| 摘要: |
| 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-pEGFP-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(0.1 mL,1×109 cfu/mL),在口服后24 h、48 h、5 d、10 d、20 d、30 d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-N1,5 d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。 |
| 关键词: 鼠伤寒沙门菌 绿色荧光蛋白 遗传载体 基因疗法 涎腺肿瘤 |
| DOI：10.3724/SP.J.1008.2013.00382 |
| 投稿时间：2012-10-24修订日期：2013-02-06 |
| 基金项目:国家自然科学基金(30572060). |
|
| Intratumoral enrichment of gene delivered by oral administration of recombinant attenuated Salmonella typhimurium in tumor-bearing nude mice |
| JIANG Zhong-ming*,YIN Xin-min,YU Yue,MIAO Liang,WEI Wei,ZHOU Yan,YIN Xue-meng,WU Ying-ying |
| (Department of Stomatology, BenQ Medical Hospital, Nanjing Medical University, Nanjing 210019, Jiangsu, China) |
| Abstract: |
| Objective To observe the distribution and expression of exogenous gene delivered by oral administration of recombinant attenuated Salmonella typhimurium in nude mouse model carrying human saliva gland adenoid cystic carcinoma. Methods The attenuated Salmonella typhimurium of green fluorescent protein (GFP) gene expression was constructed by transfecting prokaryotic and eukaryotic expression plasmid vector into the attenuated Salmonella typhimurium SL7207, and the recombinant attenuated Salmonella typhimurium SL7207-pUC-GFP for prokaryotic expression and SL7207-pEGFP-N1 for eukaryotic expression were obtained. The expression stability of GFP gene in the prokaryotic SL7207-pUC-GFP was tested for 10 times in vitro. The nude mouse models carrying human saliva gland adenoid cystic carcinoma were orally administered with prokaryotic SL7207-pUC-GFP (0.1 mL,1×109 cfu/mL), and then the distribution of the attenuated Salmonella typhimurium in the liver, spleen and tumor tissues were assessed by observing GFP expression in SL7207-pUC-GFP clones cultured out of the tissues homogenate at 24 h, 48 h, 5 d, 10 d, 20 d, and 30 d after oral administration. The nude mouse models were also orally administered with eukaryotic SL7207-pEGFP-N1, and the expression of GFP in the liver, spleen and tumor tissues was observed in frozen tissue sections 5 d later under fluorescence microscope. Results The expression of GFP harbored by the recombinant attenuated Salmonella typhimurium SL7207-pUC-GFP was not reduced or lost after 10 times of in vitro passaging. After the oral administration of prokaryotic SL7207-pUC-GFP, the attenuated Salmonella typhimurium could survive in the liver, spleen and tumor tissues for a long time, with the clone numbers in tumor tissues being significantly higher than those in the liver and spleen tissues at all the time points (P<0.05). After oral administration of eukaryotic SL7207-pEGFP-N1, the expression of GFP was higher in tumor tissues than in the liver and spleen tissues. Conclusion The recombinant attenuated Salmonella typhimurium can be enriched in human saliva gland adenoid cystic carcinoma cells and deliver the harbored exogenous gene into the tumor cells for expression, showing a double advantage for gene therapy. |
| Key words: Salmonella typhimurium green fluorescent protein genetic vectors gene therapy salivary gland neoplasms |
