药学学报  2019, Vol. 54 Issue (11): 2011-2018     DOI: 10.16438/j.0513-4870.2019-0502   PDF    
基于转移前微环境的四君子汤提高吉非替尼疗效研究
张铭予, 顾琼, 周晓伟, 李莹, 张远远, 张玉君, 王伟华, 李春雨, 李国辉     
国家癌症中心, 国家肿瘤临床医学研究中心, 中国医学科学院、北京协和医学院肿瘤医院, 北京 100021
摘要: 利用Lewis肺癌(LLC)转移小鼠模型,研究吉非替尼(gefitinib)与四君子汤(Sijunzi Tang,SJZ)对转移前微环境影响。实验方案经中国医学科学院肿瘤医院伦理委员会审议同意并批准。首先将1×106个荧光素酶标记的LLC细胞接种于小鼠右后腋下,构建肺癌转移模型。将小鼠按体重随机分为模型组、吉非替尼组(50 mg·kg-1)、SJZ组(25.74 g·kg-1)和联合给药组,造模后次日开始给药。连续给药14天后,利用小动物成像技术检测肿瘤大小;通过流式细胞技术检测小鼠外周血、脾、肺组织中单核细胞和中性粒细胞数量及其表面趋化因子受体(CXCR1、CCR2)和致癌基因c-Kit的表达水平变化情况;使用酶联免疫吸附测定(ELISA)法检测小鼠血浆及瘤组织中炎症因子(IL-1α、IL-6)的含量;给药21天后,取肿瘤组织,称重,采用游标卡尺测量肿瘤体积大小并评估联合给药的抑瘤效果;给药45天后,记录各组小鼠的生存期。流式细胞检测结果显示,在小鼠肺组织中,与模型组比较,吉非替尼组、SJZ组和联合给药组的中性粒细胞的百分比均呈显著性下降(P < 0.05或P < 0.01),但三组之间比较无显著性差异(P>0.05);在小鼠外周血和肺组织中,与模型组比较,SJZ组与联合给药组的中性粒细胞和单核细胞表面CXCR1、CCR2和c-Kit表达水平均呈下降趋势(P < 0.05或P < 0.01),但吉非替尼组单核细胞表面c-Kit表达水平呈显著性上升(P < 0.05);在小鼠脾组织中,吉非替尼组CXCR1、CCR2和c-Kit表达水平均呈上升趋势(P < 0.05),而SJZ组和联合给药组均呈下降趋势(P < 0.05或P < 0.01)。ELISA法检测结果显示,在小鼠血浆中,与模型组比较,SJZ组IL-1α含量呈显著性下降(P < 0.01),联合给药组IL-6含量呈显著性下降(P < 0.05);与吉非替尼组比较,联合给药组IL-1α含量显著性下降(P < 0.05)。在小鼠肿瘤组织中,与模型组比较,联合给药组IL-1α含量呈显著性下降(P < 0.05)。与吉非替尼组比较,联合给药组IL-1α、IL-6含量与SJZ组IL-6含量均呈显著性下降(P < 0.05)。连续给药21天后,吉非替尼组、SJZ组和联合给药组的抑瘤率分别为45.7%、38.4%和84.8%。给药45天后,模型组生存率为0%,吉非替尼组、SJZ组和联合给药组的生存率分别为40%、60%和60%。上述结果表明,SJZ可通过调控转移前微环境提高吉非替尼的疗效。
关键词: 肺癌     吉非替尼     四君子汤     肿瘤转移     转移前微环境     炎性细胞因子    
Sijunzi Tang improves the efficacy of gefitinib through pre-metastatic niche
ZHANG Ming-yu, GU Qiong, ZHOU Xiao-wei, LI Ying, ZHANG Yuan-yuan, ZHANG Yu-jun, WANG Wei-hua, LI Chun-yu, LI Guo-hui     
National Cancer Center, National Clinical Research Center for Cancer, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
Abstract: The Lewis lung carcinoma (LLC) metastatic mouse model was used to investigate the effects of gefitinib and Sijunzi Tang (SJZ) on pre-metastatic niche. The experimental protocol was approved by the Ethics Committee which belongs to Cancer Hospital, Chinese Academy of Medical Sciences. To generate spontaneous lung metastatic models, 1×106 luciferase-labeled LLC cells were injected subcutaneously in the shaved right flank of mice. One day after LLC inoculation, the mice were randomly divided into model (saline), gefitinib (50 mg·kg-1) treatment, SJZ treatment (25.74 g·kg-1), and co-treatment gefitinib with SJZ groups, with intragastrical administration. After 14 days of continuous administration, tumor size was detected by IVIS® Spectrum system. The number of monocytes and neutrophils and the expression levels of chemokine receptors (CXCR1, CCR2) and carcinogenic gene (c-Kit), in peripheral blood, spleen and lung tissues of mice were determined by flow cytometry. The contents of interleukin-IL-1α (IL-1α) and interleukin-6 (IL-6) were detected by the enzyme linked immunosorbent assay (ELISA). After 21 days of treatment, tumors were surgically removed, weighed and the tumor volume was measured with vernier caliper and the antitumor effect of co-administration was evaluated. After 45 days of administration, the survival of mice was recorded. The results of flow cytometry showed that the percentage of neutrophils in gefitinib group, SJZ group, and co-treatment group was significantly decreased in the lung tissue compared to the model group (P < 0.05 or P < 0.01), but there was no significant difference between three treatment groups (P > 0.05). In the mouse peripheral blood and lung tissue, compared with the model group, the expression levels of CXCR1, CCR2 and c-Kit on the surface of neutrophils and monocytes in SJZ group and co-treatment group decreased or decreased significantly (P < 0.01 or P < 0.05). However, there was a significant increase in the expression level of c-Kit on the surface of monocytes (P < 0.05). In the mouse spleen tissue, the expression levels of CXCR1, CCR2 and c-Kit in the gefitinib group increased significantly (P < 0.05), while decreased significantly in SJZ or co-treatment group (P < 0.05). The results of ELISA showed that the content of IL-1α in SJZ group decreased significantly in the plasma of the mice compared with the model group (P < 0.01) and the content of IL-6 in co-treatment group decreased significantly (P < 0.05). Compared with the gefitinib group, the content of IL-1 in the co-treatment group decreased significantly (P < 0.05). In the tumor tissues of mice, compared with the model group, the content of IL-1α in the co-treatment group decreased significantly (P < 0.05). Furthermore, the content of IL-1α in co-administrated group and IL-6 in SJZ or co-treatment group decreased significantly compared with the gefitinib group (P < 0.05). After 21 days of continuous administration, the tumor inhibition rates of gefitinib group, SJZ group and co-administrated group were 45.7%, 38.4%, and 84.8%, respectively. After 45 days of administration, the survival rate of the model group was 0%, whereas the gefitinib, SJZ or co-treatment group has a survival rate of 40%, 60%, or 60%, respectively. In summary, our study illustrated that Sijunzi Tang could improve the anti-tumor effect of gefitinib by regulating pre-metastatic niche.
Key words: lung cancer     gefitinib     Sijunzi Tang     tumor metastasis     pre-metastatic niche     inflammatory cytokine    

