DcR3反义RNA对肝癌的作用及与顺铂协同效应的观察
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摘要
段与诱发细胞凋亡有关。作为生物治疗重点部分的诱导凋亡治疗将成为人类征服肝癌的有力手段。
Decoy Receptor 3(DcR3)是1998年Pitti RM首先发现的一种陷阱分子,属于肿瘤坏死因子受体超家族成员,DcR3基因定位于染色体20q13.3上,该基因编码一种无明显跨膜序列的分泌型蛋白,能特异性结合FasL,阻断由Fas/FasL系统介导的细胞凋亡。研究表明,部分人类恶性肿瘤(肺癌、胃癌、肝癌等)中存在DcR3基因过度表达及扩增,而且这种过度表达仅发生于肿瘤细胞,肿瘤周围正常组织则检测不到该基因的表达。后续研究发现,DcR3基因过度表达与肿瘤免疫逃逸及抵抗机体细胞凋亡机制有关。在肿瘤发生发展中起重要作用。目前国际上对该基因研究并不充分,但是鉴于DcR3与肝癌细胞凋亡的密切关联及它在肝癌患者血清中特异地高表达,我们认为DcR3是一个有较高研究价值的基因,明确它在帮助肝癌细胞逃避免疫监视及免疫杀伤中的作用,阻断其表达对肝癌细胞之凋亡有无促进成为我们研究的目标。
第一部分 DcR3反义RNA构建及表达
目的:DcR3(decoy receptor 3)属于肿瘤坏死因子受体超家族成员,能竞争性地结合LIGHT及FasL,抑制LIGHT及FasL介导的细胞凋亡,使肿瘤细胞逃避免疫监视及免疫杀伤而得以存活。本实验利用反义RNA技术观察在DcR3基因片段与其反义DcR3基因片段结合后,凋亡有无增加?与传统化疗药顺铂比较,何者促凋亡效应更强?二者是否具有协同效应?为此,第一部分实验首先构建DcR3反义RNA,将其转染肿瘤细胞,比较其表达情况。方法:从新鲜肝癌细胞中提取总RNA,在DcR3基因序列引物限制下进行RT-PCR反应,得到大量DcR3的DNA片段,将该基因片段进行电泳鉴定并做基因测序分析,确定之后对其进行BamH Ⅰ、Xhol Ⅰ双酶切,使DcR3片段粘性末端裸露;同时对质粒PVAX Ⅰ(一)进行BamH Ⅰ、Xhol Ⅰ双酶切;将此具有粘性末端的二者在T4DNA连接酶的作用下进行反义连接,得到PVAX Ⅰ-as-DcR3,将其转染肝癌细胞株SMMC-7721,之后以β-actin为内参照,用Western Blot方法检测单纯肝癌细胞株SMMC-7721组、转染了PVAX Ⅰ-as-DcR3的肝癌细胞株SMMC-7721组、转染了空质粒PVAX Ⅰ的肝癌细胞株SMMC-7721组,比较三组蛋白表达量有无差别。结果:对DcR3基因片段进行电泳鉴定,证实在620bp处有一条符合预期结果的电泳条
PART Ⅰ CONSTRUCTION AND EXPRESSION OF THE ANTISENSE RNA OF DCR3Objective: DcR3(decoy receptor 3,DcR3) was a member belonging to the super family of tumor necrosis factor receptors,could help tumor cell surving and escaping from immunologic survival,immunologic killing through competitively combinding with LIGHT ,FasL so as to inhibite cell apoptosis induced by both LIGHT and FasL. We took use of antisense RNA technology to observe whether apoptosis of hepotoma cell group SMMC-7721 would increase after combindination of DcR3 gene fragment and antisense DcR3 gene fragment? Which was more efficient in inducing apoptosis comparing with tranditional chemical therapy medicine—cisplatin? Whether they had coordinational function when they were unitedly used? First,in part I we constructed antisense RNA of DcR3, transfected it into hepatoma cellular,then compared their protein expression in different groups. Methods: Firstly, total RNA was abstracted from fresh hepatoma tissue,RT-PCR was processed under constriction of DcR3 gene primer and DNA fragment of DcR3 were obtained,secondly the gene fragment were procedured electrophoresis and gene sequence analysis to demonstrate that it just was DcR3, after that DcR3 fragment was excised by both BamH Ⅰ and Xhol Ⅰ to make nicked end of DcR3 naked;While vecctor PVAX Ⅰ (-) was excised at the same time; PVAX I -antisense-DcR3 was obtained after the two (DcR3,PVAX Ⅰ )with nicked end were ligated in antisense direction through T4 DNA ligase , then PVAX Ⅰ -as-DcR3 was transfected into hepatoma cellular group SMMC-7721. Western Blot was used to measure differences of protein expression among 3 groups while β -actin being constrast group:group SMMC-7721,group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 transfected PVAX Ⅰ (-). Result: It was demonstrated that there was an anticipated electrophoresis belt of 620bp in electrophoresis of DcR3 fragment,while the fragment was just DcR3 fragment through gene sequencing; the recombinant vector, PVAX Ⅰ -as-DcR3,was successfully constructed and demonstrated by obtaining 2 anticipated electrophoresis belt—DcR3 (620bp) PVAX Ⅰ (3000bp) after PVAX Ⅰ -as-DcR3 was excised by both BamH Ⅰ XhoL Ⅰ and electrophoresed;Western Blot
was showed color depth of group SMMC-7721 and group SMMC-7721 transfected PVAX Ⅰ (-)was deeper comparing with group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,it revealed that protein expression was relatively decreased for translation of DcR3 mRNA being inhibited while that of the other two groups were not infiuenced,furtherly it showed that antisense RNA of DcR3 could obviously decrease the expressionof DcR3 protein. Conclusion: DcR3 gene fragment could be accurately amplificated under constriction of DcR3 primer ; The complexed vector, PVAX Ⅰ -as-DcR3,could be obtained through ligation of DcR3 fragment and vector PVAX Ⅰ (-) in antisense direction after the both were digested by BamH Ⅰ XhoL Ⅰ ; Western Blot was shown that PVAX Ⅰ -as-DcR3 could make DcR3 protein expression of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 obviously decreased.
