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【摘要】 目的 探讨构建含有Tip30与人IFN-γ基因的真核表达及共表达质粒的重组减毒伤寒沙门菌对腺样囊性癌肺转移的抑制作用。方法 将25只4周龄BALB/c nu nu雄鼠随机分成5组，每组5只，其中对照组5只（PBS）、 单纯减毒沙门菌组5只（SL7207）、携带人IFN-γ基因减毒沙门菌治疗组5只（SL7207/pCI-IFN-γ、携带Tip30基因减毒沙门菌治疗组5只（SL7207/pCI-Tip30）、携带Tip30与人IFN-（ 基因的真核共表达质粒的重组减毒沙门菌治疗组5只（SL7207/pCI-Tip30/IFN），均于尾静脉接种高转移腺样囊性癌（ACC-M）细胞。10天后对照组裸鼠口服PBS，其余各治疗组口服相应的减毒沙门菌，隔周1次，共3次。并对裸鼠肺转移肿瘤细胞内IFN-γ、Tip30的表达、肿瘤生长体积、瘤体重量、带瘤存活期进行检测。结果 治疗组裸鼠肺转移肿瘤细胞胞浆内有对应表达IFN-γ、Tip30；各治疗组均较对照组肿瘤生长慢，肿瘤体积小，重量轻，抑瘤率高，带瘤存活期长；携带基因治疗组较SL7207治疗组肿瘤生长慢，肿瘤体积小，重量轻，抑瘤率高，带瘤存活期长；联合基因治疗组较单基因治疗组肿瘤生长慢，肿瘤体积小，重量轻，抑瘤率高，带瘤存活期长。结论 减毒伤寒沙门菌不仅能作为载体介导基因对腺样囊性癌肺转移有抑制作用，而且自身对腺样囊性癌肺转移也有抑制作用。联合基因较单基因抑制效果好，有协同作用。
【关键词】 Tip30；干扰素-γ； 减毒伤寒沙门菌；联合基因治疗
A study of attenuated Salmonella-mediated gene inhibitor effect on the lungs metastasis transplant tumor in BALB/C nude mice with human salivary adenoid cystic carcinoma
JIANG Zhong-ming, ZHAO Ping,CAO Zhi-zhong,LIU Jun,ZHOU Zhong-hua,L Chun-tang
（1.Department of Stomatology, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China; 2.Department of Microbiology, The Second Military Medical University, Shanghai 200433, China）
【Abstract】 Objective To observe the constructed attenuated Salmonella typhimurium（AST） haboring an eukaryotic expression plasmid encoding Tip30, an eukaryotic expression plasmid encoding human IFN-γ and an eukaryotic co-expression plasmid encoding Tip30 and human IFN-γ gene inhibitor effect on the lungs metastasis transplant tumor growth in BALB/C nude mice with human salivary adenoid cystic carcinoma.Methods Twenty five 4 week old BALB/C male nude mice were divided randomly into 5 groups: control group （PBS group）, simple attenuated Salmonella group （SL7207 group）, AST harboring plasmid pCI-IFN group （SL7207/pCI-IFN group）, AST harboring plasmid pCI-Tip30 group （SL7207/pCI-Tip30 group）, and AST harboring co-expressing plasmid pCI-Tip30/IFN group （SL7207/pCI-Tip30/IFN group）. All groups were injected ACC-M cells suspension at the tail vein. The control group received oral PBS 10 d after injection, and the other groups received different recombinant Salmonella typhimurium 3 times weekly. The expressions of Tip30 and human IFN-γ gene in ACC-M lungs metastasis transplant tumor cells of BALB/C mice, tumor size, weight, the tumor-bearing survival times were detected. Results There were Tip30 and human IFN-γ gene expression corresponding in ACC-M lungs metastasis transplant tumor cells cytoplasm of BALB/C mice of the therapy groups; the tumor size, weight of the therapy groups were lower than that of the control group, the rate of suppressing of the therapy groups were higher than that of the control group, the tumor-bearing survival times were longer than that of the control group; the tumor size, weight of the therapy groups haboring Tip30 and human IFN-γ gene were lower than that of the SL7207 group, the rate of suppressing of the therapy groups were higher than that of the control group, the tumor-bearing survival times were longer than that of the SL7207 group; the tumor size, weight of the fusion genes therapy group were lower than that of the single gene groups, the ratio of suppressing tumor of the therapy groups were higher than that of the control group, the tumor-bearing survival times were longer than that of the single gene groups. Conclusion The attenuated Salmonella typhimurium had evident inhibitor effect as gene vector mediated gene on the lungs metastasis transplant tumor growth in BALB/C nude mice with human salivary adenoid cystic carcinoma, but also had evident inhibitor effect on the lungs metastasis transplant tumor in nude mice with human salivary adenoid cystic carcinoma. The fusion genes inhibitor effect had better than that of the single gene, indicating there is synergy between the 2 genes.
