【作者】 张琪；

【导师】 范光丽；

【作者基本信息】 西北农林科技大学 ， 神经生物学， 2010， 博士

【摘要】 目前癌症已经跃为威胁人类健康的第一杀手,治疗或消灭肿瘤成为世界医学界的难题,治疗神经系统肿瘤和疾病,诸如中脑神经母细胞瘤、帕金森氏病等成为当前最为迫切解决的热门课题之一。神经系统肿瘤疾病的治疗对于提高人类生活质量、延长人类寿命至关重要。人们应用手术、化疗、中药治疗、基因治疗、自身免疫治疗等手段虽然有一定治疗效果,但是伴随的毒副作用和昂贵的治疗费使患者倍受痛苦,甚至放弃治疗。有效地无毒副作用治疗肿瘤疾病是研究者多年的追求和愿望。因而,探索生物学因素中药康莱特(KLT)对中脑神经组织的影响,探索KLT、溶瘤病毒新城疫病毒(NDV)和豆类丝核菌代谢产物苦马豆素(SW)联合抗肿瘤疾病的作用机制是本研究的选题目的。本研究通过建立的S37动物肿瘤模型,运用H.E染色、TH染色、NeuN染色、GFAP染色技术和免疫组化SP法研究了三种药物抗肿瘤的效果及其作用机制。本研究取得以下结果:1.成功地对中脑神经组织进行了体外培养。试验表明,用1-4d胚鼠中脑组织培养运用基础培养基;5-10d,置换为N4培养基,初级突起和次级突起得到了充分的发育,并由突起形成了网络状结构。2.将KLT作用于中脑组织培养,表明添加不同浓度KLT作用后,浓度为0.001mg/ml的KLT对多巴胺能神经元具有促进生长的作用,神经元数量增加了13.3%,浓度为0.01mg/ml、0.1mg/ml、1.0mg/ml的KLT作用后,神经元的数量有所下降,表明高浓度KLT对中脑组织有轻微毒害作用,低浓度KLT能够很好地刺激中脑组织神经元的生长。3.用KLT作用于帕金森症模型中,用不同浓度KLT作用于受损的多巴胺能神经元,结果表明,各浓度KLT稀释液均能显著提升多巴胺能神经元神经元的数量,浓度为0.001mg/ml的KLT比对照度提高了88%。且细胞突起明显变长,次级突起数量增多,胞体圆滑。4.N18TG2神经母细胞瘤在N4培养基中呈现旺盛生长,胞体变大,伸出几个大的初级突起,初级突起上伸出很多细丝状的次级突起。MTT结果表明在12h,24h和48h,在N4培养基中N18TG2生长速度显著高于NBT培养基中的生长速度,在48h时,N4培养基中的细胞数是NBT培养基中的3倍。5.实现了胚鼠中脑组织与鼠源神经母细胞瘤N18TG2的共培养。将接种浓度为2.5×104、5.0×104和105 cells/ml的N18TG2神经母细胞瘤细胞接种于中脑神经组织。结果显示,各接种浓度均能在中脑组织中生长,但生长比较缓慢。N18TG2低接种浓度共培养中,神经母细胞瘤对中脑组织细胞几乎无伤害作用,只有高浓度接种的肿瘤细胞才对神经元形成毒害作用,神经元出现胞突变短,胞体肿胀,次级突起消失;神经元数量减少等变化。6.不同浓度KLT作用于N4培养基和NBT培养基的神经母细胞瘤N18TG2, MTT结果显示,各浓度KLT均对N18TG2神经母细胞瘤的有极显著的抗肿瘤效果。高浓度KLT组中可见到大量的肿瘤细胞碎片,在各种浓度KLT上清液中均能见到大量凋亡小泡,表明KLT对N18TG2神经母细胞瘤的抗肿瘤作用呈现剂量依赖关系,高浓度KLT导致肿瘤细胞发生坏死,低浓度KLT诱导肿瘤细胞凋亡。7.不同浓度KLT作用于神经母细胞瘤N18TG2细胞与中脑组织共培养中,结果显示,浓度为0.001mg/ml的KLT的肿瘤细胞数在各个时期均显著低于对照组,肿瘤抑制率为36%,且该组神经元的初级突起和次级突起均是完整的,神经元突起构成的网络结构依然清晰。在其他浓度KLT作用下,肿瘤细胞有所减少,但差异不显著。同时在共培养组织中也见到大量呈圆形无突起的神经元细胞,表明在共培养组织中低浓度组KLT具有抑制肿瘤细胞的效果。8.不同浓度KLT作用于神经母细胞瘤N18TG2细胞与中脑组织共培养中,结果表明,KLT各作用组对多巴胺能神经元、神经元和神经胶质细胞的数量大大增加,且细胞胞体和突起较平滑,突起数目较多,较长,神经元突起构成的网络较清晰。表明KLT能很好地保护中神经细胞。9.建立了S37肿瘤模型鼠,将48只S37荷瘤小鼠随机分成4组,即对照组、NDV治疗组、康莱特治疗组和NDV联合康莱特治疗组。体内抗肿瘤效果表明,随时间延长,肿瘤组织生长缓慢、肿瘤细胞发生溶解、核固缩、坏死区可见到大量的淋巴细胞,三种药物具有显著地抗肿瘤作用,其中NDV弱毒和康莱特注射液联用组的抗肿瘤效果最好,康莱特组次之,NDV组稍差。其抗肿瘤作用机制有可能是三种药物提高了机体神经系统和免疫系统机能,破坏或减弱了瘤细胞的生长繁殖速度,引起瘤细胞凋亡和死亡,起到了抗肿瘤作用。10.建立了S37肿瘤模型鼠,将48只S37荷瘤小鼠随机分成4组,即NDV治疗组、苦马豆素组治疗组、NDV联合苦马豆素治疗组和对照组。体内试验结果表明结果表明,随时间延长,肿瘤组织坏死严重、肿瘤细胞发生溶解、核固缩、坏死区可见到大量的淋巴细胞,三种药物具有显著地抗肿瘤作用,其中NDV弱毒和SW联用组的抗肿瘤效果最好,NDV组次之,SW组稍差。其抗肿瘤机制可能与三种药物均能刺激并提高机体免疫系统和神经系统机能,抑制或直接杀伤了肿瘤细胞,诱导肿瘤细胞凋亡或坏死,起到了抗肿瘤作用。更多还原

【Abstract】 Cancers are the first killer for threatening human healthes. Curing or cancelling tumors is a big problem on the world. While how to cure midbrain system disease and tumors in midbrain such as neuroblastoma and Parkinesons have become as the hottest projects to attack. It’s very important to attack nerve system tumor for leveling up the quality of the sick and extend human life. The operations, chemo-treatment, treatment by Chinese herbs, gene therapy, immunization theropy are used on the patients. Though have some effects but the toxic side effect and expensive curing fees can’t be bearable by the patients and their families. Some patients even give up treatments. To find no toxic side effects and effective new ways for curing cancer is most researchers’wishes. So to study biology factors Chinese herb Kanglaite injection (KLT) influence on the midbrain tissue. Then to study the mechanism of antitumor cooperation of Oncolytic virus New castle disease virus (NDV), KLT and secondary metabolite of Rhizoctonia leguminicola swaisonie (SW). Both researches are what our project want to do.This research we built S37 models,, H.E staining TH staining,NeuN staining and GFAP staining technical were used in our study for getting more antitumor mechanism informations