PART Ⅱ EFFECT AND COMPARISON OF INDUCING APOPTOSISBY ANTISENSE DCR3 AND CISPLATINObjective: Apoptosis was a kind of biological regulating mechanism and a kind of automatic suicide processed by gene procedure,some chemical therapy medicine ,for example cisplatin,could kill tumor cell through inducing their apoptosis.So MTT,Tunel and FCM were used to measure whether antisense DcR3 could increase inducing apoptosis of hepatoma cellular SMMC-7721 through complemental conjugation with DcR3 mRNA in nucleus? Which was more effective in inducing apoptosis comparing with cisplatin? Whether there was coordinative function when the both were united and used? Methods:On the basis of experiment Part Ⅰ ,first liposome was used to swallow and transfect PVAX I -as-DcR3 and PVAX Ⅰ (-) into hepatoma cellular SMMC-7721 respectively. MTT was used to measure OD(optical density),tumor inhibiting rate and statistical analysis was being done among the different groups: group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected by cisplatin(4mg/ml),group SMMC-7721 transfected and effected by both of PVAX Ⅰ -as-DcR3 and cisplatin(2mg/ml),some contrast groups;TuneI was used to observe picture of hepatoma cellular apoptosis in different groups.Apoptosis Index(AI) was calculated and Antomatic Picture Analysis System was used to analyse and compare Picture Unit(PU) of different groups;FCM was used to accuratively calculate apoptosis rate,observe whether cell cycle of apoptosis cell was blocked at G0G1 phase? compare apoptosis peak in different groups ;Finally statistical analysis was taken to compare whether there were statistical differences in different groups. Results:MTT shouwed that the apoptosis of group SMMC-7721 effected by cisplatin and group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin were more than that of the other two groups(p<0.05) during the first 24h ,apoptosis of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 ,group SMMC-7721 effected by cisplatin and group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin were more than that of the constrast group respectively during the second 24h(p<0.05),while there was no statistical difference of apoptosis among that of the 3 groups(p>0.05).There was no any difference in situation between that of the second and third
24h.During the forth 24h,there was no any statistical difference of apoptosis among the whole groups;Tunel showed that Apoptosis Index(AI)of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected cisplatin,group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin was much higher than that of the constrast group(p<0.05)while that of group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin was highest among those of the whole groups and there was statistical difference of apoptosis index in this group comparing with those of the other 3 groups(p<0.05);FCM showed that cell cycle of the group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 efected by cisplatin ,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin was all blocked at G0G1 phase.Apoptosis peaks appeared in different stage among the 3 groups and apoptosis rate of the 3 groups was statistical different comparing with that of the constrast group respectively(p<0.05) while there was no statistical difference of apoptosis rate among the 3 groups(p>0.05). Conclutions:MTT,Tunel,FCM were taken used to demonstrate that antisense DcR3 could obviously inhibite the expression of DcR3 gene and increase apoptosis of procedured hepatoma cellular; DcR3 acted as an important role during the apoptosis of hepatoma cell.Decrease of its protein expression and lost of its function could induce increase of apoptosis in hepatoma cell for scarcity of DcR3 protection; There was a kind of coordinating effect when antisense DcR3 and cisplatin were united and used.intensity of chemical therapy medicine could be decreased while function inducing apoptosis of hepatoma cell was not influenced,so that tolerance to chemical therapy medicine could be increased.
