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目的探讨三羟基异黄酮诱导人胃癌原代细胞裸鼠移植瘤凋亡的机制。方法建立人胃癌原代细胞裸鼠移植瘤模型并测定移植瘤的生长曲线;25只Balb/c裸鼠接种胃癌原代细胞悬液生成移植瘤后均分为五组,不同剂量的三羟基异黄酮(0.5、1.0和1.5mg/kg)、生理盐水和DMSO进行瘤旁注射,透射电镜和TUNEL法检测肿瘤组织凋亡情况,免疫组化法和RT-PCR法检测肿瘤组织凋亡相关基因bcl-2和bax的表达情况。结果三羟基异黄酮对胃癌原代细胞移植瘤有明显的抑制作用;透射电镜发现三羟基异黄酮导致移植瘤内大量细胞发生凋亡;TUNEL染色法发现三羟基异黄酮注射组瘤细胞的凋亡指数(28.7%±1.1%、33.4%±1.4%和37.1%±1.0%)明显高于生理盐水和DMSO对照组(P<0.05),而两个对照组(11.8%±0.4%和11.7%±0.4%)之间差异无统计学意义。免疫组化法发现三羟基异黄酮注射组的Bcl-2蛋白阳性细胞率(11.8%±0.9%、5.7%±0.8%和4.0%±0.8%)明显低于两个对照组(P<0.05),而两个对照组(19.5%±0.6%和17.6%±1.4%)之间差异无统计学意义;而Bax蛋白阳性细胞率(20.0%±1.2%、24.7%±0.9%和29.3%±1.6%)明显高于两个对照组(P<0.05),而两个对照组(10.3%±0.4%和10.9%±0.9%)间差异无统计学意义;RT-PCR法发现三羟基异黄酮注射组的bcl-2mRNA条带强度随剂量增大而递减,并明显低于对照组(P<0.05);而baxmRNA条带强度递增,并明显低于对照组(P<0.05),而两对照组间差异无统计学意义(P>0.05)。结论三羟基异黄酮对胃癌原代细胞移植瘤有明显的抑制作用,通过下调bcl-2和上调bax的表达而诱导胃癌裸鼠移植瘤细胞发生凋亡,是其抗胃癌作用的机制之一。
Objective To investigate the mechanism of trihydroxy isoflavone-induced apoptosis of primary human gastric cancer cell xenografts in nude mice. Methods The primary human gastric cancer xenograft model was established and the growth curve of the xenografted tumor was established. Twenty-five Balb / c nude mice were inoculated with the primary cell suspension of gastric cancer and divided into five groups. Different doses of trihydroxyiso (0.5, 1.0 and 1.5 mg / kg), normal saline and DMSO, respectively. The apoptosis of tumor tissue was detected by transmission electron microscopy and TUNEL. The expression of apoptosis related gene bcl-1 was detected by immunohistochemistry and RT- -2 and bax expression. Results Genistein had a significant inhibitory effect on the primary tumor of gastric cancer cells. Transmission electron microscopy revealed that isoflavone induced apoptosis in a large number of tumor cells. The apoptosis of tumor cells was detected by TUNEL staining The indexes (28.7% ± 1.1%, 33.4% ± 1.4% and 37.1% ± 1.0%) were significantly higher than those in the saline and DMSO control groups (P <0.05), while those in the two control groups (11.8% ± 0.4% and 11.7% ± 0.4%) between the difference was not statistically significant. Immunohistochemistry showed that the Bcl-2 protein positive cells rate (11.8% ± 0.9%, 5.7% ± 0.8% and 4.0% ± 0.8%) in the genistein group was significantly lower than that in the two control groups (P <0.05) , While there was no significant difference between the two control groups (19.5% ± 0.6% and 17.6% ± 1.4%), while the positive rate of Bax protein positive cells (20.0% ± 1.2%, 24.7% ± 0.9% and 29.3% ± 1.6 %) Was significantly higher than that of the two control groups (P <0.05), while there was no significant difference between the two control groups (10.3% ± 0.4% and 10.9% ± 0.9%). Genistein The intensity of bcl-2mRNA band decreased with the increase of dose, which was significantly lower than that of control group (P <0.05), while the intensity of bax mRNA band was increased and significantly lower than that of control group (P <0.05) There was no significant difference between the two groups (P> 0.05). Conclusions Hydroxyisoflavone can obviously inhibit the growth of primary gastric cancer cells and induce apoptosis of the transplanted tumor cells in nude mice by down-regulating the expression of bcl-2 and up-regulating bax, which is one of its mechanisms of anti-gastric cancer.