防御素mBD2基因修饰恶性黑色素瘤疫苗抗肿瘤免疫机制的实验研究

发布时间：2018-07-03 15:07

本文选题：防御素 + 黑色素瘤　；参考：《南方医科大学》2010年博士论文

【摘要】：前言: 天然免疫曾经被认为是机体免疫系统应答外界刺激的低级形式,但目前研究表明天然免疫反应不仅能够直接而快速对抗外界刺激,而且还对机体的获得性免疫反应起直接的激活和导向作用。抗菌肽是生物体天然免疫反应的重要因子,它不仅能够直接杀伤入侵的病原微生物,中和有害的微生物产物,抑制病原微生物入侵靶细胞,而且,还可通过作用于多种白细胞亚群,加强抗原特异性T淋巴细胞及B淋巴细胞的活化等免疫调节功能而增强机体的应激能力,从而间接发挥保持机体内环境稳态的作用。目前,已有若干研究表明,抗菌肽的抗菌功能对生理条件下的盐离子浓度高度敏感,而且还可受到血清成分的直接抑制作用；而其免疫调节功能却不受生理条件下,生理浓度的盐离子及血清成分等因素的影响,因此,有人主张极有可能免疫调节功能才是抗菌肽的主要功能。防御素是抗菌肽中重要的一类,它普遍存在于高等生物,不仅对病原微生物具有广谱的毒杀效应,而且还具有重要的免疫调节活性。鼠源性β-防御素2(mBD2)是防御素中具有代表性的一种,研究表明,它可通过趋化因子受体CCR6趋化未成熟树突状细胞,并作为TLR4的内源性配体促进树突状细胞的成熟,产生一系列促炎症因子、趋化因子,上调共刺激分子表达。用编码非免疫原性淋巴瘤抗原与mBD2融合蛋白的DNA质粒免疫小鼠,可在小鼠体内诱发出明显的抗肿瘤活性。可见,mBD2作为天然免疫反应的组成部分,还可协助非免疫原性抗原在小鼠体内诱导出有效的特异性获得性免疫反应,是一种天然的免疫佐剂。mBD2是连接天然免疫反应和获得性免疫反应的桥梁,提示了一种新的肿瘤免疫治疗策略。利用mBD2能够增强抗原特异性免疫反应的特性,可研究开发以mBD2为基础的肿瘤疫苗。目前,尽管人类已经研究了多种免疫佐剂,但是除了铝制剂以外,大多数免疫佐剂都由于具有严重的毒副作用而不能够直接应用于人体；而铝制剂虽然可用,但是它的免疫增强活性有限,且倾向于引发TK2型免疫反应,因此具有很大的局限性。mBD2作为一种内源性天然活性物质,具有上述强烈的免疫调节活性,我们将mBD2应用于恶性黑色素瘤的免疫治疗,期望它能够克服目前铝制剂存在的局限性。恶性黑色素瘤简称“恶黑瘤”,是一种来源于黑色素细胞的恶性肿瘤,其恶性程度高、起病隐匿、误诊率高、预后很差,是全世界范围内发病率增长最快的恶性肿瘤之一。恶性黑色素瘤对一般的综合性治疗措施：如放疗、化疗都表现为耐受,早期恶黑瘤的治疗主要以手术为主,晚期恶黑瘤预后很差,尚无有效的治疗手段,主要以个体化的综合治疗为原则。由于恶性黑色素瘤是一种免疫原性很强的肿瘤,因此特异性免疫治疗一直以来都是恶性黑色素瘤治疗的重要手段之一。尽管免疫治疗在许多恶性黑色素瘤小鼠模型中都能够诱导出明显的抗肿瘤免疫保护作用,但临床实验的结果表明,人体对恶性黑色素瘤免疫治疗的反应率远远不如实验动物有效,仅10～30%。目前,还不清楚人体对恶性黑色素瘤免疫治疗产生个体差异的确切原因,但是,普遍认为疫苗的临床有效性不仅决定于其种类和制备方法,还取决于用于增强疫苗的有效性而选择的免疫佐剂。本文将防御素mBD2的免疫调节活性应用于恶性黑色素瘤的免疫治疗,制备mBD2基因修饰的恶性黑色素瘤细胞疫苗、观察其体内抗肿瘤免疫效果,并探讨其抗肿瘤免疫保护作用的机制。实验方法： 1.采用RT-PCR技术从C57BL/6小鼠肾脏组织中扩增出小鼠β-防御素2(mBD2)成熟肽基因片段,采用overlap PCR方法将小鼠IgK信号肽序列加在mBD2成熟肽基因序列的5’端,T-A克隆后,构建含有IgK信号肽的mBD2分泌性真核表达载体pcDNA3.1(+)-IgK-mBD2,并通过PCR、酶切、测序等方法鉴定其正确性。 2.采用脂质体转染法分别将空载体pcDNA3.1(+)及含有目的基因的真核表达载体pcDNA3.1(+)-IgK-mBD2转染恶性黑色素瘤B16细胞,G418筛选后,获得稳定表达细胞株,分别命名为B16,p和B16-mBD2。RT-PCR从mRNA水平鉴定G418抗性基因neo基因的表达,从而证明pcDNA3.1(+)-IgK-mBD2和pcDNA3.1(+)等质粒的成功转染；RT-PCR从mRNA水平鉴定mBD2基因水平的表达；Western-Blot从蛋白水平鉴定mBD2分子的表达。 3.采用MTT法测定三株细胞：B16、B16-p、B16-mBD2在120小时内的生长曲线；采用PI染色流式细胞术检测对数生长期三株细胞的细胞周期分布及凋亡情况；采用FITC标记的抗小鼠CD80、CD86、MHCⅠ、MHCⅡ等抗体染色,流式细胞术检测三株细胞上述CD80、CD86、MHCⅠ、MHCⅡ等相关分子的表达变化。 4.采用辐射方法将三株细胞B16、B16-p、B16-mBD2制备成细胞疫苗,分别在C57BL/6小鼠体内进行免疫预防和免疫治疗动物实验。设立生理盐水对照组、B16对照组、B16-p对照组及B16-mBD2实验组。 5.免疫预防实验：6～8周龄雌性C57BL/6小鼠,随机分成4组,每组10～12只,分别为生理盐水对照组、B16对照组、B16-p对照组和B16-mBD2实验组。各组所有动物,按照各自的分组,分别于左腋下皮下注射给予0.1mL生理盐水及106个辐射的B16、B16-p和B16-mBD2等细胞疫苗。