PARTⅢ STUDY OF ANTISENSE DCR3 IN TUMOR TRANSPLATED SUBCUTANEOUSLY IN NUDE MICEObjective: Those were detected : invasiveness and chemotaxis motility assay of hepatoma cell in group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 ,group SMMC-7721 effected by cisplatin,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin ,and some influenced factors were compared for transplanted tumor subcutaneously injected by different treated hepatoma cell—SMMC-7721 in nude mice. Methods: On the basis of experiment Part Ⅰ and Ⅱ ,artificial basement membrane and Transwell Chamber were taken use to simulate basement membrane in vivo and being measured quatitively invasiveness and chemotaxis mobility assay of hepatoma cell SMMC-7721 treated by PVAX Ⅰ -as-DcR3,cisplatin and PVAX Ⅰ -as-DcR3+cisplatin while time of tumor forming ,tumor weight,tumor volume were calculated in different nude mice groups subcutaneously injected hepatoma cell SMMC-7721 treated by PVAX Ⅰ-as-DcR3,cisplatin(4mg/ml), PVAX Ⅰ -as-DcR3+cisplatin(2mg/ml) respectively. Results: Invasiveness statistically decreased in group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected by cisplatin,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin comparing with contrast group(p<0.05) while that of group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin decreased mostly and was statistically different comparing with those of the other groups (p<0.05);The situation in chemotaxis motility assay was the same as ininvasiveness; There was no statistical difference for time of tumor forming of nude mice between group SMMC-7721 effected by cisplatin(4mg/ml) and group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin(2mg/ml) but with statistical longer time than that of group SMMC-7721 transfected PVAX I -as-DcR3 and contrast group(p<0.05).Nude mice injected with hepatoma cell SMMC-7721 treated by PVAX I -as-DcR3+cisplatin(2mg/ml) had lightest tumor weight and there was no statistical difference from that of group SMMC-7721 effected by cisplatin(p>0.05),but statistically different from that of the both---group SMMC-7721
transfected PVAX Ⅰ -as-DcR3 and contrast group while there was no statistical difference between the two groups(p>0.05);Still,nude mice injected with hepatoma cell SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin had the smallest tumor volume,there was no statistical difference from that of group SMMC-7721 effected by cisplatin (p>0.05),while contrast group nude mice had the biggest tumor volume and there was no statistical difference from that of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,but tumor volume of the two groups were statistically different from that of the other two groups(p<0.05). Conclusions: The effect of antisense DcR3 was not very satisfied in vivo experiment and was not different from that in vitro experiment,maybe because antisense DcR3 was degraded more in vivo or had relatively lower transfecting efficiency; Enviroment and factors in vivo were much complicated than that in vitro,it couldn't be concluded that antisense DcR3 was not fit to clinical need,but a very hopeful candidate treatment when antisense DcR3 were decorated to escape from being degraded or were improved transfecting efficiency; The union of antisense DcR3 and cisplatin showed more effective function in inhibiting hepatoma cell while elevating tolerance to chemical therapy and decreasing intensity and toxicity of chemical therapy medicine.
