中国实验动物学报
中國實驗動物學報
중국실험동물학보
ACTA LABORATORIUM ANIMALIS SCIENTIA SINICA
2015年
2期
153-158
,共6页
严磊%李晶%赵婷婷%王会娟%王蕾%赖国旗
嚴磊%李晶%趙婷婷%王會娟%王蕾%賴國旂
엄뢰%리정%조정정%왕회연%왕뢰%뢰국기
CB1基因%HEK293细胞%CaSki细胞%细胞凋亡
CB1基因%HEK293細胞%CaSki細胞%細胞凋亡
CB1기인%HEK293세포%CaSki세포%세포조망
rCB1%HEK293 cells%CaSki cells%Cell apoptosis
目的：构建大鼠CB1（ rCB1）基因真核表达载体，检测rCB1基因在细胞中的表达，研究rCB1对人宫颈癌CaSki细胞凋亡的影响。方法从大鼠脑组织中提取总RNA，RT-PCR扩增rCB1基因，通过酶切、纯化、连接PCR纯化产物与pCDNA 3.1（＋）质粒，构建pcDNA3.1（＋）-rCB1。脂质体法将其转染到HEK293和CaSki细胞， Western blot及细胞免疫荧光联合激光扫描共聚焦方法检测rCB1的表达及定位，流式细胞仪检测细胞凋亡率， Western blot与实时荧光定量PCR检测rCB1、Bcl-2、Bax、Bad表达。结果酶切重组质粒获得5300 bp的载体片段和1500 bp的目的片段，测序结果和rCB1基因序列（ NM＿012784.4）一致。转染HEK293细胞后， rCB1在HEK293细胞细胞膜和细胞质表达。转染CaSki细胞后， rCB1使细胞凋亡率增加（ P＜0.05）；rCB1基因上调Bax、Bad的表达，同时抑制Bcl-2的表达，与空白组比较，其差异具有显著性（ P＜0.05）。结论成功构建了pcDNA3.1（＋）-rCB1真核表达载体， rCB1表达于细胞膜和细胞质，rCB1可以明显促进宫颈癌CaSki细胞凋亡，其机制是上调Bax、Bad和抑制Bcl-2表达。
目的：構建大鼠CB1（ rCB1）基因真覈錶達載體，檢測rCB1基因在細胞中的錶達，研究rCB1對人宮頸癌CaSki細胞凋亡的影響。方法從大鼠腦組織中提取總RNA，RT-PCR擴增rCB1基因，通過酶切、純化、連接PCR純化產物與pCDNA 3.1（＋）質粒，構建pcDNA3.1（＋）-rCB1。脂質體法將其轉染到HEK293和CaSki細胞， Western blot及細胞免疫熒光聯閤激光掃描共聚焦方法檢測rCB1的錶達及定位，流式細胞儀檢測細胞凋亡率， Western blot與實時熒光定量PCR檢測rCB1、Bcl-2、Bax、Bad錶達。結果酶切重組質粒穫得5300 bp的載體片段和1500 bp的目的片段，測序結果和rCB1基因序列（ NM＿012784.4）一緻。轉染HEK293細胞後， rCB1在HEK293細胞細胞膜和細胞質錶達。轉染CaSki細胞後， rCB1使細胞凋亡率增加（ P＜0.05）；rCB1基因上調Bax、Bad的錶達，同時抑製Bcl-2的錶達，與空白組比較，其差異具有顯著性（ P＜0.05）。結論成功構建瞭pcDNA3.1（＋）-rCB1真覈錶達載體， rCB1錶達于細胞膜和細胞質，rCB1可以明顯促進宮頸癌CaSki細胞凋亡，其機製是上調Bax、Bad和抑製Bcl-2錶達。
목적：구건대서CB1（ rCB1）기인진핵표체재체，검측rCB1기인재세포중적표체，연구rCB1대인궁경암CaSki세포조망적영향。방법종대서뇌조직중제취총RNA，RT-PCR확증rCB1기인，통과매절、순화、련접PCR순화산물여pCDNA 3.1（＋）질립，구건pcDNA3.1（＋）-rCB1。지질체법장기전염도HEK293화CaSki세포， Western blot급세포면역형광연합격광소묘공취초방법검측rCB1적표체급정위，류식세포의검측세포조망솔， Western blot여실시형광정량PCR검측rCB1、Bcl-2、Bax、Bad표체。결과매절중조질립획득5300 bp적재체편단화1500 bp적목적편단，측서결과화rCB1기인서렬（ NM＿012784.4）일치。전염HEK293세포후， rCB1재HEK293세포세포막화세포질표체。전염CaSki세포후， rCB1사세포조망솔증가（ P＜0.05）；rCB1기인상조Bax、Bad적표체，동시억제Bcl-2적표체，여공백조비교，기차이구유현저성（ P＜0.05）。결론성공구건료pcDNA3.1（＋）-rCB1진핵표체재체， rCB1표체우세포막화세포질，rCB1가이명현촉진궁경암CaSki세포조망，기궤제시상조Bax、Bad화억제Bcl-2표체。
Objective To construct rCB1 gene eukaryotic expression vector, detect its expression in the cell, and explore its influence on apoptosis in human cervical cancer CaSki cells.Methods The total RNA was extracted from rat brains.The rCB1gene was amplified by RT-PCR.The pcDNA3.1(+)-rCB1 was constructed by enzyme digestion, purifi-cation, bind the PCR purification products and pcDNA3.1 (+) DNA.The pcDNA3.1 (+)-rCB1 plasmid was transfect-ed into HEK293 and CaSki cells by liposomes.The expression and localization of rCB1 were detected by Western blot and immunofluorescence combined with confocal laser scanning microscopy.The apoptosis rate of CaSki cells was detected by flow cytometry.The expression of rCB1, Bcl-2, Bax and Bad was detected by Western blot and real-time fluorescence quantitative RT-PCR (qRT-PCR).Results The 5300 bp pcDNA3.1(+) and 1500 bp rCB1 were obtained after diges-ting the pcDNA3.1 ( +)-rCB1.The result of sequencing was in agreement with the sequence of rCB1 gene ( NM_012784.4 ) .The rCB1 expressed in the membrane and cytoplasm when pcDNA3.1 (+)-rCB1 plasmid was transfected into HEK293 cells.The apoptosis rate of rCB1 group was increased compared with the blank group when pcDNA3.1 (+)-rCB1 plasmid was transfected into CaSki cells (P<0.05).Compared with the blank group, rCB1 gene upregulated the expres-sion of Bax and Bad, and suppressed the expression of Bcl-2.The statistical difference was significant ( P <0.05). Conclusions The pCDNA3.1(+)-rCB1 eukaryotic expression vector is constructed successfully.It is found that rCB1 is expressed in membrane and cytoplasm of HEK293 cells.rCB1 can significantly promote the apoptosis in cervical cancer CaSki cells by up-regulating the expression of Bax and Bad, and down-regulating the expression of Bcl-2 as well.
