CAJ | 학술논문

目的: 研究携带Slug-siRNA的rAAV对裸鼠体内胰腺癌的作用.方法: 建立人胰腺癌裸鼠皮下移植瘤模型,2wk后随机将建模成功的裸鼠分为3组,每组6只. 构建携带Slug-siRNA的rAAV载体,采用瘤体内多点注射的方法,阴性对照组裸鼠按1×109 puf(50 μL)标准注射rAAV-EGFP,空白对照组裸鼠注射等量生理盐水,实验组裸鼠按1×109 puf(50 μL)标准注射rAAV2-EGFP-slugsiRNA,只注射1次. 观察裸鼠的一般情况,摄食,活动能力,每周用电子天平秤体质量,绘制裸鼠生长曲线. 治疗6 wk后二氧化碳吸入法处死裸鼠,切取肿瘤称质量;免疫组织化学SP法检测slug蛋白的表达,TUNEL检测皮下移植瘤细胞凋亡,透射电镜检测细胞超微结构.结果: 3组裸鼠分别接受不同的处理后,在开始的2 wk皮下移植瘤的生长无明显差别,2 wk以后,实验组裸鼠皮下移植瘤的体积增长明显滞后于阴性对照组和空白对照组,差异有统计学意义( P<0.05). 治疗6 wk后,实验组裸鼠皮下移植瘤的质量明显轻于阴性对照组和空白对照组差异有统计学意义(3.26±0.48 g vs7.56±1.25 g,7.50±1.23 g,均P<0.05). 实验组裸鼠皮下移植瘤的AI值明显高于阴性对照组和空白对照组,差异有统计学意义(0.27±0.06vs 0.024±0.01,0.025±0.01,均P<0.05);实验组的抑瘤率明显高于阴性对照组,差异有统计学差异(71.4% vs 0.04%,P<0.01). 透射电镜下,实验组肿瘤细胞可见细胞器结构模糊,核固缩,染色质边集等现象. 免疫组织化学检测示实验组Slug表达明显减少.结论: Slug基因被有效沉默,Slug基因沉默后促进细胞凋亡,抑制胰腺癌实体瘤增殖和生长.
목적: 연구휴대Slug-siRNA적rAAV대라서체내이선암적작용.방법: 건립인이선암라서피하이식류모형,2wk후수궤장건모성공적라서분위3조,매조6지. 구건휴대Slug-siRNA적rAAV재체,채용류체내다점주사적방법,음성대조조라서안1×109 puf(50 μL)표준주사rAAV-EGFP,공백대조조라서주사등량생리염수,실험조라서안1×109 puf(50 μL)표준주사rAAV2-EGFP-slugsiRNA,지주사1차. 관찰라서적일반정황,섭식,활동능력,매주용전자천평칭체질량,회제라서생장곡선. 치료6 wk후이양화탄흡입법처사라서,절취종류칭질량;면역조직화학SP법검측slug단백적표체,TUNEL검측피하이식류세포조망,투사전경검측세포초미결구.결과: 3조라서분별접수불동적처리후,재개시적2 wk피하이식류적생장무명현차별,2 wk이후,실험조라서피하이식류적체적증장명현체후우음성대조조화공백대조조,차이유통계학의의( P<0.05). 치료6 wk후,실험조라서피하이식류적질량명현경우음성대조조화공백대조조차이유통계학의의(3.26±0.48 g vs7.56±1.25 g,7.50±1.23 g,균P<0.05). 실험조라서피하이식류적AI치명현고우음성대조조화공백대조조,차이유통계학의의(0.27±0.06vs 0.024±0.01,0.025±0.01,균P<0.05);실험조적억류솔명현고우음성대조조,차이유통계학차이(71.4% vs 0.04%,P<0.01). 투사전경하,실험조종류세포가견세포기결구모호,핵고축,염색질변집등현상. 면역조직화학검측시실험조Slug표체명현감소.결론: Slug기인피유효침묵,Slug기인침묵후촉진세포조망,억제이선암실체류증식화생장.
AIM: To study the effect on pancreatic cancer in rats of rAAV carrying Slug-siRNA to interfere Slug gene expression. METHODS: A model of subcutaneous tumor was generated through injection of human pancreatic cancer cells BxPC-3 into the nude mice,two weeks later,the nude mice were divided into 3 groups randomly with 6 per group. A recombinant adeno-associated virus vector containing Slug-siRNA gene (rAAV-Slug-siRNA) was constructed. By continuous infusions over 14 d,in negative controls,1×109 puf (50 μL) rAAV-EGFP was injected,while in black controls,50 μL normal saline was given,in experimental groups,1×109 puf (50 μL) rAAV2-EGFPslug-siRNA was injected. The general condition,feeding,activities of the rats,and weight were observed. The growth curve of the rats was drawn. After 6 wk,the rats were killed with CO2,and tumor tissues were cut off and weighed. The expression of Slug protein was detected using the IHC SP method,the apoptosis was detected by TUNEL,and the ultrastructure was detected with TEM.RESULTS: In the beginning of 2 wk,not any apparent growth change was found in three groups. But 2 wk later,the tumor grew slowly in experimental groups than in negative controls and blank controls (P < 0.05). After treatment of 6 wk,the tumor weight in experimental groups was lower than in negative control group or than blank controls group (3.26 ± 0.48 g vs 7.56 ± 1.25 g,7.50 ± 1.23 g,all P < 0.05). The AI was significant higher in experimental group than in the negative control group or blank control group (0.27 ± 0.06vs 0.024 ± 0.01,0.025 ± 0.01,all P < 0.05). The inhibitory rate was 71.4% in the experimental group,and 0.04% in the negative group,showing a significant difference (P < 0.01). Under TEM,the organelle had fuzzy structure,and karyopyknosis,chromatin margination was found in tumor cells in experimental group. The expression of Slug was down-regulated in experimental groups.CONCLUSION: RNAi-mediated Slug gene with rAAV silencing could transfect to pancreatic cancer cells and silence Slug effectively and selectively,which results in the growth and proliferation inhibition in pancreatic cancer in vivo.