肺癌是我国最常见的恶性肿瘤, 其发病率和死亡率一直位居我国恶性肿瘤榜首。根据国家癌症中心最新的研究统计显示, 2015年发病例数约为78.7万例, 死亡病例约63.0万例[1]。据报道, 约80%肺癌是属于非小细胞肺癌(non-small-cell carcinoma, NSCLC), 目前针对NSCLC主要治疗手段包括化疗、放疗、手术治疗和免疫治疗等[2]。近年来, 分子靶向治疗显著提高了晚期NSCLC患者的生活质量, 延长其生存期, 尤其是表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)的代表药物吉非替尼(gefitinib), 现已成为NSCLC的一线治疗药物[3]。然而, 肺癌患者使用吉非替尼的中位无进展生存期(PFS)仅1年左右[4], 最终不可避免出现耐药, 或肺肿瘤发生远处转移[5], 且复发转移已成为肺癌相关死亡的主要原因。转移前微环境的形成是促进肿瘤转移的重要免疫学机制, 免疫抑制和炎症是转移微环境形成的重要特征[6]。研究表明, 吉非替尼治疗肺癌的同时, 产生炎症和免疫抑制因子, 这可能是其疗效降低、最终产生耐药的重要原因[7]。临床研究发现四君子汤(Sijunzi Tang, SJZ)在中西医结合治疗肺癌、肠癌和胃癌等临床治疗中发挥着扶正培本的作用, 可抑制肿瘤复发转移、增强抗肿瘤药物疗效、延长患者生存期、改善生活质量[8, 9]。本课题组前期证实, 在小鼠Lewis肺癌(Lewis lung cancer, LLC)转移模型上, SJZ与吉非替尼可协同抑制肿瘤的生长, 且抑制癌细胞向肺部转移[10]。然而, SJZ如何提高吉非替尼疗效的生物学机制尚不清楚, 难以形成有效的临床用药对策和方案。本研究采用LLC转移模型, 探索SJZ和吉非替尼对转移前微环境的影响, 以期为SJZ提高吉非替尼疗效的免疫学机制的阐明提供参考, 为中西医联合治疗肺癌的临床用药对策和方案提供科学依据。

