蓝萼乙素对两种宫颈癌细胞裸鼠移植瘤的抑制作用及其机制

目的 探讨蓝萼乙素(GLB)对两种宫颈癌细胞株HeLa、SiHa裸鼠移植瘤的抑制作用及其机制.方法 将HeLa、SiHa细胞分别种植至BALB/C nu裸小鼠右大腿根部背侧皮下,建立裸小鼠移植瘤模型;待移植瘤体积约为0.1 cm3时,于第10天将HeLa组随机分为4组,第14天将SiHa组随机分为4组,分别为生理盐水组、10%乙醇组、GLB低剂量组和高剂量组,每组5只;经腹腔注射给药,HeLa组给予生理盐水0.4 mL/只、10%乙醇0.4 mL/只、GLB 0.15μmol/g 0.4 mL/只和0.20μmol/g 0.4 mL/只,隔日1次,共20 d;SiHa组给予生理盐水0.4 mL/只、10%乙醇0.4 mL/只、GLB 0.18μmol/g 0.4 mL/只和0.24μmol/g 0.4 mL/只,隔日1次,共20 d.腹腔注射给药的同时,隔天用标准体重秤称量体质量并做好记录,每隔2d专人用游标卡尺精确测量移植瘤的最大长径和短径,至少测量两次取平均值.给药结束后处死裸鼠前,眼球取血,测生化指标和肿瘤标志物,观察各组荷瘤鼠生化指标和肿瘤标志物的变化;通过移植瘤生长抑制试验,组织HE染色,观察各组移植瘤的大体形态、组织形态学的变化;免疫组化法检测移植瘤组织中自噬相关蛋白PTEN、Beclin 1、LC3的表达变化,Western blot法进一步检验相关蛋白变化.结果 裸鼠处死后,HeLa、SiHa组中生理盐水组、10%乙醇组、GLB低剂量组和高剂量组生化指标比较差异均无统计学意义(P>0.05);GLB低剂量组和高剂量组的肿瘤标志物较生理盐水组、10%乙醇组降低,差异均有统计学意义(P<0.05);GLB低剂量组和高剂量组的移植瘤体积均明显小于生理盐水组和10%乙醇组,差异均有统计学意义(P<0.05);HE染色显示:GLB低剂量组和高剂量组移植瘤组织出现凋亡、坏死改变;HeLa和SiHa组心肝脾肺肾均未发现转移瘤细胞;免疫组化法、Western-blot法结果示:GLB低剂量组和高剂量组的肿瘤组织PTEN、Beclin 1及LC3蛋白表达水平较生理盐水组和10%乙醇组明显升高,差异均具有统计学意义(P<0.05).结论 GLB对人宫颈癌HeLa和SiHa细胞移植瘤的生长具有明显的抑制作用,其机制可能与上调PTEN、Beclin 1、LC3的表达,诱导细胞自噬凋亡有关.

Inhibitory effect of GLB on two kinds of cervical cancer cell lines transplanted tumor in nude mice and its mechanism

Objective To study the inhibitory effect of Glaucocalyxin B (GLB) on two kinds of cervical cancer cell lines HeLa and SiHa transplanted tumor in nude mice and its mechanism. Methods Human cervical cancer HeLa and SiHa cells were transplanted into nude mice in right thigh dorsal subcutaneously to establish the transplanted tumor model of HeLa and SiHa cell. When the transplanted tumor volume was about 0.1 cm3, HeLa cell were randomly divided into 4 groups at the tenth day. At the fourteenth day, SiHa group were randomly divided into 4 groups, namely normal sa-line group, 10%ethanol group, GLB low dose group and high dose group, with 5 mice in each group. Through intraperito-neal injection, HeLa group was injected with 0.4 mL physiological saline, 0.4 mL 10%ethanol, 0.4 mL GLB (0.15μmol/g) and 0.4 mL GLB (0.20μmol/g), once every other day for 20 days. SiHa group was injected with 0.4 mL physiological sa-line, 0.4 mL 10%ethanol, 0.4 mL GLB (0.18μmol/g) and 0.4 mL GLB (0.24μmol/g), once every other day for 20 days. At the same time as intraperitoneal injection, the maximum diameter and minor diameter of the transplanted tumor were accurately measured at least two times for average by a Vernier caliper every 2 days. Before the administration of the nude mice, the nude mice were sacrificed and the blood samples were taken from the naked eye to measure and observe changes of biochemical indexes and tumor markers. Through the tumor growth inhibition test and transplanted tumor tis-sue HE staining, transplanted tumor gross morphology and histomorphological changes of each group were observed. The expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN), Beclin 1 and LC3 in transplant-ed tumor tissues was detected by immunohistochemistry, and the changes of related proteins were detected by West-ern-blot. Results There was no significant difference in biochemical indexes for physiological saline group, 10%etha-nol group, GLB low dose group and high dose group (P>0.05). The tumor markers of GLB low dose group and high dose group were significantly lower than those of saline group and 10%ethanol group (P<0.05). The tumor volume of GLB low dose group and high dose group were significantly lower than those in the saline group and 10%ethanol group (P<0.05). HE staining showed more apoptosis and necrosis were observed in transplanted tumor tissue of GLB low dose group and high dose group, and no metastatic tumor cells were found in heart, liver, spleen, kidney and lung of HeLa and SiHa group. The results of immunohistochemistry and Western-blot showed that the expression levels of PTEN, Beclin 1 and LC3 protein in tumor tissues of GLB low dose group and high dose group were significantly higher than those in sa-line group and 10% ethanol group (P<0.05). Conclusion GLB can inhibit the growth of human cervical cancer cell line HeLa and SiHa cells in nude mice, and its mechanism may be related to the up-regulation expression of tumor sup-pressor gene PTEN, microtubule associated protein LC3, autophagy gene Beclinl protein expression and induction of au-tophagy and apoptosis.