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目的观察重组减毒鼠伤寒沙门菌作为基因载体在人涎腺腺样囊性癌裸鼠移植瘤中的富集情况和对外源基因的呈递能力。方法以绿色荧光蛋白基因(GFP)为报告基因,以减毒鼠伤寒沙门菌SL7207为转基因载体,分别构建原核启动GFP表达的重组减毒鼠伤寒沙门菌SL7207-pUC-GFP和真核启动GFP表达的重组减毒鼠伤寒沙门菌SL7207-pEG-FP-N1。原核菌SL7207-pUC-GFP在体外连续传代,观察GFP基因表达的稳定性;同时,对荷人涎腺腺样囊性癌皮下移植瘤裸鼠模型口服给予原核菌SL7207-pUC-GFP(0.1mL,1×109cfu/mL),在口服后24h、48h、5d、10d、20d、30d处死荷瘤裸鼠,获取肝、脾及肿瘤组织并制成匀浆液进行重组菌培养及GFP表达检测,观察重组菌在瘤体细胞内富集情况。对荷瘤裸鼠模型口服给予真核菌SL7207-pEGFP-N1,5d后取肝、脾及肿瘤组织进行冰冻组织切片,荧光显微镜下观察GFP的表达,了解重组菌携带外源基因在肿瘤细胞内的表达。结果携带GFP原核表达的重组减毒鼠伤寒沙门菌SL7207-pUC-GFP在体外连续传代10次未见表达减少或缺失。荷瘤裸鼠口服原核表达GFP基因重组菌SL7207-pUC-GFP菌液实验表明,重组菌SL7207-pUC-GFP在肝、脾及肿瘤组织中能长期存活,以肿瘤组织中聚集明显(P<0.05)且维持时间较长。荷瘤裸鼠口服真核表达GFP基因重组菌SL7207-pEGFP-N1菌液实验表明,相对肝脏和脾脏组织,外源基因在肿瘤细胞内表达量最高。结论重组减毒鼠伤寒沙门菌可以在瘤体细胞内富集存活,并且携带的外源基因可以释放到肿瘤细胞内表达,具有作为基因治疗载体的双重优势。
Objective To observe the enrichment of recombinant Salmonella typhimurium as gene carrier in human salivary adenoid cystic carcinoma xenografts in nude mice and the presentation of foreign gene. Methods GFP was used as reporter gene and Salmonella typhimurium SL7207 was attenuated to construct the recombinant attenuated Salmonella typhimurium SL7207-pUC-GFP with prokaryotic initiation GFP expression and eukaryotic initiation GFP expression Of recombinant attenuated Salmonella typhimurium SL7207-pEG-FP-N1. The prokaryotic bacterium SL7207-pUC-GFP was serially passaged in vitro to observe the stability of GFP gene expression. At the same time, the prokaryotic strain SL7207-pUC-GFP was orally administered to human subcutaneous xenografted adenoid cystic carcinoma in nude mice , 1 × 109cfu / mL). The nude mice were sacrificed 24h, 48h, 5d, 10d, 20d and 30d after oral administration to obtain the liver, spleen and tumor tissues and the homogenates were harvested for culture and GFP expression. Recombinant bacteria enrichment in the tumor cells. Eukaryotic bacterium SL7207-pEGFP-N1 was orally administered to the tumor-bearing nude mice model. After 5 days, the liver, spleen and tumor tissues were harvested for frozen sections. The expression of GFP was observed under a fluorescence microscope to find out whether the recombinant bacterium carries the exogenous gene in the tumor cells expression. Results The recombinant attenuated Salmonella typhimurium SL7207-pUC-GFP harboring prokaryotic expression of GFP showed no decrease or deletion of expression in 10 consecutive passages in vitro. The recombinant plasmid SL7207-pUC-GFP was successfully transfected into the tumor-bearing nude mice by oral administration. The results showed that the recombinant strain SL7207-pUC-GFP could survive for a long time in liver, spleen and tumor tissues, ) And maintain a longer time. Tumor-bearing nude mice orally eukaryotic expression of recombinant GFP gene SL7207-pEGFP-N1 bacterial liquid experiments showed that relative to the liver and spleen tissue, exogenous gene expression in tumor cells in the highest. Conclusion Recombinant attenuated Salmonella typhimurium can accumulate in tumor cells, and the foreign gene can be released into the tumor cells to express, which has the dual advantage of being gene therapy vector.