【作者】 陈伟；

【导师】 陈谦学；

【作者基本信息】 武汉大学 ， 神经外科学， 2016， 博士

【摘要】 研究背景脑胶质瘤约占各种颅内肿瘤的50%,其发病原因尚不十分清楚。各级别胶质瘤的中位生存时间差别很大,其中低级别胶质瘤(WHO Ⅰ、Ⅱ级)的中位生存时间为5-8年；高级别胶质瘤(WHOⅢ、Ⅳ级)又称为恶性胶质瘤,星形胶质细胞瘤(WHO Ⅲ级)的中位生存时间为3年;Ⅳ级的中位生存时间仅有12～18个月,高龄(60岁以上)患者中位生存期更短。目前对于胶质瘤的起源有两种学说：1、肿瘤细胞由正常神经胶质细胞起源：当其分化失调后变成不成熟的胶质细胞；2、肿瘤细胞起源于神经干细胞。1992年,Reynolds[3]等首次在成年小鼠纹状体中分离得到神经干细胞,神经肿瘤干细胞的学说逐渐引起学术界科研人员重视。后来的研究发现人类成熟或发育中的中枢神经组织中存在部分休眠状态的神经干细胞,这类细胞同样具有无限增殖、自我更新和分化潜能,这就决定着肿瘤的发生、发展、侵袭、转移、复发和对各种治疗敏感性等。当中枢神经受损时,这些休眠状态下的神经干细胞会发生一定程度的增殖分化,但却不能完全修复损伤,其具体机制不明。事实上,对于胶质瘤的真正起源,目前还没有定论,在脑的胶质瘤动物模型研究中发现,经过特定基因改造处理的成熟的星形胶质细胞和神经胶质祖细胞(星形胶质细胞的前体细胞)均有可能发展成为胶质瘤。肿瘤细胞一旦发生,其就具有了高发病率、高复发率、高死亡率和低治愈率的特点。几乎所有胶质瘤都具备侵袭和转移的内在特性。肿瘤细胞能够经血管和有髓纤维路径向周围或者更远的地方迁徙。胶质瘤的生物学特性使得绝大部分手术患者的术后生存期极为短暂。现在,虽然显微手术技术已经极大改善了胶质瘤患者的预后,但目前的手术方式还只能达到影像学意义上的肿瘤全切,但无法彻底清除肿瘤细胞,手术后放化疗仍是最为普遍接受的治疗方式。然而肿瘤细胞对放疗的耐受性很可能造成残余病灶再复发。此外,由于常规化疗药物效果不佳,免疫疗法、基因疗法等治疗新策略仍处于实验阶段等原因,临床仍然缺乏满意的治疗手段。如何提高胶质瘤的综合治疗效果,降低肿瘤复发率,延长患者生存时间,是当前胶质瘤研究的关键问题。中药在肿瘤发生、发展等多个环节具有显著作用,具有多靶点、多环节、多效应等特点,它在抑制、杀伤肿瘤细胞、改善患者症状与体征,以及减轻患者放化疗不良反应、延长患者生存期等方面表现出重要的作用,同时,还具有不良反应少,改善机体免疫力,不易产生耐药性等特点,中药的抗肿瘤作用研究已受到越来越多学者的重视。近年来,从细胞增殖抑制和凋亡等角度来探讨中药抗肿瘤的作用机制已成为研究的热点。藤梨根是临床上治疗大肠癌的常用中药,有较确切的抗癌症效果,其抗肿瘤活性倍受人们关注,对肝癌、食管癌、胃癌也有明显的抑制作用,但对脑胶质瘤的作用目前研究较少,因此开展藤梨根对脑胶质瘤细胞影响的研究,并阐明其作用机制,可以为藤梨根在抗脑胶质瘤方面的进一步开发利用提供依据。由于藤梨根分布广泛、易于种植、资源丰富、价格低廉,研究开发其抗肿瘤新药生产成本较低,故具有较好的社会效益和经济效益。因此,本课题进行藤梨根对人脑胶质瘤U251及U87细胞的影响及其机制的研究。本课题分为三部分进行研究：1)体内外实验研究藤梨根对人脑胶质瘤U251及U87细胞凋亡的作用； 2)藤梨根对人脑胶质瘤U251细胞细胞周期的作用；3)藤梨根对人脑胶质瘤U251及U87细胞STAT3信号通路的作用。第一部分藤梨根提取物对胶质瘤的抑制作用目的：探讨藤梨根提取物对人脑胶质瘤的作用。方法：1)采用倒置显微镜观察不同浓度藤梨根提取物对培养孔内的脑胶质瘤U251及U87细胞形态结构变化的影响,并采用Hoechst33258荧光染色观察细胞核内染色质改变、核固缩、细胞膜皱缩、凋亡小体等。2)采用MTT法检测不同浓度藤梨根提取物对脑胶质瘤U251及U87细胞的抑制作用。3)采用CCK-8法检测经不同浓度藤梨根提取物作用48小时后的脑胶质瘤U251及U87细胞活性改变。4)体内实验研究藤梨根提取物对胶质瘤的抑制作用。结果：1、藤梨根提取物对胶质瘤细胞增殖有抑制作用在U251及U87细胞中,使用藤梨根组细胞增殖率明显低于对照组(P<0.05)2、藤梨根提取物对胶质瘤细胞增殖抑制作用具有浓度依赖性藤梨根提取物在单位时间内处理胶质瘤细胞后细胞增殖率明显降低,且随着药物浓度增加,增殖率逐渐降低(P<0.05)。3、藤梨根提取物对胶质瘤细胞增殖抑制作用无时间依赖性同一浓度藤梨根提取物作用胶质瘤细胞24h、48h、72h后,细胞增殖率变化无统计学意义(P>0.05)。4、藤梨根提取物对胶质瘤生长有抑制作用接种U251细胞于裸鼠脑内,使用藤梨根提取物及对照治疗小鼠,使用藤梨根提取物组肿瘤明显小于对照组(P<0.05)。结论：藤梨根提取物可以抑制胶质瘤细胞增殖及肿瘤生长。第二部分藤梨根提取物通过调节胶质瘤细胞周期调节胶质瘤细胞增殖目的：探讨藤梨根提取物对脑胶质瘤U251细胞周期的作用。方法：藤梨根提取物处理脑胶质瘤U251细胞24h,使用流式细胞术检测不同浓度藤梨根提取物对胶质瘤细胞周期的影响。免疫印迹技术检测BCL-2和BAX表达量的改变。结果：1、不同浓度藤梨根提取物处理胶质瘤细胞24h对细胞周期的作用藤梨根提取物处理胶质瘤细胞24h后细胞周期发生改变,细胞被阻滞在G1期,且随着藤梨根提取物浓度增加,阻滞增强,差异有统计学意义(P<0.05)。2、不同浓度藤梨根提取物处理胶质瘤细胞24h对BCL-2及BAX的作用不同浓度藤梨根提取物处理胶质瘤细胞24h后BCL-2表达量降低,BAX表达量升高,且随着药物浓度增加,BCL-2/BAX比值变小(P<0.05)。3、BCL-2及BAX可以调节胶质瘤细胞的增殖调节胶质瘤细胞中BCL-2及BAX表达量,可以调节胶质瘤细胞增殖。4、在藤梨根提取物处理的U251细胞中高表达BCL-2或者低表达BAX,可以逆转藤梨根提取物对胶质瘤的作用使用myc-BCL-2高表达BCL-2或者siBAX敲低BAX后,藤梨根提取物对胶质瘤细胞增殖的作用消失了。结论：藤梨根提取物通过调控BCL-2和BAX调控胶质瘤细胞周期,使其阻滞于G1期。第三部分藤梨根提取物通过调节STAT3信号通路调节胶质瘤细胞周期目的：探讨藤梨根提取物调控脑胶质瘤U251及U87细胞增殖的机制。方法：免疫印迹技术检测不同浓度藤梨根提取物处理脑胶质瘤U251及U87细胞后STAT3及P-STAT3蛋白表达量变化。荧光定量PCR检测不同浓度藤梨根提取物处理脑胶质瘤U251细胞后STAT3的mRNA表达量变化。结果：1、不同浓度藤梨根提取物处理胶质瘤细胞24h后STAT3及P-STAT3蛋白表达量变化藤梨根提取物处理胶质瘤细胞24h后,细胞的STAT3及P-STAT3蛋白表达量降低,且随着药物浓度增加逐渐降低,差异有统计学意义(P<0.05)。2、不同浓度藤梨根提取物处理胶质瘤细胞24h后STAT3的mRNA表达量变化藤梨根提取物处理胶质瘤细胞24h后,细胞的STAT3的mRNA表达量降低,且随着药物浓度增加逐渐降低,差异有统计学意义(P<0.05)。3、STAT3可以调节胶质瘤细胞的增殖及BCL-2和BAX表达量高表达STAT3,胶质瘤细胞的增殖能力增强,低表达STAT3,胶质瘤细胞的增殖能力减弱4、在藤梨根提取物处理的U251细胞中高表达STAT3可以逆转藤梨根提取物对胶质瘤细胞增殖及BCL-2和BAX的作用使用Flag-STAT3高表达STAT3后,藤梨根提取物对胶质瘤细胞的增殖作用及对BCL-2和BAX的作用消失了。结论：藤梨根提取物通过调控STAT3磷酸化及STAT3mRNA表达调控胶质瘤细胞周期,进而调控细胞增殖。更多还原

