【作者】 何克菲；

【导师】 张积仁；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 研究背景肿瘤,作为一种严重影响人群健康的慢性疾病,发病率和死亡率逐年上升。在发展中的中国,日益增长的发病人数及治疗费用,已经成为制约社会进步和发展的绊脚石。肿瘤治疗经历了传统的手术、化疗、放疗,到分子靶向治疗,似乎人类对肿瘤的认识还非常有限,尽管目前已经公认肿瘤是一种全身系统性的疾病,肿瘤学家也试图通过系统地综合治疗,但目前尚未取得理想的效果。显然,传统的治疗手段不足以消灭肿瘤,我们能否从肿瘤的生物学行为中寻找新的方法呢?肿瘤学家正试图通过干预肿瘤免疫来寻找答案,免疫治疗应用于肿瘤治疗,并成为目前临床关注的热点之一。肿瘤免疫治疗的目的是激发或调动机体的免疫系统,增强肿瘤微环境抗肿瘤免疫力,从而控制和杀伤肿瘤细胞。肿瘤免疫学治疗的方法种类繁多,已与现代生物高科技结合,发展成为继手术、化疗和放疗之后的第四种肿瘤治疗模式。免疫增强剂是一类具有广泛生物学活性和抗肿瘤活性的生物制剂,既包括一大类天然产生的生物物质,又包括能改变体内宿主和肿瘤平衡状态的方法和手段,被广泛用于抗肿瘤治疗。虽然作用机制多种多样,但不外乎两大方面：通过干扰细胞生长、转化或转移的直接抗瘤作用或通过激活免疫系统的效应细胞及其所分泌的细胞因子来增强特异性免疫,达到对肿瘤杀伤或抑制的目的。在临床治疗中已经被广泛的应用。Okamoto H等最早用链球菌制备出抗肿瘤制剂OK-432。该制剂不仅具有直接杀伤肿瘤细胞的作用,亦能增强机体的抗肿瘤免疫功能,如：激活NK细胞、巨噬细胞及补体系统,已应用于临床抗肿瘤治疗。越来越多的研究表明在实体肿瘤和血液恶性肿瘤患者中外周血和肿瘤局部微环境中Treg数目增多,比例增高,且数目与患者肿瘤进展程度、预后和生存率呈负相关。本中心前期实验表明采取太空灭活链球菌免疫序贯瘤内注射的治疗方法发现,经过灭活链球菌免疫后小鼠的效应细胞更快地被募集,肿瘤MHC-I类抗原上调,减少了肿瘤局部调节性T细胞的数量,可以有效地在改善肿瘤局部免疫状况,有良好的抗肿瘤作用。序贯疗法目前尚无明确的定义,药物的序贯疗法通常是指连续用药达到药物稳定的抗肿瘤血药浓度后改用口服制剂或暂停用药,以维持有效药物在血浆中的浓度。序贯疗法可缩短住院时间,降低治疗费用,特别对于需要长期维持给药的患者,可以预防长期静脉输液引起的感染,减少药物副作用所致的死亡,是值得临床推广的一种治疗方法。针对免疫制剂长期应用,本研究在前人实验结论的基础上,设计使用太空灭活链球菌作为异种抗原来探索不同的瘤内给药模式对小鼠S180肿瘤的抗肿瘤作用,荷瘤小鼠对太空灭活链球菌治疗的耐受性以及重要器官病理学改变,重点分析太空灭活链球菌瘤内注射后对Bcl-2蛋白和Bax蛋白表达的影响。目的研究太空灭活链球菌瘤内注射对S180荷瘤小鼠抗肿瘤疗效、荷瘤小鼠肿瘤组织内凋亡相关Bcl-2蛋白和Bax蛋白表达的影响以及长期用药后对肝脏和肾脏毒性作用的初步探索。方法1.S180实体瘤小鼠模型构建4-6周龄雌性昆明小鼠40只,获赠S180腹水小鼠,观察后五日后无菌抽取腹水,培养3-4代后细胞生长旺盛,制成癌细胞悬液,调整细胞浓度为1×107个/ml,取0.1ml用于小鼠背部皮下接种以制作实体瘤模型,成瘤率近100%。2.实验动物分组：造模成功后第8天按大小均一性原则选取30只分成三组,每组10只：A、太空灭活链球菌连续瘤内注射组；B、太空灭活链球菌序贯组瘤内注射组；C、生理盐水瘤内注射空白对照组。3.瘤内注射药物及安慰剂按照分组开始进行瘤内注射(A组小鼠连续每天瘤内注射浓度为1mg/ml的太空灭活链球菌0.1ml/只；B组小鼠连续5天瘤内注射浓度为1mg/ml的太空灭活链球菌0.1ml/只,然后休息2天,C组瘤内注射生理盐水0.1ml/只)。研究以荷瘤小鼠的死亡为观察终点。所有瘤内注射均严格遵守无菌操作,采取多点注射。4.观察和测量实验数据注射后每周测量肿瘤大小,计算肿瘤体积,肿瘤体积按公式：长径×宽径×宽径/2。记录各组小鼠治疗后生存时间。5.免疫组织化学技术测肿瘤组织Bcl-2、Bax蛋白表达6.图片的数字化处理每组免疫组化染色切片按随机原则选取5个不同显色部位,NIKON TE显微镜×400下拍照,保存图片资料,使用Image-Pro Plus 6.0进行染色光密度分析。7.数据统计使用SPSS 13.0统计软件,数据使用x±s表示,肿瘤体积使用重复测量数据的方差分析,生存期使用Kaplan-Meier法分析,免疫组化图片积分光密度使用单因素方差分析。8.获取荷瘤小鼠肝脏和肾脏标本,经HE染色观察组织病理学改变。结果1.小鼠S180肿瘤细胞培养后生长旺盛,接种皮下成瘤率高。2.连续注射组和序贯注射组用药后每周的肿瘤平均体积均呈下降趋势,与对照组比较均有统计学差异(P=0.000)。连续注射组和序贯注射组之间差异不明显(P=0.81),无统计学意义。3.各组小鼠生长曲线表明,连续注射组、序贯注射组和对照组荷瘤小鼠治疗后中位生存时间分别为38.0天、37.0天、34.0天。连续组与序贯组比较,P>0.05,差异无统计学意义；连续组与对照组比较,P<0.05,差异有统计学意义；序贯组与对照组比较,P<0.05,差异具有统计学意义(P<0.05)。4.瘤内注射太空灭活链球菌组与对照组比较,太空灭活链球菌瘤内注射组的Bcl-2蛋白表达明显减低,Bax蛋白表达明显增加(P<0.05)。5.太空灭活链球菌作为异种抗原瘤内注射主要作用于肿瘤局部,不增加重要器官中的毒性产物和免疫复合物,持续用药未出现肝脏、肾脏的免疫损伤。使用安全性好。本文结论：太空灭活链球菌持续瘤内注射方法有明显抑制小鼠S180肿瘤细胞生长的作用,其作用可能是通过降低Bcl-2蛋白表达和增强Bax蛋白表达,从而促进肿瘤细胞凋亡来实现的。瘤内注射太空灭活链球菌能提高小鼠的中位生存时间。序贯注射能取得和连续注射同样的效果。持续瘤内注射太空灭活链球菌对肝脏和肾脏组织无损伤,使用安全性好。更多还原

【Abstract】 Cancer as a serious chronic disease affects human health, the morbidity and mortality increases year by year. In the development of China, the growing incidence and treatment costs, has restricted the stumbling block to social progress and development. Cancer treatment through traditional surgery, chemotherapy, radiotherapy, molecular targeted therapy to the current seems to understand human tumors is still very limited. Although cancer has been recognized as a systemic disease, oncologists are trying to systematically integrated treatment, but the achievement is not satisfactory.Clearly, not