鼻咽癌的靶向性放射增敏基因治疗
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摘要
目的:构建放射敏感的融合启动子E6hTERTp,观察在不同放射剂量诱导下放射敏感增强子增强hTERTp(人端粒酶催化亚基启动子)启动下游基因转录活性及对特异性的影响;并进一步构建含E6hTERTp以及融合UL49片段的自杀基因CD/UPRT.UL49的质粒载体,放射干预诱导其在鼻咽癌CNE-2细胞表达,观察放射条件下该自杀基因/前药系统在鼻咽癌细胞中的体内以及体外放射增敏效应,为探索新的鼻咽癌放射基因治疗奠定实验基础。
方法和结果:
第一部分:放射诱导型增强子E6对hTERTp靶向性增敏效应研究
方法:overlap PCR扩增融合启动子E6hTERTp以及CD/UPRT.UL49片段。基因重组技术构建融合启动子E6hTERTp的报告基因载体PGL3—E6hTERTp,以EGR-1p(早期生长反应因子1启动子)以及hTERTp为对照,经脂质体介导重组质粒与pRL-SV40质粒共转染鼻咽癌CNE-2细胞以及正常人成纤维细胞(HDF),不同放射剂量(0,2,6,10gy)诱导下通过双荧光素酶分析系统检测报告基因的表达效率,研究不同放射剂量对各启动子启动下游基因转录活性及对其特异性的影响;构建自杀基因载体pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49以及pcDNA3.1(-)E6.hTERTp.CD/UPRT,脂质体介导转染鼻咽癌CNE-2细胞,在0gy,2Gy,6gy,10gy等不同放射条件下,半定量RT-PCR法检验其转录活性,免疫印迹法检测CD/UPRT.UL49以及CD/UPRT蛋白表达:
结果:双荧光素酶分析系统检测显示,正常HDF细胞系中,转染PGL3-E6.hTERTp组以及PGL3-hTERTp组无论照射与否,其启动活性均维持在极低水平;在给予0Gy,2Gy,6Gy,以及10Gy放射干预之后,转染CNE-2细胞的各启动子活性均随放射剂量的增加而增加,其中,转染E6.hTERTp各放射剂量组启动活性分别为18.8184±4.1969,102.6512±20.5879,291.7274±35.4752.407.5505±27.1526,放射组比较未放射组分别增加了5.4倍,15.2倍和21.7倍,各放射组两两间同样有显著性差异(p＜0.05)。E6.hTERTp2Gy组、6Gy组以及10Gy组启动活性分别是hTERTp相同放射剂量组的2.4倍,3.5倍和2.8倍,两个启动子组间差异具有统计学意义(p＜0.05),证实鼻咽癌CNE-2细胞中,放射诱导型增强子E6能够明显提高hTERTp放射敏感性,且不影响其肿瘤特异性。
自杀基因载体pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49以及pcDNA3.1(-)E6.hTERTp.CD/UPRT转染CNE-2细胞后,WESTERNBLOTTING显示检测到的特异性蛋白条带。RT-PCR检测显示所有经照射后的实验组mRNA量均要显著高于未照射组(p＜0.05),其中CD/UPRT在6Gy时出现最高值,而CD/UPRT.UL49在6Gy和10Gy处理下,mRNA量没有显著差别(p＞0.05),但均高于0Gy,2Gy时mRNA转录量(p＜0.05);证实放射干预可激发融合启动子E6hTERTp启动下游自杀基因表达。
第二部分启动子E6hTERTp介导的自杀基因CD/UPRT.UL49以及CD/UPRT表达对鼻咽癌放射—基因治疗增敏效应的体外实验
方法:自杀基因载体pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49以及pcDNA3.1(-)E6.hTERTp.CD/UPRT经脂质体介导转染鼻咽癌CNE-2细胞,在0gy,2Gy,6gy,10gy等不同放射条件下,高效液相色谱法检测其促进前药系统5-Fc(5-氟胞嘧啶)转化为5-FU(5-氟尿嘧啶)的效率,MTT(四甲基偶氮唑蓝)法检测不同放射剂量下,不同浓度5-Fc对转染自杀基因载体的的CNE-2细胞的杀伤效应并进行两种不同基因治疗组之间以及各基因治疗组与单纯放射治疗组之间细胞存活率对比,用SPSS统计分析软件进行分析。
结果:给予前药5-Fc以及放射干预后,高效液相色谱法检测发现,经过照射后转染自杀基因载体pcDNA3.1(-)E6.hTERTp.CD/UPRT以及pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49载体的孔中均检测到5-Fu,未照射孔没有检测到5-Fu;
MTT检测,单纯前药干预浓度为200ug/ml时,细胞生存率为93.04;转染自杀基因载体后,前药浓度为200(ug/ml),给予2Gy,6Gy,10Gy放射干预,CNE-2细胞存活率较野生CNE-2细胞组均明显下降(p＜0.05);放射剂量为2Gy以及10Gy时转染pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49组比较转染pcDNA3.1(-)E6.hTERTp.CD/UPRT组放射增敏效果更明显(p＜0.05)。
第三部分前体药物/自杀基因系统对鼻咽癌放射增敏的原位基因治疗实验研究