Decoy Receptor 3(DcR3)是1998年Pitti RM首先发现的一种陷阱分子,属于肿瘤坏死因子受体超家族成员,DcR3基因定位于染色体20q13.3上,该基因编码一种无明显跨膜序列的分泌型蛋白,能特异性结合FasL,阻断由Fas/FasL系统介导的细胞凋亡。研究表明,部分人类恶性肿瘤(肺癌、胃癌、肝癌等)中存在DcR3基因过度表达及扩增,而且这种过度表达仅发生于肿瘤细胞,肿瘤周围正常组织则检测不到该基因的表达。后续研究发现,DcR3基因过度表达与肿瘤免疫逃逸及抵抗机体细胞凋亡机制有关。在肿瘤发生发展中起重要作用。目前国际上对该基因研究并不充分,但是鉴于DcR3与肝癌细胞凋亡的密切关联及它在肝癌患者血清中特异地高表达,我们认为DcR3是一个有较高研究价值的基因,明确它在帮助肝癌细胞逃避免疫监视及免疫杀伤中的作用,阻断其表达对肝癌细胞之凋亡有无促进成为我们研究的目标。
第一部分 DcR3反义RNA构建及表达
目的:DcR3(decoy receptor 3)属于肿瘤坏死因子受体超家族成员,能竞争性地结合LIGHT及FasL,抑制LIGHT及FasL介导的细胞凋亡,使肿瘤细胞逃避免疫监视及免疫杀伤而得以存活。本实验利用反义RNA技术观察在DcR3基因片段与其反义DcR3基因片段结合后,凋亡有无增加?与传统化疗药顺铂比较,何者促凋亡效应更强?二者是否具有协同效应?为此,第一部分实验首先构建DcR3反义RNA,将其转染肿瘤细胞,比较其表达情况。方法:从新鲜肝癌细胞中提取总RNA,在DcR3基因序列引物限制下进行RT-PCR反应,得到大量DcR3的DNA片段,将该基因片段进行电泳鉴定并做基因测序分析,确定之后对其进行BamH Ⅰ、Xhol Ⅰ双酶切,使DcR3片段粘性末端裸露;同时对质粒PVAX Ⅰ(一)进行BamH Ⅰ、Xhol Ⅰ双酶切;将此具有粘性末端的二者在T4DNA连接酶的作用下进行反义连接,得到PVAX Ⅰ-as-DcR3,将其转染肝癌细胞株SMMC-7721,之后以β-actin为内参照,用Western Blot方法检测单纯肝癌细胞株SMMC-7721组、转染了PVAX Ⅰ-as-DcR3的肝癌细胞株SMMC-7721组、转染了空质粒PVAX Ⅰ的肝癌细胞株SMMC-7721组,比较三组蛋白表达量有无差别。结果:对DcR3基因片段进行电泳鉴定,证实在620bp处有一条符合预期结果的电泳条
PART Ⅰ CONSTRUCTION AND EXPRESSION OF THE ANTISENSE RNA OF DCR3Objective: DcR3(decoy receptor 3,DcR3) was a member belonging to the super family of tumor necrosis factor receptors,could help tumor cell surving and escaping from immunologic survival,immunologic killing through competitively combinding with LIGHT ,FasL so as to inhibite cell apoptosis induced by both LIGHT and FasL. We took use of antisense RNA technology to observe whether apoptosis of hepotoma cell group SMMC-7721 would increase after combindination of DcR3 gene fragment and antisense DcR3 gene fragment? Which was more efficient in inducing apoptosis comparing with tranditional chemical therapy medicine—cisplatin? Whether they had coordinational function when they were unitedly used? First,in part I we constructed antisense RNA of DcR3, transfected it into hepatoma cellular,then compared their protein expression in different groups. Methods: Firstly, total RNA was abstracted from fresh hepatoma tissue,RT-PCR was processed under constriction of DcR3 gene primer and DNA fragment of DcR3 were obtained,secondly the gene fragment were procedured electrophoresis and gene sequence analysis to demonstrate that it just was DcR3, after that DcR3 fragment was excised by both BamH Ⅰ and Xhol Ⅰ to make nicked end of DcR3 naked;While vecctor PVAX Ⅰ (-) was excised at the same time; PVAX I -antisense-DcR3 was obtained after the two (DcR3,PVAX Ⅰ )with nicked end were ligated in antisense direction through T4 DNA ligase , then PVAX Ⅰ -as-DcR3 was transfected into hepatoma cellular group SMMC-7721. Western Blot was used to measure differences of protein expression among 3 groups while β -actin being constrast group:group SMMC-7721,group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 transfected PVAX Ⅰ (-). Result: It was demonstrated that there was an anticipated electrophoresis belt of 620bp in electrophoresis of DcR3 fragment,while the fragment was just DcR3 fragment through gene sequencing; the recombinant vector, PVAX Ⅰ -as-DcR3,was successfully constructed and demonstrated by obtaining 2 anticipated electrophoresis belt—DcR3 (620bp) PVAX Ⅰ (3000bp) after PVAX Ⅰ -as-DcR3 was excised by both BamH Ⅰ XhoL Ⅰ and electrophoresed;Western Blot
was showed color depth of group SMMC-7721 and group SMMC-7721 transfected PVAX Ⅰ (-)was deeper comparing with group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,it revealed that protein expression was relatively decreased for translation of DcR3 mRNA being inhibited while that of the other two groups were not infiuenced,furtherly it showed that antisense RNA of DcR3 could obviously decrease the expressionof DcR3 protein. Conclusion: DcR3 gene fragment could be accurately amplificated under constriction of DcR3 primer ; The complexed vector, PVAX Ⅰ -as-DcR3,could be obtained through ligation of DcR3 fragment and vector PVAX Ⅰ (-) in antisense direction after the both were digested by BamH Ⅰ XhoL Ⅰ ; Western Blot was shown that PVAX Ⅰ -as-DcR3 could make DcR3 protein expression of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 obviously decreased.
