CAJ | 학술논문

目的：构建 BAG-1、Bcl-2双靶区反义 RNA 重组载体 pVITRO2-AsBAG-1-Bcl-2及单表达重组载体 pVITRO2-As-BAG-1和 pVITRO2-AsBcl-2，并初步研究它们对人胃癌细胞株 SGC-7901增殖活性的影响，为进一步探讨该重组载体对肿瘤细胞的影响打下基础。方法从胃癌细胞株 SGC-7901总 RNA 中逆转录扩增包括全部编码序列的 BAG-1和 Bcl-2 cDNA，TA 克隆到pMD18-T Simple 载体，分别经 BamHⅠ和 ClaⅠ、EcoRⅠ和 NheⅠ双酶切并回收纯化目的片段后，反向插入真核细胞双表达载体pVITRO2的 mcs1和 mcs2中，经酶切、测序鉴定，确定已建立单表达重组载体 pVITRO2-AsBAG-1和 pVITRO2-AsBcl-2，再将Bcl-2反向插入单表达重组载体 pVITRO2-AsBAG-1的 mcs2中，构建双表达重组载体 pVITRO2-AsBAG-1-Bcl-2，酶切、测序鉴定。然后分别转染 SGC-7901细胞，MTT 法检测细胞的增殖活性，半定量 RT-PCR 检测基因 BAG-1 mRNA 和 Bcl-2 mRNA 的表达，流式细胞术检测 SGC-7901的细胞周期变化情况。结果酶切、测序鉴定表明，单表达重组载体 pVITRO2-AsBAG-1和pVITRO2-AsBcl-2及共表达重组载体 pVITRO2-AsBAG-1-Bcl-2构建成功。与对照组比较，MTT 法检测显示重组载体抑制SGC-7901细胞增殖，并呈时间依赖性，其中以72 h 转染组抑制作用最显著（P ＜0.01）；RT-PCR 结果显示 BAG-1 mRNA 和 Bcl-2 mRNA 表达水平明显下降（P ＜0.01），而且共表达载体组比单表达载体组更为显著（P ＜0.05），但 pVITRO2-AsBcl-2组对 BAG-1 mRNA 表达水平无明显影响（P ＞0.05）；流式细胞术检测重组载体组凋亡细胞比例升高，与 pVITRO2组和对照组比较均有统计学意义（P ＜0.01），并且共表达重组载体组的效应比单表达重组载体组更为显著（P ＜0.01），凋亡率由对照组的0.57％增加至15.75％。结论成功构建双靶区反义 RNA 重组载体 pVITRO2-AsBAG-1-Bcl-2及单表达重组载体 pVITRO2-AsBAG-1和pVITRO2-AsBcl-2，并发现其能抑制 SGC-7901细胞增殖并引起细胞凋亡，且以 pVITRO2-AsBAG-1-Bcl-2重组载体最为明显。目的：構建 BAG-1、Bcl-2雙靶區反義 RNA 重組載體 pVITRO2-AsBAG-1-Bcl-2及單錶達重組載體 pVITRO2-As-BAG-1和 pVITRO2-AsBcl-2，併初步研究它們對人胃癌細胞株 SGC-7901增殖活性的影響，為進一步探討該重組載體對腫瘤細胞的影響打下基礎。方法從胃癌細胞株 SGC-7901總 RNA 中逆轉錄擴增包括全部編碼序列的 BAG-1和 Bcl-2 cDNA，TA 剋隆到pMD18-T Simple 載體，分彆經 BamHⅠ和 ClaⅠ、EcoRⅠ和 NheⅠ雙酶切併迴收純化目的片段後，反嚮插入真覈細胞雙錶達載體pVITRO2的 mcs1和 mcs2中，經酶切、測序鑒定，確定已建立單錶達重組載體 pVITRO2-AsBAG-1和 pVITRO2-AsBcl-2，再將Bcl-2反嚮插入單錶達重組載體 pVITRO2-AsBAG-1的 mcs2中，構建雙錶達重組載體 pVITRO2-AsBAG-1-Bcl-2，酶切、測序鑒定。然後分彆轉染 SGC-7901細胞，MTT 法檢測細胞的增殖活性，半定量 RT-PCR 檢測基因 BAG-1 mRNA 和 Bcl-2 mRNA 的錶達，流式細胞術檢測 SGC-7901的細胞週期變化情況。結果酶切、測序鑒定錶明，單錶達重組載體 pVITRO2-AsBAG-1和pVITRO2-AsBcl-2及共錶達重組載體 pVITRO2-AsBAG-1-Bcl-2構建成功。與對照組比較，MTT 法檢測顯示重組載體抑製SGC-7901細胞增殖，併呈時間依賴性，其中以72 h 轉染組抑製作用最顯著（P ＜0.01）；RT-PCR 結果顯示 BAG-1 mRNA 和 Bcl-2 mRNA 錶達水平明顯下降（P ＜0.01），而且共錶達載體組比單錶達載體組更為顯著（P ＜0.05），但 pVITRO2-AsBcl-2組對 BAG-1 mRNA 錶達水平無明顯影響（P ＞0.05）；流式細胞術檢測重組載體組凋亡細胞比例升高，與 pVITRO2組和對照組比較均有統計學意義（P ＜0.01），併且共錶達重組載體組的效應比單錶達重組載體組更為顯著（P ＜0.01），凋亡率由對照組的0.57％增加至15.75％。結論成功構建雙靶區反義 RNA 重組載體 pVITRO2-AsBAG-1-Bcl-2及單錶達重組載體 pVITRO2-AsBAG-1和pVITRO2-AsBcl-2，併髮現其能抑製 SGC-7901細胞增殖併引起細胞凋亡，且以 pVITRO2-AsBAG-1-Bcl-2重組載體最為明顯。