方法:CNE-2细胞接种裸鼠皮下建立鼻咽癌动物模型,进行脂质体包裹质粒DNA瘤内注射的原位基因(in situ)治疗实验,待肿瘤长宽径达到0.7-1.0cm时,将裸鼠随机分组,瘤内注射脂质体包裹的DNA质粒,以及腹腔注射5-FC,并给予肿瘤局部每天3Gy照射,总剂量9Gy。记录肿瘤体积,比较不同处理组的抑瘤效应;病理切片证实肿块性质。抽提肿瘤组织RNA,RT-PCR反应,鉴定CD/UPRT.UL49基因在肿瘤组织内是否有表达。
结果:1×10~7CNE-2细胞接种裸鼠后,9天内均顺利成瘤。未经放射的自杀基因pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49/前药治疗组没有明显抑瘤效果;放射-自杀基因pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49/前药治疗组生长抑制率93.7579,优于其他各放射组(p＜0.05),而放射-自杀基因pcDNA3.1(-)E6.hTERTp.CD/UPRT/前药治疗组生长抑制率88.86427,较其放射治疗对照组没有差别(p＞0.05)。病理切片检查各放射组均出现严重坏死。
结论:E6.hTERTp.CD/UPRT.UL49自杀基因载体具有放射诱导和肿瘤特异性双重特性,融合了VP22基因片段可以明显增强放射干预下CD/UPRT/5-FC前药系统对鼻咽癌CNE-2细胞的杀伤效应,为鼻咽癌的基因-放射治疗开辟了新思路。
Objective: Construct radiosensitive novel fusion promoter E6hTERTp. Observe the effect of radiosensitive enhancer to hTERT promoter's specifity and radiosensitivuty after different dose of radiation; construct vector carrying E6hTERTp and fusion suicide gene CD/UPRT.UL49, induce the expression of the suicide gene in CNE-2 cells with different dose of radiation to find the radiosensitization effect of the radio -suicide gene /prodrug system in vitro and vivo to lay a solid experimental foundation for a new stratety for radio-gene therapy.
Method and results
The first part: Research on hTERTp's tumor-specific promotive effect enhanced by radiosensitive enhancer E6
Method: The fusion gene E6.hTERTp and CD/UPRT.UL49 were constructed by overlap PCR. The combined promoter was inserted into PGL3 basic plasmid. PGL3-hTERTp and pGL3-EGR-lp were also constructed for comparison. All the plasmids together with pRL-SV40 plasmids were transfected into CNE-2 cells and HDF cells with liposome .The promotive effect of recombinant promoter E6.hTERTp in CNE-2 cells and HDF cells were measured by Dual Luciferase Reporter Gene Assay system and was compared with EGR-1p and hTERTp for difference between every two groups under different dose of radiation to find the enhancing effect of radiosensitive enhancer to hTERT promoter and its influence to specifity after different dose of radiation. Construct suicide gene vector pcDNA3.1(-)E6.hTERTp. CD/UPRT and pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49; transfect the plasmids into CNE-2 cells with liposome . after different dose (0Gy, 2Gy,6Gy,10Gy ) of radiation, the expression of suicide gene CD/UPRT.UL49 and CD/UPRT were analyzed by half -quantitive RT-PCR and WEST BLOTTING.