enough to eliminate tumor by the traditional treatment, then can we find new ways from the biological behavior of tumors? No one knows the answer, but some oncologists are trying to interfere with tumor immune to find the answer, a new immune therapy has began to be used in cancer patient. Immunotherapy become the focus of attention of tumor clinical. The purpose of tumor immunotherapy is to stimulate or mobilize the immune system, regularing the tumor microenvironment to enhance anti-tumor immunity. Immunotherapy have tured into the fourth treatement methods following the surgery, chemotherapy and radiotherapy for cancer treatment. Immunostimulants are a class of broad biological activity and antitumor activity of biological agents, both in a large class of naturally occurring biological substances, but also the host and the tumor can change the balance of the body’s ways and means, are widely used anti-cancer therapy. Although the mechanism are various, but no more than two major aspects:nhibition tumor by interfering with cell growth, transformation, or transfer directly and enhance specific immune by activating effector cells and secreting cytokines. Okamoto H first use streptococcus to prepare OK-432. The agent, not only directly kill tumor cells, can also enhance the body’s anti-tumor immune function, such as:activated NK cells, macrophages and complement system, has been used in clinical anti-cancer therapy. More research showed that the number of regulatory T cell (Treg) increase in patients with solid tumors and blood malignancies, and the number of Treg with tumor progression and prognosis, the survival rate was negative correlate. Early results show that the Centre take inactivated Streptococcus sequential intratumoral injection to enhance the effect of immune cells in mice, then tumor MHC-Ⅰantigen increases, and the number of tumor local regulatory T cells decreases,which can be effective in improving local tumor immune status. Sequential therapy is currently no clear definition, the sequential drug treatment usually refers to the continuous administration of drug to achieve a stable blood concentration of anti-tumor agents or suspended after the switch to oral medication, in order to maintain effective drug concentrations in plasma. Use of sequential therapy can be shortened hospital stay and reduce the cost of treatment, preventing the side effects caused by long-term intravenous infusion,so sequential therapy is worthy of a clinical treatment. Preparations for the long-term application of immunopotentiator, we designed for use a heteroantigen to explore the anti-tumor effect with different patterns of intratumoral administration of mice bearing S180 saroma, and the tolerance of injecting inactivated streptococcus by intratumoral way, and the pathological changes of vital organs.We focus on analyzing the expression of Bcl-2 protein and Bax protein after intratumoral injection in tissue of S180 saroma.Purpose ·To study the antitumor effect of the inactivated streptococcus intratumoral injection on mice bearing S180 saroma and the expression of Bcl-2 and Bax protein in tumor