血管内皮抑素转基因口服制剂联合5-Fu、IFN-α 2a治疗结肠癌的实验研究

Effects on Experimental Colon Cancer by Combination of Endostatin Transfected Oral Powder Preparation (ETB-2) and 5-Fu or Recombinant IFN-α 2a

目的探讨血管内皮抑素转基因双歧杆菌口服制剂(ETB-2,E)联合5-Fu(F)、IFNα-2a(I)对裸鼠结肠癌的治疗效应,并探讨其作用机制。方法将人结肠癌细胞LoVo接种于BALB/c裸鼠皮下建立动物模型,随机分为6组,每组6只动物,除对照组用等量生理盐水灌胃外,余分别给予E、F、I、E+F、E+I等药物。6组均于接种后第22天处死动物,计算抑瘤率。观察病理变化,用免疫组化法检测微血管密度(MVD)、增殖细胞核抗原指数(PCNA指数)和血管内皮生长因子(VEGF)阳性率,用RT-PCR法检测VEGF mRNA、TGFβ-1mRNA,用DNA原位末端标记(TUNEL)法检测凋亡率。结果(1)对照组抑瘤率为0,E组、F组、I组、E+F组、E+I组抑瘤率分别为47.26%、60.69%、34.68%、71.23%、63.09%,E+F组、E+I组抑瘤率分别与E组、I组相比,差异有显著性(P<0.05)。(2)E+F组和E+I组MVD与对照组相比,差异有显著性(P<0.05);E+F组、E+I组VEGF阳性率分别与对照组、F组相比,差异有显著性(P<0.05);PCNA指数各治疗组有降低趋势,但各组间比较,差异无显著性(P>0.05)。(3)E+F组、E+I组VEGF mRNA、TGFβ-1mRNA较对照组、E组、F组、I组明显降低(P<0.05)。(4)E组、I组、E+F组、E+I组凋亡率与对照组相比,差异均有显著性(P<0.05)。(5)常规病理检查发现,E组血管明显减少,E+F组和E+I组则坏死较为严重,血管明显减少。结论ETB-2分别联合5-Fu、IFN-α2a可更有效地抑制裸鼠结肠癌的生长,具有协同作用。

Objective To investigate the effects on experimental colon cancer by combination of ETB-2（E） and 5-Fu（F） or recombinant IFN-α 2a（I） in BALB/c nude mice, and its mechanism. Methods The human colon cancer line LoVo was implanted subcutaneously in BALB/c nude mice. Then the mice were allocated into 6 groups： the control group（ n = 6）, E（4.5 g/kg· d^-1, n = 6）, F （every time 140 mg/kg, n = 6）, I（every time 1.5 million IU/kg, n = 6）, E ＋ F（ n = 6） and E ＋ I（ n = 6）, the dosage similar to above. The control groups were given saline water, E was given by drenching 7 days after being implanted, F and I were given by intraperitoneally injection 10, 12 and 14 days after being implanted. The drenching was conducted for successive 14 days, then the mice were sacrificed on the 22nd day and the rate of tumor inhibition was calculated. The pathologic changes were observed by conventional pathological method, the microvessel density （ MVD ） , proliferating cell nuclear antigen（PCNA） index and the positivity rate of vascular endothelial growth factor（VEGF） were measured immunohistochemically; apoptosis rate was analyzed with TDT-mediated dUTP nick end labeling （TUNEL） technique;and the VEGF mRNA and TGF-β1 mRNA were measured by RT-PCR. Results （1）The tumor inhibition rate in E（47.26%）, F（60.69%）, 1（34.68%）, E ＋ F（71.23%）, E ＋ I （63.09%） was significantly higher than that in the controls（0%, P 〈（0.05）. The tumor inhibition rate in E ＋ F and E ＋ I also was significantly higher than that in E and I, respectively（P 〈（0.05）. （2） The MVD in E ＋ F and E ＋ I was significantly decreased than that in the controls（ P 〈（0.05）, the difference was significant when the positivity rate of VEGF in E ＋ F and E ＋ I was respectively compared with that in the control groups and F（P 〈 0.05）. Although PCNA index in experimental groups tended to decreasing, comparing with the control groups, the difference was not found. （3）The VEGF mRNA in the combined therapy was significantly lower than that in the controls, E, F and I （P 〈0.05）, the TGF-β1 mRNA in the combined therapy was also significantly lower than that in the controls, E and F（P 〈（0.05）. （4）The apoptosis rate in E, I, E ＋ F and E ＋ I was significantly higher than that in the controls （P 〈 0.05）, the apoptosis rate in I, E ＋ F and E ＋ I was significantly higher than that in F （ P 〈0.05）, the difference was found between E ＋ I and E （ P 〈0.05）. （5） The microvessal decreased in E and the necrosis much serevely, significantly-lessened microvessal in combined therapies was seen by conventional pathological method. Conclusion Combinating ETB-2 with 5-Fu or IFN-α 2a can synergistically restrain the growth of experimental human colon cancer in BALB/c nude mice.