31P磁共振波谱分析技术监测兔肝移植瘤及白细胞介素12基因的表达

Experimental study on monitoring gene expression by noninvasive method in rabbit VX2 liver tumor model

目的探讨31P磁共振波谱分析（31P MRS）技术监测AdCMVIL12-IRES—CKb腺病毒基因转导治疗兔VX2肝肿瘤的可行性。方法19只新西兰大白兔，随机取1只麻醉后，取1ml VX2瘤细胞悬液（1×10 7个／ml）注射于兔后腿外侧肌肉。待肿瘤长到鸡蛋大时，切开肿瘤，剪成1mm×1mm×1mm大小的组织块，将组织块以开腹包埋法接种到18只大白兔肝脏中，超声监测肿瘤的生长晴况，取肿瘤组织，行HE染色。18只大白兔随机分为治疗组（6只）、对照组（6只）和空白组（6只），经耳缘静脉分别注射等量的重组腺病毒AdCMVIL12-IRES—CKb颗粒（5×10 12粒／kg）、AdCMV-Empty颗粒（5×10 12粒／kg）和等渗盐水，注射处理后，肌酸水溶液饲养5d行31P MRS扫描。采用免疫组织化学和酶联免疫吸附法检测各组白细胞介素12（IL-12）水平，Western blot法检测脑型肌酸激酶（CKB）水平，超声法检测注射处理前后肿瘤的大小。采用统计软件SPSS 17．0，通过t检验、单因素方差分析、LSD法作统计学处理。结果肿瘤接种后15d左右，直径增至1．5～1．7cm，彩色多普勒超声检查血流成像显示肿瘤周边可见供血小动脉直通肿瘤内部。兔肝脏肿瘤模型表面可见突出的瘤体，质地偏硬，标本切面见肿瘤组织呈灰白色、鱼肉状，与周围正常肝组织分界欠清楚。HE染色显示肿瘤呈浸润性生长，可见明显异型性。治疗组、空白组和对照组注射处理前、后肿瘤直径分别为（1．63±0．04）cm和（1．62±0．03）cm、（1．59±0．05）cm和（1．84±0．11）cm、（1．60±0．02）cm和（2．07±0．12）cm，与注射处理前比较，空白组和对照组大白兔肿瘤直径增大，t值分别为-5．291、-9．475，P值均〈0．05，差异有统计学意义，治疗组注射处理前后肿瘤直径差异无统计学意义。兔肝肿瘤经AdCMVIL12-IRES—CKb腺病毒治疗后，肝组织内IL—12相关信号获得表达，但对照组、空白组未见IL—12相关信号表达；治疗组、空白组、对照组大白兔血清IL-12的浓度分别为（65．96±3．67）pg／ml、（1．83±0．81）pg／ml、（1．60±0．76）pg／ml，与对照组和空白组比较，治疗组IL-12浓度高，t值分别为48．893和36．548，P〈0．01，差异有统计学意义；对照组与空白组IL-12水平相比较，差异无统计学意义。治疗组肝组织CKB获得表达，但空白组和对照组未见CKB表达。与注射处理前比较，治疗组注射处理后有异常增高的典型的Pcr峰。治疗组、对照组、空白组注射处理前后的Pcr值分别为（0．23±0．14）mmol／L和（0．88±0．52）mmol／L；（0．69±0．21）mmol／L和（0．28±0．29）mmol／L；（0．19±0．19）mmol／L和（0．25±0．36）mmol／L，与处理前比较，治疗组注射处理后Pcr值增大，对照组注射处理后Pcr值减少，t值分别为-2．629、3．505，P值均〈0．05，差异有统计学意义。空白组注射处理前后Pcr值差异无统计学意义。3组大白兔注射处理前后Pcr值之差的单因素方差分析结果显示，F=6．235，P〈0．05。采用LSD法对注射处理前后3组Pcr差值进行两两比较，治疗组与对照组及空白组比较，P=0．004和0．049，差异均有统计学意义；对照组与空白组间差异无统计学意义。结论兔肝脏肿瘤模型的建立是成功的。肝内CKB活性可预测IL-12的表达，31P MRS技术可用于监测AdCMVIL12-IRES—CKb腺病毒基因转导治疗兔VX2肝肿瘤。

Objective To investigate the feasibility of monitoring therapeutic effect of adenovirus vector containing IL12-IRES-CKb gene on a rabbit VX2 liver tumor model by using phosphorous-31 magnetic resonance spectroscopy （31p MRS）. Methods A total of 18 healthy New Zealand White rabbits were used to generate animal models by implanting VX2 tumor chips into livers through laparotomy. Tumorbearing animals were randomly divided into three groups and were injected with AdCMVIL12-IRES-CKb, AdCMV-Empty and salinerespectively via ear veins. 31p MRS scan was performed after animals were fed with creatine solution for five days. Animals were euthanized thereafter and tumors were removed for pathological examination, immunohistochemistry （IHC） staining and protein analysis （Western blot）. Results The intrahepatic and seral expressions of creatine kinase （CKb） and IL-12 were detected only in AdCMVIL12- IRES-CKb group. Tumor diameters pre- and post- treatment in three groups were 1.63 ± 0.04 vs 1.62 ±0.03 in AdCMVIL12-IRES-CKb group （P = 0.229）, 1.59 ± 0.05 vs 1.84 ± 0.11 in AdCMV-Empty group （P = 0.003） and 1.60 ± 0.02 vs 2.07 ± 0.12 in saline group （P = 0.001）, respectively. Per Changes between pre- and post- treatment among the three groups were compared （F = 6.235, P 〈 0.05）. PCr increased significantly in AdCMVIL12-IRES-CKb group as compared to AdCMV-Empty （P = 0.004） and saline group （P = 0.049）, whereas no change found between AdCMV-Empty and saline group （P = 0.153）. Conclusion 31p MRS, an effective and non-invasive functional imaging method, can be used to monitor the therapeutic effect of adenovirus vector containing IL 12-IRES-CKb gene on rabbit VX2 liver tumor model through detecting metabolic product of imaging reporter gene CKb （pCr）.