【Abstract】 BackgroundBrain glioma accounts for 50% of brain tumors and its risk factors are unclear. The median survival time was 5 to 8 years in low grade glioma (WHO grade Ⅰ/Ⅱ). However, in high grade glioma (WHO grade III/IV, also called malignant glioma), median survival time of astrocytic glioma (WHO grade III) was 3 years and merely 12 to 18 months in grade IV glioma. Survival time of glioma patients with an age of 60 years or older was even shorter[1，2]. Presently, two kinds of theories about origin of glioma occurred. The first theory held a view that glioma cell originated from normal glial cell which could become immature glial cells when its proliferation and differentiation were out of control. The other theory which attracting more and more attention since glial stem cells were separated from the striatum of mouse by Reynolds et al in 1992, supported that glioma cell originated from neural stem cell[3] After that, a few researches found some amount of glial stem cells with a state of dormancy existed in human mature or developing central nervous tissue. These glial stem cells in dormancy have a potential of infinite proliferation, self-renewal and multidirectional differentiation, which determines the characteristics of tumors including occurrence, development, invasion, metastasis, dissemination, recurrence and sensitivity to many kinds of treatments[4]. When central nerves damaged, these dormant glial stem cells would proliferate and differentiate finitely, which could not repair the damage completely. And the specific mechanism was unclear yet[5,6]. In the animal models of brain glioma, both mature astrocyte and glial progenitor cell (precursor cell of mature astrocyte) with a treatment of specific genes could develop to be glioma.Thus, the origin of glioma was unclear[7]. Glioma formatted to be with characteristics of high incidence, recurrence and death rate and low curability[8,9]. And invasion is the characteristic of almost all kinds of glioma. Glioma cells migrated to nearby or more distant tissues following vessels and myelinated fibers path. These biological characteristics of glioma made the survival time of most part of glioma patients extremely short. Although the prognosis of glioma patients was improved greatly by microscopic surgery technology, the present operation methods could not remove all glioma tissue completely. Postoperative radiation and chemotherapy were generally accepted. However, resistance to radiation of tumor cells usually made residual lesions recur[10]. In addition, due to the poor curative effect of conventional chemotherapy drugs and experimental stage of immunotherapy and gene therapy, there is still a lack of satisfactory treatment in clinical practice. At present, the key problem in the research of glioma was how to improve the therapeutic effect of glioma, reduce