMD2．G包装产生所需的慢病毒，再用含慢病毒的上清液感染 Hela 细胞。结果 miR‐424基因与慢病毒载体连接成功，测序结果证明了插入到质粒载体中的 miR‐424前体序列完全正确，成功构建 pLentis‐CMV‐GFP‐miR424重组慢病毒载体，用其感染宫颈癌Hela 细胞后上调 miR‐424的表达近60倍。采用 M TT 法检测增殖结果提示：感染 miR‐424慢病毒的宫颈癌 Hela 细胞株均存在细胞增殖减慢。结论成功的构建了 miR‐424慢病毒载体，建立了高效稳定表达 miR‐424的细胞株，并用含慢病毒的上清液感染宫颈癌 Hela 细胞，成功抑制了 Hela 细胞的增殖，为后续相关的研究奠定了良好的基础。
목적：구건hsa‐miR‐424기인만병독표체재체，병감정miR‐424재세포내표체수평，연구hsa‐miR‐424대궁경암 He‐la 세포증식적영향。방법이인기인조 DNA 위모판，설계합성 miR‐424적상하유인물，PCR 확증목적편단，회수산물장기련입pMD18T 재체중，진행측서。재이 pMD18T‐miR424위모판진행 PCR 확증，장기중표체 pMD18T‐miR424적결구경매절후삽입pLentis‐CMV‐GFP‐MCS‐PGK‐PURO 재체，구건성 pLentis‐CMV‐GFP‐miR424‐PGK‐PURO ，재293T 세포중여 pSPAX2、pMD2．G포장산생소수적만병독，재용함만병독적상청액감염 Hela 세포。결과 miR‐424기인여만병독재체련접성공，측서결과증명료삽입도질립재체중적 miR‐424전체서렬완전정학，성공구건 pLentis‐CMV‐GFP‐miR424중조만병독재체，용기감염궁경암Hela 세포후상조 miR‐424적표체근60배。채용 M TT 법검측증식결과제시：감염 miR‐424만병독적궁경암 Hela 세포주균존재세포증식감만。결론성공적구건료 miR‐424만병독재체，건립료고효은정표체 miR‐424적세포주，병용함만병독적상청액감염궁경암 Hela 세포，성공억제료 Hela 세포적증식，위후속상관적연구전정료량호적기출。
Objective To construct the lentivirus vector containing the hsa‐miR‐424 gene ,and identify the expression level of miR‐424 in cells .Research the influence of hsa‐miR‐424 on proliferation of cervical cancer Hela cell line .Methods Using the human genomic DNA as template to design the upper and lower primers for synthesis of miR‐424 ,and amplifying the target fragment by polymerase chain reaction (PCR) .Recover the products and conduct sequencing after connecting it into the pMD18T vector .Ampli‐fy the product by PCR template as pMD18T‐miR424 ,and insert the fragment expressing pMD18T‐miR424 into the vector of pLen‐tis‐CMV‐GFP‐MCS‐PGK‐PURO after enzyme cutting to construct the pLentis‐CMV‐GFP‐miR424‐PGK‐PURO .Package the com‐pound with pMD2 .G and pSPAX2 in 293T cell to produce the lentivirus ,and using the supernatant containing lentivirus to infect the Hela cell line .Results The sequencing result proved the sequence of miR‐424 in plasmid vector was correct ,which proved the construction of lentivirus was successful and the target lentivirus was obtained .The expression of miR‐424 almost rise 60 times af‐ter infected the cervical cancer Hela cell by the carrier .The result of M TT method suggested :the cervical cancer Hela cell lines have slowed proliferation with infection miR‐424 lentivirus .Conclusion The miR‐424 lentivirus vector was constructed successfully and the high efficacy expression miR‐424 cell line was established and stable .The cervical cancer Hela cell were infected with the super‐natant containing lentivirus ,inhibited the proliferation of Hela cell successfully ,and laid a good foundation for subsequent research .