天津医药
天津醫藥
천진의약
TIANJIN MEDICAL JOURNAL
2014年
2期
109-112
,共4页
路灿%徐惠君%贾勇圣%佟仲生
路燦%徐惠君%賈勇聖%佟仲生
로찬%서혜군%가용골%동중생
蛋白质类%蛋白质构象%乳腺肿瘤%质粒%坏死%细胞凋亡%肿瘤坏死因子α%存活率
蛋白質類%蛋白質構象%乳腺腫瘤%質粒%壞死%細胞凋亡%腫瘤壞死因子α%存活率
단백질류%단백질구상%유선종류%질립%배사%세포조망%종류배사인자α%존활솔
proteins%protein conformation%breast neoplasms%plasmids%necrosis%apoptosis%tumor necrosis factor-alpha%survival rate
目的：建立稳定过表达RIP3基因的乳腺癌细胞株，并证实融合蛋白在细胞内的表达、定位及对MCF7细胞死亡方式的影响。方法逆转录聚合酶链反应(RT-PCR)检测4种乳腺癌细胞及正常乳腺上皮细胞中RIP3 mRNA的表达。以正常乳腺上皮细胞MCF10A cDNA为模板，PCR扩增RIP3基因cDNA全长，将合成的RIP3编码区序列，克隆入mCherry载体的N末端，构建重组质粒mCherry-RIP3，对重组质粒进行酶切鉴定及DNA测序。钙法转染293T细胞，收集病毒感染MCF7细胞，杀稻瘟菌素(4 mg/L)维持筛选，构建稳定表达细胞株。Western blot、荧光显微镜等检测目的基因表达效率及蛋白定位。显微镜下观察肿瘤坏死因子（TNF）-α及Caspase抑制剂Z-VAD-FMK处理下mCherry-RIP3-MCF7细胞的死亡形态及比例。结果 RIP3 mRNA在乳腺癌细胞中普遍低表达。RIP3基因成功克隆入载体。过表达mCherry-RIP3基因的MCF7细胞可见红色荧光蛋白表达，定位于胞质。目的基因RIP3在转染细胞中为过表达。mCherry-RIP3转染后增强MCF7细胞对TNF-α联合Z-VAD-FMK诱导的细胞坏死的敏感性。结论成功构建RIP3基因过表达重组质粒，获得外源性RIP3稳定过表达的乳腺癌MCF7细胞株，mCherry-RIP3定位于胞质，并在TNF-α介导的程序性坏死中起作用。
目的：建立穩定過錶達RIP3基因的乳腺癌細胞株，併證實融閤蛋白在細胞內的錶達、定位及對MCF7細胞死亡方式的影響。方法逆轉錄聚閤酶鏈反應(RT-PCR)檢測4種乳腺癌細胞及正常乳腺上皮細胞中RIP3 mRNA的錶達。以正常乳腺上皮細胞MCF10A cDNA為模闆，PCR擴增RIP3基因cDNA全長，將閤成的RIP3編碼區序列，剋隆入mCherry載體的N末耑，構建重組質粒mCherry-RIP3，對重組質粒進行酶切鑒定及DNA測序。鈣法轉染293T細胞，收集病毒感染MCF7細胞，殺稻瘟菌素(4 mg/L)維持篩選，構建穩定錶達細胞株。Western blot、熒光顯微鏡等檢測目的基因錶達效率及蛋白定位。顯微鏡下觀察腫瘤壞死因子（TNF）-α及Caspase抑製劑Z-VAD-FMK處理下mCherry-RIP3-MCF7細胞的死亡形態及比例。結果 RIP3 mRNA在乳腺癌細胞中普遍低錶達。RIP3基因成功剋隆入載體。過錶達mCherry-RIP3基因的MCF7細胞可見紅色熒光蛋白錶達，定位于胞質。目的基因RIP3在轉染細胞中為過錶達。mCherry-RIP3轉染後增彊MCF7細胞對TNF-α聯閤Z-VAD-FMK誘導的細胞壞死的敏感性。結論成功構建RIP3基因過錶達重組質粒，穫得外源性RIP3穩定過錶達的乳腺癌MCF7細胞株，mCherry-RIP3定位于胞質，併在TNF-α介導的程序性壞死中起作用。
목적：건립은정과표체RIP3기인적유선암세포주，병증실융합단백재세포내적표체、정위급대MCF7세포사망방식적영향。방법역전록취합매련반응(RT-PCR)검측4충유선암세포급정상유선상피세포중RIP3 mRNA적표체。이정상유선상피세포MCF10A cDNA위모판，PCR확증RIP3기인cDNA전장，장합성적RIP3편마구서렬，극륭입mCherry재체적N말단，구건중조질립mCherry-RIP3，대중조질립진행매절감정급DNA측서。개법전염293T세포，수집병독감염MCF7세포，살도온균소(4 mg/L)유지사선，구건은정표체세포주。Western blot、형광현미경등검측목적기인표체효솔급단백정위。현미경하관찰종류배사인자（TNF）-α급Caspase억제제Z-VAD-FMK처리하mCherry-RIP3-MCF7세포적사망형태급비례。결과 RIP3 mRNA재유선암세포중보편저표체。RIP3기인성공극륭입재체。과표체mCherry-RIP3기인적MCF7세포가견홍색형광단백표체，정위우포질。목적기인RIP3재전염세포중위과표체。mCherry-RIP3전염후증강MCF7세포대TNF-α연합Z-VAD-FMK유도적세포배사적민감성。결론성공구건RIP3기인과표체중조질립，획득외원성RIP3은정과표체적유선암MCF7세포주，mCherry-RIP3정위우포질，병재TNF-α개도적정서성배사중기작용。
Objective To construct the recombinant RIP3 over-expressed plasmids and transfect them in breast cancer MCF7 cells, and identify the expression and localization of fusion protein, as well as its effect on the death way of MCF7 cells. Methods The expression levels of RIP3 mRNA in four breast cancer cell lines and normal mammary epithelial cells were detected by reverse transcription polymerase chain reaction (RT-PCR). The RIP 3 coding sequence was amplified by polymerase chain reaction and subcloned into mCherry vector to construct recombinant plasmids. The plasmids were transfected into MCF7 cells by lentivirus after DNA sequencing, then screened by basticidin (4 mg/L) for 1 week. The efficiencies of RIP3 expression were validated by Western blotting assay. The death way of mCherry-RIP3-MCF7 cells was observed under the treatment of TNF-αand Z-VAD-FMK. Results The lowest expression of RIP3 mRNA was found in MCF7 cells. The sequencing results validated the well recombinant plasmids. The expression of mCherry-RIP3 fusion pro-tein with a molecular weight of 85 ku was detected by Western blot assay. The mCherry-RIP3 expression enhanced the sensi-tivity of MCF7 cells to TNF-αand Z-VAD-FMK induced cell death. Conclusion The recombinant RIP3 over-expressed plasmids were successfully constructed, and the stable MCF7 cells with ectopic RIP3 transfection were obtained. The mCher-ry-RIP3 fusion protein was expressed in the cytoplasm and was conformed to mediate TNF-αinduced necroptosis.
