【作者】 柴小姝；

【导师】 吴万垠；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2011， 博士

【摘要】 研究背景：肺癌是全球常见的恶性肿瘤之一,并成为目前人类因癌症死亡的主要原因,是对人类健康与生命危害最大的恶性肿瘤之一。PI3K/AKT/mTOR信号通路在肺癌的发生发展中起着重要的作用,该通路的活化可以抑制多种刺激诱发的细胞凋亡,促进细胞周期进展,从而促进细胞的生长和增殖,同时参与血管形成,在肺癌的形成中扮演重要角色,并参与肿瘤的侵袭和转移。维持治疗作为延长肺癌患者长期生存的新的重要手段,已引起临床医生的极大重视。晚期非小细胞肺癌的维持治疗临床研究表明,患者经过4-6周期含铂类药物的一线标准联合化疗方案后,疾病无进展且体力状况良好,选择毒副作用较小的药物如细胞毒单药维持化疗或分子靶向药物进行维持治疗可使晚期非小细胞肺癌(NSCLC)患者受益。而中药在维持治疗晚期非小细胞肺癌的领域中的研究尚为数不多,基于长期化疗药物及分子靶向药物维持治疗的毒副反应,中医药可发挥自身优势,探索最佳维持治疗方案。消积饮作为广东省中医院名老中医刘伟胜教授的多年经验方,已取得了良好的临床疗效,同时亦有多个实验证明该药能够抑制小鼠Lewis肺癌的生长和自发性肺转移、延长荷瘤小鼠的生存期,其作用机制与消积饮降低小鼠Lewis肺癌细胞cyclinDl的表达,影响其细胞周期G1/S调控点,有效地阻止肿瘤细胞周期于G0、G1期,使其不能进入S期进行DNA复制有关。此外,消积饮能有效下调肿瘤细胞PCNA(增殖细胞核抗原)、survivin蛋白及bcl-2基因的表达强度,诱导肿瘤细胞凋亡,降低其转移潜能,从而稳定病灶、减少转移。研究目的：为探索一条中药维持治疗晚期肺癌的治疗方案,同时深层次挖掘名老中医的学术内涵,从PI3K/AKT/mTOR信号通路探讨消积饮抑制肺癌生长的分子机制亟待进一步研究。研究内容与方法：1临床部分根据纳入标准选入64例经4～6周期化疗后1个月以上,RECIST评价稳定的ⅢB及Ⅳ期NSCLC患者,入组前患者卡氏评分≥60分,无脑转移或脑转移无症状,骨髓、心、肝、肾功能正常。按信封法随机分为治疗组和对照组各32例,治疗组给予消积饮(60ml/m2, po, qd)维持治疗,对照组给予支持治疗,治疗3月后对照统计两组维持治疗的有效率(CR+PR+SD)、临床证候及体力状况、疾病无进展生存期(PFS)及总生存期(OS)。治疗组评价无疾病进展继续治疗及随访观察至疾病进展或死亡。应用SPSS12.0统计分析软件,组间临床特征均衡性评估采用方差分析和PearsornX2检验；组间OS、PFS比较采用Kaplan-Meier分析；治疗后临床证候、体力状况评分取治疗后评估分。组间症状缓解率、体力状况比较采用两个独立样本Mann-Whitney U Test。安全性评价采用描述性统计。2实验部分选取人A549肺癌细胞株作为研究对象,生长于RPMI-1640培养液中,细胞传代24h进入对数生长期后分别加入低浓度、中浓度、高浓度含药血清(含消积饮的大鼠血清)及胎牛血清对照组进行细胞诱导。MTS法测定消积饮对A549细胞增殖的抑制作用,Western Blot印迹法检测PI3K/AKT通路p-AKT蛋白的表达,RT-PCR法检测含药血清对PI3K/AKT通路下游分子BAD、Caspase-9、FKHR等基因的mRNA的表达。应用SPSS12.0统计分析软件,组间对照采用单因素方差分析和t检验。研究结果：1临床部分1.1临床特征64例PSO-2的ⅢB/Ⅳ期非小细胞肺癌患者均来自于广东省中医院肿瘤科住院部,患者于2008年9月到2010年12月纳入研究,治疗前两组基线资料,性别、吸烟史、年龄、分期、KPS评分、入组前化疗等差异均无显著性,组间分布均衡(P>0.05)。共4例出组,治疗组2例因未完成既定治疗出组,对照组2例因主观违背治疗方案出组。实际可评价疗效病例60例,已死亡41例,其余19例正在随访中,按预期计划,经统计学样本数估算,符合疗效评价要求。1.2近期疗效治疗3个月后疗效评估,消积饮治疗组有效率达76.7%,对照组有效率达43.3%,两组之间相比具有显著差异(P=0.00)。1.3生存时间截止最后随访时间,治疗组和对照组OS分别为6.43个月和3.27个月,组间比较,治疗组的OS延长了3.16个月,两组有明显差异(P=0.00)；治疗组和对照组PFS分别为5.07个月和2.53个月,组间比较,治疗组的PFS延长了2.54个月,两组有明显差异(P=0.00)。1.4临床证候评分治疗3个月后,与对照组比较,治疗组临床证候评估改善率提高了43.3%,差异有统计学意义(Z=2.362,P=0.009)。1.5体力状况治疗3个月后,与对照组比较,治疗组体力状况改善率提高了26.6%,差异有统计学意义(Z=2.128,P=0.033)。1.6安全性评价安全性评价表明受试者服用消积饮前后血常规,肝、肾功能及心电图检查均未见明显变化。按WHO《实体瘤疗效评定标准和急性和亚急性毒副反应评定标准》,本组病例中没有出现因为不能耐受不良反应而出组的情况。未见相关血液学毒性。未见明显呕吐、腹泻等胃肠道症状、脱发及严重的心、肝、肾功能损害。治疗组3例受试者第2天出现恶心,按WHO毒性分级1级,未经处理,持续约10min,后症状消失；1例受试者服药后约30min出现轻度腹泻,未经处理自行缓解。未出现与治疗相关的死亡。2实验部分2.1 MTS法检测含不同浓度消积饮血清对细胞增殖影响的研究不同浓度的消积饮血清均可抑制人A549肺癌细胞的增殖；高浓度消积饮血清培养人A549肺癌细胞72小时增殖抑制率与对照组相比具有显著差异。2.2消积饮调控人肺癌细胞p-PI3K/p-AKT/mTOR基因表达的研究人A549肺癌细胞中加入不同浓度的消积饮血清作用48小时、72小时后记录基因表达量,结果示BAD、Caspase-9基因mRNA的表达量明显增加,作用48小时的高浓度消积饮组2-△△Ct较对照组明显升高,并具有统计学意义(P<0.01)；各实验组之间比较无明显差异。同时,mTOR、NF-κB、FKHR基因mRNA的表达量也明显增加,较对照组也具有统计学意义(P<0.01)；各实验组之间比较无明显差异(P>O.05)。2.3 Western Blotting检测消积饮对PTEN、PI3K及p-AKT蛋白表达的影响经图像处理分析,以GAPDH的灰度值为参照,PTEN、P13K、磷酸化AKT的蛋白表达减少,PI3K的蛋白表达量增加；其中低、中浓度消积饮组可使p-PTEN表达下调,与对照组相比有明显差异(P<0.01)；低、高浓度消积饮组可使P-PI3K表达上调,与对照组相比有明显差异(P<0.01)；中、高浓度消积饮均可使磷酸化AKT蛋白表达水平下调,与对照组相比具有统计学意义(P<0.01)。研究结论：1临床部分本研究通过前瞻性随机对照试验,消积饮治疗组的有效率较对照组明显升高,中位生存时间及疾病无进展时间较对照组明显延长,临床证候与体力状况较对照组明显改善,毒副反应轻微,安全性评价高。因此,消积饮作为晚期非小细胞肺癌的中药维持治疗方案具有有效低毒的优势,可使晚期NSCLC患者的生存时间及生活质量获益。2实验部分消积饮含药血清可在体外有效抑制人肺癌细胞株A549细胞的增殖,具有一定的细胞毒性作用；消积饮可有效抑制PI3K/AKT信号转导通路的AKT位点,增加PI3K/AKT信号转导通路的下游分子BAD、Casepase-9基因mRNA的表达,促进A549肺癌细胞的凋亡,从而有效抑制A549肺癌细肺癌的生长。更多还原

