磁感应加热和HSV—tk自杀基因及核素内照射联合治疗乳腺癌的实验研究

目的 观察纳米磁粒磁感应加热、单纯疱疹病毒-胸苷激酶（HSV-tk）基因及核素内照射联合治疗MCF-7乳腺癌的作用,探讨联合治疗的有效性与可行性.方法 制备转染试剂脂质体（Lipofectamine）/含热休克蛋白启动子的HSV-tk基因表达质粒（pHSP-HSV-tk）、四氧化三铁纳米磁流液、188Re-丙氧鸟苷白蛋白纳米微球（GCV-BSA-NP）;进行四氧化三铁纳米磁流液的体内加热实验;建立MCF-7乳腺癌模型,并用单纯随机抽样法将60只荷瘤鼠分成6组,每组10只.A组为空白对照组,B组为单纯基因转染治疗组,C组为单纯热疗组,D组为基因转染联合核素内照射治疗组,E组为基因转染联合热疗组,F组为基因、热疗、核素内照射联合治疗组.各组经不同方法处理后,观察肿瘤生长情况,测肿瘤质量,观察肿瘤组织病理变化;RT-PCR法检测B、D、E、F组HSV-tk基因表达（与内参β-actin mRNA吸光度比值）情况.各组与对照组比较采用泊松分布及单因素方差分析,所用统计软件为SPSS 10.0.结果 在动物体内加热实验中,交变磁场（AMF）作用下,不同注射剂量磁粒都引起肿瘤组织温度快速升高,2,4和6 mg磁粒分别使肿瘤温度升至39.6 ℃,43.2 ℃和48.1 ℃,且持续40 min.当AMF作用停止后,肿瘤组织温度10 min内分别降至36.8 ℃,37.5 ℃和37.8 ℃.成功建立MCF-7乳腺癌模型,治疗结果显示：C、D、E、F组肿瘤质量（452.50±30.29）、（240.98±35.32）、（231.87±27.41）、（141.55±23.78）mg]较A组（719.12±22.65）mg]差异有统计学意义（F=800.007,P＜0.01）,且以F组疗效最好;C组与E组肿瘤质量大于F组,差异有统计学意义（t=25.533,7.872,P均＜0.05）;D组较B组肿瘤质量明显减小,且差异有统计学意义（t=32.805,P＜0.05）.RT-PCR半定量分析显示：B、D组的表达量（0.33±1.30,0.46±0.12）与E、F组（0.66±0.13,0.74±0.11）相比差异有统计学意义（F=21.573,P＜0.05）.结论 热疗、基因治疗联合核素治疗能有效抑制MCF-7乳腺癌的生长,对乳腺癌治疗有潜在的应用前景.

Combined anti-tumor therapeutic effect of targeted gene, hyperthermia, radionuclide brachytherapy in breast carcinoma

Objective To investigate the antitumor therapeutic effect of combined therapy of magnetic induction heating by nano-magnetic particles, herpes simplex virus thymidine kinase gene(HSV-tk suicide gene) and internal radiation in mice bearing MCF-7 breast carcinoma. Methods The transfection reagents, plasmids heat shock protein-HSV-tk (pHSP-HSV-tk), ferroso-ferric oxide nano-magnetic fluid flow and 188Re-ganciclovir-bovine serum albumin-nanopaticles (GCV-BSA-NP) were prepared. The heating experiments in vivo were carried out using ferroso-ferric oxide nano-magnetic fluid flow. Sixty mice tumor models bearing MCF-7 breast carcinoma were established and randomly divided into six groups. Group A was the control group, B was gene transfection therapy group, C was hyperthermia group, D was gene transfection therapy combined with radionuclide brachytherapy group, E was gene therapy combined with hyperthermia group, and F was gene therapy, hyperthermia combined with radionuclide brachytherapy group. The tumor growth, tumor mass and histopathological changes were evaluated. The expression of HSV-tk in the groups of B, D, E and F was detected by RT-PCR. Poisson distribution and one-way analysis of variance (ANOVA) were used for statistical analysis by SPSS 10.0 software. Results In the animal heating experiments, the temperature of tumor increased up to 39.6 ℃, 43.2 ℃, and 48.1 ℃ quickly with different injected doses (2, 4 and 6 mg respectively) of nano-magnetic particles and maintained for 40 min. The temperature of tumor tissue reduced to 36.8 ℃, 37.5 ℃ and 37.8 ℃ in 10 min when alternating magnetic field (AMF) stopped. The tumor mass in Groups C ((452.50 ±30.29) mg), D ((240.98 ±35.32)mg), E((231.87 ±27.41) mg) and F ((141.55 ±23.78) mg) were much lower than that in Group A ((719.12±22.65) mg) (F=800.07, P＜0. 01), with the most significant treatment effect in Group F.The tumor mass in Group B((684.05 ±24.02) mg) was higher than that in Group D (t =32. 805, P ＜0. 05). Semi-quantitative RT-PCR analysis showed that the expression of HSV-tk in Groups B and D (0.33 ±0. 13 and 0. 46 ±0.12) was significantly different from that in Groups E and F (0.66 ±0.13 and 0.74 ±0. 11)(F = 21. 573, P ＜ 0.05). Conclusion Combined use of hyperthermia, gene therapy and radionuclide brachytherapy could effectively depress the growth of MCF-7 breast carcinoma, thus possessing treatment potential for this tumor.