【Key words】 Tip30; interferon-γ; attenuated Salmonella typhimurium; fusion genes therapy
The human salivary adenoid cystic carcinoma was one of the common malignancy tumor in oral and maxillofacial region, its morbidity was the second salivary malignancy tumor, it was common occurred in large and small salivary in clinical, its morbidity was ascendant in the recent year. The human salivary adenoid cystic carcinoma had higher local invasion and metastasis. The routine therapy methods were the integrated therapy conclude operation, actinotheraphy and chemotherapy at the present time, but the effect of therapy did not satisfaction and about 30%～40% patients had relapsed in 2 years after therapy, mostly modality were the local soakage, lungs metastasis, bones metastasis and local lymph nodes metastasis. The metastasis of salivary adenoid cystic carcinoma had characteristic indulge in lungs that had different from the metastasis of oral squamous cell carcinoma had mostly in local lymph nodes. Fordice J［1］ studied a lot of cases, then found that although the human salivary adenoid cystic carcinoma was passed by integrated therapy but about 21.9% patients had lungs metastasis, lungs metastasis were the crucial factor that induced the ratio of the survive evidently decline after operation. Then, this experiment was studied that attenuated Salmonella-mediated Tip30 and human IFN-γ gene inhibitor effect on the lungs metastasis transplant tumor in BALB/C nude mice with human salivary adenoid cystic carcinoma.
1 Meterials
1.1 Tumor cell lines ACC-M tumor cell lines purchased from laboratory room of Oral and Maxillofacial Surgery, Affiliated 9th People's Hospital, Shanghai Second Medical University.
1.2 Mice BALB/c male nude mice purchased from Shanghai Central of Experimental Animal, Academy of Science, China. Mice were used between 4 weeks of age.
1.3 Plasmid Plasmid pcDNA-Tip30 encoding Tip30 gene was kindly provided by Professor Xiao Hua （Nebraca University, American）
1.4 Bacterial strains The S.typhimurium stain LB5000 and the attenuated S.typhimurium AroA-stain SL7207 was kindly provided by Dr.B.A.D.Stocker （Stanford University, Stanford, CA）
1.5 Reagent The monoclonal antibody anti-Tip30 was kindly provided by Professor Xiao Hua （Nebraca University, American）;
The monoclonal antibody anti-human IFNγ- purchased from Kang Cheng biological product company limited.
1.6 Instrument YWY781B microwave instrument for medicine produced by Zhejing Lin-an Ai-di electron instrument factory.
2 Methods
2.1 Construction of the genetically engineered Salmonella typhimurium Three recombinant expression plasmids pCI-tip30, pCI-IFN and co-expression plasmid pCI-tip30/IFN were transformed into an attenuated AroA- autotrophic mutant of Salmonella typhimurium SL7207 according to Jiang Zhongming's［2］ methods, named SL7207/pCI-IFN、SL7207/ pCI-Tip30、SL7207/pCI-Tip30/IFN respectively.
2.2 Construction of the model of the lungs metastasis transplant tumor in BALB/C nude mice with human salivary adenoid cystic carcinoma The higher metastasis human salivary adenoid cystic carcinoma （ACC-M）cells were routine revived, generation cultured, modulated the cells concentration to 1.5×107 cells ml-1L, all mice applied 0.2 ml ACC-M cells suspension injection at the tail vein of BALB/C mice, altogether 25 mice, divided randomly into 5 groups, each group had 5 mice.
2.3 Cultured and measured the attenuated Salmonella typhimurium SL7207、SL7207/pCI-IFN、SL7207/pCI-Tip30 and SL7207/pCI-Tip30/IFN bacterial colonies were inoculated in Luria-bertani（LB） medium supplemented with 100mg/L ampicillin, the bacteria were grown at 37℃ with continuous shaking overnight, 30μl bacteria were fetched to inoculate in 2×YT medium respectively next day, cultured to 1.0 in A600nm, bacteria were centrifuged at 4℃4000rpm 10min, abandoned the supernatants, diluted to 1ml in PBS, inhaled 50μl and diluted 60 times, measured optical density at 600nm, calculated the bacteria concentration（1 OD600＝2×107cfu/ml）.
2.4 Mice with human salivary adenoid cystic carcinoma were cured by oral gavage with recombinant attenuated Salmonella typhimurium After 10 days of the ACC-M cells inoculated, every mouse was administered by oral gavage with 5% NaHCO3 0.2ml. 30mins later, 4 therapy groups mice were cured by oral gavage with 1×108cfu recombinant attenuated Salmonella typhimurium SL7207, SL7207/pCI-IFN, SL7207/pCI-Tip30 and SL7207/pCI-Tip30/IFN respectively, and the another group mice were administered by oral gavage with 0.2 ml PBS as the control group, three times at 1-wk intervals.
2.5 Immunoassay Mice were put to death, lungs metastasis transplant tumors were taken out, portions of the tumor were fixed in 10% formalin, dehydration with gradient alcohol, embedded in paraffin, sectioned, sections were immunostained routinely with anti-Tip30 Ab or anti-human IFN-γ Ab. Judgement standards: the positive stained was the brown grains appeared in tumor cells cytoplasm.