Results:
Dual Luciferase Reporter Gene Assay system revealed that the promotive effects of hTERT promoter and E6.hTERTp remains low in HDF cells with any dose of radiation ;the value between all radiation groups are not significantly different (p>0.05) ; While in CNE-2 cells ,increase of promotive effects in accordance with the radiation dose can be observed in all groups . The promotive effect of E6.hTERTp group were 18.8184±4.1969 , 102.6512±20.5879 , 291.7274±35.4752 , 407.5505±27.1526, increased by5.4, 15.2 and 21.7 times compared with baseline(OGy), the promotive effect of different radiation groups are statisticaly significant (p<0.05) . When the dose of radiation were 2Gy, 6Gy and 10Gy , the promotive effect of E6.hTERTp increased to2.4, 3.5 and 2.8 times the value of hTERTp group ,which proved that radiosensitive enhancer E6 increase the radiosensitive promotive effect of hTERTp in CNE-2 cells and do not affect its NPC specific character. When Suicide gene pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 and pcDNA3.1 (-) E6.hTERTp.CD/UPRT were transfected into CNE-2 cells, expression of the novel fusion suicide gene can be detected by western blotting test .RT-PCR shows that after radiation ,the amount of mRNA were significantly higher than that of 0Gy group (p<0.05). the amount of mRNA of pcDNA3.1 (-) E6.hTERTp.CD/UPRT reach the peak in 6Gy group; The amount is not signifieantly different for pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 in 6Gy and 10Gy group (p>0.05) but is significantly higher than 2Gy group (p<0.05) . The result revealed that radiation can evoke the activity of E6hTERTP and enhance the expression of downstream suicide gene.
The second part: radiosensitization effect by enhanced expression of novel fusion gene CD/UPRT.UL49 and CD/UPRTgene mediated by E6hTERTp in NPC
Method: Suicide gene pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 and pcDNA3.1 (-) E6.hTERTp.CD/UPRT were transfected into CNE-2 cells , and the transform rate of prodrug 5-Fc to 5-FU for the two suicide gene groups under different dose of radiation (0Gy, 2Gy,6Gy, 10Gy )were measured by HPLC.
The killing effect of both suicide gene/prodrug system were tested by MTT after different dose of radiation with different 5-Fc concentration and were analyzed for difference between the two treatment groups and simple radiation -prodrug groups with Spss software.
Results: After treated with prodrug 5-Fc and radiation, 5-FU can be detected from the supernatant of the culture media of CNE-2 transfected with both suicide gene ,which shows that after incorporation of VP22 segment ,the CD/UPRT protein maintained its bioactivity of transforming 5-Fc.
The MTT analysis showed that when the prodrug concentration was 200 (ug/ml) , the survival rate of wild CNE-2 group was 93.04,;while after radiation the survival rate of gene therapy group decrease significantly, the tendency of decrease is more obvious in CNE-2/pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 group (p<0.05) in 2Gy and 10Gy group .
The third part: Research on the radiosensitization effect of prodrug suicide gene therapy in situ
Method : CNE-2 cells in log phase were inoculated subcutaneously in nude mice to construct a nude mouse model of NPC ,and an in situ gene therapy was performed with plasmid packed by plasmid. When the size of tumors reaching 1.0-0.7cm, the nude mice were randomized to different grouped, and was given radiation , system administration of produg and in situ gene injection, the suppression of tumor growth was observed. Biopsy of the tumor were performed to find the difference between treatment groups .Then targeted RNA was isolated from tumor tissues and RT-PCR was performed to define the expression of suicide gene.
Results 9 days after inoculation, all the nude mouse developed transplanted tumor. In situ gene therapy experiment showed no suppressing effect of gene /prodrug system without radiation; compared with other radiated groups , growth of transplant tumor of radio-pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 /prodrug group was distinctly suppressed(P<0.05) ,the growth suppressing rate is 93.7579 While the radio-pcDNA3.1 (-) E6.hTERTp.CD/UPRT /prodrug group showed no sign of more suppressing effect compared with other radiation groups (p>0.05) ,the growth suppressing rate is88.86427.biopsy shows severe necrosis of all radiated group .