the recurrence rate and prolong survival time.Traditional Chinese medicines (TCMs) play roles in the occurrence and developing of tumor with multiple targets, links and effects. They played important roles in inhibiting and killing tumor cells, improving clinical symptoms and signs, reducing radiation and chemotherapy adverse events and prolonging patients’survival time. And TCMs could regulate the body’s immune system and have a characteristic of low adverse event and drug resistance. So, antitumor TCMs researches received more and more scholars’attention[11]. In recent years, exploring the antitumor mechanism of TCMs from tumor cells proliferation inhibition and apoptosis became the research hot spot. Radix actinidiae chinensis was commonly used TCM in the treatment of colorectal cancer with an exact anticancer effect and its antitumor activity was high-profiled[12-16]. Radix actinidiae chinensis could significantly inhibit liver cancer, esophageal cancer and gastric cancer. However, researches about its roles in glioma were very few. Thus, to carry out the radix actinidiae chinensis on human brain U251 glioblastoma cell and its mechanism research would provide basis for further development and utilization of radix actinidiae chinensis in the treatment of brain glioma. Due to widely distribution, easy to grow, rich resources, low price and drug production cost, radix actinidiae chinensis would bring good social and economic benefits. So, we researched the effect of radix actinidiae chinensis on human brain U251 glioblastoma cell and its mechanism research. Our research consisted of three parts:I. The effect of radix actinidiae chinensis on human brain U251 glioblastoma cell proliferation in vivo and in vitro;Ⅱ. Effect of radix actinidiae chinensis on cells cycle in U251 and U87 cells; III. Effect of radix actinidiae chinensis on STAT3 signal pathway in U251 and U87 cells.Part Ⅰ. Effect of radix actinidiae chinensis on human U251 and U87 glioblastoma cell proliferation inhibitionObjective:to explore the effect of radix actinidiae chinensis on human U251 glioblastoma cell proliferation inhibition.Methods:(1) Observe the effect of different concentrations of radix actinidiae chinensis on human brain U251 and U87 glioblastoma cells morphological structure in culture plate under inverted microscope. And then dye U251 and U87 glioblastoma cell with Hoechst33258 fluorescent staining to observe the change of chromatin in nucleus and the phenomenon of nucleus pycnosis, cell membrane shrivel apoptotic body. (2) Detect the effect of different concentrations of radix actinidiae chinensis on human brain U251 and U87 glioblastoma cell proliferation inhibition by MTT method. (3) Detect the effect of different concentrations of radix actinidiae chinensis on human brian U251 and U87 glioblastoma cell activity after treatment of 48 hours by CCK-8 method. (4) Detect the effect of radix actinidiae chinensis on glioblastoma growth in vivo.Results:(1) Radix Actinidiae extractive could inhibit glioblastoma cell proliferationBoth in U251 and U87 glioblastoma cells, the cell proliferation rate in group of radix actinidiae chinensis is significantly lower than that in group of control.