PART Ⅱ EFFECT AND COMPARISON OF INDUCING APOPTOSISBY ANTISENSE DCR3 AND CISPLATINObjective: Apoptosis was a kind of biological regulating mechanism and a kind of automatic suicide processed by gene procedure,some chemical therapy medicine ,for example cisplatin,could kill tumor cell through inducing their apoptosis.So MTT,Tunel and FCM were used to measure whether antisense DcR3 could increase inducing apoptosis of hepatoma cellular SMMC-7721 through complemental conjugation with DcR3 mRNA in nucleus? Which was more effective in inducing apoptosis comparing with cisplatin? Whether there was coordinative function when the both were united and used? Methods:On the basis of experiment Part Ⅰ ,first liposome was used to swallow and transfect PVAX I -as-DcR3 and PVAX Ⅰ (-) into hepatoma cellular SMMC-7721 respectively. MTT was used to measure OD(optical density),tumor inhibiting rate and statistical analysis was being done among the different groups: group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected by cisplatin(4mg/ml),group SMMC-7721 transfected and effected by both of PVAX Ⅰ -as-DcR3 and cisplatin(2mg/ml),some contrast groups;TuneI was used to observe picture of hepatoma cellular apoptosis in different groups.Apoptosis Index(AI) was calculated and Antomatic Picture Analysis System was used to analyse and compare Picture Unit(PU) of different groups;FCM was used to accuratively calculate apoptosis rate,observe whether cell cycle of apoptosis cell was blocked at G0G1 phase? compare apoptosis peak in different groups ;Finally statistical analysis was taken to compare whether there were statistical differences in different groups. Results:MTT shouwed that the apoptosis of group SMMC-7721 effected by cisplatin and group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin were more than that of the other two groups(p<0.05) during the first 24h ,apoptosis of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 ,group SMMC-7721 effected by cisplatin and group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin were more than that of the constrast group respectively during the second 24h(p<0.05),while there was no statistical difference of apoptosis among that of the 3 groups(p>0.05).There was no any difference in situation between that of the second and third
24h.During the forth 24h,there was no any statistical difference of apoptosis among the whole groups;Tunel showed that Apoptosis Index(AI)of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected cisplatin,group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin was much higher than that of the constrast group(p<0.05)while that of group SMMC-7721 effected by PVAX Ⅰ -as-DcR3+cisplatin was highest among those of the whole groups and there was statistical difference of apoptosis index in this group comparing with those of the other 3 groups(p<0.05);FCM showed that cell cycle of the group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 efected by cisplatin ,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin was all blocked at G0G1 phase.Apoptosis peaks appeared in different stage among the 3 groups and apoptosis rate of the 3 groups was statistical different comparing with that of the constrast group respectively(p<0.05) while there was no statistical difference of apoptosis rate among the 3 groups(p>0.05). Conclutions:MTT,Tunel,FCM were taken used to demonstrate that antisense DcR3 could obviously inhibite the expression of DcR3 gene and increase apoptosis of procedured hepatoma cellular; DcR3 acted as an important role during the apoptosis of hepatoma cell.Decrease of its protein expression and lost of its function could induce increase of apoptosis in hepatoma cell for scarcity of DcR3 protection; There was a kind of coordinating effect when antisense DcR3 and cisplatin were united and used.intensity of chemical therapy medicine could be decreased while function inducing apoptosis of hepatoma cell was not influenced,so that tolerance to chemical therapy medicine could be increased.
