中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2010年
5期
414-419
,共6页
申聪香%文忠%钱宇虹%关小芳%牟少凤
申聰香%文忠%錢宇虹%關小芳%牟少鳳
신총향%문충%전우홍%관소방%모소봉
胸苷激酶%鼻咽肿瘤%巨细胞病毒%端粒,末端转移酶
胸苷激酶%鼻嚥腫瘤%巨細胞病毒%耑粒,末耑轉移酶
흉감격매%비인종류%거세포병독%단립,말단전이매
Thymidine kinase%Nasopharyngeal neoplasms%Cytomegalovirus%Telomerase
目的 探讨改良构建人端粒酶催化亚单位(human telomerase catalytic subunit promoter,hTERT)启动子及巨细胞病毒原核(cytomegalovirus,CMV)增强子联合调控单纯疱疹病毒胸苷激酶基因(thymidine kinase,TK)的增强型表达载体在鼻咽癌细胞体内外实验中的靶向杀伤效应及体内应用的安全性评价.方法 以pGL3-basic空载体为模板,分别酶切连接hTERT启动子、CMV增强子及TK基因构建靶向性增强型表达载体pGL3-basic-EGFP-TK-hTERTp-CMV(以hTERT单启动子调控TK基因表达载体作为对照组),转染端粒酶阳性的人鼻咽癌5-8F细胞及用做对照组的人乳腺癌MCF-7细胞和端粒酶阴性的正常人脐静脉内皮细胞ECV-304,分别采用荧光显微镜观察绿色荧光蛋白的表达差异、实时荧光定量PCR检测转染后TK基因mRNA表达差异、TeloChaser法检测上述细胞中的端粒酶活性、四甲基偶氮唑蓝(MTt)联合Boyden小室实验分析增强型载体对鼻咽癌细胞增殖及侵袭抑制的作用.将鼻咽癌细胞接种于裸鼠腋部皮下,构建裸鼠鼻咽癌移植瘤模型,研究增强型载体对裸鼠移植瘤的体内生长抑制作用及对全身的毒副反应情况.结果 ①成功改良构建hTERT/CMV双调控TK基因的增强型表达载体.②转染增强型载体组及单启动子载体组的鼻咽癌5-8F细胞均有绿色荧光表达,但前者荧光数量及强度均较后者强,对照组端粒酶阳性的乳腺癌细胞也有很强的荧光表达,而ECV-304细胞几乎无绿色荧光表达.实时荧光定量PCR结果显示,增强型载体组的TK基因mRNA表达量是单启动子组的2-5倍.③ TeloChaser法结果显示,两种肿瘤细胞(5-8F细胞、MCF-7细胞)端粒酶活性均为阳性,正常对照ECV-304细胞端粒酶活性为阴性.④MTT联合Boyden小室侵袭实验结果显示,增强型表达载体对5-8F细胞体外增殖及侵袭力均有明显抑制作用.相对细胞存活率为37.0%,较对照组明显低(P<0.01).⑤体内实验结果显示,增强型靶向表达载体对裸鼠鼻咽癌移植瘤生长具有明显抑制作用,抑瘤率达56.3%,与对照组比较,差异有统计学意义(P<0.01),而裸鼠肝肾病理检查未发现明显损害.结论 以hTERT/CMV双调控机制介导TK基因的增强型表达载体能够高效、靶向杀伤鼻咽癌细胞及移植瘤,实验动物体内应用无明显毒副作用.
目的 探討改良構建人耑粒酶催化亞單位(human telomerase catalytic subunit promoter,hTERT)啟動子及巨細胞病毒原覈(cytomegalovirus,CMV)增彊子聯閤調控單純皰疹病毒胸苷激酶基因(thymidine kinase,TK)的增彊型錶達載體在鼻嚥癌細胞體內外實驗中的靶嚮殺傷效應及體內應用的安全性評價.方法 以pGL3-basic空載體為模闆,分彆酶切連接hTERT啟動子、CMV增彊子及TK基因構建靶嚮性增彊型錶達載體pGL3-basic-EGFP-TK-hTERTp-CMV(以hTERT單啟動子調控TK基因錶達載體作為對照組),轉染耑粒酶暘性的人鼻嚥癌5-8F細胞及用做對照組的人乳腺癌MCF-7細胞和耑粒酶陰性的正常人臍靜脈內皮細胞ECV-304,分彆採用熒光顯微鏡觀察綠色熒光蛋白的錶達差異、實時熒光定量PCR檢測轉染後TK基因mRNA錶達差異、TeloChaser法檢測上述細胞中的耑粒酶活性、四甲基偶氮唑藍(MTt)聯閤Boyden小室實驗分析增彊型載體對鼻嚥癌細胞增殖及侵襲抑製的作用.將鼻嚥癌細胞接種于裸鼠腋部皮下,構建裸鼠鼻嚥癌移植瘤模型,研究增彊型載體對裸鼠移植瘤的體內生長抑製作用及對全身的毒副反應情況.結果 ①成功改良構建hTERT/CMV雙調控TK基因的增彊型錶達載體.②轉染增彊型載體組及單啟動子載體組的鼻嚥癌5-8F細胞均有綠色熒光錶達,但前者熒光數量及彊度均較後者彊,對照組耑粒酶暘性的乳腺癌細胞也有很彊的熒光錶達,而ECV-304細胞幾乎無綠色熒光錶達.實時熒光定量PCR結果顯示,增彊型載體組的TK基因mRNA錶達量是單啟動子組的2-5倍.③ TeloChaser法結果顯示,兩種腫瘤細胞(5-8F細胞、MCF-7細胞)耑粒酶活性均為暘性,正常對照ECV-304細胞耑粒酶活性為陰性.④MTT聯閤Boyden小室侵襲實驗結果顯示,增彊型錶達載體對5-8F細胞體外增殖及侵襲力均有明顯抑製作用.相對細胞存活率為37.0%,較對照組明顯低(P<0.01).⑤體內實驗結果顯示,增彊型靶嚮錶達載體對裸鼠鼻嚥癌移植瘤生長具有明顯抑製作用,抑瘤率達56.3%,與對照組比較,差異有統計學意義(P<0.01),而裸鼠肝腎病理檢查未髮現明顯損害.結論 以hTERT/CMV雙調控機製介導TK基因的增彊型錶達載體能夠高效、靶嚮殺傷鼻嚥癌細胞及移植瘤,實驗動物體內應用無明顯毒副作用.
