肿瘤研究与临床
腫瘤研究與臨床
종류연구여림상
CANCER RESEARCH AND CLINIC
2009年
1期
4-6
,共3页
刘燕%邓志华%杨长青%刘京龙%贾静%郭素雅%杨强
劉燕%鄧誌華%楊長青%劉京龍%賈靜%郭素雅%楊彊
류연%산지화%양장청%류경룡%가정%곽소아%양강
hTERT启动子%单纯疱疹病毒%胸苷激酶%腺病毒载体%肝肿瘤%基因%治疗
hTERT啟動子%單純皰疹病毒%胸苷激酶%腺病毒載體%肝腫瘤%基因%治療
hTERT계동자%단순포진병독%흉감격매%선병독재체%간종류%기인%치료
hTERT promoter%Simplexvirus%Thymidine kinase%Adenovirus vector%Liver neoplasms%Gone%Therapy
目的 构建hTFRT启动子调控的HSV-TK基因重组腺病毒载体系统,观察Ad-hTERTp-HSV-TK/GCV自杀基因系统对肝癌细胞的杀伤作用.方法 将穿梭质粒pSU-Tp-TK与腺病毒骨架质粒pBHGE3共转导至HEK293细胞,利用细胞内同源重组法构建出hTERT启动子调控的携带TK基因的复制缺陷型腺病毒Ad-hTERTp-HSV-TK载体系统;将不同感染复数(MOI-1、10、100、1000)的重组腺病毒Ad-hTERTp-HSV-TK感染肝癌细胞HepG2及正常肝细胞L-02,加入不同浓度的GCV(0、1、10、100、1000 μg/ml),MTT法检测细胞活性.结果 HEK293细胞出现病毒空斑,提取病毒DNA,经PCR鉴定正确后扩增、纯化,滴度为1.5×1010pfu/ml;MTT法检测到Ad-hTERTp-HSV-TK/GCV能靶向杀死肝癌细胞HepG2,且细胞存活率随着病毒滴度和GCV浓度的增加而降低.结论 该实验构建的Ad-hTERTp-HSV-TK载体系统,可靶向抑制肝癌细胞HepG2,面对正常肝细胞L-02几乎无影响.
目的 構建hTFRT啟動子調控的HSV-TK基因重組腺病毒載體繫統,觀察Ad-hTERTp-HSV-TK/GCV自殺基因繫統對肝癌細胞的殺傷作用.方法 將穿梭質粒pSU-Tp-TK與腺病毒骨架質粒pBHGE3共轉導至HEK293細胞,利用細胞內同源重組法構建齣hTERT啟動子調控的攜帶TK基因的複製缺陷型腺病毒Ad-hTERTp-HSV-TK載體繫統;將不同感染複數(MOI-1、10、100、1000)的重組腺病毒Ad-hTERTp-HSV-TK感染肝癌細胞HepG2及正常肝細胞L-02,加入不同濃度的GCV(0、1、10、100、1000 μg/ml),MTT法檢測細胞活性.結果 HEK293細胞齣現病毒空斑,提取病毒DNA,經PCR鑒定正確後擴增、純化,滴度為1.5×1010pfu/ml;MTT法檢測到Ad-hTERTp-HSV-TK/GCV能靶嚮殺死肝癌細胞HepG2,且細胞存活率隨著病毒滴度和GCV濃度的增加而降低.結論 該實驗構建的Ad-hTERTp-HSV-TK載體繫統,可靶嚮抑製肝癌細胞HepG2,麵對正常肝細胞L-02幾乎無影響.
목적 구건hTFRT계동자조공적HSV-TK기인중조선병독재체계통,관찰Ad-hTERTp-HSV-TK/GCV자살기인계통대간암세포적살상작용.방법 장천사질립pSU-Tp-TK여선병독골가질립pBHGE3공전도지HEK293세포,이용세포내동원중조법구건출hTERT계동자조공적휴대TK기인적복제결함형선병독Ad-hTERTp-HSV-TK재체계통;장불동감염복수(MOI-1、10、100、1000)적중조선병독Ad-hTERTp-HSV-TK감염간암세포HepG2급정상간세포L-02,가입불동농도적GCV(0、1、10、100、1000 μg/ml),MTT법검측세포활성.결과 HEK293세포출현병독공반,제취병독DNA,경PCR감정정학후확증、순화,적도위1.5×1010pfu/ml;MTT법검측도Ad-hTERTp-HSV-TK/GCV능파향살사간암세포HepG2,차세포존활솔수착병독적도화GCV농도적증가이강저.결론 해실험구건적Ad-hTERTp-HSV-TK재체계통,가파향억제간암세포HepG2,면대정상간세포L-02궤호무영향.
Objective To construct the recombinant adenovirus vector with hTERT-HSV-TK and observe the killing effect of Ad-hTERTp-HSV-TK/GCV system on hepatocellular carcinoma cells. Methods A recombinant replication defective adenoviral vector of Ad-hTERTp-HSV-TK was constructed via homologous recombination which both shuttle plasmid pSU-Tp-TK and adenovirus backbone plasmid pBHGE3 transfected into the HEK293 packaging cells. Then the Ad-hTERTp-HSV-TK was amplified and purified through PCR. The activity of the HepG2 cells and the L-02 cells were tested by methyl thiazolyl terazolium (MTT) after they were transfected by the recombinant adenovirus of different multiplicities of infection (MOI) and then were added GCV of different conc.entration. Results The recombinant replication defective adenoviral vector of Ad-hTERTp-HSV-TK were identified by PCR successfully. The viral titer was 1.5×1010 pfu/ml after amplification and purification. The HepG2 cells were targetedly suppressed by Ad-hTERTp-HSV-TK/GCV system. The survival rate of cells decreased gradually along with the increase of the MOI and the GCV' s concentration. Conclusion The recombinant replication defective adenoviral vector of Ad-hTERTp-HSV-TK can inhibit the HepG2 cells significantly, but has not influence on the L-02 cells.
