【作者】 高芳；

【导师】 陈学良；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 研究背景和研究目的恶性淋巴瘤是淋巴结或淋巴结外部分淋巴组织的免疫细胞肿瘤,一般分为霍奇金淋巴瘤(Hodgkin’s Lymphoma)和非霍奇金淋巴瘤(non-Hodgkin’sLymphoma,NHL)两大类。我国霍奇金淋巴瘤的发病率总体较低,非霍奇金淋巴瘤是最常见的淋巴系统恶性肿瘤。据世界卫生组织统计,在我国近年来非霍奇金淋巴瘤的发病率呈上升趋势,在过去20年,它的发病率增加了75%,在发病率增长最快的肿瘤中位居第3位。因此NHL的防治工作一直是我国医药卫生领域的重点,寻找安全有效的药物是肿瘤治疗中的重要课题。近年来动物来源药物越来越受重视,充分利用我国动物资源开发新的抗肿瘤药物,成为肿瘤研究领域的重要内容。蝎毒作为中医传统药材已经应用几千年了。在中国第一部药典《本草纲目》中,全蝎被用于治疗多种疾病。蝎毒为传统药材全蝎的主要活性物质,存在于蝎尾部毒囊内,鳌刺时由鳌针排出。现代药理学研究表明,蝎毒主要由蛋白质和非蛋白质两部分组成,蝎毒的主要活性成分是蛋白质。蝎毒具有广泛的药理学性质,近年来国外对蝎毒的研究主要集中在膜通道阻滞的作用和抗毒素方面,罕见关于蝎毒抗肿瘤方面的研究报道,国内的研究侧重其抗肿瘤、抗风湿、抗癫痫、抗炎、纤溶、镇痛以及对心血管病的作用等方面;国内学者对蝎毒的抗肿瘤作用进行了较多研究,从蝎及蝎毒的抗肿瘤作用、蝎毒的成分和主要成分的药理作用、全蝎及蝎毒抗肿瘤的临床应用等方面分析,结果与结论显示,蝎毒可抑制多种人类肿瘤细胞生长,诱导肿瘤细胞凋亡。但是,蝎毒素对肿瘤细胞杀伤或生长抑制具有一定的肿瘤差异性,有一定的肿瘤谱;而且有关蝎毒抗肿瘤作用与靶细胞基因表达的关联问题目前未见文献报道,其诱导细胞凋亡的具体分子机制至今尚未完全阐明,这是一个值得探讨的领域。肿瘤的发生通常被认为是由于细胞原癌基因的激活和抑癌基因的失活,扰乱了正常的增殖、分化和凋亡的调控,导致细胞增殖过度而凋亡不足。PTEN(第10号染色体缺失性磷酸酶-张力蛋白同源性基因)基因是一种新发现的肿瘤抑制基因。它的蛋白产物具有脂磷酸酶活性,其主要作用底物是磷脂酰肌醇-3,4,5-三磷酸(PIP3)。PIP3是PI3K/Akt信号转导通路的关键性组分,负责刺激正常细胞生长以及抑制肿瘤细胞的生长。PTEN使PIP3形成PIP2,降低PIP3水平,抑制了Akt的活性,因此降低了Bad磷酸化,导致细胞的凋亡;促进了p27的表达,引起细胞周期停滞。已发现这种基因在淋巴瘤细胞中5%可发生突变,导致对肿瘤的生长失去抑制作用。非霍奇金淋巴瘤(NHL)作为我国最常见淋巴系统的恶性肿瘤,近年来虽然放疗、化疗及造血干细胞移植的有了极大发展,淋巴瘤的死亡率并没有降低。人们致力于寻找新的、疗效肯定、毒副作用小的化疗药物,尤其是作用于肿瘤发生环节的药物。为了探讨蝎毒在NHL淋巴瘤治疗中的应用价值,我们研究了蝎毒对NHL淋巴瘤细胞株Raji和Jurkat细胞及正常人外周血淋巴细胞增殖、细胞周期和凋亡的影响。通过采用分子生物学技术检测凋亡相关蛋白PTEN、Akt、p-Akt、Bad、p-Bad、p27和PTEN mRNA的改变,以进一步阐明蝎毒的抗肿瘤作用的分子机制。研究内容1、蝎毒素诱导NHL细胞株Raji和Jurkat细胞凋亡的作用;2、蝎毒素对Raji和Jurkat细胞周期分布的影响;3、蝎毒素诱导Raji和Jurkat细胞凋亡的重要分子及信号通路;4、蝎毒素对Raji和Jurkat胞周期相关蛋白的影响。研究方法1.甲基四唑蓝(MTT)法检测不同浓度、不同时间点蝎毒素(BmK)对NHL细胞株及人正常外周血淋巴细胞的抑制作用选用NHL细胞株Raji和Jurkat及人正常外周血淋巴细胞为研究对象,采用台盼蓝染色,细胞计数仪计数,MTT法检测细胞存活率,由此确定各组的的IC50值。2.细胞凋亡检测方法如下2.1倒置相差显微镜观察药物作用后细胞形态学变化;2.2 Hoechst3342荧光染色并利用荧光显微镜,观察细胞核形态学变化;2.3 Annexin V-FITC和PI染色,流式细胞仪(FCM)检测细胞凋亡情况,计算细胞凋亡率;2.4 LY294002联合蝎毒素对细胞凋亡的影响在蝎毒素作用细胞前20分钟加入终浓度对20μM的LY294002,然后蝎毒素处理细胞48 h,FCM检测细胞凋亡率。3.PI染色流式细胞仪(FCM)检测细胞周期分布情况。4.Western blot进一步检测蝎毒素处理前后细胞周期、细胞凋亡及信号转导途径的相关蛋白PTEN、Akt、p-Akt、Bad、p-Bad、p27蛋白水平变化。5.半定量逆转录聚合酶链反应(PT-PCR)检测PTEN mRNA的表达。6.统计学处理:本实验所有数据用均数±标准差表达,实验组与对照组比较用t检验,组间均数比较采用单因素方差分析。研究结果1.台盼蓝染色,细胞计数仪计数和MTT法均显示:蝎毒素对NHL细胞株Raji和Jurkat细胞具有生长的抑制作用,呈浓度、时间相关性。蝎毒素对NHL细胞株和人外周血淋巴细胞(PBLs)作用48后,IC50分别为275.40μg/ml(Raji)、360.60μg/ml(Jurkat)和1110μg/mL(PBLs),各组间经统计学处理有显著性差异(p＜0.05)。以上结果提示:蝎毒素对NHL细胞株Raji和Jurkat细胞的增殖抑制作用强于人外周血淋巴细胞(PBLs),低浓度(≤500μg/ml)时对PBLs细胞的生长抑制作用不明显。两种NHL细胞株中,Raji细胞对蝎毒素较敏感,强于Jurkat细胞。2.