材料与方法

实验细胞  荧光素酶标记的LLC细胞由中国中医科学院广安门医院肿瘤研究所赠与。

药物与试剂  人参(Panax ginseng C.A.Mey)、茯苓(Wolfiporia Cocos)、白术(Atractylodesmacrocephala Koidz)、炙甘草(Glycyrrhizauralensis Fisch)均购自北京华邈药业有限公司, 以上药品皆鉴定为合格药材。吉非替尼(批号: Lot#5-SXG-56-1, Toronto Research Chemicals公司); 胶原酶V (批号: C9263-100MG, 美国Sigma公司); 抗体Ly-6G、Ly-6C、CD45、CD11b、Firable Viabiity Stain 510、CD181 (CXCR1)、CD192 (CCR2)、CD117、CD16/CD32 (美国Biolegend公司); xenolight D-luciferin potassium Sal [珀金埃尔默企业管理(上海)有限公司]; Mouse IL-6 Elisa kit、Mouse IL-1α Elisa kit (深圳市达科为生物工程有限公司)。

主要仪器  全功能微孔板检测仪(SynergyH2, 美国BioTek公司); 生物显微镜(E200, 日本尼康公司); IVIS® Spectrum小动物活体光学成像系统(珀金埃尔默公司, NO.124262);倒置显微镜(莱卡DMIL-LED, 北京冠普佳科技有限公司); 低速台式大容量离心机(L-550, 湖南湘仪离心机仪器有限公司); 流式细胞术检测仪(BD FACSCantoTM Ⅱ, 美国BD Bioscience公司)。

动物  雄性C57BL/6小鼠, 体重(20 ± 2) g, SPF级, 购于北京维通利华有限责任公司, 合格证号: SYXK 2014-0003。分笼饲养于中国人民解放军第五医学中心实验动物中心, 自由饮水及进食, 饲养环境保持安静, 通风良好, 并定期消毒, 温度(25 ± 2) ℃, 湿度(45 ± 15)%, 人工光照约12 h/12 h明暗周期。实验方案经中国医学科学院肿瘤医院伦理委员会审议同意并批准。

细胞培养  LLC细胞加入新鲜DMEM培养液中, 内含青霉素(1×105 u·L-1)、链霉素(100 mg·L-1)、10%胎牛血浆, 5% CO2、37 ℃恒温全湿培养箱中进行培养, 每隔48 h进行换液传代, 取处于对数生长期的细胞进行实验。