2.6 Comparison of the ratio of suppressing tumor Mice were put to death, lungs metastasis transplant tumors were taken out, weighed amount, the ratio of suppressing tumor was calculated according to the follow formula.
The ratio of suppressing tumor = ［ average tumor weight of PBS group（mg）- average tumor weight of therapy group（mg）］ / average tumor weight of PBS group（mg）×100%.
2.7 Statistical analysis Significance of differences was calculated according to Student's t test. P＜0.05 was considered significant.
3 Results
3.1 There were 24 lungs metastasis transplant tumor formed in 25 BALB/C nude mice bearing ACC-M cell, the ratio of forming tumor was 96%.
3.2 Human IFN-γ gene expression in ACC-M lungs metastasis transplant tumor cells cytoplasm of BALB/C mice （Fig 1, 2）
3.3 Tip30 gene expression in ACC-M lungs metastasis transplant tumor cells cytoplasm of BALB/C mice （Fig 3, 4）
3.4 The survival time of lungs metastasis transplant tumor of BALB/C nude mice （Fig 5）
3.5 Comparison the weight of lungs metastasis transplant tumor and the ratio of suppressing tumor （Fig 6）
The average tumor weight was 0.516g in PBS group; it was 0.398g in SL7207 group, the ratio of suppressing tumor was 22.87%, there was significant difference（P＜0.05 vs PBS group）. The average tumor weight was 0.28g in SL7207/pCI-IFN group; it was 0.254g in SL7207/pCI-Tip30 group, the ratios of suppressing tumor were 45.74% and 50.78% respectively, there were significant difference（P＜0.05 vs SL7207 group，P＜0.01 vs PBS group，respectively）. The average tumor weight was 0.15g in SL7207/pCI-Tip30/IFN group, the ratio of suppressing tumor was 70.93%, there was significant difference（P＜0.01 vs PBS group , SL7207 group and SL7207/pCI-IFN group，P＜0.05 vs SL7207/pCI-Tip30 group）.
4 Discussion
Tip30 was a new found suppressing carcinoma gene. Tip30 as a cellular cofactor, which stimulates HIV- I Tat-activated transcription by interacting with both Tat and RNA polymerase Ⅱ, Tip30 is identical to a novel anti-tumor activity for the metastasis suppressor gene CC3 indicates that Tip30 might facilitate suppression of tumor metastasis and growth by up-regulating the transcription of genes involved in apoptosis and up-regulating the expression of angiogenesis suppressing gene, meanwhile down- regulating the expression of angiogenesis stimulation gene［3～5］. Thus, it had potential applied prospect on tumor prevention and therapy.
IFN-γ was multifunctional cell factor major secreted by T cell and NK cell, it had an effect of direct suppressing the DNA synthesis and cell division, and it could arrest or slow the conversion process of normal cell to tumor cell by suppressing the expression of carcinoma gene, furthermore, it could activate direct kill tumor cell such as macrophagocyte and NK cell. At present, IFN-γ was widely applied on therapy malignant tumor in clinic［6,7］, it was widely applied and studied on carcinoma of tongue, salivary adenoid cystic carcinoma, mucoepidermoid carcinoma and carcinoma of gingival in oral and maxillofacial region, and acquired some better effect［8］.
The fusion genes therapy was a developing direction of gene therapy, at present, the study was mostly focus on fusion application of some different purpose genes, in order to seek a best model of tumor therapy. In view of IFN-γ had an effect of direct and indirect kill tumor cell and Tip30 suppressing tumor angiogenesis and inducing tumor cell apoptosis, this study fusion this two genes suppressing lungs metastasis of salivary adenoid cystic carcinoma, in order to suppressing tumor metastasis though different patterns to inducing tumor cell apoptosis and suppressing tumor angiogenesis.
In the current study, we have demonstrated that Tip30 and IFN-γ genes could express in tumor cell and effective suppress tumor cell metastasis in model of ACC-M mice by oral administration of recombinant attenuated Salmonella typhimurium haboring an eukaryotic expression plasmid encoding Tip30, an eukaryotic expression plasmid encoding IFN-γ and an eukaryotic co-expression plasmid encoding Tip30 and IFN-γ gene, the two genes had obvious synergistic action.
Previous reports have demonstrated that attenuated Salmonella typhimurium itself could suppress tumor metastasis by eliciting a strong antitumor immunity［9］, the current study also demonstrated this results, but this model was the T cell deficiency ACC-M nude mice, thus, the mechanism had to be for further study.
This results original presented that fusion Tip30 and IFN-γ genes could suppress lungs metastasis of salivary adenoid cystic carcinoma of ACC-M nude mice by attenuated Salmonella typhimurium vector via the oral route, which may provide a new gene therapy on salivary adenoid cystic carcinoma.
（The figs on inside back cover）
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基金项目：国家自然基金资助项目（编号：30171055,30572060）
作者单位： 1 200433 上海，第二军医大学长海医院口腔科
2 200433 上海，第二军医大学基础医学部微生物教研室
（编辑：黄杰）