蝎毒对淋巴瘤细胞凋亡的影响:2.1倒置相差显微镜观察发现:蝎毒处理Raji和Jurkat细胞48 h后,细胞大小不一,呈现细胞碎片及胞浆内颗粒增多等特征,并最终死亡。2.2 Hoechst3342染色荧光显微镜检测发现:与对照组相比,蝎毒素处理组中Raji和Jurkat细胞核均呈现碎块状致密浓染,荧光染色增强;随着浓度增高,凋亡小体产生,符合凋亡的形态学改变。2.3 Annexin V-FITC/PI染色FCM检测显示:蝎毒素能够诱导Raji和Jurkat细胞株凋亡,其凋亡率随用药剂量增加而增加,并且Raji细胞的早期凋亡率(43±7%)明显高于Jurkat细胞(24±5%),有统计学意义(P＜0.05)。蝎毒素作用PBLs细胞48小时后,各浓度组凋亡率较空白对照组的差异未显示统计学意义(P＞0.05)。以上结果提示:蝎毒素可选择性作用于NHL细胞株诱导其凋亡,而对人正常外周血淋巴细胞影响较小。3.PI染色流式细胞仪(FCM)检测发现:蝎毒素引起NHL细胞株Raji和Jurkat细胞G0/G1期阻滞。4.Western blot检测发现:蝎毒素促使Rajii细胞PTEN蛋白表达增加,同时p-Akt、p-Bad蛋白表达减少;而对Jurkat细胞以上蛋白表达无影响。提示蝎毒素诱导PTEN蛋白表达上调,且该PTEN蛋白为功能性蛋白,可以下调Akt、Bad磷酸化水平,从而影响PI3K/Akt信号转导通路,这可能是蝎毒素诱导Raji细胞凋亡的重要机制。5.为了进一步验证PI3K/Akt信号转导通路在蝎毒素诱导Raji细胞凋亡中的作用,LY294002(PI3K特异抑制剂)联合蝎毒素对Raji细胞的影响。发现联合用药呈现叠加效应,不仅使Raji细胞凋亡率明显增加,同时使Akt磷酸化水平进一步降低。由此我们判断,蝎毒素通过上调PTEN表达水平,激活PI3K/Akt信号转导通路是蝎毒素诱导Raji细胞凋亡的重要机制之一。6.RT-PCR法测PTEN mRNA的表达水平,Raji细胞中发现PTEN mRNA表达呈蝎毒素浓度依赖性增加,说明蝎毒素是在PTEN基因转录水平影响PTEN蛋白表达。7.Western blot检测发现:蝎毒素作用Raji和Jurkat细胞48 h后,伴随着G1期阻滞,p27蛋白表达呈蝎毒素浓度依赖性增加。提示蝎毒素促使Raji和Jurkat细胞凋亡的机制还与上调p27蛋白进而导致G1期细胞阻滞有关。结论蝎毒素能够抑制NHL细胞株Raji和Jurkat细胞的增殖,并且呈时间、浓度依赖性。可以诱导Raji和Jurkat细胞凋亡和细胞周期G1期阻滞的能力。可能是通过以下的机制发挥作用的:1、在PTEN基因转录水平促进Raji细胞PTEN蛋白表达,抑制Akt、Bad的磷酸化,从而激活PI3K/Akt信号转导通路,这可能是蝎毒素诱导Raji细胞凋亡的重要机制之一。2、还可以通过非PTEN依赖途径,上调p27蛋白的表达,引起G1期细胞周期停滞,这是蝎毒诱导Raji和Jurkat细胞凋亡的共同通道。更多还原

【Abstract】 BACKGROUND AND OBJECTIVE:Malignant tumors are serious diseases endangering human health.E very year about 1,600,000 patients with cancers were newly increased in our country and about 1,300,000 patients died from tumors.With the growth of population and the acceleration of aging population,the incidence and mortality of tumors are on the rise.Therefore,the prevention and treatment of tumors have long been the focal work in the Chinese medicine.It is then an important subject to find medicines both safe and effective in the treatment of tumors.Recently more attentions have been attached to the drugs from animals,and antineoplastics have been developed in using fully the animal sources of our country,which has then become the important content in the research work of tumors.The scorpion Buthus martensii Karsch（BmK） had been used as a Chinese traditional medicine for thousands of years.In the first authoritative Chinese pharmacopoeia "Compendium of Material Medica" edited by Li Shizhen in 1578, dried scorpion bodies were used to treat a variety of diseases.Scorpion venoms are complex