各组动物免疫七天后,各组所有小鼠均给予5×104个对数生长期的野生型B16细胞致瘤,逐日观察各组小鼠生存状态、制作肿瘤体内生长曲线、进行生存期分析及HE染色观察各免疫预防组肿瘤组织及重要脏器,如：肝脏、肾脏、脾脏、肺脏等的组织学变化。 6.免疫治疗实验：6～8周龄雌性C57BL/6小鼠,随机分成4组,每组10～12只,分别为生理盐水对照组、B16对照组、B16-p对照组和B16-mBD2实验组.,各组所有动物分别于左腋下皮下注射给予105个对数生长期野生型B16细胞致瘤,同时各组所有小鼠按照各自的分组,于同一天开始,左腋下皮下注射分别给予O.1mL生理盐水及106个辐射的B16、B16-p和B16-mBD2等细胞疫苗进行治疗,每周两次,连续两周。逐日观察各组小鼠生存状态、制作肿瘤体内生长曲线、进行生存期分析及HE染色观察各免疫治疗组肿瘤组织及重要脏器,如：肝脏、肾脏、脾脏、肺脏等的组织学变化。 7.采用ELISA方法测定B16、B16-p和B16-mBD2等细胞疫苗免疫小鼠后,各组小鼠脾脏淋巴细胞培养上清中IFN-γ,、IL-12、IL-4等含量的变化；采用非放射性细胞毒性分析方法测定B16、B16-p和B16-mBD2等细胞疫苗免疫小鼠后,各组小鼠NK细胞杀伤活性及CTL细胞杀伤活性。 8.采用SPSS 13.0进行统计学分析。细胞生长曲线、动物实验中的肿瘤体内生长曲线等采用重复测量因素的方差分析；细胞周期分布、凋亡率、细胞膜表面免疫相关分子的表达变化等采用单向方差分析；方差齐性时,多重比较采用LSD法,方差不齐时,多重比较采用Dunnett T3法。CTL细胞杀伤活性、NK细胞杀伤活性等采用析因设计,运用单因素方差检验(one-way ANOVA)LSD法进行数据分析。P0.05表示有统计学意义。实验结果： 1.成功构建含有鼠源性IgK信号肽序列的mBD2真核分泌性表达载体pcDNA3.1(+)-IgK-mBD2,并鉴定正确。 2.pcDNA3.1(+)及pcDNA3.1(+)-IgK-mBD2等载体转染B16细胞后,G418筛选得到稳定表达细胞株,分别命名为B16-p和B16-mBD2。 3.细胞生长曲线实验证实,与野生型B16细胞及B16-p相比较,B16-mBD2细胞的增殖速度显著减慢(F=144.256,P0.05)；与野生型B16细胞相比较,B16-p的增殖速度没有显著变化。 4.流式细胞仪检测结果显示,接种24小时后,与对数生长期的野生型B16细胞及B16-p相比较,对数生长期的B16-mBD2细胞的细胞周期发生S期轻度阻滞(F=8.952,P0.05)；三株细胞B16、B16-p和B16-mBD2的细胞膜表面分子CD80、CD86、MHCⅠ、MHCⅡ等表达没有显著性差异。 5.在免疫预防实验中,生理盐水对照组、B16对照组、B16-p对照组所有小鼠在49天内全部死亡,组间没有显著性差异,中位生存期分别为35天、37天、33天；而B16-mBD2免疫预防组小鼠肿瘤体内生长速度显著缓慢(F=118.387,P0.05),小鼠生存期显著延长(X2=18.857,P0.05),中位生存期为55天,同期生存状态比其他各个对照组明显良好。至实验结束,即致瘤后第150天为止,仍有37.5%的小鼠实现无瘤生存。 6.在免疫治疗实验中,生理盐水对照组、B16对照组及B16-p对照组所有小鼠均在44天内全部死亡,组间没有显著性差异,中位生存期分别为32天、34天、31天；B16-mBD2免疫治疗组小鼠肿瘤体内生长速度显著缓慢(F=289.615,P0.05),小鼠生存期显著延长(X2=22.006,P0.05),中位生存期为59天,同期生存状态比其他各个对照组明显良好。至实验结束,即致瘤后第150天为止,仍有25%的小鼠实现无瘤生存。 7.在免疫预防和免疫治疗实验中,B16-mBD2疫苗免疫组均诱导淋巴细胞大量浸润到肿瘤组织,脾脏淋巴小结增多,生发中心反应性增生、增大。生理盐水对照组、B16对照组、B16-p对照组均不能诱导这种主动性抗肿瘤免疫反应。 8.B16-mBD2细胞疫苗免疫后,可促进IFN-γ,产生显著增多(F=506.814,P0.05),促进IL-12产生显著增多(F=83.637,P0.05),与B16对照组、B16-p对照组比较差异有统计学意义,但对IL-4的含量没有影响。B16-mBD2细胞疫苗可诱导小鼠针对B16细胞的特异性CTL杀伤活性显著增强(F=44.376, P0.05)、NK细胞杀伤活性显著增强(F=119.750,P0.05),与B16对照组、B16-p对照组比较差异有统计学意义。 9.B16-mBD2细胞疫苗免疫小鼠后,对小鼠的主要脏器如：肝脏、肾脏、脾脏、肺脏等的HE染色形态学观察显示,各个主要脏器未见明显的病理改变,提示B16-mBD2疫苗相对安全。结论： mBD2基因转染恶性黑色素瘤B16细胞后,制备的转基因细胞疫苗具有明显的抗小鼠恶性黑色素瘤B16效应,该疫苗免疫后,通过诱导小鼠NK细胞杀伤活性增强、CTL杀伤活性增强、促进IFN-γ、IL-12等Thl型细胞因子产生增加、诱导大量淋巴细胞浸润到肿瘤组织内部以及诱发小鼠脾脏淋巴小结增生、增大等多种机制发挥抗恶性黑色素瘤免疫保护作用,且安全无毒副作用。mBD2基因修饰的B16细胞疫苗免疫后对Th2型细胞因子IL-4的含量没有影响,提示该疫苗抗肿瘤免疫保护作用可能与Th2型免疫反应无关。总之,mBD2基因修饰的B16细胞疫苗能够同时激活天然免疫(NK细胞活性)和获得性免疫(CTL细胞杀伤活性)、肿瘤局部免疫(肿瘤组织内淋巴细胞浸润)和系统免疫(诱导IFN-γ、IL-12等细胞因子含量增高)来发挥抗恶性黑色素瘤作用,为恶性黑色素瘤的免疫治疗开拓了新的思路。
