以“甘润濡养”立法的启膈方抑制裸鼠食管癌肺转移的研究

Qige Decoction for the Inhibition of Lung Metastasis from Esophageal Cancer Based on Theory of Ganrunruyang

目的建立裸鼠食管癌肺转移模型,探讨启膈方对尾静脉注射食管癌细胞裸鼠肺转移的影响。方法 2017年2—10月,选取BABL/c裸鼠21只,将低分化食管癌细胞株KYSE-150-Luc用1 ml注射器接种于BABL/c裸鼠尾静脉,每只100μl,建立裸鼠肺转移模型,随机分为对照组(7只)、卡培他滨组(7只)和启膈方组(7只)。接种后第3天,对照组给予0.9%氯化钠溶液灌胃0.2 ml/d,卡培他滨组给予卡培他滨溶液(浓度400 mg/kg)灌胃0.2ml/d,启膈方组给予启膈方提取物(QGF)溶液(浓度3 913 mg/kg)灌胃0.2 ml/d。其中卡培他滨组每灌胃2周停药1周。3组均灌胃给药12周。应用动物活体成像系统观察各组裸鼠肺转移情况;记录裸鼠体质量;处死裸鼠后记录裸鼠肺转移结节个数;Western blotting法检测裸鼠肺转移结节组织中E钙黏蛋白(E-cad)、角蛋白(KRT)8、snail、间隙连接蛋白(Connexin)43、WNT2表达水平;HE染色法检测各组裸鼠肺转移结节组织镜下表现。结果接种后第12周在肺部区域检测到肿瘤细胞的生物发光信号,提示出现肺转移,其中启膈方组和卡培他滨组光子强度均低于对照组;裸鼠肺呈灰白色肺转移结节。对照组、启膈方组第4、8、12、16周体质量与初始体质量百分比大于卡培他滨组(P<0.05);对照组第4、8、12、16周体质量与初始体质量百分比小于启膈方组(P<0.05)。对照组肺转移结节数多于启膈方组、卡培他滨组(P<0.05)。启膈方组及卡培他滨组E-cad、KRT8、Connexin43表达水平高于对照组,snail、WNT2表达水平低于对照组(P<0.05);卡培他滨组E-cad、KRT8、Connexin43表达水平低于启膈方组,snail、WNT2表达水平高于启膈方组(P<0.05)。观察肺转移组织结节HE染色切片,3组裸鼠肿瘤组织均呈巢状生长,肿瘤细胞排列密集,异质性明显;3组均未见明显肿瘤细胞坏死征象。结论启膈方能够抑制食管癌肺转移发生,可能与其增强裸鼠间隙连接、抑制上皮细胞间质化有关,且启膈方在控制裸鼠体质量减轻上效果明显优于卡培他滨。

Objective To investigate the effect of Qige decoction on the incidence of lung metastasis from esophageal cancer(EC)among a nude mouse model of EC established by injecting EC cells into the tail vein.Methods This study was implemented from February to October 2017.21 BABL/c nude mice were selected and injected 100μl KYSE-150-Luc poorly differentiated esophageal carcinoma cell lines via the tail vein using 1 ml syringes for establishing nude mice models of EC.They were equally randomized into the control group,Qige decoction group,and Capecitabine group.From the third day after the modeling,the former two groups received intragastric administration of 0.2 ml of 0.9%sodium chloride solution once per day,0.2 ml of Qige decoction(3 913 mg/kg)once per day,respectively,for 12 consecutive weeks,while the Capecitabine group received 0.2 ml of Capecitabine(400 mg/kg)once per day,for 12 weeks but 2 weeks on and 1 week off.The lung metastasis of nude mice was observed by using in vivo small animal imaging.The weight of the mice was measured and recorded.After the sacrifice,the number of lung metastastic nodules was counted,the expression levels of E-cad,KRT8,snail,Connexin43 and WNT2 in lung metastases were detected by Western blotting,and lung metastases were examined by microscopy with H&E staining.Results During the 12th week after modeling,bioluminescence signal of tumor cells was detected in the lung area,suggesting there were spontaneous lung metastases.Capecitabine group showed much lower ratios of baseline weight to the weight at the end of 4,8,12,16 weeks of intervention when compared to other two groups,as did the control group when compared to the Qige decoction group(P<0.05).For more detail,the control group showed significantly lower photon intensity compared with other two groups(P<0.05).All groups demonstrated gray-white lung metastastic nodules,and the control group owned more nodules than other groups(P<0.05).Western blotting results showed that the control group owned significantly lower expression levels of E-cad,KRT8 and Connexin43 and much higher expression levels of snail and WNT2 when compared to other two groups,so did the Capecitabine group when compared to the Qige decoction group(P<0.05).Microscopy examination of H&E staining pathological sections of lung metastastic nodules showed that all groups had tumor tissues with a nested growth pattern,and the tumor cells were dense and heterogeneous.No obvious signs of tumor necrosis were found in the 3 groups.Conclusion Qige decoction could inhibit the lung metastasis from EC,which may be associated with enhanced gap junction inhibition of epithelial-mesenchymal transition.Moreover,Qige decoction was superior to Capecitabine in prolonging the weight control.