【作者】 高军；

【导师】 姜达；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的:通过构建小鼠H22肝癌移植瘤模型,观察沙利度胺联合表阿霉素对小鼠H22肝癌移植瘤生长的影响,并初步探讨沙利度胺抗肿瘤的作用机制。方法:取40只健康雄性昆明种小鼠,无菌条件下将小鼠H22肝癌腹水瘤细胞接种到小鼠右腋皮下,0.2ml/只(2.0×106个细胞),构建荷瘤肝癌小鼠模型。接种第二天将荷瘤小鼠随机分为4组,每组10只:沙利度胺组(灌胃沙利度胺200mg/kg/日,连续给药10天)、沙利度胺联合表阿霉素组(灌胃沙利度胺200mg/kg/日,连续给药10天;腹腔注射表阿霉素50mg/m2,接种第二天给药一次)、表阿霉素组(腹腔注射表阿霉素50mg/m2,接种第二天给药一次)、生理盐水对照组(灌胃生理盐水0.3ml/日,连续给药10天),停药次日脱臼处死,完整剥离瘤组织,称重,计算抑瘤率及两药相互作用系数。分别提取各组瘤细胞总RNA,鉴定其完整性及含量,采用半定量RT-PCR方法检测肿瘤组织内bFGF mRNA、MMP-2 mRNA的表达水平,应用免疫组化方法检测肿瘤组织内bFGF、MMP-2蛋白的表达水平。用SPSS15.0统计学软件进行数据处理,各组数据以均值±标准差的形式表示。结果:1沙利度胺组瘤重为(3.67±0.44)g,抑瘤率为19.52%,沙利度胺联合表阿霉素组瘤重为(1.95±0.24)g,抑瘤率为57.24%,表阿霉素组瘤重为(2.45±0.31)g,抑瘤率为46.27%,均与生理盐水对照组相比有统计学意义(P<0.01)。生理盐水对照组瘤重为(4.56±0.46)g。两药相互作用系数(CDI)=0.9889<1,沙利度胺联合表阿霉素组平均瘤重最低,与沙利度胺组、表阿霉素组相比均有统计学意义(P<0.01)。2采用半定量RT-PCR的方法检测各组肿瘤组织内bFGF mRNA、MMP-2 mRNA的表达水平。沙利度胺组bFGF mRNA表达相对值为0.3307±0.0463,沙利度胺联合表阿霉素组bFGF mRNA表达相对值为0.3054±0.0539,均与生理盐水对照组相比有统计学差异(P<0.01)。生理盐水对照组bFGF mRNA表达相对值为0.6732±0.1244,表阿霉素组bFGF mRNA表达相对值为0.6113±0.0629。生理盐水对照组和表阿霉素组相比无统计学意义(P>0.05)。沙利度胺组MMP-2 mRNA表达相对值为0.2835±0.0569,沙利度胺联合表阿霉素组MMP-2 mRNA表达相对值为0.2502±0.069,均与生理盐水对照组相比有统计学意义(P<0.01)。生理盐水对照组MMP-2 mRNA表达相对值为0.716±0.1126,表阿霉素组MMP-2 mRNA表达相对值为0.6847±0.1002,生理盐水对照组和表阿霉素组相比无统计学意义(P>0.05)。3应用免疫组化方法检测各组肿瘤组织内bFGF、MMP-2蛋白的表达水平。沙利度胺组bFGF表达值为2.4±0.9661,沙利度胺联合表阿霉素组bFGF表达值为2.3±0.675,均与生理盐水对照组相比有统计学意义(P<0.01)。生理盐水对照组bFGF表达值为5.2±0.7888,表阿霉素组bFGF表达值为4.5±1.1785,表阿霉素组和生理盐水对照组相比无统计学意义(P>0.05)。沙利度胺组MMP-2表达值为2.0±0.6667,沙利度胺联合表阿霉素组MMP-2表达值为1.9±0.7379,均与生理盐水对照组相比有统计学意义(P<0.01)。生理盐水对照组MMP-2表达值为3.8±1.3166,表阿霉素组MMP-2表达值为3.2±1.0328,表阿霉素组和生理盐水对照组相比无统计学意义(P>0.05)。结论:1沙利度胺具有抑制小鼠H22肝癌移植瘤增殖的作用。2沙利度胺可协同表阿霉素对小鼠H22肝癌移植瘤增殖的抑制作用。3沙利度胺可能通过下调bFGF、MMP-2的表达而发挥抑制小鼠H22肝癌移植瘤增殖作用。4本实验证实了沙利度胺具有抗肝癌作用,并通过沙利度胺联合抗肿瘤药物表阿霉素的实验研究丰富了肝癌的治疗,为肝癌的综合治疗提供了新的选择。更多还原

【Abstract】 Objective: Established an H22 mice hepatocellular carcinoma model, the effict of Thalidomide(Tha) combined with Epirubicine(Epi) on H22 murine hepatocellular carcinoma was observed, the aim was to explore the possible mechanism of thalidomide anti-tumor.Methods: Fourty healthy male kunming mice were transplanted by right armpit injection of H22 tumor cells in asepsis conditions, 0.2ml per mice(2.0×106 cells). Then these mice were randomly divided into four groups, ten mice in each group. Tha group (oral Tha 200mg/kg/d, d1-10), Tha+Epi group (oral Tha 200mg/kg/d, d1-10; Epirubicine intraperitoneal injection, 50mg/m2, d1), Epi group (Epirubicine intraperitoneal injection,50mg/m2, d1), Normal Saline group (oral normal saline 0.3ml/d, d1-10). Ten days later all mice were killed and the tumor were taken and weighed. Then the ratio of tumor suppression could be calculated, coefficient of drug interaction(CDI) was calculated. The expression of bFGF mRNA and MMP-2 mRNA were examined by reverse transcription polym-erase