PARTⅢ STUDY OF ANTISENSE DCR3 IN TUMOR TRANSPLATED SUBCUTANEOUSLY IN NUDE MICEObjective: Those were detected : invasiveness and chemotaxis motility assay of hepatoma cell in group SMMC-7721 transfected PVAX Ⅰ -as-DcR3 ,group SMMC-7721 effected by cisplatin,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin ,and some influenced factors were compared for transplanted tumor subcutaneously injected by different treated hepatoma cell—SMMC-7721 in nude mice. Methods: On the basis of experiment Part Ⅰ and Ⅱ ,artificial basement membrane and Transwell Chamber were taken use to simulate basement membrane in vivo and being measured quatitively invasiveness and chemotaxis mobility assay of hepatoma cell SMMC-7721 treated by PVAX Ⅰ -as-DcR3,cisplatin and PVAX Ⅰ -as-DcR3+cisplatin while time of tumor forming ,tumor weight,tumor volume were calculated in different nude mice groups subcutaneously injected hepatoma cell SMMC-7721 treated by PVAX Ⅰ-as-DcR3,cisplatin(4mg/ml), PVAX Ⅰ -as-DcR3+cisplatin(2mg/ml) respectively. Results: Invasiveness statistically decreased in group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,group SMMC-7721 effected by cisplatin,group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin comparing with contrast group(p<0.05) while that of group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin decreased mostly and was statistically different comparing with those of the other groups (p<0.05);The situation in chemotaxis motility assay was the same as ininvasiveness; There was no statistical difference for time of tumor forming of nude mice between group SMMC-7721 effected by cisplatin(4mg/ml) and group SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin(2mg/ml) but with statistical longer time than that of group SMMC-7721 transfected PVAX I -as-DcR3 and contrast group(p<0.05).Nude mice injected with hepatoma cell SMMC-7721 treated by PVAX I -as-DcR3+cisplatin(2mg/ml) had lightest tumor weight and there was no statistical difference from that of group SMMC-7721 effected by cisplatin(p>0.05),but statistically different from that of the both---group SMMC-7721
transfected PVAX Ⅰ -as-DcR3 and contrast group while there was no statistical difference between the two groups(p>0.05);Still,nude mice injected with hepatoma cell SMMC-7721 treated by PVAX Ⅰ -as-DcR3+cisplatin had the smallest tumor volume,there was no statistical difference from that of group SMMC-7721 effected by cisplatin (p>0.05),while contrast group nude mice had the biggest tumor volume and there was no statistical difference from that of group SMMC-7721 transfected PVAX Ⅰ -as-DcR3,but tumor volume of the two groups were statistically different from that of the other two groups(p<0.05). Conclusions: The effect of antisense DcR3 was not very satisfied in vivo experiment and was not different from that in vitro experiment,maybe because antisense DcR3 was degraded more in vivo or had relatively lower transfecting efficiency; Enviroment and factors in vivo were much complicated than that in vitro,it couldn't be concluded that antisense DcR3 was not fit to clinical need,but a very hopeful candidate treatment when antisense DcR3 were decorated to escape from being degraded or were improved transfecting efficiency; The union of antisense DcR3 and cisplatin showed more effective function in inhibiting hepatoma cell while elevating tolerance to chemical therapy and decreasing intensity and toxicity of chemical therapy medicine.
引文
1. Pitti RM, Marsters SA, Lawrence DA, et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature. 1998; 396(6712): 699-703.
2. Kang-Yeol Yu, Know B, Ni J, et al. A newly identified member of Tumor Necrosis Factor Receptor superfamily suppresses LIGHT-mediated Apoptosis. [J] The Journal of Biological Chemistry. 1999 May vol 274, no 20, 13733-13736.
3. Feldmann G, Lamboley C, Moreau A, et al. Fas-mediated apoptosis of hepatocytes Ⅰ in viral hepatisis patients. Hepatology, 1995; 22: 230.
4. Bai C, Connolly B, Metzker ML, et al. Overexpression of M68/DcR3 in four gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster. Proc-Natl-Acad-Sci-U-S-A. 2000; 97(3): 1230-5.
5. Roth W, Isenmanns S, Nakamura M, et al. Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Research. 2001; 61: 2759-2765.
6. Yu KY, Xwon B, Ni J, et al. A newly identified member of tumor necrosis factor receptor superfamily(TR6) suppresses LIGHT-mediated apoptosis. J Bio Chem. 1992; 274(20): 13733-6.
7.沈宏伟,吴育连,彭淑傭。原发性肝癌组织中陷阱受体3基因表达与扩增的临床意义。中华医学杂志,2003,83(9):744—747。
8. Bayever E, Iversen P, Smith L, et al. Guest editorial: systemic human antisense therapy begins. Antisense Res Dev, 1992, 2: 109-110
9. Schwendel A, Richard F, Langreck H. et al. Chromosome alterations in breast carcinomas: frequent involvement of DNA losses including chomosomes 4q and 21q. Br J Cancer. 1998; 78: 806-811.
10. Ibrahim SM, Ringel J, Schmidt C, et al. Pancreatic adenocarcinoma cell lines show variable susceptibility to TRAIL-mediated cell death. Pancrease 2001 Jul; 23(1): 72-79.
11. Maeta T, Hao C, Tron VA. Ultraviolet Light(UV) Regulation of the TNF family Decoy Receptors DcR2 and DcR3 in Human Keratinocytes. J Cutan Med Surg 2001; 5(4): 294-8.
12.Jodo S, Kobayashi S, Nakajima Y,et al. Elevated serum levels of soluble Fas/Apo-1(CD95) in patient with hepatocelluler carcinoma. Clin Exp Immunol, 1998; 111(2):166.