[Abstract]:Preface:
Natural immunity has been considered as a low-grade form of immune response to external stimuli. However, the present study shows that the natural immune response can not only directly and rapidly antagonize external stimuli, but also directly activate and guide the immune response of the body. Antimicrobial peptides are an important factor in the natural immune response of organisms. It can not only kill the pathogenic microorganism of the invasion directly, neutralize the harmful microbial products, inhibit the pathogenic microorganism to invade the target cells, but also can enhance the stress of the body by strengthening the immune regulation function such as the activation of the antigen specific T lymphocyte and the B lymphocyte. At present, some studies have shown that the antibacterial function of antimicrobial peptides is highly sensitive to the concentration of salt ions under physiological conditions, and can also be directly inhibited by the serum components, but the immune regulation function is not affected by physiological conditions, the physiological concentration of salt ions and serum components. Therefore, it has been suggested that immunomodulation function is the main function of antimicrobial peptides.
Defensin is an important class of antimicrobial peptides. It exists widely in higher organisms. It not only has broad-spectrum toxicity to pathogenic microorganisms, but also has important immunoregulatory activity. The mouse derived beta defensin 2 (mBD2) is a representative of the defensin. It is shown that it can be used to chemotaxis the immature tree through the chemokine receptor CCR6. Protrusion cells, which act as endogenous ligands for TLR4, promote the maturation of dendritic cells and produce a series of pro-inflammatory factors, chemokines and up regulation of the expression of CO stimulator molecules. Immunization of mice with DNA plasmid encoding non immunogenic lymphoma antigen and mBD2 fusion protein can induce obvious antitumor activity in mice. Visible, mBD2 The component of natural immune response can also assist non immunogenic antigen in inducing effective specific acquired immune response in mice. A natural immune adjuvant,.MBD2, is a bridge linking natural immune response and acquired