from threemedicines. The results as follows:1. The midbrain cultures were successfully built from 14-day old mouse embryos. From 1-5 day the midbrain cultures were maintained in the basic medium and the cell bodies grew bigger and bigger. There are some short processes appeared. From 5-10 days, the midbrain culture medium was changed by N4 medium. Many long processes were sprayed out from the cell bodies and the second processes can be found. And the processes were formed very big network in the cultures. All these mean that midbrain tissue could be grown well outside body.2. It’s the first time that KLT were used in the midbrain cultures to detect if there is some cytotoxixity on the midbrain cultures. The different dilutions of KLT were added. The results showed that the concentration of 0.001mg/ml could stimulate neurons growing faster and the number of the neurons increased 13.3%. While the number of the neurons in other KLT solutions had some little decrease. All means that higher concentrations of KLT do some harms to midbrain cultures. However the lower concentrations could well stimulate the growth of the neurons.3. The 200μM MPP+ were used to build Parkinsonismus mould. Different concentrations of KLT were used to do some treatments on the hurted dopaminergic neurons. The numbers of the dopaminergic neurons were counted and statistics and the morphology changes can be seen on the dopaminergic neurons. The concentration of 0.001mg/ml leveled up the number of the dopaminergic neurons 88% in contrast to the control. And the processes of the dopaminergic neurons became more longer and the neutrites possess more to the control. The results showed that KLT could be used to cure the Parkinsonismus.4. The N18TG2 neuroblastoma cells was the first time to grow in the midbrain culture medium N4 medium to detect if the N18TG2 can be growed in the N4 medium. The morphology changes and MTT assay showed that the N18TG2 can grow very well inside the N4 medium. Many big primary processes showed up and many second processes on the primary. The bodies of the N18TG2 become more bigger and bigger. MTT results suggested that the N18TG2 cells grew more quickly than the N18TG2 inside NBT medium in 12h,24h and 48h. Especially the number of N18TG2 cell in N4 medium is triple to the NBT medium. So the N18TG2 cells can grow in the midbrain medium.5. It’s the first time that N18TG2 neuroblastoma cells were cocultured with midbrain tissue. The seeding densities of the N18TG2 cells are 2.5×104,5.0×104 and 105 cells/ml and were seeded in the midbrain culture. Take pictures at 12h,24h,36h and 48h separately. The numbers of the tumor cells were counted and statistics and the morphology changes could be found. The results showed that the N18TG2 could grow in the midbrain culture in all seeding densities but grew very slowly. Lower seeding densities N18TG2 cells can’t do cytotoxicity on neurons in midbrain culture. While higher seeding densities made the neuron swell and the processes became shorter and the second processes disappeared. The number of the neurons was decreased.6. To detect the antitumor effects of KLT on N18TG2 neuroblastoma in N4 medium and NBT medium. The different concentrations of KLT treatments on N18TG2 neuroblastoma were added. The antitumor effect values were detected by MTT assay. The results showed that the KLT treatments have significant antitumor effects contrast to the control either in N4 medium or in NBT medium. Plenty of fragments and