中国普外基础与临床杂志

中国普外基础与临床杂志

曲妥珠单抗对 SW-620 人结肠癌细胞增殖凋亡的影响及其与奥沙利铂协同作用的研究

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目的 观察不同浓度曲妥珠单抗单独或联合奥沙利铂对 SW-620 人结肠癌细胞增殖、凋亡及细胞周期的影响,并探讨其作用机制。 方法 体外培养 SW-620 人结肠癌细胞。① 细胞增殖实验:将细胞分为 2 个大组:曲妥珠单抗组和曲妥珠单抗+奥沙利铂组,每个大组内设 8 个浓度小组(每个小组设 5 个复孔),曲妥珠单抗组的浓度依次为 0、0.001、0.01、0.1、1、10、100 及 1 000 μg/mL,对应的曲妥珠单抗+奥沙利铂组曲妥珠单抗的浓度与前相同,不同之处在于均加入了奥沙利铂(10 μmol/L)。采用 CCK-8 法检测各组细胞的吸光度(OD)值。② 细胞凋亡实验:组别设置同增殖实验,只是曲妥珠单抗的浓度仅包括 0、0.1、1 及 10 μg/mL。采用流式细胞仪检测各组细胞的细胞凋亡率及细胞周期分布。③ her-2 蛋白表达检测:分别以 0、100 及 1 000 μg/mL 曲妥珠单抗,以及 0、1 及 10 μg/mL 曲妥珠单抗联合奥沙利铂(10 μmol/L)处理 SW-620 细胞,采用 Western blot 法检测各细胞中 her-2 蛋白的表达。 结果 ① 细胞增殖实验:同种浓度下曲妥珠单抗+奥沙利铂组的 OD 值均低于曲妥珠单抗组(P<0.05),且随着曲妥珠单抗浓度的增加,OD 值在逐渐降低。② 细胞凋亡实验:同种曲妥珠单抗浓度下,曲妥珠单抗组+奥沙利铂组的细胞凋亡率均较曲妥珠单抗组高(P<0.05)。流式细胞检测:不同浓度曲妥珠单抗+奥沙利铂处理后,随浓度增加,G1 期细胞比例呈下降趋势,S 期细胞比例呈上升趋势,且呈剂量依赖性;至 1 μg/mL 和 10 μg/mL 时,与 0 μg/mL 时比较,前者的 G1 期细胞比例较低,S 期细胞比例较高(P<0.05);在 0.1、1 和 10 μg/mL 浓度的曲妥珠单抗条件下,曲妥珠单抗联合奥沙利铂组的 G2 期细胞比例较曲妥珠单抗单药组升高(P<0.01)。③ her-2 蛋白的表达水平:1、100 及 1 000 μg/mL 曲妥珠单抗组细胞中 her-2 蛋白的表达水平逐渐降低,3 组间两两比较差异均有统计学意义(P<0.05);0、1 及 10 μg/mL 曲妥珠单抗+奥沙利铂组中 her-2 蛋白的表达水平也逐渐降低,3 组间两两比较差异也有统计学意义(P<0.05)。 结论 高浓度曲妥珠单抗可抑制 SW620 人结肠癌细胞的增殖,诱导其凋亡。曲妥珠单抗和奥沙利铂具有协同抑制细胞增殖、促进细胞凋亡的作用。

Objective To observe the effects of different concentrations of trastuzumab alone or in combination with oxaliplatin on proliferation, apoptosis, and cell cycle of SW-620 human colon cancer cell, and to explore its mechanism. Methods SW-620 human colon cancer cell were culturedin vitro. ① Cell proliferation experiment: the cells were divided into two large groups: trastuzumab group and trastuzumab combined with oxaliplatin group. There were 8 concentration groups in each large group (5 holes for each group). The concentration of the trastuzumab group was 0, 0.001, 0.01, 0.1, 1, 10, 100, and 1 000 μg/mL, corresponding to the trastuzumab combined with oxaliplatin group. The concentration of the antibiotic was the same as before, except that oxaliplatin (10 μmol/L) was added. The absorbance (OD) value of each group of cells was measured by CCK-8 method. ② Apoptosis experiment: the same proliferation experiment was performed in the group, except that the concentration of trastuzumab only included concentrations of 0, 0.1, 1 and 10 μg/mL. Flow cytometry was used to detect the proportion of apoptotic cells and cell cycle distribution in each group. ③ Determination of her-2 protein. The SW-620 cells were divided into 2 large groups, the concentration of trastuzumab group conclude 0, 100, and 1 000 μg/mL, as well as the concentration of trastuzumab in trastuzumab combined with oxaliplatin group conclude 0, 1, and 10 μg/mL. Expressions of her-2 protein in SW-620 cells were detected by Western blot method. Results ① Cell proliferation assay: the OD values at 100 μg/mL and 1 000 μg/mL were significantly lower than those at 0 μg/mL (P<0.05). At the same concentration, the OD value of the trastuzumab combined with oxaliplatin group was lower than that of the trastuzumab group (P<0.05 ), and the OD value gradually decreased with the increase of the concentration of trastuzumab. ② Apoptosis experiment: the proportion of apoptotic cells in the trastuzumab group combined with oxaliplatin group was higher than that in the trastuzumab group (P<0.05). Flow cytometry: after treatment with different concentrations of trastuzumab combined with oxaliplatin, cells in G1 phase showed a downward trend, and cells in S phase showed an upward trend in a dose-dependent manner. At 1 and 10 μg/mL concentration of trastuzumab, the trastuzumab plus oxaliplatin group significantly reduced the proportion of cells in the G1 phase of SW-620 cell cycle compared with the trastuzumab monotherapy group (P<0.05). The proportion of G2 phase cells was significantly higher in trastuzumab combined with oxaliplatin group than trastuzumab monotherapy group at 0.1, 1 and 10 μg/mL concentrations of trastuzumab (P<0.01). ③ Expressions of her-2 protein: the expression level of her-2 protein gradually decreased at 1, 100, and 1 000 μg/mL trastuzumab group (P<0.05). The expression levels of her-2 protein in 0, 1 and 10 μg/mL trastuzumab combined with oxaliplatin group also gradually decreased (P<0.01). Conclusions High concentration of trastuzumab can inhibit the proliferation of SW-620 human colon cancer cells and induce apoptosis. Trastuzumab combined with oxaliplatin have synergistic effects on inhibiting cell proliferation and promoting apoptosis.

关键词: 结肠癌; 曲妥珠单抗; 奥沙利铂; her-2 蛋白; SW-620 细胞; 体外实验

Key words: colon cancer; trastuzumab; oxaliplatin; her-2 protein; SW-620 cell; experiment in vitro

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