摘要目的 观察胰腺癌移植瘤不可逆电穿孔 (IRE)术后肿瘤细胞凋亡情况,探讨IRE技术对胰腺癌移植瘤的抗肿瘤效果及其安全性.方法 12只荷载VX2胰腺癌移植瘤的新西兰大白兔随机分为IRE治疗组(10只)和对照组(2只).治疗组胰腺移植瘤行IRE治疗,对照组不进行处理.治疗后第1、3、5、7、14 d各处死2只,取血分离血浆或血清检测血肝肾功能及淀粉酶、肌酸激酶、caspose-3、TNF-α、VEGF、肌钙蛋白Ⅰ水平.取移植瘤行常规病理学检查,采用TUNEL法检测细胞凋亡,免疫组化法检测瘤组织Bcl-2、HSP70、VEGF表达.结果 对照组及IRE 1、3、5、7、14 d组移植瘤平均最大径分别为2.0、2.0、1.8、1.7、1.5、1.3 cm.治疗组移植瘤的治疗坏死区域和正常区域边界清晰,坏死区边缘可见大量红细胞及炎症细胞,治疗后14 d治疗区边缘出现肿瘤再生迹象.对照组瘤组织及IRE 1、3、5、7、14 d组移植瘤治疗区边缘组织TUNEL阳性细胞数分别为52、78、98、135、196、217个/每200倍视野,随时间延长而增加;Bcl-2阳性细胞百分数分别为39.3％、37.5％、51.9％、17.7％、31.0％、34.5％,总体呈下降趋势;HSP70阳性细胞百分数分别为12.6％、33.6％、27.4％、24.3％、31.4％、23.4％,术后均升高;VEGF阳性细胞百分数分别为28.3％、27.2％、12.3％、8.7％、32.2％、12.2％,术后有所下降;血caspase-3水平分别为0.185、0.345、0.312、0.210、0.558、0.173 μg/L,术后不同程度升高,7d达峰值;血TNF-α水平分别为24.48、60.54、61.83、28.59、44.71、64.78 ng/L,术后维持在较高水平,14 d达峰值;血VEGF水平分别为75.18、123.76、178.66、58.2、68.68、64.09 ng/L,术后3d达峰值,之后下降;血淀粉酶活性于术后1d升高,3d开始下降,14 d恢复正常;血肌酸激酶有不同程度升高;肝肾功能均未见明显异常;血浆肌钙蛋白Ⅰ水平在正常范围内.结论 IRE通过诱导细胞凋亡、产生特异抗肿瘤免疫效应及抑制血管生成来抑制胰腺移植瘤的生长,治疗方法安全、有效.

abstractsObjective To investigate the apoptosis of pancreatic cancer xenograft after irreversible electroporation (IRE) treatment, and observe the antitumor effect and safety of IRE technique.Methods Twelve New Zealand white rabbits with pancreatic cancer xenograft were randomly divided into IRE treatment group (n =10) and control group (n =2).The treatment group received IRE;the control group received no treatment.Two rabbits were sacrificed at 1 st, 3rd, 5th, 7th, 14th day after IRE treatment, the blood plasma or blood serum was separated to detect the liver and renal function, amylase, creatine kinase, caspase-3, TNF-α,VEGF, CTnI.Pancreatic cancer xenograft was taken for routine pathological examination.TUNEL assay was used to detect the apoptosis of tumor cells.Immunohistochemistry was applied to detect Bcl-2, HSP70 and VEGF protein expression.Results The median maximum pancreatic cancer xenograft of control group and IRE 1st, 3rd, 5th, 7th, 14th d group was 2.0, 2.0, 1.8, 1.7, 1.5, 1.3 cm.HE staining showed a clear borderline between the necrotic and normal area of IRE treated tumor, and with a large number of red blood and inflammatory cells infiltrated near the necrotic area's edge.After 14 days of treatment, regeneration appeared in the treatments edge.The number of TUNEL positive cell near the treatment's edge was 52, 78, 98, 135,196, 217/per 200 magnification in control group and IRE 1st, 3rd, 5th, 7th, 14th d group, and the number increased with time;the percent of positive cell of Bcl-2 was 39.3％, 37.5％, 51.9％, 17.7％, 31.0％,34.5％, and it showed a decreasing trend;the percent of positive cell of HSP70 was 12.6％, 33.6％,27.4％, 24.3％, 31.4％, 23.4％, and it all increased when compared with that of control group;the percent of positive cell of VEGF was 28.3％ , 27.2％, 12.3％, 8.7％, 32.2％, 12.2％ , which decreased when compared with that of control group;the serum caspase 3 level was 0.185, 0.345, 0.312, 0.210, 0.558,0.173 μg/L, which increased when compared with that of control group and reached the peak at 7 d;the serum TNF-α level was 24.48, 60.54, 61.83, 28.59, 44.71, and 64.78 ng/L, which remained at high level and reached the peak at 14d;the serum VEGF level was 75.18, 123.76, 178.66, 58.2, 68.68,64.09 ng/L, which reached the peak at 3rd day and gradually decreased.The serum amylase level increased at the 1st day, and decreased at 3rd day, and it returned to normal at 14th day.The liver and kidney functions,and serum CTnI levels were within the normal range, higher level of serum creatine kinase was found.Conclusions IRE can inhibit the growth of pancreatic tumors via inducing apoptosis, producing specific antitumor immune response, and inhibiting angiogenesis, which is a safe and effective approach for pancreatic cancer.