immune response, suggesting a new strategy for tumor immunotherapy. The use of mBD2 can enhance resistance. The characteristics of the primary specific immune response can be used to develop a mBD2 based tumor vaccine. At present, although a variety of immune adjuvant has been studied, most of the immune adjuvant, in addition to aluminum, can not be directly applied to the human body because of its serious toxic and side effects; although aluminum is available, it is immune to it. The enhanced activity is limited and tends to trigger the TK2 type immune response. Therefore, it has a great limitation of.MBD2 as an endogenous natural active substance, which has the above strong immunomodulatory activity. We apply mBD2 to the immunotherapy of malignant melanoma, and expect it to overcome the limitations of the present aluminum preparation.
Malignant melanoma, called "evil black tumor", is a malignant tumor derived from melanocytes. Its malignant degree is high, the disease is concealed, the misdiagnosis rate is high, and the prognosis is poor. It is one of the fastest growing malignant tumors in the world. The malignant melanoma is resistant to general comprehensive treatment, such as radiotherapy and chemotherapy. The treatment of early malignant melanoma is mainly based on operation, and the prognosis of late malignant melanoma is very poor. There is no effective treatment. It is mainly based on the principle of individualized comprehensive treatment. Because malignant melanoma is a kind of tumor with strong immunogenicity, specific immunotherapy has always been one of the important means for the treatment of malignant melanoma. Although immunotherapy can induce obvious antitumor immune protection in many malignant melanoma mice models, the results of clinical trials show that the response rate of the human body to malignant melanoma is far less effective than that of experimental animals. At present, it is not clear that the human immune treatment for malignant melanoma is not clear at the present time of 10 to 30%.. The exact cause of individual differences is produced, but it is generally believed that the clinical effectiveness of the vaccine is not only determined by its species and preparation methods, but also depends on the immune adjuvant used to enhance the effectiveness of the vaccine.