【Abstract】 ObjectivesTo investigate a treatment regime of Traditional Chinese Medicine (TCM) as a maintenance therapy for advanced Non-small cell Lung Cancer (NSCLC), to deepen the discovery in the academic content of renowned old TCM practitioner Liu Wei-sheng, and to further investigate the molecular mechanism of XiaoJiYin in suppressing lung cancer growth through the PI3K/AKT/mTOR pathway.Methods1 Clinical studySixty-four patients of advanced NSCLC were included according to the inclusion criteria, and were randomly divided into treatment group and control group, with 32 patients respectively. In the treatment group, XiaoJiYin was given as a maintenance therapy (60ml/m2, po, qd), while only follow-up and observation was given in the control group (Best support care). After 3 months treatment, the response rate, clinical syndrome scores, KPS scores, overall survival (OS) and progression-free survival (PFS) were compared between the two groups. Variance analysis was performed for the clinical characteristics between groups, while Kaplan-Meier analysis was conducted for the comparison of OS and PFS.2 Experimental studyHuman A549 lung cancer cell line was selected. It was cultivated in RPMI-1640, and XiaoJiYin containing rat serum of low, medium and high concentrations, and fetal bovine serum (FBS) as a control, were added to the cells in logarithmic phase for cell induction. MTS method was used for the suppressive effect of XiaoJiYin in A549 cell proliferation. Western-blot was used for the detection of p-AKT protein in PI3K/AKT pathway, and RT-PCR was used in detecting the expression of downstream mRNA, such as BAD, Caspase-9, FKHR, mTOR and NF-K B in the PI3K/AKT pathway. Variance analysis was performed between groups.Results1 Clinical study1.1 Patient characteristicsThe 64 cases of stage IIIB/IV NSCLC patients with PSO-2 were recruited from the In-patient Oncology Department of Guangdong Provincial Hospital of TCM. Patients were enrolled during September 2008 and December 2010. The pre-enrolment baseline characteristics, such as sex, smoking history, age, staging, KPS score and chemotherapy regimens received, had no significant difference and was balanced between groups (P>0.05). Four cases were drop-off, in which the 2 cases in treatment group withdrew due to discontinuation of treatment, while the 2 cases in the control group withdrew because of change in self-decision. There were 60 evaluable cases, 41 cases deceased, while follow-up was conducting. According to the plan, through statistical evaluation of sample number, the requirement of therapeutic evaluation was reached.1.2 Short-term EfficacyTherapeutic evaluation was conducted after 3 months of treatment. The response rate (RR) was 76.6% in the treatment group, while the disease control rate (DCR) was 43.3% in the control group. There was significant difference between the 2 groups (P=0.00).1.3 SurvivalUp to the last follow-up, the overall survival (OS) was 6.43 months and 3.27 months in the treatment and control groups respectively. Intra-group comparison suggested that the OS was extended for 3.16 months in the treatment group, indicating significant difference between the 2 groups (P=0.00). The progression-free survivals (PFS) were 5.07 months and 2.53 months in the treatment group and control group respectively. Intra-group comparison suggested that the PFS was extended for 2.54 months in the treatment group, indicating significant difference between the 2 groups (P=0.00). 