목적：구건 BAG-1、Bcl-2쌍파구반의 RNA 중조재체 pVITRO2-AsBAG-1-Bcl-2급단표체중조재체 pVITRO2-As-BAG-1화 pVITRO2-AsBcl-2，병초보연구타문대인위암세포주 SGC-7901증식활성적영향，위진일보탐토해중조재체대종류세포적영향타하기출。방법종위암세포주 SGC-7901총 RNA 중역전록확증포괄전부편마서렬적 BAG-1화 Bcl-2 cDNA，TA 극륭도pMD18-T Simple 재체，분별경 BamHⅠ화 ClaⅠ、EcoRⅠ화 NheⅠ쌍매절병회수순화목적편단후，반향삽입진핵세포쌍표체재체pVITRO2적 mcs1화 mcs2중，경매절、측서감정，학정이건립단표체중조재체 pVITRO2-AsBAG-1화 pVITRO2-AsBcl-2，재장Bcl-2반향삽입단표체중조재체 pVITRO2-AsBAG-1적 mcs2중，구건쌍표체중조재체 pVITRO2-AsBAG-1-Bcl-2，매절、측서감정。연후분별전염 SGC-7901세포，MTT 법검측세포적증식활성，반정량 RT-PCR 검측기인 BAG-1 mRNA 화 Bcl-2 mRNA 적표체，류식세포술검측 SGC-7901적세포주기변화정황。결과매절、측서감정표명，단표체중조재체 pVITRO2-AsBAG-1화pVITRO2-AsBcl-2급공표체중조재체 pVITRO2-AsBAG-1-Bcl-2구건성공。여대조조비교，MTT 법검측현시중조재체억제SGC-7901세포증식，병정시간의뢰성，기중이72 h 전염조억제작용최현저（P ＜0.01）；RT-PCR 결과현시 BAG-1 mRNA 화 Bcl-2 mRNA 표체수평명현하강（P ＜0.01），이차공표체재체조비단표체재체조경위현저（P ＜0.05），단 pVITRO2-AsBcl-2조대 BAG-1 mRNA 표체수평무명현영향（P ＞0.05）；류식세포술검측중조재체조조망세포비례승고，여 pVITRO2조화대조조비교균유통계학의의（P ＜0.01），병차공표체중조재체조적효응비단표체중조재체조경위현저（P ＜0.01），조망솔유대조조적0.57％증가지15.75％。결론성공구건쌍파구반의 RNA 중조재체 pVITRO2-AsBAG-1-Bcl-2급단표체중조재체 pVITRO2-AsBAG-1화pVITRO2-AsBcl-2，병발현기능억제 SGC-7901세포증식병인기세포조망，차이 pVITRO2-AsBAG-1-Bcl-2중조재체최위명현。
Objective To construct the recombinant co-expression vector carrying antisense RNA to dual-target BAG-1 and Bcl-2 genes and recombinant single expression vector carrying antisense RNA to target BAG-1 and Bcl-2 gene respectively,then to pre-liminarily investigate their effect on the proliferation of gastric cancer cell SGC-7901 in order to lay the foundation for further study the effect of this recombinant vector on the tumor cells.Methods RT-PCR was used to amplify the full length of BAG-1 and Bcl-2 cDNA from total RNA of gastric cancer cell line SGC-7901.The BAG-1 cDNA fragment and the Bcl-2 cDNA fragment were insert-ed into pMD18-T simple vector respectively.The pMD18-T-BAG-1 was digested with BamH Ⅰ and Cla Ⅰ and the pMD18-T-Bcl-2 was digested with EcoR Ⅰ and Nhe Ⅰ.Then the BAG-1 cDNA fragment and the Bcl-2 cDNA fragment were inserted into the mcs1 and mcs2 of the eukaryotic co-expression vector pVITRO2 in the antisense orientation respectively.The construction of the single expression vector pVITRO2-AsBAG-1 and pVITRO2-AsBcl-2 was confirmed by restriction endonuclease treatment and se-quence identification.Then the Bcl-2 cDNA fragment was inserted into the mcs2 of the recombinant vector pVITRO2-AsBAG-1 in the antisense orientation to construct the co-expression vector pVITRO2-AsBAG-1-Bcl-2,and the recombinant vector was