In this paper, the immunomodulatory activity of defensin mBD2 was applied to the immunotherapy of malignant melanoma, to prepare the mBD2 gene modified melanoma cell vaccine, to observe the anti-tumor immune effect in the body and to explore the mechanism of its anti-tumor immunity protection.
Experimental methods:
1. RT-PCR technique was used to amplify the mouse beta defensin 2 (mBD2) mature peptide gene fragment from the C57BL/6 mouse kidney tissue, and the IgK signal peptide sequence of mice was added to the 5 'end of the mBD2 mature peptide gene sequence by overlap PCR method. After T-A was cloned, the mBD2 secreting eukaryotic expression vector containing IgK signal peptide was constructed, and pcDNA3.1 (+) -IgK-mBD2 was constructed. PCR, enzyme digestion, sequencing and other methods were used to identify their correctness.
2. liposome transfection was used to transfect the empty carrier pcDNA3.1 (+) and the eukaryotic expression vector containing the target gene pcDNA3.1 (+) -IgK-mBD2 into the malignant melanoma B16 cells. After G418 screening, the stable expression cell lines were obtained, which were named B16, P and B16-mBD2.RT-PCR, respectively, to identify the neo gene of the G418 resistance gene from mRNA level, thus proving PC. DNA3.1 (+) -IgK-mBD2 and pcDNA3.1 (+) and other plasmids were transfected successfully; RT-PCR was identified from mRNA level and the expression of mBD2 molecules was identified from the protein level by Western-Blot.
3. MTT method was used to determine the growth curve of three cells: B16, B16-p, B16-mBD2 in 120 hours. The cell cycle distribution and apoptosis of three cells in the logarithmic growth period were detected by PI staining flow cytometry; the anti body staining of CD80, CD86, MHC I and MHC II was used as a FITC marker, and the CD80 of three cells was detected by flow cytometry. C cells were tested for CD80, C Expression changes of D86, MHC I, MHC II and other related molecules.
4. the three cells B16, B16-p, and B16-mBD2 were prepared by radiation method, and the immunotherapy and immunotherapy in C57BL/6 mice were carried out. The control group of physiological saline, the B16 control group, the B16-p control group and the B16-mBD2 experimental group were set up.
5. immunoprophylaxis experiment: 6~8 weeks old female C57BL/6 mice were randomly divided into 4 groups, 10~12 in each group, respectively, the saline control group, the B16 control group, the B16-p control group and the B16-mBD2 experimental group. All the animals were subsubcutaneously injected into the left axillary saline and 106 radiation B16, B16-p and B16-mBD2 under the left axillary subaxillary injection. Seven days after seven days of immunization, all mice in each group were given 5 * 104 logarithmic long-term wild type of wild type of tumor. The survival state of each group was observed day by day, the growth curve of the tumor in vivo was made, the survival time analysis and HE staining were used to observe the tumor tissues and important organs of the immunization prevention groups, such as liver, kidney and spleen. Histological changes of the dirty, lung and so on.
6. immunotherapy experiment: 6~8 weeks old female C57BL/6 mice were randomly divided into 4 groups, with 10~12 rats in each group, respectively, the saline control group, the B16 control group, the B16-p control group and the B16-mBD2 experimental group. All the animals in each group were subcutaneously injected under the left axillary subcutaneous injection to induce the tumor of the wild type B16 cells of the 105 logarithmic growth period, and all the mice in each group were in accordance with each group. Each group, on the same day, was injected under the left subaxillary subaxillary injection to give O.1mL saline and 106 radiation B16, B16-p and B16-mBD2 cell vaccines for two times a week for two weeks. The survival state of the mice in each group was observed day by day, the growth curve of the tumor in vivo was made, the survival time analysis and HE staining were used to observe the immunotherapy. Histological changes of tumor tissues and important organs, such as liver, kidney, spleen and lungs.
7. ELISA method was used to determine the changes of IFN- gamma, IL-12, IL-4 in the spleen lymphocyte culture supernatant of mice after immunization with B16, B16-p and B16-mBD2 cells. The cytotoxicity of B16, B16-p and B16-mBD2 vaccines in mice were determined by non radioactive cytotoxicity analysis method. Cell killing activity.
