以LSC为靶标的复方浙贝颗粒抗AML临床研究

【作者】 赖宗浪；

【导师】 李冬云；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 本文由两部分组成,通过两篇文献综述回顾了白血病干细胞(leukemia stem cell,LSC)具有自我更新和无限增殖潜能,是造成白血病发生发展的根源。而且LSC处于静止期,对化疗药物不敏感,易产生对多种化疗药物的交叉耐药,一般细胞周期化疗药物很难对其发挥杀伤效果。加之LSC集中于药物难以达到的骨髓区域以及具有天然耐药特性,正好逃逸了抗癌药物的攻击,为急性白血病复发潜伏了祸根。当多数白血病患者处于临床完全缓解状态时,体内仍残留包括LSC在内的微小残留病灶会重新活跃进入细胞分裂周期,并很快自我更新,无限增殖,造成急性白血病复发。临床观察还发现,部分难治性急性白血病患者一旦使用化疗既会出现严重的骨髓抑制,但停药后外周血或骨髓却很快显现大量的白血病细胞,且具有耐药相关蛋白高表达特征,支持LSC具有高度增殖潜能与多药耐药的生物学特征。因此针对LSC源头上的靶向干预治疗是克服白血病多药耐药最有效途径。我们前期研究发现,复方浙贝颗粒联合化疗能够提高难治性急性白血病患者临床缓解率,效应机制与诱导细胞凋亡、降低膜耐药蛋白高表达、调节酶异常变化相关,但均以白血病细胞为靶标,是否能针对LSC起到逆转耐药效应缺乏深入研究。临床研究主要为：1.研究目的通过观察复方浙贝颗粒联合化疗对难治性急性髓系白血病(acute myeloid leukemia, AML)患者骨髓LSC表面分子标志物的影响及难治性AML患者白血病细胞PI3K/Akt/mTOR信号通路和NF-κB信号通路中相关分子标记物(PI3K、mTOR、PTEN、 NF-κB)与骨髓周围微环境相互作用的细胞表面标记物CD44表达变化,试图明确复方浙贝颗粒逆转多药耐药效应靶点。2.研究方法全部病例资料来自北京中医药大学东直门医院血液肿瘤科,北京大学人民医院血液科,廊坊市中医医院血液科于2011年12月至2013年8月观察的难治性AML患者。治疗采用化疗方案有HAE, MA, CAG, AA, EA, FLAG等,复方浙贝颗粒(浙贝母12克,川芎9克,防已9克)由北京康仁堂药业有限公司制成中药配方颗粒,于化疗前3天开始服用复方浙贝颗粒,每次1袋,每日2次,共服用14天,21天为一疗程。以流式细胞术分别检测骨髓有核细胞中LSC表面分子标志物(CD34+CD123+、 CD33+CD123+)细胞表达比例。有30例患者完成治疗前后对照,分别检测了2次LSC表达比例；另外23例患者仅检测了1次LSC表达比例。20例次在北京中医药大学东直门医院血液肿瘤科住院的难治性AML患者行骨髓活检术采集组织标本,以免疫组化技术检测了CD44、PI3K、mTOR、PTEN、NF-κB表达情况。3.研究结果①难治AML中CD34+与CD33+CD123+细胞比例分别为11.78%、12.86%,两者相关性分析显示相关系数为0.953,两者具有明显相关性(p<0.001)。②按诊疗标准分类将难治性AML分为两组,其中处于缓解状态时患者的LSC表达比例分别为0.66%,0.92%,较处于复发状态时LSC比例(20.73%,22.47%)明显降低,差异具有统计学意义(p<0.001)。③30例完成前后治疗对照的难治性AML患者治疗后LSC表面分子标志物细胞表达比例(5.49%,5.94%)较治疗前明显降低(17.05%,17.71%),差异有统计学意义(p<0.05)。其中25例处于复发状态时完成治疗前后对照的患者进一步分析发现,治疗后LSC比例(6.82%,7.39%)也较治疗前LSC比例(21.74%,22.48%)明显降低,差异有统计学意义(p<0.01)。治疗后达到完全缓解有10例患者(有效率40%),且LSC表面分子标志物细胞表达比例分别为1.50%,1.65%,较未缓解患者明显下降(11.65%,12.61%),差异具有统计学意义(p<0.01)。④20例取骨髓活检患者中9例处于完全缓解的白血病细胞相关分子标记物CD44、PI3K、mTOR.PTEN.NF-κB阳性细胞表达率分别为22.2%、0%、33.3%、33.3%、77.8%,与11例未缓解的63.6%、0%、18.2%、63.6%、90.9%相比,CD44表达差异有统计学意义(p<0.05),阳性表达强度也提示CD44表达在两组间有统计学差异。完全缓解的白血病细胞相关分子标记物CD44.PI3K.mTOR.PTEN.NFκB平均光密度分别为441.78、217.28、393.58、91.16、642.66,与未缓解的840.06、316.21、223.00、292.51、775.35相比,CD44表达差异有统计学意义(p<0.05)。4.研究结论①CD34’CD123+与CD33+CD123+细胞具有显著相关,可以用来定位LSC。②LSC比例可以反映急性白血病患者是否处于持续缓解状态。③复方浙贝颗粒是通过降低LSC表面分子标记物CD123、CD33表达比例,提高了难治性AML化疗缓解率。④白血病细胞中CD44高表达可作为难治性AML预后判定的重要参考指标,PI3K/Akt/mTOR信号通路和NF-κB信号通路在白血病细胞中的作用需要进一步研究。更多还原

【Abstract】 This article contains two parts. From literature review, leukemia stem cell (LSC) which has self renewal capacity and infinite proliferation potential is the root of acute leukemia. LSC is in stationary stage, reluctant to most chemotherapy drugs, and easily causes multidrug resistance in leukemia cells. LSC concentrates in bone marrow niche where chemotherapy drugs can’t reach and owns natural resistance characteristics, so LSC can avoid drug’s attack to be a risk factor of relapsed acute leukemia. When most acute leukemia patients are in clinical complete remission stage, minimal residual diseases including LSC could repeatedly activate into cell cycles, realize self renewal and infinitely