13.Scheikh MS, Fomace AJ. Death and decoy receptors and p53-mediated apoptosis leukemia. 2000 Aug; 14(8):1509-13.
14.Zhang J,Salcedo TW, Wan X,et al. Modulation of T-cell responses to alloantigens by TR6/DcR3. Clin Invest.2001; 107(11): 1459-68.
15.Roughton SA, Lareu RR, Bittles AH., et al. Fas and Fas Ligand Messenger Ribonucleic Acid and Protein Expression in the Rat Corpus Luteum during Apoptosis-Mediated Luteolysis. Biology of Reproduction.1999;60:797-804.
16.Conolly K, Cho YH, Duan R, et al. In vivo inhibition of Fas ligand-mediated killing by TR6, a Fas ligand decoy receptor. J Pharmaco Exp Ther.2000;298(l):25-33.
17.Zhai Y, Guo R, Hus TL, et al. LIGHT, A Novel Ligand for Lymphotoxin b Receptor and TR2/HVEM Induces Apoptosis and Suppresses In Vivo Tumor Formation Via Gene Transfer. J Clin Invest. 1998;102:1142-1151.
18.Harrop JA, Mcdonnell PC, Brigham Burke M, et al. Antibodies to TR2(Herpesvirus Entry Mediator), a New Member of the TNF Receptor Superfamily,Block T Cell Proliferation,Expression of Activation Markers, and Production of Cytokines. J Immuno. 1998,161:1786-1794.
19.Altura RA, Maris JM,Li H, et al. Novel regions of chromosomal loss in familial neuroblastoma by comparative genomic hybridization. Genes Chromosome Cancer. 1997; 19:176-184
20.Mild G, Bachmann F, Boulay JL, et al. DcR3 locus is a predictive marker for 5-fluorouracil-based adjuvant chemotherpy in colorectal cancer. Int J Cancer, 2002, 102: 254-257.
21.Victor J,Witcher DR,Becker GW,et al.Decoy receptor 3 (DcR3) is proteolytically processed to a metabolic fragment having differential activities against Fas ligand and LIGHT. Biochem Pharmacol .2003 ;65;657-667.
22.Yu lian Wu,Bing Han,Sheng H,et al.Clinical significance of detecting elevated serum DcR3/TR6 / M68 in malignant tumor patients. Int J Cancer,2003,105:724-732.
23.Ohshima K,Haraoka S, Sugihara M, et al. Amplification and expression of a decoy receptor for Fas ligand (DcR3) in virus (EBV or HTLV-1) associated lymphomas.Cancer Lett.2000;160(1):89-97.
24.Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA, 1978. 75 (1):280-4
25.Izant J, Weintraub H. Inhibition of thymidine kinase gene expression by antisense RNA: a molecular approach to genetic analysis.Cell.1984;36(4):1007-15.
26Uhlmann E. Peptide nucleic acids (PNA) and PNA-DNA chimeras:from high binding affinity towards biological function. Bio Chem. 1998;379(8-9): 1045-52.
27.Connor DS, Schechner JS, Adida C, et al. Control of apoptosis during angiogenesis by survin express in endothelial cell. Am J Pathol.2000;156(2):393-398.
28.Blanc Brude OP,Yu J, Simosa H, et al. Inhibitor of apoptosis protein survin regulates vascular injury. Nat Med.2002Sep;8(9):987-9.
29.Deguchi M, Shiraki K, Inoue H, et al. Expression of survivin during liver regeneration. Biochem Biophys Res.2002 Sep13;297(1):59.
2. Kang-Yeol Yu, Know B, Ni J, et al. A newly identified member of Tumor Necrosis Factor Receptor superfamily suppresses LIGHT-mediated Apoptosis. [J] The Journal of Biological Chemistry. 1999 May vol 274, no 20, 13733-13736.
3. Feldmann G, Lamboley C, Moreau A, et al. Fas-mediated apoptosis of hepatocytes Ⅰ in viral hepatisis patients. Hepatology, 1995; 22: 230.
4. Bai C, Connolly B, Metzker ML, et al. Overexpression of M68/DcR3 in four gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster. Proc-Natl-Acad-Sci-U-S-A. 2000; 97(3): 1230-5.
5. Roth W, Isenmanns S, Nakamura M, et al. Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Research. 2001; 61: 2759-2765.
6. Yu KY, Xwon B, Ni J, et al. A newly identified member of tumor necrosis factor receptor superfamily(TR6) suppresses LIGHT-mediated apoptosis. J Bio Chem. 1992; 274(20): 13733-6.
