超声靶向微泡破坏联合溶瘤腺病毒治疗胰腺癌的实验研究

Ultrasound targeted microbubble destruction combined with oncolytic adenovirus therapy in treatment of pancreatic cancer in mice

目的探讨超声靶向微泡破坏(UTMD)联合溶瘤腺病毒(oAd)治疗胰腺癌的疗效及相关机制。方法在C57BL/6小鼠右前肢皮下注射Panc-02细胞(2×10^(6)个/只),选取肿瘤体积在100~150 mm3的小鼠55只,随机分为NC组(注射PBS 100μL)、UTMD组(注射微泡溶液100μL,并行超声微泡破坏处理5 min)、oAd组(注射10^(8)PFU/mL oAd溶液100μL)、oAd+微泡组(注射10^(8)PFU/mL微泡oAd混合液100μL)、oAd+UTMD组(注射10^(8)PFU/mL微泡oAd混合液100μL,并行超声微泡破坏处理5 min),每组11只。2 d给药1次,共给药5次,2 d记录1次肿瘤体积。第3次给药24 h后每组随机取6只小鼠颈椎脱臼法处死,剥离肿瘤制成肿瘤切片及肿瘤细胞悬液。肿瘤切片行HE染色比较各组肿瘤组织坏死情况并计算坏死面积,E1A抗体染色观察oAd在各组肿瘤组织内分布情况,CD3抗体染色比较各组肿瘤内CD3+T细胞数,Tunel染色及流式细胞术分析各组小鼠肿瘤细胞凋亡情况。当小鼠肿瘤体积超过2000 mm3时终止实验处死所有小鼠。结果在14 d时终止实验,oAd+UTMD组和oAd组相比肿瘤体积增长显著减缓(P<0.05)。oAd+UTMD组细胞质内oAd的E1A蛋白染色最深,oAd组染色最浅。oAd+UT-MD组和oAd组坏死面积与NC组比值分别是9.50±0.60和3.51±0.24,差异有统计学意义(P<0.05)。含oAd的3组小鼠肿瘤内CD3+T细胞数均增加,oAd+UTMD组肿瘤内的CD3+T细胞数(196.33±12.58)明显高于oAd组(120.67±12.90,P<0.05)。oAd+UTMD组的细胞总凋亡率及Tunel染色荧光分布比oAd组多。结论UTMD增强oAd对胰腺肿瘤细胞的杀伤作用,促进oAd在肿瘤内转染,协助CD3+T细胞在肿瘤内富集,同时诱导肿瘤细胞凋亡和坏死增加。

Objective To explore the efficacy of ultrasound targeted microbubble destruction(UTMD)combined with oncolytic adenovirus therapy(oAd)for pancreatic cancer in mice.Methods Subcutaneous injection of Panc-02 cells(2×10^(6)cells per mouse)was performed on the right forelimb of C57BL/6 mice.A total of 55 mouses with tumor volumes ranging from 100-150 mm3 were selected and randomly divided into 5 groups,with 11 mouses in each group.The grouping and intratumoral administration were as follows:NC group(phosphate solution 100μL),UTMD group(microbubble solution 100μL and ultrasonic microbubble destruction treatment for 5 min),oAd group(10^(8)PFU/mL oncolytic adenovirus solution 100μL),oAd+MBs group(10^(8)PFU/mL oncolytic adenovirus microbubble mixture 100μL),oAd+UTMD group(10^(8)PFU/mL oncolytic adenovirus microbubble mixture 100μL and ultrasound microbubble destruction treatment for 5 min).All groups were treated every alternative days for 5 times,and the tumor volumes were measured also every alternative days.After 24 h of the third administration,6 mouses in each group were randomly selected and sacrificed.The tumors were obtained and tumor tissue sections and cell suspensions were prepared.HE staining was used to observe the necrosis of tumor tissues,E1A antibody staining was used to observe the distribution of oAds in tumor tissues in each group,CD3 antibody staining was used to count the number of CD3+T cells in tumor tissues,Tunel staining and flow cytometry were used to analyze the apoptosis of tumor tissues.Results When the experiment was terminated at 14 days,the tumor volume growth in the oAd+UTMD group was significantly slower than that in the oAd group(P<0.05).The E1A protein staining of oAds in the cytoplasm was the deepest in the oAd+UTMD group and the lightest in the oAd group.The ratios of necrotic area in the oAd+UTMD group and the oAd group were 9.50±0.60 and 3.51±0.24,respectively,which were significantly different from those in the NC group(P<0.05).CD3+T cells were accumulated in the tumors of the three groups of mice treated with oAd.The number of CD3+T cells in the tumors of the oAd+UTMD group(196.33±12.58)was significantly higher than that of the oAd group(120.67±12.90,P<0.05).The total apoptosis rate and Tunel staining fluorescence range in the oAd+UTMD group were significantly higher than those in the oAd group.Conclusion UTMD can enhance the killing effect of oAd on pancreatic tumor,promote the transfection of oAd in tumor,assist the enrichment of CD3+T cells in tumor,and induce apoptosis and necrosis of tumor cells.