Conclusions:
The suicide gene E6.hTERTp.CD/UPRT.UL49/prodrug system, is both radiosensitive and tumor specifically effective in anticancer therapy, and exert powerful killing effect to CNE-2 cells than E6.hTERTp.CD/UPRT / prodrug system under radiation. From this study, we established a new strategy for radio-gene therapy of NPC.
方法和结果:
第一部分:放射诱导型增强子E6对hTERTp靶向性增敏效应研究
方法:overlap PCR扩增融合启动子E6hTERTp以及CD/UPRT.UL49片段。基因重组技术构建融合启动子E6hTERTp的报告基因载体PGL3—E6hTERTp,以EGR-1p(早期生长反应因子1启动子)以及hTERTp为对照,经脂质体介导重组质粒与pRL-SV40质粒共转染鼻咽癌CNE-2细胞以及正常人成纤维细胞(HDF),不同放射剂量(0,2,6,10gy)诱导下通过双荧光素酶分析系统检测报告基因的表达效率,研究不同放射剂量对各启动子启动下游基因转录活性及对其特异性的影响;构建自杀基因载体pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49以及pcDNA3.1(-)E6.hTERTp.CD/UPRT,脂质体介导转染鼻咽癌CNE-2细胞,在0gy,2Gy,6gy,10gy等不同放射条件下,半定量RT-PCR法检验其转录活性,免疫印迹法检测CD/UPRT.UL49以及CD/UPRT蛋白表达:
结果:双荧光素酶分析系统检测显示,正常HDF细胞系中,转染PGL3-E6.hTERTp组以及PGL3-hTERTp组无论照射与否,其启动活性均维持在极低水平;在给予0Gy,2Gy,6Gy,以及10Gy放射干预之后,转染CNE-2细胞的各启动子活性均随放射剂量的增加而增加,其中,转染E6.hTERTp各放射剂量组启动活性分别为18.8184±4.1969,102.6512±20.5879,291.7274±35.4752.407.5505±27.1526,放射组比较未放射组分别增加了5.4倍,15.2倍和21.7倍,各放射组两两间同样有显著性差异(p＜0.05)。E6.hTERTp2Gy组、6Gy组以及10Gy组启动活性分别是hTERTp相同放射剂量组的2.4倍,3.5倍和2.8倍,两个启动子组间差异具有统计学意义(p＜0.05),证实鼻咽癌CNE-2细胞中,放射诱导型增强子E6能够明显提高hTERTp放射敏感性,且不影响其肿瘤特异性。
自杀基因载体pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49以及pcDNA3.1(-)E6.hTERTp.CD/UPRT转染CNE-2细胞后,WESTERNBLOTTING显示检测到的特异性蛋白条带。RT-PCR检测显示所有经照射后的实验组mRNA量均要显著高于未照射组(p＜0.05),其中CD/UPRT在6Gy时出现最高值,而CD/UPRT.UL49在6Gy和10Gy处理下,mRNA量没有显著差别(p＞0.05),但均高于0Gy,2Gy时mRNA转录量(p＜0.05);证实放射干预可激发融合启动子E6hTERTp启动下游自杀基因表达。
第二部分启动子E6hTERTp介导的自杀基因CD/UPRT.UL49以及CD/UPRT表达对鼻咽癌放射—基因治疗增敏效应的体外实验
方法:自杀基因载体pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49以及pcDNA3.1(-)E6.hTERTp.CD/UPRT经脂质体介导转染鼻咽癌CNE-2细胞,在0gy,2Gy,6gy,10gy等不同放射条件下,高效液相色谱法检测其促进前药系统5-Fc(5-氟胞嘧啶)转化为5-FU(5-氟尿嘧啶)的效率,MTT(四甲基偶氮唑蓝)法检测不同放射剂量下,不同浓度5-Fc对转染自杀基因载体的的CNE-2细胞的杀伤效应并进行两种不同基因治疗组之间以及各基因治疗组与单纯放射治疗组之间细胞存活率对比,用SPSS统计分析软件进行分析。
结果:给予前药5-Fc以及放射干预后,高效液相色谱法检测发现,经过照射后转染自杀基因载体pcDNA3.1(-)E6.hTERTp.CD/UPRT以及pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49载体的孔中均检测到5-Fu,未照射孔没有检测到5-Fu;
MTT检测,单纯前药干预浓度为200ug/ml时,细胞生存率为93.04;转染自杀基因载体后,前药浓度为200(ug/ml),给予2Gy,6Gy,10Gy放射干预,CNE-2细胞存活率较野生CNE-2细胞组均明显下降(p＜0.05);放射剂量为2Gy以及10Gy时转染pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49组比较转染pcDNA3.1(-)E6.hTERTp.CD/UPRT组放射增敏效果更明显(p＜0.05)。
第三部分前体药物/自杀基因系统对鼻咽癌放射增敏的原位基因治疗实验研究