chain reaction(RT-PCR). The expression of bFGF, MMP-2 in tumor tissue was detected by using immunohistochemical (IHC) method. All data was showed by Mean±SD. The data was analyzed with the SPSS15.0 software.Results:1 The tumor weight of Tha group was (3.67±0.44)g, the inhibition rate was 19.52%; The tumor weight of Tha+Epi group was (1.95±0.24)g, the inhibition rate was 57.24%; The tumor weight of Epi group was (2.45±0.31)g, the inhibition rate was 46.27%, all groups have significant difference compared to NS group(P<0.01). The tumor weight of NS group was (4.56±0.46)g. CDI=0.9889<1. The average tumor weight of Tha+Epi group was lighter than other groups, the difference has statitistic significance .2 The expression of bFGF mRNA and MMP-2 mRNA in tumor tissue were detected by reverse transcription polymerase chain reaction(RT-PCR) method. The expression of bFGF mRNA in Tha group, Tha+Epi group, Epi group, NS group was 0.3307±0.0463, 0.3054±0.0539, 0.6115±0.0629, 0.6732±0.1244 respectively. Comparing to NS group, there was remarkable difference in Tha group and Tha+Epi group(P<0.01), there was no difference between Epi group and NS group (P>0.05). The expression of MMP-2 mRNA in Tha group, Tha+Epi group, Epi group, NS group was 0.2835±0.0569, 0.2502±0.069, 0.6847±0.1002, 0.716±0.1126 respectively. Comparing to NS group, there was remarkable difference in Tha group and Tha+Epi group (P<0.01), there was no difference between Epi group and NS group (P>0.05).3 The expression of bFGF protein and MMP-2 protein in tumor tissue were detected by immunohistochemical (IHC) method. The expression of bFGF protein in Tha group, Tha+Epi group, Epi group, NS group was 2.4±0.9661, 2.3±0.675, 4.5±1.1785, 5.2±0.7888 respectively. Comparing to NS group, there was significant difference in Tha group and Tha+Epi group(P<0.01), there was no difference between Epi group and NS group(P>0.05). The expression of MMP-2 protein in Tha group, Tha+Epi group, Epi group, NS group was 2.0±0.6667, 1.9±0.7379, 3.2±1.0328, 3.8±1.3166 respectively. Comparing to NS group, there was significant difference in Tha group and Tha+Epi group(P<0.01), there was no difference between Epi group and NS group(P>0.05).Conclusions:1 Thalidomide can inhibit the proliferation of H22 murine hepatocellular carcinoma.2 Thalidomide can strengthen Epirubicine on H22 murine hepatocellular carcinoma proliferation inhibition, they show synergistic action.3 Thalidomide can inhibit the proliferation in H22 murine hepatocellular carcinoma by down regulating the expression of bFGF and MMP-2.4 This research proved Thalidomide has the effect of anti-tumor on H22 murine hepatocellular carcinoma, and inriched the treatment of H22 murine hepatocellular carcinoma through combining with Epirubicine, provided new choice in the comprehensive therapy.更多还原