목적 탐토개량구건인단립매최화아단위(human telomerase catalytic subunit promoter,hTERT)계동자급거세포병독원핵(cytomegalovirus,CMV)증강자연합조공단순포진병독흉감격매기인(thymidine kinase,TK)적증강형표체재체재비인암세포체내외실험중적파향살상효응급체내응용적안전성평개.방법 이pGL3-basic공재체위모판,분별매절련접hTERT계동자、CMV증강자급TK기인구건파향성증강형표체재체pGL3-basic-EGFP-TK-hTERTp-CMV(이hTERT단계동자조공TK기인표체재체작위대조조),전염단립매양성적인비인암5-8F세포급용주대조조적인유선암MCF-7세포화단립매음성적정상인제정맥내피세포ECV-304,분별채용형광현미경관찰록색형광단백적표체차이、실시형광정량PCR검측전염후TK기인mRNA표체차이、TeloChaser법검측상술세포중적단립매활성、사갑기우담서람(MTt)연합Boyden소실실험분석증강형재체대비인암세포증식급침습억제적작용.장비인암세포접충우라서액부피하,구건라서비인암이식류모형,연구증강형재체대라서이식류적체내생장억제작용급대전신적독부반응정황.결과 ①성공개량구건hTERT/CMV쌍조공TK기인적증강형표체재체.②전염증강형재체조급단계동자재체조적비인암5-8F세포균유록색형광표체,단전자형광수량급강도균교후자강,대조조단립매양성적유선암세포야유흔강적형광표체,이ECV-304세포궤호무록색형광표체.실시형광정량PCR결과현시,증강형재체조적TK기인mRNA표체량시단계동자조적2-5배.③ TeloChaser법결과현시,량충종류세포(5-8F세포、MCF-7세포)단립매활성균위양성,정상대조ECV-304세포단립매활성위음성.④MTT연합Boyden소실침습실험결과현시,증강형표체재체대5-8F세포체외증식급침습력균유명현억제작용.상대세포존활솔위37.0%,교대조조명현저(P<0.01).⑤체내실험결과현시,증강형파향표체재체대라서비인암이식류생장구유명현억제작용,억류솔체56.3%,여대조조비교,차이유통계학의의(P<0.01),이라서간신병리검사미발현명현손해.결론 이hTERT/CMV쌍조공궤제개도TK기인적증강형표체재체능구고효、파향살상비인암세포급이식류,실험동물체내응용무명현독부작용.
Objective To construct a modified and enhanced thymidine kinase (TK) vector regulated by human telemerase catalytic subunit promoter (hTERT) promoter and cytomegaiovirus (CMV) enhancer and its killing effect on nasopharyngeal carcinoma in vitro and in vivo and its safety in vivo. Methods The pGL3-basic,as basic vector template,was linked and constructed into TK vector regulated by hTKRT promoter and CMV enhancer with mono-promoter vector as control. Enhanced TK expression was confirmed by fluorescent microscopy and real time fluorescent quantitative PCR. Telomerase activity was measured by stretch PCR. Tumour killing effects were examined by MTT and Boyden areole. The effects of enhanced TK on the invasiveness of tumor cell NPC 5-8F and the growth of xenograft implanted in nude mice were investigated. Results Compared with non-enhanced vector, TK expressed by the enhanced vector significantly increased in NPC 5-8F and MCF-7 cells,telomerase activity was positive in human in NPC 5-8F cells and breast cancer MCF-7 cells and negative in control human blood vessel endothelium ECV-304 cells. After ganciclovir (GCV) treatment, NPC 5-8F cell survival rate and invasiveness decreased and tumor progress of NPC xenograft implanted in nude mice was inhibited, without obvious toxicity effects on mouse liver and kidney. Conclusions The enhanced TK vector regulated by hTERT promoter and CMV enhancer can obviously and specifically inhibit and kill nasopharyngeal carcinoma cells in culture and nasopharyngeal carcinoma xenograft in nude mice in vivo, without obviously toxic side effects on nude mice. The targeted and enhanced TK gene vector with high performance may be a new tumour targeted gene therapy strategy clinically to aim directly at most malignant tumours including nasopharyngeal carcinoma, with more extensive anti-cancer spectrum.