(2) The inhibition role of Radix Actinidiae extractive on proliferation of glioma cells was of concentration dependenceAfter certain time treatment of radix actinidiae chinensis, the proliferation rate of human brain glioblastoma cell siginificantly reduced. And with the increase of concentration, the proliferation rate reduced (P<0.05).(3) The inhibition role of Radix Actinidiae extractive on proliferation of glioma cells was not time-dependentAfter 24h,48h and 72h,at the the same concentration of radix actinidiae chinensis,the cell proliferation rates have no obvious change(P>0.05)(4) radix actinidiae chinensis inhibited glioblastoma growth in vivoGlioblastoma can be seen in all the ctrl groups, however, there was only one glioblastoma can be seen in radix actinidiae chinensis group. There was significant difference between ctrl group and radix actinidiae chinensis group(P<0.05).Conclusion:Radix actinidiae chinensis could significantly inhibit U251 and U87 glioblastoma cell proliferation in vitro and in vivo.Part II. Radix actinidiae chinensis regulated glioblastoma cell proliferation via regulating its cell cycleObjective:to explore the effect of radix actinidiae chinensis on human glioblastoma cell cycle.Methods:Detect the effect of different concentrations of radix actinidiae chinensis on human brian U251 glioblastoma cell cycle after treatment of 24 hours by flow cytometry method. And detect the expression level of BCL-2 and BAX by western bloting.Results:(1) effect of different concentrations of radix actinidiae chinensis on brain U251 glioblastoma cell cycle after treatment of 24 hourThe human brain U251 glioblastoma cell cycle was changed after 24 hours1 treatment of radix actinidiae chinensis. Cell cycle was arrested in G1 phase. And with the increase of concentration, the cell cycle arresting enhanced (P<0.05).(2) expression level of BCL-2 and BAX in human brain U251 glioblastoma cell after 24 hours’ treatment of different concentrations of radix actinidiae chinensisThe BCL-2 expression level decreased and BAX increased after 24 hours’ treatment of radix actinidiae chinensis. And with the increase of concentration, differences became more significant (P<0.05).(3)Regulating BCL-2 and BAX can regulate glioblastoma proliferationThe cell number increased when Flag-BCL-2 plasmid was transfected into U251 cells, and there was significant difference compared to its control (P<0.05). The cell number decreased when siBCL-2 was transfected into U251 cells and there was significant difference compared to its control (P<0.05).The cell number increased when si-BAX plasmid was transfected into U251 cells, and there was significant difference compared to its control (P<0.05). The cell number decreased when Flag-BAX was transfected into U251 cells and there was significant difference compared to its control (P<0.05).