方法:CNE-2细胞接种裸鼠皮下建立鼻咽癌动物模型,进行脂质体包裹质粒DNA瘤内注射的原位基因(in situ)治疗实验,待肿瘤长宽径达到0.7-1.0cm时,将裸鼠随机分组,瘤内注射脂质体包裹的DNA质粒,以及腹腔注射5-FC,并给予肿瘤局部每天3Gy照射,总剂量9Gy。记录肿瘤体积,比较不同处理组的抑瘤效应;病理切片证实肿块性质。抽提肿瘤组织RNA,RT-PCR反应,鉴定CD/UPRT.UL49基因在肿瘤组织内是否有表达。
结果:1×10~7CNE-2细胞接种裸鼠后,9天内均顺利成瘤。未经放射的自杀基因pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49/前药治疗组没有明显抑瘤效果;放射-自杀基因pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49/前药治疗组生长抑制率93.7579,优于其他各放射组(p＜0.05),而放射-自杀基因pcDNA3.1(-)E6.hTERTp.CD/UPRT/前药治疗组生长抑制率88.86427,较其放射治疗对照组没有差别(p＞0.05)。病理切片检查各放射组均出现严重坏死。
结论:E6.hTERTp.CD/UPRT.UL49自杀基因载体具有放射诱导和肿瘤特异性双重特性,融合了VP22基因片段可以明显增强放射干预下CD/UPRT/5-FC前药系统对鼻咽癌CNE-2细胞的杀伤效应,为鼻咽癌的基因-放射治疗开辟了新思路。
Objective: Construct radiosensitive novel fusion promoter E6hTERTp. Observe the effect of radiosensitive enhancer to hTERT promoter's specifity and radiosensitivuty after different dose of radiation; construct vector carrying E6hTERTp and fusion suicide gene CD/UPRT.UL49, induce the expression of the suicide gene in CNE-2 cells with different dose of radiation to find the radiosensitization effect of the radio -suicide gene /prodrug system in vitro and vivo to lay a solid experimental foundation for a new stratety for radio-gene therapy.
Method and results
The first part: Research on hTERTp's tumor-specific promotive effect enhanced by radiosensitive enhancer E6
Method: The fusion gene E6.hTERTp and CD/UPRT.UL49 were constructed by overlap PCR. The combined promoter was inserted into PGL3 basic plasmid. PGL3-hTERTp and pGL3-EGR-lp were also constructed for comparison. All the plasmids together with pRL-SV40 plasmids were transfected into CNE-2 cells and HDF cells with liposome .The promotive effect of recombinant promoter E6.hTERTp in CNE-2 cells and HDF cells were measured by Dual Luciferase Reporter Gene Assay system and was compared with EGR-1p and hTERTp for difference between every two groups under different dose of radiation to find the enhancing effect of radiosensitive enhancer to hTERT promoter and its influence to specifity after different dose of radiation. Construct suicide gene vector pcDNA3.1(-)E6.hTERTp. CD/UPRT and pcDNA3.1(-)E6.hTERTp.CD/UPRT.UL49; transfect the plasmids into CNE-2 cells with liposome . after different dose (0Gy, 2Gy,6Gy,10Gy ) of radiation, the expression of suicide gene CD/UPRT.UL49 and CD/UPRT were analyzed by half -quantitive RT-PCR and WEST BLOTTING.