many apoptotic vesicles can be found in the culture suspension especially in the higher KLT concentrations. The results suggest that KLT treatments on N18TG2 neuroblastoma are dose dependent on the antitumor effects. And higher KLT solutions can make cancer cells depressed and lower KLT solutions induce apoptosis on N18TG2 neuroblastoma. The inhibition rate of 1.0mg/ml KLT is 48.7%.7. KLT was used in the coculture for studying the antitumor effects on the N18TG2 neuroblastoma cells. The different KLT solutions were added into the coculture separately for another 48h. The numbers of the tumor cells were counted and statistics and the morphology changes could be found. The results showed that 0.001mg/ml KLT solution possessed significant lowest tumor number in all the tests in different periods to the control. The inhibition rate is 36%. The morphology showed that the primary processes and second processes didn’t been hurted and the networks made by processes were very clear. In other KLT solution treatment, the number of the tumor cells was decreased but not significantly to the control. Many round, no processes neuron could be found in other KLT solutions. These suggest that 0.001mg/ml KLT solution possess antitumor effect in the coculture of midbrain neurons and N18TG2 neuroblastoma.8.After different solutions of KLT treatments on the coculture of neuroblastoma cells and midbrain cells, the numbers of the dopaminergic neurons, all neurons and astrocytes were counted and statistics and the morphology changes could be found. The morphology showed that the primary processes and second processes didn’t been hurted and the networks made by processes were very clear. The numbers of the dopaminergic neurons, all neurons and astrocytes were increased quickly and the bodies and the processes are very smooth. The decrease of the dopaminergic neurons, all neurons and astrocytes in the control was easy to find. Many apoptotic vesicle can be found in the bodies and the processes. KLT could level up the numbers of the dopaminergic neurons, all neurons and astrocytes and improve the lives of the neurons.9 After building the cancer models, 48 cancer models were divided into three groups. Those are control group, Kanglaite injection group and NDV+Kanglaite group. The results suggested that the tumors grew very slowly, the tumor cells were dissolved and karyopyknosis when time expand. Plenty of lymphocytes could be found in the depression area. All treatment groups markedly do effects on the cancer models. The best group is NDV+Kanglaite group, then KLT, last NDV. The antitumor mechanism of these three medcines may be that they could level up the functions of immune system and nervous system. Those can inhibit or weak the cancer cell growth.10. After building the cancer models, 48 cancer models were divided into three groups. Those are NDV group, SW group, NDV+Kanglaite group and control group. The results suggested that the tumors were necrosis, the tumor cells were dissolved and karyopyknosis when time expand. Plenty of lymphocytes could be found in the depression area. All treatment groups markedly do effects on the cancer models. The best group is SW+Kanglaite group, then NDV, last SW. The antitumor mechanism of these three medcines may be that they could level up the functions of immune system and nervous system. Those can inhibit or weak the cancer cell growth rate, make apoptosis or necrosis on tumor cells. So they have effects on the tumors.更多还原