CAJ | 학술논문

目的：评价携带肿瘤增殖基因Ki-67小干扰RNA（siRNA）的表达质粒pSliencer-Ki-67对人宫颈癌细胞株Hela细胞的基因治疗功效。方法将构建的携带肿瘤增殖基因Ki-67 siRNA表达质粒pSliencer-Ki-67，用G250单克隆抗体（G250 mAb）与非病毒基因载体聚乙烯亚胺（PEI）偶联制成新型基因转染载体感染Hela细胞以及人肝癌细胞株HepG2细胞、小鼠胚胎成纤维细胞株NIH3T3细胞，通过四甲基偶氮唑蓝比色法（MTT法）检测其抑制肿瘤细胞增殖的效能，应用膜联蛋白Ⅴ-碘化丙啶（AnnexinⅤ-PI）法、4′，6-二脒基-2-苯基吲哚（DAPI）法检测pSliencer-Ki-67对细胞早、晚期凋亡的影响，并应用逆转录-聚合酶链反应（RT-PCR）、蛋白质印迹（Western blotting）法检测Ki-67 mRNA和Ki-67蛋白的表达。结果在RT-PCR与Western blotting检测中，用G250 mAb-PEI/pSliencer-Ki-67转染细胞后，Ki-67 mRNA、Ki-67蛋白表达较对照组明显降低；MTT法检测发现，G250 mAb/PEI-pSliencer-Ki-67对Hela细胞（G250抗原阳性）有明显抑制作用，而对HepG2细胞、NIH3T3细胞（G250抗原阴性）无明显抑制作用。通过荧光显微镜检测也发现，在早、晚期凋亡实验中，G250 mAb/PEI-pSliencer-Ki-67转染Hela细胞后，可诱导细胞凋亡，但对正常细胞的凋亡诱导作用非常弱。结论pSliencer-Ki-67能够诱导Hela细胞（G250抗原阳性）凋亡，而对HepG2细胞及NIH3T3细胞（G250抗原阴性）无明显抑制作用，pSliencer-Ki-67在Hela细胞的基因治疗方面有潜在应用前景。
목적：평개휴대종류증식기인Ki-67소간우RNA（siRNA）적표체질립pSliencer-Ki-67대인궁경암세포주Hela세포적기인치료공효。방법장구건적휴대종류증식기인Ki-67 siRNA표체질립pSliencer-Ki-67，용G250단극륭항체（G250 mAb）여비병독기인재체취을희아알（PEI）우련제성신형기인전염재체감염Hela세포이급인간암세포주HepG2세포、소서배태성섬유세포주NIH3T3세포，통과사갑기우담서람비색법（MTT법）검측기억제종류세포증식적효능，응용막련단백Ⅴ-전화병정（AnnexinⅤ-PI）법、4′，6-이미기-2-분기신타（DAPI）법검측pSliencer-Ki-67대세포조、만기조망적영향，병응용역전록-취합매련반응（RT-PCR）、단백질인적（Western blotting）법검측Ki-67 mRNA화Ki-67단백적표체。결과재RT-PCR여Western blotting검측중，용G250 mAb-PEI/pSliencer-Ki-67전염세포후，Ki-67 mRNA、Ki-67단백표체교대조조명현강저；MTT법검측발현，G250 mAb/PEI-pSliencer-Ki-67대Hela세포（G250항원양성）유명현억제작용，이대HepG2세포、NIH3T3세포（G250항원음성）무명현억제작용。통과형광현미경검측야발현，재조、만기조망실험중，G250 mAb/PEI-pSliencer-Ki-67전염Hela세포후，가유도세포조망，단대정상세포적조망유도작용비상약。결론pSliencer-Ki-67능구유도Hela세포（G250항원양성）조망，이대HepG2세포급NIH3T3세포（G250항원음성）무명현억제작용，pSliencer-Ki-67재Hela세포적기인치료방면유잠재응용전경。
Objective To evaluate the therapeutic efficacy of pSliencer-Ki-67—the expression plasmid of carry tumor proliferation gen Ki-67 small interfering RNA (siRNA)—on human cervical carcinoma line Hela gene. Methods The constructed pSliencer-Ki-67—the expression plasmid of carry tumor proliferation gen Ki-67 siRNA,was coupled with G250 monoclonal anti-body(G250 mAb) and non-viral vector gene polyethyleneimine(PEI) to form new gene transfection vector to infect human cervi-cal cancer cell line Hela,human hepatoma cell line HepG2 and rats embryonic fibroblast cell line NIH3T3,the methyl thiazolyl te-trazolium (MTT) assay was adopted to determine the efficacy of pSliencer-Ki-67 on inhibiting carcinoma cells proliferation, Annexin V-PI and DAPI(4′,6-diamidino-2-phenylindole) were used to detect the influence of pSliencer-Ki-67 on early and late stage of cells,and the reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting were employed to detect the protein expression of Ki-67mRNA and Ki-67. Results By RT-PCR and Western blotting,the protein expression of Ki-67mR-NA and Ki-67 decreased obviously after transfected by G250 mAb-PEI/pSliencer-Ki-67;MTT assay showed that G250 mAb-PEI/pSliencer-Ki-67 inhibited Hela cell(G250 antigen positive) obviously,but it had on obvious inhibitory effect on HepG2 and NIH3T3(G250 antigen negative). Fluorescence microscope detection exhibited that in the experiment of apoptosis in early and late stage,after transfected by G250 mAb-PEI/pSliencer-Ki-67,Hela cell could induce cell apoptosis,but the induction to normal cells was extremely weak. Conclusion pSliencer-Ki-67 can induce Hela cell apoptosis(G250 antigen positive),but it had no ob-vious inhibitory effect on NIH3T3 cells(G250 antigen negative),therefore,the pSliencer-Ki-67 has potential application prospect in the gene therapy of Hela cell.