(4) radix actinidiae chinensis regulated proliferation by negatively regulating Bcl-2 and positively regulating BAX.The cell number decreased when radix actinidiae chinensis treated U251 cells and there was significant difference between ctrl group and radix actinidiae chinensis group(P<0.001), however, when we transfected BCL-2 or siBAX to U251 cells, the cell number of radix actinidiae chinensis +Flag-BCL-2 and radix actinidiae chinensis +si-BAX group was increased and there is no significant difference compared to Ctrl group(P=0.291,0.319);Conclusion:Radix actinidiae chinensis arrested human brain U251 glioblastoma cell cycle at Gl phase by decreased BCL-2 expression level and increased BAX expression level.Part Ⅲ. Radix actinidiae chinensis regulated glioblastoma cell cycle via STAT3 signal pathwayObjective:To explore the mechanism of radix actinidiae chinensis regulating human brain U251 glioblastoma cell proliferation.Methods:Detect human brain U251 glioblastoma cell STAT3 and p-STAT3 protein expression level after treatment of different concentrations of radix actinidiae chinensis by western bloting. And detect STAT3 mRNA by q-PCR.Results:(1) STAT3 and p-STAT3 protein expression level in human brain U251 glioblastoma cell after 24 hours’treatment of different concentrations of radix actinidiae chinensisSTAT3 and p-STAT3 protein expression level decreased in human brain U251 glioblastoma cell after 24 hours’ treatment of different concentrations of radix actinidiae chinensis. And with the increase of concentration, STAT3 and p-STAT3 protein expression level decreased (P<0.05).(2) STAT3 and p-STAT3 mRNA expression level in human brain U251 glioblastoma cell after 24 hours’treatment of different concentrations of radix actinidiae chinensisSTAT3 and p-STAT3 mRNA expression level decreased in human brain U251 glioblastoma cell after 24 hours’ treatment of different concentrations of radix actinidiae chinensis. And with the increase of concentration, STAT3 and p-STAT3 mRNA expression level decreased (P<0.05).(3)Regulating STAT3 signaling can regulate glioblastoma proliferationThe cell number increased when STAT3 signaling was actived by Flag-STAT3, and there was significant difference compared to its control (P<0.05); Cell number decreased when STAT3 signaling was inhibited by siSTAT3 and there was significant difference compared to their control groups(P<0.05);(4)RND3 regulated proliferation by negatively regulating Notchl signalingThe cell number decreased when radix actinidiae chinensis treated U251 cells and there was significant difference between radix actinidiae chinensis group and Ctrl group(P< 0.001), however, when we transfected Flg-STAT3 to active STAT3 signaling, the cell number of radix actinidiae chinensis +Flag-STAT3 group was increased and there is no significant difference compared to Ctrl group(P=0.191);Conclusion:Radix actinidiae chinensis regulated glioblastoma cell cycle via regulating STAT3 phosphorylation and mRNA expression, and then regulated glioblastoma cell proliferation.更多还原