四君子汤制备  称取处方量人参、茯苓、白术、甘草(9:9:9:6) 4种中药饮片适量[11], 加入10倍量水浸泡30 min后, 回流提取1 h, 过滤并收集滤液, 药渣加入8倍量的水再次回流提取40 min, 过滤, 合并两次的滤液, 减压浓缩至所需体积, 四君子汤质量浓度为0.858 g·mL-1。药材的含量均符合中国药典要求, 质量可控。

模型建立、动物分组及给药  采用文献[12]报道的方法制备LLC肺癌转移模型, 取LLC细胞悬液(每毫升1×107个) 0.1 mL接种于小鼠的右后腋下。将C57BL/6小鼠按体重随机分为模型组、吉非替尼组、SJZ组和联合给药组, 每组15只。造模后次日灌胃给药, SJZ给药剂量为25.74 g·kg-1 (该剂量参考本课题组前期实验给药剂量)[10], 吉非替尼给药剂量为50 mg·kg-1, 模型组灌胃给予相同体积的生理盐水, 联合给药组中两种药物给药时间间隔为1 h, 每日1次, 连续给药至45天。

小动物成像分析  小鼠腹腔注射Luciferin生物发光底物溶液(15 g·L-1) 0.2 mL, 10 min后置于成像暗箱平台中, 通过成像软件进行拍照、ROI (region of interest)测量, 计算选定区域绝对光子数(p/sec/cm2/sr), 红色代表高强度信号, 紫色代表低强度信号。

肿瘤体积与质量测定  末次给药24 h后, 颈椎脱臼法处死各组小鼠, 剥离肿瘤组织, 测量肿瘤最长径(a)和最短径(b), 肿瘤称重, 计算给药组平均瘤质量(m1)和模型组平均瘤质量(m2), 分别按公式(1)和(2)计算肿瘤体积(V)和抑瘤率(Z%)[13, 14]

$ V=\text{ }{{\left( a\times b \right)}^{2}}/2 $ (1)
$ Z=\text{ }\left( 1-{{m}_{1}}/{{m}_{2}} \right)\text{ }\times 100\% $ (2)

酶联免疫吸附测定分析  取小鼠外周血, 离心(3 000 r·min-1, 15 min), 取上清; 取肿瘤组织50 mg, 加入磷酸盐缓冲溶液(PBS) 500 μL匀浆后, 离心(10 000 r·min-1, 10 min), 取上清, 备用。然后, 按照酶联免疫吸附测定分析试剂盒说明书测定IL-6和IL-1α的含量。

流式细胞技术分析  取小鼠外周血、脾和肺组织, 脾组织加入PBS研磨, 经200目筛网过滤后得脾单细胞悬液; 使用无菌手术剪刀将肺组织剪成小块, 加入胶原酶V溶液(2 mg·mL-1) 5 mL, 37 ℃环境下消化后得肺单细胞悬液。分别取外周血、脾单细胞悬液、肺单细胞悬液各100 μL, 依次加入抗体、中性粒细胞(CD45+ Ly6G+ Ly6C+)、单核细胞(CD45+Ly6G-Ly6C+)、趋化因子受体(CCR2+、CXCR1+)和c-Kit+。孵育20 min后离心(5 000 r·min-1, 5 min), 加入多聚甲醛(5%) 100 μL, 固定, 以待检测。

统计学方法  实验结果使用Graphpad Primer 7.0统计软件进行数据统计。采用计量资料多组间比较采用单因素方差分析(one-wayANOVA), 两组间比较采用t检验, 以P < 0.05表示具有显著性差异。

