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目的观察超声微泡携单酰基甘油脂肪酶沉默基因(monoacylglycerol lipase short hairpin RNA,MAGL-shR-NA)在大鼠肝细胞肝癌(hepatocellular carcinoma,HCC)组织中转染及对HCC转移的作用。方法建立大鼠肝细胞肝癌模型,病理解剖和二维超声验证肝脏成瘤情况。40只SD大鼠完全随机分成4组,分别为PBS液组、MAGL-shRNA质粒微泡组(MAGL-shRNA+microbubble,MB)、空白质粒微泡+超声辐照组(microbubbles+ultrasound,MB+US)、MAGL-shRNA质粒微泡+超声辐照组(MAGL-shRNA+MB+US)。每只注射1 mL,对MB+US组和MAGL-shRNA+MB+US组大鼠肝区同时给予超声辐照,辐照条件为300 kHz,2 W/cm2,辐照10 s,间隔10 s,共20 min。Western blot检测大鼠HCC组织MAGL蛋白的表达,免疫组化检测MAGL和基质金属蛋白酶2(matrixmetalloproteinase-2,MMP-2)的表达。比较各组动物的肿瘤转移情况。结果 MAGL在HCC组织中表达明显高于正常肝组织(P<0.05);微泡携MAGL-shRNA可以在HCC组织被超声辐照击破后靶向释放,在各组HCC组织中均有MAGL蛋白的表达,其中MAGL-shRNA+MB+US组表达量明显低于其他组(P<0.01);免疫组化检测MAGL-shRNA+MB+US组MMP-2表达均低于其他组(P<0.01);各组动物均见肿瘤转移,但MAGL-shRNA+MB+US组转移率最低(P<0.05)。结论超声辐照可破坏携MAGL-shRNA的微泡使之靶向释放并增强了MAGL-shRNA的转染效率,MAGL-MMP-2通路可能与HCC的转移相关。
Objective To observe the effect of transfection of monoacylglycerol lipase short hairpin RNA (MAGL-shR-NA) in HCC tissue and its effect on HCC metastasis in rats. Methods The model of hepatocellular carcinoma in rats was established. Pathological anatomy and two-dimensional ultrasonography were used to verify the liver tumorigenesis. Forty SD rats were randomly divided into 4 groups: PBS solution group, MAGL-shRNA + microbubble group (MB), blank plasmid microbubbles + ultrasound group (MB + US), MAGL-shRNA plasmid microbubbles + ultrasound irradiation group (MAGL-shRNA + MB + US). Each injection of 1 mL, ultrasound irradiation was given simultaneously to the liver of the MB + US group and the MAGL-shRNA + MB + US group under the irradiation conditions of 300 kHz, 2 W / cm2 and irradiation time of 10 s , A total of 20 min. The expression of MAGL protein in HCC tissues was detected by Western blot. The expression of MAGL and matrix metalloproteinase-2 (MMP-2) were detected by immunohistochemistry. The tumor metastasis of each group was compared. Results The expression of MAGL in HCC tissues was significantly higher than that in normal liver tissues (P <0.05). The microvesicles carrying MAGL-shRNA could be targeted released after the HCC tissues were broken by ultrasonic irradiation. In all HCC tissues, MAGL protein The expression of MMP-2 in MAGL-shRNA + MB + US group was significantly lower than that in other groups (P <0.01) The tumor metastasis was found in all the groups, but the metastasis rate was the lowest in MAGL-shRNA + MB + US group (P <0.05). Conclusion Ultrasound irradiation can destroy the micro-vesicles carrying MAGL-shRNA to target the release and enhance the transfection efficiency of MAGL-shRNA. MAGL-MMP-2 pathway may be related to the metastasis of HCC.