proliferated, then acute leukemia patients will become relapsed. In clinical observation, some refractory acute leukemia patients turn out to serious myelosuppression after chemotherapy, but they will relapse quickly with mass leukemia cells possessing over expression of drug-resistant related proteins in peripheral blood or bone marrow after drugs withdrawl. This indicates that LSC has biological characteristics of high proferation potential and multidrug resistance. Targeting on leukemia stem cell may be one of best therapies to overcome leukemia cells’multidrug resistance. In previous studies, Compound Zhebei Granule plus chemotherapy can improve clinical efficacy of refractory acute leukemia, which curative mechanisms rely on inducing cell apoptosis, down regulating over expression resistant proteins on membrane surface and adjusting abnormal changes of enzymes about multidrug resistance. These studies focus on leukemia cells and lack further researching on its function of leukemia stem cell.Objective: this thesis is to investigate the efficacy of Compound Zhebei Granule plus chemotherapy on acute myeloid LSC’s surface markers, and to detect the expression of PI3K/Akt/mTOR and NF-κB signal pathway biomarkers (PTEN, PI3K, mTOR, NF-κB) and bone marrow peripheral microenviroment’ scell surface marker (CD44) related with bone marrow cells in patients with refractory acute myeloid leukemia.Methods:From December,2011to August,2013, all cases who were candidates for refractory acute myeloid leukemia criteria were included from department of oncology and hematology in Dongzhimen hospital affiliated Beijing University of Traditional Chinese Medicine, department of hematology in Renmin hospital affiliated Beijing University and department of hematology in Langfang hospital of Chinese medicine. The common chemotherapy has HAE, MA, CAG, AA, EA, FLAG, et al. Compound Zhebei Granules are composed of fritillaria12grams,ligust icum9grams and tetrandr ine9grams, and is made into granule by Beijing tcmages pharmaceutical company limited. Compound Zhebei Granules were taken before chemotherapy in three days, once one bag, and twice a day, lasting fourteen days, twenty-one days a treatment cycle. Two milliliters bone marrow were analyzed by flow cytometry to evaluate the quantity of CD34+CD123+、CD33+CD123+leukemia stem cell. Thirty patients had undergone Compound Zhebei Granules plus chemotherapy and were abstracted bone marrow twice, the other twenty three patients were abstracted bone marrow once. Twenty patients in department of oncology and hematology in Dongzhimen hospital affiliated Beijing University of Traditional Chinese Medicine have abstracted bone marrow biopsy, then to do immunohis tochemistry to detect PTEN, PI3K, mTOR, NF-κB and CD44in all the samples. Results:①The proportions of CD34+CD123+, CD33+CD123+cells in refractory AML respectively are11.78%,12.86%, the correlation coefficient between them is0.953, both of them have significant correlation (p<0.001).