结果 1 吉非替尼联合四君子汤对小鼠肿瘤生长的影响

构建LLC肺癌转移模型后, 次日给药, 分别在14和21天取材检测, 小鼠生存期观察至45天(图 1A)。给药14天后, 通过IVIS Spectrum系统获得的肿瘤细胞的荧光发光图片及其平均光子数, 检测肿瘤大小。结果显示, 与模型组比较, 吉非替尼组、SJZ组与联合给药组均能抑制肿瘤生长(P < 0.01), 其中联合给药组抑制肿瘤生长最明显(图 1BC)。第21天, 末次给药后小鼠称体重, 眼眶取血, 将肿瘤分离出来并称重, 采用游标卡尺测量肿瘤体积大小, 计算瘤体积。结果表明, 与模型组比较, 吉非替尼组、SJZ组与联合给药组均能降低肿瘤重量(P < 0.01), 与吉非替尼组比较, 联合给药组的肿瘤重量和体积均呈显著性下降(P < 0.05), 且各组小鼠体重无显著性差异(P > 0.05) (图 1D~F)。计算给药21天时吉非替尼组、SJZ组和联合给药组的抑瘤率分别为45.7%、38.4%和84.8%。给药45天, 模型组生存率为0%, 其余3组生存率分别为40%、60%和60% (图 1G)。给药期间, 各给药组对肿瘤生长均有不同抑制作用, 联合给药组的抑瘤效果最好(图 1H)。

Figure 1 Anti-tumor effect of Sijunzi Tang (SJZ) and gefitinib. A: Experimental arrangement for the spontaneous metastatic model in this study; B, C: Representative pictures of tumor fluorescence luminescence obtained by the IVIS spectrum system and their average radiance number statistics on 14 days of administration; D, E: Tumor's weight and volume on 21 days of administration; F: The trend of changes in body weight of mice; G: Survival rate situation of mice during 45 days of administration; H: Tumor volume change trend. n = 15, x± s. **P < 0.01, ***P < 0.001, ****P < 0.000 1 vs model group; ##P < 0.01 vs gefitinib group
2 吉非替尼联合四君子汤对转移前微环境中性粒细胞和单核细胞的影响

图 2可见, 与模型组比较, 吉非替尼组、SJZ组和联合给药组的肺组织的中性粒细胞百分比均呈显著性下降(P < 0.01);与模型组比较, SJZ组外周血的中性粒细胞数量呈显著性下降(P < 0.01), 吉非替尼组和联合给药组无显著性变化(P > 0.05);吉非替尼组、SJZ组和联合给药组脾组织的中性粒细胞有下降趋势, 但无显著性变化(P > 0.05);各给药组对外周血、脾、肺组织中单核细胞的数量无明显影响(P > 0.05)。结果表明, SJZ联合吉非替尼能够显著抑制中性粒细胞在肺中募集, 对转移前微环境中单核细胞的数量作用不明显。

Figure 2 Effects of gefitinib and SJZ on percentage of immune cells of CD45+ cells in blood, spleen and lung in Lewis lung carcinoma (LLC) mouse. n = 15, x± s. *P < 0.05, **P < 0.01 vs model group; #P < 0.05 vs gefitinib group
3 吉非替尼联合四君子汤对CXCR2、CCR2和c-Kit表达水平的影响

给药后, 转移前微环境中中性粒细胞表面c-Kit、CCR2和CXCR1表达水平变化情况如图 3所示。在小鼠肺组织中, 与模型组比较, 吉非替尼组的c-Kit、CCR2和CXCR1表达水平无明显变化(P > 0.05), 而SJZ组的CXCR1、c-Kit表达水平呈显著性下降(P < 0.05), 联合给药组的CXCR1表达水平也呈显著性下降(P < 0.05);在小鼠外周血中, 与模型组比较, 吉非替尼组、联合给药组的c-Kit、CCR2和CXCR1表达水平无明显变化(P > 0.05), 而四君子汤组c-Kit表达水平呈显著性下降(P < 0.05), 四君子汤组、联合给药组CXCR1的表达水平也呈显著性下降(P < 0.05);在小鼠脾组织中, 与模型组比较, SJZ组的CXCR1、c-Kit表达水平均呈显著性下降(P < 0.05), 联合给药组的CXCR1表达水平也呈显著性下降(P < 0.05), 吉非替尼组的CXCR1、CCR2、c-Kit表达水平均无显著性差异(P > 0.05)。