1.4 Clinical syndrome differentiationThe clinical syndrome evaluation improvement was elevated for 43.3% in the treatment group when compared with control, indicating significant statistical difference (Z=-2.362, P=0.009).1.5 Physical statusThe KPS score in the treatment group was elevated for 26.6% compared with control group, with statistical difference, (Z=-2.128, P=0.033).1.6 Safety evaluationThe safety evaluation suggested that the blood routine, liver and kidney function and ECG examination showed no significant different before and after XiaoJiYin treatment. According to the WHO RESIST standard, there was no drop-off case due to intolerable adverse event. There were no hematotoxicity, gastrointestinal symptoms, alopecia or severe cardio, renal or hepato toxicity. Nausea of WHO toxicity grade 1 was observed on the second day of treatment for 3 patients in the treatment group. The symptom disappeared after 10 minutes without handling. Slight diarrhea was observed in on patient after administration of drug for 30 minutes, and was relieved without handling. No treatment-related death was observed.2 Experimental study2.1 Effect of different concentration of XiaoJiYin on cell proliferation by MTS methodDifferent concentration of XiaoJiYin containing serum can suppress proliferation of A549 lung cancer cells. The 72 hours anti-proliferation rate of high concentration XiaoJiYin containing serum on A549 had a significant difference compared with control group.2.2 The effect of XiaoJiYin in regulating the p-PI3K/p-AKT/mTOR gene expression in human lung cancer cellsThe gene expression quantity was recorded after adding different concentration of XiaoJiYin containing serum to A549 cells for 48 and 72 hours. The mRNA expression on BAD and Caspase-9 genes were significantly increased. The 2-ΔΔCt in the high concentration serum for 48 hours group was significantly elevated compared with the control group and had statistical significance (P<0.01). There was no significant difference between experimental groups. In addition, the mRNA expression of mTOR, FKHR and NF-K B was also obviously elevated, and had statistical difference compared with control group (P<0.01) There was no significant difference between experimental groups.2.3 The effect of XiaoJiYin on the expression of PTEN, PI3K, ATF2 and p-AKT protein through Western BlottingThrough image processing analysis, using GAPDH grey scale as reference, the protein expression of PTEN and phosphor-AKT reduced, PI3K increased yet. Low and medium concentration led to p-PTEN down-regulating, showing significant difference compared with control group (P<0.01). Low and high concentration of XiaoJiYin led to up-regulation of p-PI3K, showing significant difference compared with control group (P<0.01). Medium and high concentration of XiaoJiYin led to down-regulation of p-AKT, showing significant difference compared with control group (P<0.01).Conclusion1 Clinical studyThrough a prospective randomized control trial (RCT), it was found that the response rate, overall survival and progression-free survival were obviously elevated in XiaoJiYin group compared with the control. The clinical symptoms and physical status were obviously improved, with slight adverse side effects and high safety. Therefore, XiaoJiYin as a maintenance therapy for advanced NSCLC can improve the survival and quality of life.2 Experimental studyXiaoJiYin containing serum can effectively suppress the proliferation of human lung cancer cell line A549 in vitro, and has certain cytotoxicity. XiaoJiYin can effectively suppress the AKT point in the PI3K/AKT signal transduction pathway, increasing the mRNA expression of downstream molecules like BAD and Caspase-9, enhancing apoptosis of A549 and therefore effectively suppress the growth of A549.更多还原