tissue, and the long-term safety of using the inactivated streptococcus. adjusting the cell concentration of 1×107 cells/ml. Use 0.1ml cell suspension inoculated in 40 mice of 4-6 weeks old subcutaneously. Tumor formation rate close to 100%.2. Animal groups After the modeling, the first 8 days, according to the principle of uniformity of size selected 30 mice were divided into three groups of 10 each:A, continuous intratumoral injection of inactivated Streptococcus group; B, sequential intratumoral injection of inactivated Streptococcus group; C, continuous intratumoral injection of normal saline control group.3. Intratumoral injection of drugs and placebo In accordance with the groups began:Group A of consecutive daily intratumoral injection of mice with the concentration of inactivated Streptococcus 1mg/ml 0.1ml/only; Group B, tumors in mice injected for 5 days to eliminate the concentration of lmg/ml inactivated Streptococcus 0.1ml/only, and then rest 2 days; Group C, continuous intratumoral injection of saline 0.1ml/only. All intratumoral injection of sterile operation are strictly adhered to, to multi-point injection.4. Observation and measurement of experimental data Weekly measurement of tumor size after injection, then we compute tumor size. Tumor volume by the formula: diameter×width2/2.5. Immunohistochemical measurement of tumor tissue Bcl-2, Bax protein expression6. Digital picture information Using Image-pro plus software processing immunohistochemical picture7. Statistical analysis Using the SPSS 13.0 statistical software, choose the appropriate statistical analysis of data.8. The safety of drugs Collecting the liver and kidney specimens, then observing histological changes by H.E staining.Results Compared with the control group, continuous treatment group and the sequential treatment group had significant anti-tumor effect (P<0.05), but there was no significantly difference between the continuous group and the sequential group (P> 0.05). Growth curves of mice in each group showed that the median survival time of three groups of mice after treatment was 38.0 days,37.0 days,34.0 days. With the control group, the expression of Bcl-2 protein reduced significantly on the group of intratumoral injection of inactivated Streptococcus, and the expression of Bax protein increased significantly (P<0.05). There was no significantly difference between the continuous group and the sequential group on the expression of Bcl-2 and Bax protein (P>0.05). As a xenoantigen, inactivated Streptococcus does not increase immune damage of liver and kidney in tumor-bearing mice.Conclusion Intratumoral injection of inactivated Streptococcus continuous method has significantly inhibit the S180 tumor growth in mice, significantly increased the median survival time of mice, and the sequential injection can achieve the same effect with a continuous injection. The antitumor effect of inactivated Streptococcus may be through the promotion of apoptosis of tumor cells by regulating the expression of Bcl-2 protein and Bax protein. Continuous medication don’t damage to the liver and kidneys, using good security.更多还原