②Eighty-three cases of refractory acute leukemia had been tested the proportions of CD34+CD123+, CD33+CD123+cel Is. According to diagnostic criteria and therapeutic standards, the proportions of LSC in refractory AML patients who were under remission stations are0.66%,0.92%, which have a significant decrease when compared with the proportions of leukemia stem cell in refractory AML patients who were under relapsed stations.③Thirty refractory AML patients had gone through Compound Zhebei Granules plus chemotherapy. Compared with the prior chemotherapy group (17.05%,17.71%), the proportions of LSC cells in post-treatment group (5.49%,5.94%) have a significant decrease (p<0.05). To further investigation, the proportions of leukemia stem cell in twenty-five patients who were under relapsed stations also had a significant decrease when compared with the prior chemotherapy group. Ten patients in twenty-five who were refractory AML patients under relapsed stations had accomplished completely release with the effects of Compound Zhebei Granules plus chemotherapy, and the remission rate is40%. When compared with the unreleased group, the proportions of CD34+CD123+, CD33+CD123+cel Is have a statistical significant reduction (p<0.01)in clinical remission.④Twenty refractory AML pat ients who have biopsy bone marrow were divided into nine patients who were in remission stations and eleven patients who were unreleased stations. The positive expression rates of PTEN, mTOR, NF- kB, CD44and PI3K in nine remission patients were33.3%,33.3%,77.8%,22.2%and0%, meanwhile in eleven relapsed patients, the positive expression rates were63.6%,18.2%,90.9%,63.6%and0%. There were statistical differences in the percentage and mean optical density for PTEN, CD44between the two groups (p<0.05). Also the average optical density of PTEN, mTOR, NF-κB, CD44and PI3K in nine remission patients were91.16,393.58,642.66,441.78,217.28, meanwhile in eleven relapsed patients, average optical density of PTEN, mTOR, NF-κB, CD44and PI3K were292.51,223.00,775.35,840.06,316.21. There were statistical differences in the percentage and mean optical density for PTEN, CD44between the two groups (p<0.05). Conclusions:①The proportions of CD34+CD123+, CD33+CD123+cells have significant correlation and could be defined as LSC②The proportion of LSC could be utilized to judge acute leukemia patients station of sustained remission.③Compound Zhebei Granule can reduce the ratio of CD34+CD123+,CD33+CD123+LSC to improve the efficacy of refractory and relapse acute leukemia when combining chemotherapy, and Compound Zhebei Granule plus chemotherapy has a brilliant application prospect in the efficacy of refractory and relapse acute leukemia.④High expreesion of CD44could be an important index for diagnosis and prognosis in acute myeloid leukemia. PI3K/Akt/mTOR and NF-κB signal pathway in leukemia cells need further investigation.更多还原