Figure 3 Effects of SJZ and gefitinib on the expression of receptor protein on neutrophils in blood, lung and spleen of LLC mouse. n = 15, x± s. *P < 0.05, **P < 0.01 vs model group; #P < 0.05 vs gefitinib group

给药后, 转移前微环境中单核细胞表面c-Kit、CCR2和CXCR1表达水平变化情况如图 4所示。在小鼠肺组织中, 与模型组比较, 吉非替尼组、SJZ组和联合给药组的CXCR1、c-Kit表达水平均呈显著性下降(P < 0.05), 但各给药组CCR2表达水平均无明显变化(P > 0.05)。在小鼠外周血中, 与模型组比较, 吉非替尼组c-Kit表达水平呈显著性上升(P < 0.05), CXCR1、CCR2表达水平无明显变化(P > 0.05), 而SJZ组和联合给药组c-Kit、CXCR1表达水平均呈显著性下降(P < 0.05)。在小鼠脾组织中, 与模型组比较, 吉非替尼组CCR2表达水平呈显著性上升(P < 0.05), 但联合给药组呈显著性下降(P < 0.05), SJZ组c-Kit和CXCR1表达水平均呈显著性下降(P < 0.01);与吉非替尼组比较, SJZ组CCR2表达水平显著性下降(P < 0.05), 联合给药组c-Kit、CXCR1表达水平均呈显著性下降(P < 0.05或P < 0.01)。

Figure 4 Effects of SJZ and gefitinib on the expression of receptor protein on monocytes in blood, lung and spleen of LLC mouse. n = 15, x± s. *P < 0.05, **P < 0.01, ***P < 0.001 vs model group; #P < 0.05, ##P < 0.01, ####P < 0.000 1 vs gefitinib group

总之, 吉非替尼可使转移前微环境中趋化因子受体上调, 推测其可能与治疗后肿瘤出现复发、转移有关; 而四君子汤可显著下调其表达, 尤其是中性粒细胞和单核细胞表面CXCR1、CCR2、c-Kit的表达, 结果表明, SJZ与吉非替尼合用发挥协同抗肿瘤作用, 是抑制肺癌转移的潜在机制。

4 吉非替尼联合四君子汤对IL-1α和IL-6含量的影响

采用ELISA法测定小鼠血浆和肿瘤组织中IL-1α、IL-6表达水平(图 5)。在小鼠血浆中, 与模型组比较, 吉非替尼组与联合给药组IL-1α含量无显著性差异(P > 0.05), SJZ组表达水平呈显著性下降(P < 0.01);与吉非替尼组比较, 联合给药组IL-1α含量呈显著性下降(P < 0.05)。在小鼠肿瘤组织中, 与模型组或吉非替尼组比较, 联合给药组IL-1α含量均呈显著性下降(P < 0.05)。在小鼠血浆中, 与模型组比较, 吉非替尼组和SJZ组IL-6含量均无明显变化(P > 0.05), 而联合给药组呈显著性下降(P < 0.05)。在小鼠肿瘤组织中, 与模型组比较, 吉非替尼组IL-6含量呈显著性上升(P > 0.05);与吉非替尼组比较, SJZ组与联合给药组IL-6含量均呈显著性下降(P < 0.05)。结果表明, 吉非替尼可使转移前微环境中的炎性细胞因子含量升高, 而四君子汤可以降低炎性细胞因子含量。因此, 提示吉非替尼联合四君子汤可改善肿瘤免疫炎性微环境, 提高吉非替尼的疗效。

Figure 5 Effects of SJZ and gefitinib on cytokine (IL-1α, IL-6) levels in blood and tumor tissues of LLC mouse. n = 15, x± s. *P < 0.05 vs model group; #P < 0.05, ##P < 0.01 vs gefitinib group
讨论