mixtures of molecules,most of which are peptides displaying various kinds of biological activity.During the past two decades,several of these compounds have been identified.Toxins modulating Na⁺,K⁺,Ca²⁺ and Ckl^- currents across cell membranes have been described.Some enzymes like proteases and hyaluronidases as well as chlorotoxin-like peptides from this venom may have therapeutic potential,including anticancer activity.Recent studies have demonstrated that BmK venom can induce apoptotic cell death,and possesses anti-tumor effects.However,the molecular mechanisms that underlie the induction of apoptosis by BmK venom are poorly understood,and require further exploration.PTEN（phosphatase and tensin homolog deleted from chromosome 10） gene is a new tumor suppressor gene proved to be a lipid phosphatase.The target lipid, called phosphatidylinositol-3,4,5-trisphosphate,PIP3,and it is a key component of the major cell growth control pathways,acting both to stimulate cell growth and to inhibit tumor cell proliferation blocking apoptosis.PTEN mutations have been linked to a variety of human cancers.Mutations of PTEN during tumorigenesis allow the mutated cells to grow unchecked when they should die.Lymphoma is a cancer in the lymphatic system.Despite recent advances in radiotherapy,chemotherapy,and stem cell transplantation,the severe morbidity from lymphoma has not been alleviated.Much effort has been focused on the discovery and development of new chemopreventive agents,especially agents targeted at mechanisms known to be involved in the process of carcinogenesis. Therefore,we have sought to identify novel agents that can effectively prevent lymphoma carcinogenesis but have minimal toxicity to healthy cells.In this study, we examined the effects of BmK venom on two human lymphoma cell lines（Raji and Jurkat） compared with peripheral blood lymphocytes（PBLs）,and focused on changes in the PTEN and the PI3K/Akt signal pathway and to further investigate the molecular mechanisms involved in these effects.STUDY CONTENTS:1.Identification of apoptosis induction in Raji and Jurkat cells by BmK venom.2.Analysis of the effects of BmK venom on cell cycle distribution and p27 protein level.3.Analysis of the apoptosis pathways activated by the small molecules in Raij and Jurkat cells.METHODS:1.MTT assay for cell viability,by which small molecules were screened initially.2.Methods for apoptosis identification:2.1 Observation of cell morphological changes by Phase Contrast Microscope. 