7.沈宏伟,吴育连,彭淑傭。原发性肝癌组织中陷阱受体3基因表达与扩增的临床意义。中华医学杂志,2003,83(9):744—747。
8. Bayever E, Iversen P, Smith L, et al. Guest editorial: systemic human antisense therapy begins. Antisense Res Dev, 1992, 2: 109-110
9. Schwendel A, Richard F, Langreck H. et al. Chromosome alterations in breast carcinomas: frequent involvement of DNA losses including chomosomes 4q and 21q. Br J Cancer. 1998; 78: 806-811.
10. Ibrahim SM, Ringel J, Schmidt C, et al. Pancreatic adenocarcinoma cell lines show variable susceptibility to TRAIL-mediated cell death. Pancrease 2001 Jul; 23(1): 72-79.
11. Maeta T, Hao C, Tron VA. Ultraviolet Light(UV) Regulation of the TNF family Decoy Receptors DcR2 and DcR3 in Human Keratinocytes. J Cutan Med Surg 2001; 5(4): 294-8.
12.Jodo S, Kobayashi S, Nakajima Y,et al. Elevated serum levels of soluble Fas/Apo-1(CD95) in patient with hepatocelluler carcinoma. Clin Exp Immunol, 1998; 111(2):166.
13.Scheikh MS, Fomace AJ. Death and decoy receptors and p53-mediated apoptosis leukemia. 2000 Aug; 14(8):1509-13.
14.Zhang J,Salcedo TW, Wan X,et al. Modulation of T-cell responses to alloantigens by TR6/DcR3. Clin Invest.2001; 107(11): 1459-68.
15.Roughton SA, Lareu RR, Bittles AH., et al. Fas and Fas Ligand Messenger Ribonucleic Acid and Protein Expression in the Rat Corpus Luteum during Apoptosis-Mediated Luteolysis. Biology of Reproduction.1999;60:797-804.
16.Conolly K, Cho YH, Duan R, et al. In vivo inhibition of Fas ligand-mediated killing by TR6, a Fas ligand decoy receptor. J Pharmaco Exp Ther.2000;298(l):25-33.
17.Zhai Y, Guo R, Hus TL, et al. LIGHT, A Novel Ligand for Lymphotoxin b Receptor and TR2/HVEM Induces Apoptosis and Suppresses In Vivo Tumor Formation Via Gene Transfer. J Clin Invest. 1998;102:1142-1151.
18.Harrop JA, Mcdonnell PC, Brigham Burke M, et al. Antibodies to TR2(Herpesvirus Entry Mediator), a New Member of the TNF Receptor Superfamily,Block T Cell Proliferation,Expression of Activation Markers, and Production of Cytokines. J Immuno. 1998,161:1786-1794.
19.Altura RA, Maris JM,Li H, et al. Novel regions of chromosomal loss in familial neuroblastoma by comparative genomic hybridization. Genes Chromosome Cancer. 1997; 19:176-184
20.Mild G, Bachmann F, Boulay JL, et al. DcR3 locus is a predictive marker for 5-fluorouracil-based adjuvant chemotherpy in colorectal cancer. Int J Cancer, 2002, 102: 254-257.
21.Victor J,Witcher DR,Becker GW,et al.Decoy receptor 3 (DcR3) is proteolytically processed to a metabolic fragment having differential activities against Fas ligand and LIGHT. Biochem Pharmacol .2003 ;65;657-667.
22.Yu lian Wu,Bing Han,Sheng H,et al.Clinical significance of detecting elevated serum DcR3/TR6 / M68 in malignant tumor patients. Int J Cancer,2003,105:724-732.
23.Ohshima K,Haraoka S, Sugihara M, et al. Amplification and expression of a decoy receptor for Fas ligand (DcR3) in virus (EBV or HTLV-1) associated lymphomas.Cancer Lett.2000;160(1):89-97.
24.Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA, 1978. 75 (1):280-4
25.Izant J, Weintraub H. Inhibition of thymidine kinase gene expression by antisense RNA: a molecular approach to genetic analysis.Cell.1984;36(4):1007-15.
26Uhlmann E. Peptide nucleic acids (PNA) and PNA-DNA chimeras:from high binding affinity towards biological function. Bio Chem. 1998;379(8-9): 1045-52.
27.Connor DS, Schechner JS, Adida C, et al. Control of apoptosis during angiogenesis by survin express in endothelial cell. Am J Pathol.2000;156(2):393-398.
28.Blanc Brude OP,Yu J, Simosa H, et al. Inhibitor of apoptosis protein survin regulates vascular injury. Nat Med.2002Sep;8(9):987-9.
29.Deguchi M, Shiraki K, Inoue H, et al. Expression of survivin during liver regeneration. Biochem Biophys Res.2002 Sep13;297(1):59.