8. the SPSS 13 was used for statistical analysis. The cell growth curve, the growth curve of the tumor in the animal experiment, the variance analysis of the repeated measurement factors, the cell cycle distribution, the apoptosis rate, the change of the expression of the immune related molecules on the surface of the cell membrane were analyzed by one-way ANOVA; when the variance was homogeneous, the multiple comparison was compared with the LSD method and variance. In the absence of homogeneity, multiple comparison used Dunnett T3.CTL cell killing activity, NK cell killing activity by factorial design and statistical significance of data analysis using single factor variance test (one-way ANOVA) LSD method for data analysis.P0.05.
Experimental results:
1. mBD2 eukaryotic secretory expression vector pcDNA3.1 (+) -IgK-mBD2 containing the IgK signal peptide sequence of rat origin was successfully constructed and identified correctly.
After transfection of B16 cells with 2.pcDNA3.1 (+) and pcDNA3.1 (+) -IgK-mBD2, G418 was screened for stable expression of cell lines, named B16-p and B16-mBD2. respectively.
The 3. cell growth curve experiment showed that the proliferation rate of B16-mBD2 cells was significantly slower than that of wild type B16 cells and B16-p (F=144.256, P0.05), and the proliferation rate of B16-p was not significantly changed compared with that of wild type B16 cells.
4. flow cytometry showed that, after 24 hours of inoculation, compared with the wild type B16 cells and B16-p of the logarithmic growth period, the cell cycle of B16-mBD2 cells in logarithmic growth period had a mild block of S phase (F=8.952, P0.05), and the expression of the membrane surface molecules CD80, CD86, MHC I, etc. of the three cells B16, B16-p and B16-mBD2 were not significant. Sexual differences.
5. in the immunoprophylaxis experiment, all the mice in the saline control group, the B16 control group and the B16-p control group were all dead within 49 days. There was no significant difference between the groups and the median survival period was 35 days, 37 days and 33 days respectively. The growth rate of the mice in the B16-mBD2 immunization group was significantly slow (F=118.387, P0.05), and the survival period of the mice was significantly prolonged (X 2=18.857, P0.05), the median survival period was 55 days, and the survival state of the same period was better than that of the other control groups. Until the end of the experiment, 37.5% of the mice were still alive without tumor until 150th days after the tumor.
6. in the immunotherapy experiment, all the mice in the saline control group, the B16 control group and the B16-p control group all died within 44 days, and there was no significant difference between the groups. The median survival period was 32 days, 34 days and 31 days respectively. The growth rate of the mice in the B16-mBD2 immunotherapy group was significantly slow (F=289.615, P0.05), and the survival period of the mice was significantly prolonged. (X2=22.006, P0.05), the median survival period was 59 days, and the survival state of the same period was better than that of the other control groups. Until the end of the experiment, 25% of the mice were still alive without tumor at 150th days after the tumor.
7. in the immunoprophylaxis and immunotherapy experiments, the B16-mBD2 immunization group induced a large number of lymphocytes infiltrating into the tumor tissue, the increase of the lymph nodes in the spleen, the reactive hyperplasia of the germinal center, and the increase. The control group of the physiological saline, the B16 control group and the B16-p control group all did not induce this active anti-tumor immune response.
After immunization of 8.B16-mBD2 cell vaccine, IFN- gamma could be promoted and increased significantly (F=506.814, P0.05), and IL-12 production was significantly increased (F=83.637, P0.05). Compared with B16 control group, the B16-p control group had a significant difference, but the content of IL-4 did not affect the specific activity of.B16-mBD2 cell vaccine to induce specific killing activity against B16 cells. Sex increased significantly (F=44.376, P0.05), and NK cell killing activity increased significantly (F=119.750, P0.05), compared with B16 control group and B16-p control group, the difference was statistically significant.
After the 9.B16-mBD2 cell vaccine was immunized with mice, the HE staining of the main organs of the mice, such as liver, kidney, spleen and lung, showed that there was no obvious pathological changes in the main organs, suggesting that the B16-mBD2 vaccine was relatively safe.
Conclusion:
After transfection of mBD2 gene to malignant melanoma B16 cells, the transgenic cell vaccine prepared by the vaccine has obvious anti murine melanoma B16 effect. After immunization, the mouse NK cells are induced by the vaccine.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.5

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