2.2 Observation of nuclear morphological changes by Hoechst 3342 staining combined with fluorescence microscopy.2.2 Analysis of the changes in apoptosis by flow cytometry.3.Analysis of the changes in cell cycle distribution by flow cytometry.4.Analysis of the changes in PTEN、Akt、p-Akt、Bad、p-Bad protein level by Western blot assay.5.Analysis of the changes in PTEN mRNA level by PT-PCR assay.RESULTS:1.BmK venom inhibited the viability of Raji and Jurkat cells in a dose-and time-dependent manner,and the IC50 value calculated to be 275.40μg/ml（Raji） and 360.60μg/ml（Jurkat）.2.BmK venom-induced apoptosis:When Raji and Jurkat cells were exposed to BmK venom for 24-48 h,cell shrinkage,membrane blebbing and apoptotic bodies occurred.Ultimately,many cells underwent death;Hoechst 3342 staining combined with Fluorescence Microscopy showed that compared with the nuclei in the control group,the nuclei were stained into brightly blue after treated by BmK venom for 48h.Flow cytometry showed that in Raji cells,the levels of p-Akt and p-Bad clearly decreased in a dose-dependent manner,while the levels of total Akt and Bad protein were stable.PTEN expression was inversely correlated with phosphorylation of Akt, indicating that the PTEN was functional.3.Flow cytometry showed that with increasing concentrations of BmK venom,the number of Jurkat and Raji cells in the G₀/G₁ phase increased significantly compared with control cells,while the cells in S and G₂/M phase decreased accordingly.4.In Raji cells,BmK venom up regulated the expression of PTEN accompanied by decreased levels of Akt and Bad phosphorylation.Treatment with BmK venom and LY294002（an inhibitor of Akt） synergistically enhanced apoptosis. The expression of p27 was increased in both PYEN-positive Raji and PTEN-negative Jurkat cells exposed to BmK venom.CONCLUSION:In summary,it was demonstrated that BmK venom could inhibit cell proliferation and induces apoptosis on human NHLs cell lines,Raji and Jurkat cells. The cytostatic effect was dose- and time-dependent.One possible mechanism may be suppression of Akt signal pathway via PTEN and up regulation of p27 with affecting auxiliary pathways.These data suggest that BMK may be effective in the treatment of non-Hodgkin’s lymphoma.更多还原