also iden-tified by restriction endonucleases digestion and sequence identification.Then the recombinant vector was transfected into SGC-7901 cell respectively.The proliferation of the cell was determined by the MTT assay.The level change of BAG-1 and Bcl-2 mRNA in SGC-7901 cell was detected by the semi quantitative RT-PCR and the change situation of cell cycle was detected by the flow cytom-etry(FCM).Results The restriction endonucleases digestion and sequencing identification indicated that the eukaryotic co-expres-sion vector pVITRO2-AsBAG-1-Bcl-2 and the single gene expression vector pVITRO2-AsBAG-1 and pVITRO2-AsBcl-2 were con-structed successfully.The MTT assay method demonstrated that compared with the control group,the recombinant vector could in-hibit the proliferation of the cells in time dependent manner,and the inhibiting effect was most notably in the 72 h transfection group(P <0.01);inhibition ratio of recombinant vector groups was significantly higher than that of control group and pVITRO2 group(P <0.01).BAG-1 and Bcl-2 mRNA expression in recombinant vector groups were significantly decreased compared with that in the control group and pVITRO2 group(P <0.01),The RT-PCR results showed that the expression level of BAG-1 mRNA and Bcl-2 mRNA was significantly decreased(P <0.01),moreover the co-expression vector group was more notably than the single expression vector groups(P <0.05),but the pVITRO2-AsBcl-2 had no obvious effect on the expression of BAG-1 mRNA(P >0. 05);the FCM detection results showed that the apoptosis rate of the recombinant vector groups was significantly higher than that of the control group and the pVITRO2 group with statistical difference(P <0.01),and the co-expression vector group was more nota-bly than single expression vector groups(P <0.01).In addition,the effect of the co-expression recombinant vector group was more significant than that of the single expression co-expression vector(P <0.01 ),the apoptosis rate was increased from 0.57% to 15.75%.Conclusion The co-expression recombinant vector pVITRO2-AsBAG-1-Bcl-2 and single expression vector pVITRO2-As-BAG-1 and pVITRO2-AsBcl-2 are successfully constructed and they can inhibit the proliferation of SGC-7901 cell and induce cell apoptosis,moreover the pVITRO2-AsBAG-1-Bcl-2 vector is most notably.