转移前微环境是指肿瘤在尚未发生转移之前, 先诱导转移器官中微环境发生改变, 以营造一个适宜转移的“沃土”, 肿瘤细胞可通过血管或淋巴管转移, 在此处种植、生长, 并形成继发性转移灶[15]。原发性肿瘤可分泌肿瘤源性因子, 动员骨髓来源的免疫细胞, 进而改变远端器官基质成分, 发生转移[16]。在肺转移微环境形成过程中, 中性粒细胞和单核细胞在肺中募集, 趋化因子受体表达增加[12]。因此, 抑制免疫细胞的募集和炎性因子的产生, 可以破坏转移前微环境的形成与功能。趋化因子受体CXCR1、CCR2均属于G蛋白偶联受体(GPCR)超家族成员, 主要表达于中性粒细胞、单核细胞等免疫细胞的表面, 可促进肿瘤转移, 介导炎症反应, 并在调控免疫细胞募集过程中发挥重要作用[17, 18]。c-Kit致癌基因的突变及异常表达同样在肿瘤发生发展过程中发挥重要作用[19], 有研究表明c-Kit过表达可阻断吉非替尼下调PI3K/AKT信号传导途径, 致使肿瘤细胞对吉非替尼产生耐药[20]。本研究发现, 与模型组比较, 吉非替尼组、SJZ组与联合给药组对肺组织中的中性粒细胞的募集均有不同程度的抑制作用, 联合给药组抑制作用最明显。在血和脾组织中, 吉非替尼对中性粒细胞数量无明显作用, 而SJZ可明显降低中性粒细胞数量。值得注意的是, 与模型组比较, 吉非替尼给药后, 外周血和脾组织中单核细胞表面CXCR1、CCR2和c-Kit的表达均有不同程度上升, 外周血中c-Kit及脾组织中CCR2表达水平上升最明显, 而SJZ组和联合给药组均可抑制中性粒细胞和单核细胞表面CXCR1、CCR2、c-Kit的表达。趋化因子受体和c-Kit作为潜在的治疗靶标, 目前已成为拟攻克肿瘤转移及耐药的研究热点, 如sitravatinib[21]。本研究显示, 四君子汤可显著下调免疫细胞表面受体的表达水平, 提示四君子汤的某些化学成分, 能通过靶向作用该受体, 调控肺癌转移前微环境, 进而改善吉非替尼耐药。

免疫细胞与肿瘤细胞的多种生物学行为的密切联系是通过炎性细胞因子的交互作用而实现的[22-24]。IL-1通过影响血管黏附因子的表达, 进而诱导中性粒细胞穿过血管进入肿瘤转移前微环境[22]。IL-6可协同粒细胞集落刺激因子(human granulocyte colony stimulating factor, G-CSF), 增强中性粒细胞的促肿瘤血管生成能力[25]。此外, 肿瘤细胞过度分泌IL-6可能是导致埃克替尼、厄洛替尼等酪氨酸抑制剂疗效下降, 进而产生耐药的机制之一[26, 27]。本研究通过对IL-1α和IL-6炎性细胞因子的检测发现, 吉非替尼可提高肿瘤组织中IL-1α、IL-6炎性细胞因子含量, SJZ组和联合给药组可降低炎性细胞因子含量, 提示吉非替尼在发挥治疗作用的同时, 也会使某些炎性细胞因子升高, 加剧了肿瘤免疫抑制微环境, 该结果与文献报道一致[7, 28-30]。但是, 四君子汤可抑制吉非替尼在肿瘤微环境中释放炎性细胞因子, 调控吉非替尼及肿瘤发展所致的炎性微环境, 提示四君子汤具有改善吉非替尼耐药的潜力。

总之, 吉非替尼可使转移前微环境趋化因子和炎性因子表达增加, 进而诱发机体炎症反应, 加剧肿瘤微环境免疫抑制现象, 而四君子汤可抑制吉非替尼所致趋化因子受体或炎性因子表达上调。因此, 四君子汤可提高吉非替尼疗效, 抑制肿瘤转移, 改善吉非替尼耐药, 调控机体转移前微环境可能是其重要免疫学机制, 但具体作用机制需要进一步研究。

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