贝伐单抗通过抑制mTOR通路对卵巢癌腹水模型裸鼠的改善作用

Ameliorating Effect of Bevacizumab on Nude Mice with Ovarian Cancer Ascites Model by Inhibiting mTOR Pathway

目的探讨贝伐单抗通过抑制哺乳动物雷帕霉素靶蛋白(mTOR)通路对卵巢癌腹水模型裸鼠的改善作用。方法选取40只无特定病原体级8周龄雌性BALB/c裸鼠,将卵巢癌细胞HO-8910接种于裸鼠腹腔,建立卵巢癌腹水模型,并将建模成功的36只裸鼠按随机数字表法分为3组,各12只。模型组:给予等量0.9%氯化钠溶液;3-苄基-5-(2-硝基苯氧甲基)-γ-丁内酯(3BDO)+贝伐单抗组给予0.1 nmol/L 3BDO+5 mg/kg贝伐单抗;贝伐单抗组给予5 mg/kg贝伐单抗;各组均连续腹腔注射给药28 d后处死。测量各组腹水平均体积及腹膜渗透性,苏木精-伊红染色观察各组肿瘤组织病理改变,并以反转录聚合酶链反应检测mTOR及真核起始因子4E结合蛋白1(4E-BP1)信使RNA(mRNA)表达水平,蛋白免疫印迹法检测mTOR、磷酸化mTOR(p-mTOR)和4E-BP1、磷酸化4E-BP1(p-4E-BP1)的表达水平。结果与模型组比较,3BDO+贝伐单抗组和贝伐单抗组腹水平均体积均减少、腹膜渗透性均降低,且贝伐单抗组的腹水平均体积最小、腹膜渗透性最低(P<0.05)。苏木精-伊红染色镜下观察发现,模型组肿瘤细胞胞质饱满,细胞核大深染;3BDO+贝伐单抗组部分肿瘤细胞皱缩减小,核分裂象减少,且有小片状坏死区;贝伐单抗组肿瘤组织内可见凋亡小体以及片状坏死区。贝伐单抗组mTOR mRNA、4E-BP1 mRNA表达及p-mTOR/mTOR、p-4E-BP1/4E-BP1均低于模型组和3BDO+贝伐单抗组[(0.97±0.16)比(3.08±0.75)、(1.14±0.31),(0.74±0.09)比(2.12±0.53)、(1.03±0.14),(0.57±0.03)比(0.78±0.07)、(0.64±0.05),(0.33±0.02)比(0.79±0.06)、(0.51±0.05)](P<0.05)。结论贝伐单抗可能通过抑制mTOR通路减少肿瘤细胞增殖,减少卵巢癌腹水模型裸鼠腹水量,降低腹膜渗透性。

Objective To explore the ameliorating effect of bevacizumab on nude mice with ovarian cancer ascites model by inhibiting the mammalian target of rapamycin(mTOR)pathway.Methods A total of 408-week-old female BALB/c nude mice with specific pathogen-free were selected,and ovarian cancer cells HO-8910 were inoculated into the abdominal cavity of the nude mice to establish a model of ovarian cancer ascites,and 36 nude mice which were successfully modeled according to random numbers were divided into 3 groups,12 each.Model group:given the same amount of 0.9%sodium chloride solution;3-benzyl-5-(2-nitrophenoxymethyl)-γ-butyrolactone(3BDO)+bevacizumab group given 0.1 nmol/L 3BDO+5 mg/kg bevacizumab;the bevacizumab group was given 5 mg/kg bevacizumab;each group was given continuous intraperitoneal injection for 28 days and then sacrificed.The average volume of ascites fluid and peritoneal permeability of each group were measured.Hematoxylin-eosin staining method was used to observe the pathological changes of tumor tissues in each group,and reverse transcription-polymerase chain reaction was performed to detect the mTOR and eukaryotic initiation factor 4E binding protein 1(4E-BP1)messenger RNA(mRNA)expression level,and Western blotting was used to detect the expression level of mTOR,phosphorylated mTOR(p-mTOR)and 4E-BP1,phosphorylated 4E-BP1(p-4E-BP1).Results Compared with the model group,the average volume of ascites and the peritoneal permeability in the 3BDO+bevacizumab group and the bevacizumab group decreased,while in the bevacizumab group the average volume of ascites was the least and the peritoneal permeability lowest(P<0.05).Observation under the microscope with hematoxylin-eosin staining showed that the tumor cells in the model group had full cytoplasm and large and deep stained nuclei;some tumor cells in the 3BDO+bevacizumab group had reduced and shrunk,with reduced mitotic images and small patches of necrosis;apoptotic bodies and flaky necrosis were seen in the tumor tissues in the bevacizumab group.mTOR mRNA,4E-BP1 mRNA expression and p-mTOR/mTOR,p-4E-BP1/4E-BP1 in the bevacizumab group were lower than those in the 3BDO+bevacizumab group and the model group[(0.97±0.16)vs(3.08±0.75),(1.14±0.31);(0.74±0.09)vs(2.12±0.53),(1.03±0.14);(0.57±0.03)vs(0.78±0.07),(0.64±0.05);(0.33±0.02)vs(0.79±0.06),(0.51±0.05)](P<0.05).Conclusion Bevacizumab may reduce tumor cell proliferation by inhibiting the mTOR pathway,reduce the amount of ascites in nude mice with ovarian cancer ascites model,and reduce peritoneal permeability.