Results:
Dual Luciferase Reporter Gene Assay system revealed that the promotive effects of hTERT promoter and E6.hTERTp remains low in HDF cells with any dose of radiation ;the value between all radiation groups are not significantly different (p>0.05) ; While in CNE-2 cells ,increase of promotive effects in accordance with the radiation dose can be observed in all groups . The promotive effect of E6.hTERTp group were 18.8184±4.1969 , 102.6512±20.5879 , 291.7274±35.4752 , 407.5505±27.1526, increased by5.4, 15.2 and 21.7 times compared with baseline(OGy), the promotive effect of different radiation groups are statisticaly significant (p<0.05) . When the dose of radiation were 2Gy, 6Gy and 10Gy , the promotive effect of E6.hTERTp increased to2.4, 3.5 and 2.8 times the value of hTERTp group ,which proved that radiosensitive enhancer E6 increase the radiosensitive promotive effect of hTERTp in CNE-2 cells and do not affect its NPC specific character. When Suicide gene pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 and pcDNA3.1 (-) E6.hTERTp.CD/UPRT were transfected into CNE-2 cells, expression of the novel fusion suicide gene can be detected by western blotting test .RT-PCR shows that after radiation ,the amount of mRNA were significantly higher than that of 0Gy group (p<0.05). the amount of mRNA of pcDNA3.1 (-) E6.hTERTp.CD/UPRT reach the peak in 6Gy group; The amount is not signifieantly different for pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 in 6Gy and 10Gy group (p>0.05) but is significantly higher than 2Gy group (p<0.05) . The result revealed that radiation can evoke the activity of E6hTERTP and enhance the expression of downstream suicide gene.
The second part: radiosensitization effect by enhanced expression of novel fusion gene CD/UPRT.UL49 and CD/UPRTgene mediated by E6hTERTp in NPC
Method: Suicide gene pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 and pcDNA3.1 (-) E6.hTERTp.CD/UPRT were transfected into CNE-2 cells , and the transform rate of prodrug 5-Fc to 5-FU for the two suicide gene groups under different dose of radiation (0Gy, 2Gy,6Gy, 10Gy )were measured by HPLC.
The killing effect of both suicide gene/prodrug system were tested by MTT after different dose of radiation with different 5-Fc concentration and were analyzed for difference between the two treatment groups and simple radiation -prodrug groups with Spss software.
Results: After treated with prodrug 5-Fc and radiation, 5-FU can be detected from the supernatant of the culture media of CNE-2 transfected with both suicide gene ,which shows that after incorporation of VP22 segment ,the CD/UPRT protein maintained its bioactivity of transforming 5-Fc.
The MTT analysis showed that when the prodrug concentration was 200 (ug/ml) , the survival rate of wild CNE-2 group was 93.04,;while after radiation the survival rate of gene therapy group decrease significantly, the tendency of decrease is more obvious in CNE-2/pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 group (p<0.05) in 2Gy and 10Gy group .
The third part: Research on the radiosensitization effect of prodrug suicide gene therapy in situ
Method : CNE-2 cells in log phase were inoculated subcutaneously in nude mice to construct a nude mouse model of NPC ,and an in situ gene therapy was performed with plasmid packed by plasmid. When the size of tumors reaching 1.0-0.7cm, the nude mice were randomized to different grouped, and was given radiation , system administration of produg and in situ gene injection, the suppression of tumor growth was observed. Biopsy of the tumor were performed to find the difference between treatment groups .Then targeted RNA was isolated from tumor tissues and RT-PCR was performed to define the expression of suicide gene.
Results 9 days after inoculation, all the nude mouse developed transplanted tumor. In situ gene therapy experiment showed no suppressing effect of gene /prodrug system without radiation; compared with other radiated groups , growth of transplant tumor of radio-pcDNA3.1 (-) E6.hTERTp.CD/UPRT.UL49 /prodrug group was distinctly suppressed(P<0.05) ,the growth suppressing rate is 93.7579 While the radio-pcDNA3.1 (-) E6.hTERTp.CD/UPRT /prodrug group showed no sign of more suppressing effect compared with other radiation groups (p>0.05) ,the growth suppressing rate is88.86427.biopsy shows severe necrosis of all radiated group .
Conclusions:
The suicide gene E6.hTERTp.CD/UPRT.UL49/prodrug system, is both radiosensitive and tumor specifically effective in anticancer therapy, and exert powerful killing effect to CNE-2 cells than E6.hTERTp.CD/UPRT / prodrug system under radiation. From this study, we established a new strategy for radio-gene therapy of NPC.
引文
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