四川大学学报（医学版）
四川大學學報（醫學版）
사천대학학보（의학판）
JOURNAL OF SICHUAN UNIVERSITY(MEDICAL SCIENCE EDITION)
2009年
6期
983-987
,共5页
双自杀基因%癌胚抗原基因启动子%CD%TK%大肠癌
雙自殺基因%癌胚抗原基因啟動子%CD%TK%大腸癌
쌍자살기인%암배항원기인계동자%CD%TK%대장암
Suicide gene%CEA promoter%CD%TK%Colorectal carcinoma
目的 构建由癌胚抗原启动子(CEA promoter,Cp)驱动的靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK,并观察其在CEA阳性大肠癌细胞中专一性表达和对增殖的影响.方法 采用PCR分别扩增出Cp、CD、TK三种目的 基因并采用双酶切、连接依次将Cp、CD、TK基因插入pcDNA3.1(-)质粒;将靶向性载体pcDNA3.1(-)Cp-CD-TK分别转染CEA阳性的人大肠癌SW480细胞和CEA阴性的Hela细胞,采用RT-PCR检测CD-TK基因的表达.用MTT法检测转染pcDNA3.1(-)Cp-CD-TK后的SW480细胞对化疗前药5-氟胞嘧啶(5-Fc)和丙氧鸟苷(GCV)的敏感性.结果 Cp基因片段、CD基因片段和TK基因均克隆正确.靶向性基因治疗载体pcDNA3.1(-)Cp-CD-TK经凝胶电泳和DNA测序证实构建正确.转染了靶向性载体pcDNA3.1(-)Cp-CD-TK的SW480细胞经过RT-PCR鉴定证实有CD-TK基因的表达,CEA阴性Hela细胞则没有CD-TK基因的表达.细胞培养试验中,转染了靶向性载体pcDNA3.1(-)Cp-CD-TK的SW480细胞对前药5-Fc和GCV敏感.结论 正确构建了靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK,靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK能够使CD-TK基因在CEA阳性细胞中专一性表达,达到靶向杀伤大肠癌细胞目的 .
目的 構建由癌胚抗原啟動子(CEA promoter,Cp)驅動的靶嚮性雙自殺基因治療載體pcDNA3.1(-)Cp-CD-TK,併觀察其在CEA暘性大腸癌細胞中專一性錶達和對增殖的影響.方法 採用PCR分彆擴增齣Cp、CD、TK三種目的 基因併採用雙酶切、連接依次將Cp、CD、TK基因插入pcDNA3.1(-)質粒;將靶嚮性載體pcDNA3.1(-)Cp-CD-TK分彆轉染CEA暘性的人大腸癌SW480細胞和CEA陰性的Hela細胞,採用RT-PCR檢測CD-TK基因的錶達.用MTT法檢測轉染pcDNA3.1(-)Cp-CD-TK後的SW480細胞對化療前藥5-氟胞嘧啶(5-Fc)和丙氧鳥苷(GCV)的敏感性.結果 Cp基因片段、CD基因片段和TK基因均剋隆正確.靶嚮性基因治療載體pcDNA3.1(-)Cp-CD-TK經凝膠電泳和DNA測序證實構建正確.轉染瞭靶嚮性載體pcDNA3.1(-)Cp-CD-TK的SW480細胞經過RT-PCR鑒定證實有CD-TK基因的錶達,CEA陰性Hela細胞則沒有CD-TK基因的錶達.細胞培養試驗中,轉染瞭靶嚮性載體pcDNA3.1(-)Cp-CD-TK的SW480細胞對前藥5-Fc和GCV敏感.結論 正確構建瞭靶嚮性雙自殺基因治療載體pcDNA3.1(-)Cp-CD-TK,靶嚮性雙自殺基因治療載體pcDNA3.1(-)Cp-CD-TK能夠使CD-TK基因在CEA暘性細胞中專一性錶達,達到靶嚮殺傷大腸癌細胞目的 .
목적 구건유암배항원계동자(CEA promoter,Cp)구동적파향성쌍자살기인치료재체pcDNA3.1(-)Cp-CD-TK,병관찰기재CEA양성대장암세포중전일성표체화대증식적영향.방법 채용PCR분별확증출Cp、CD、TK삼충목적 기인병채용쌍매절、련접의차장Cp、CD、TK기인삽입pcDNA3.1(-)질립;장파향성재체pcDNA3.1(-)Cp-CD-TK분별전염CEA양성적인대장암SW480세포화CEA음성적Hela세포,채용RT-PCR검측CD-TK기인적표체.용MTT법검측전염pcDNA3.1(-)Cp-CD-TK후적SW480세포대화료전약5-불포밀정(5-Fc)화병양조감(GCV)적민감성.결과 Cp기인편단、CD기인편단화TK기인균극륭정학.파향성기인치료재체pcDNA3.1(-)Cp-CD-TK경응효전영화DNA측서증실구건정학.전염료파향성재체pcDNA3.1(-)Cp-CD-TK적SW480세포경과RT-PCR감정증실유CD-TK기인적표체,CEA음성Hela세포칙몰유CD-TK기인적표체.세포배양시험중,전염료파향성재체pcDNA3.1(-)Cp-CD-TK적SW480세포대전약5-Fc화GCV민감.결론 정학구건료파향성쌍자살기인치료재체pcDNA3.1(-)Cp-CD-TK,파향성쌍자살기인치료재체pcDNA3.1(-)Cp-CD-TK능구사CD-TK기인재CEA양성세포중전일성표체,체도파향살상대장암세포목적 .
Objective To construct the targeting double suicide gene therapy vector pcDNA3. 1( -) Cp-CD-TK driven by carcino-embryonic antigen promoter (CEA promoter,Cp), and to investigate whether the vector could control the expression of CD-TK gene specificity and impact on proliferation of CEA positive colon cancer cells. Methods Three kinds of target gene, Cp, CD, and TK were obtained by PCR, the products were double digested and inserted into pcDNA3. 1(-), then the targeting vector pcDNA3. 1 (-) Cp-CD-TK was transfected into CEA positive human colon cancer SW480 cells and CEA negative Hela cells, the expression of CD-TK was examined by RT-PCR. The sensitivities of SW480 cells transfected with pcDNA3. 1 (-) Cp-CD-TK to pro-drug 5-Fluorocytosine (5-Fc) and Ganciclovir (GCV) were detected by MTT analysis. Results Recombinants with Cp, CD and TK insert were obtained. Constructed targeting gene therapy vector pcDNA3. 1 (-) Cp-CD-TK was confirmed by gel electrophoresis and sequencing. RT-PCR analysis further confirmed CD-TK gene was expressed in SW480 cells and was not expressed in CEA negative Hela cells. MTT assay demonstrated that SW480 cells transfected with targeting vector pcDNA3.1(-)Cp-CD-TK were sensitive to pro-drug 5-Fc and GCV. Conclusion Targeting double suicide gene therapy vector pcDNA3. 1 (- ) Cp-CD-TK was constructed correctly. The vector could make CD-TK gene express specifically in CEA positive cells for the purpose of targeting killing colon cancer.
