【作者】 彭细毛；

【导师】 曾曙光；

【作者基本信息】 南方医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 恶性黑素瘤(malignant melanoma)是来源于黑色素细胞的恶性肿瘤,是一类起源于神经嵴黑色素细胞的恶性肿瘤,可发生于皮肤、粘膜,且发生于口腔粘膜的恶性黑色素瘤是口腔恶性肿瘤中恶性程度最高的肿瘤,占全身恶性肿瘤的1%～2%。而在我国与东南亚地区发生于口腔黏膜者反比面部皮肤多,占约80%以上。近年来许多调查表明,恶性黑色素瘤的发病率不断地增高,且约70%早期转移至区域性淋巴结,还可经血流远处转移至肺、肝、骨、脑等器官,其远处转移率可高达40%以上。传统治疗肿瘤的方法：手术、放疗、化疗以及辅助疗法虽取得了不错的成效,但恶性黑素瘤细胞对化疗药物相对不敏感,对放射存在一定的抗拒性；并且B16黑色素瘤是基于黑色素瘤肿瘤细胞,具有高致瘤性以及低免疫原性,目前尚无治疗的良策,故寻求一种安全有效的防治恶性黑素瘤的方法迫在眉睫。随着对免疫学与分子生物学的深入了解,肿瘤基因免疫靶向治疗已成为目前肿瘤治疗的研究热点。自20世纪90年代基因(DNA)疫苗这一概念提出后,DNA疫苗已进入多个领域并取得了很大发展,而在肿瘤基因防治方面,肿瘤基因疫苗种类众多,其中自杀基因疗法被认为是目前最有前景的肿瘤基因治疗方法。肿瘤的自杀基因疗法(suicide gene therapy),又称作基因导向性酶前体药物疗法(gene-directed enzyme prodrug therapy, GDEPT),是近年来新发展的针对实体瘤的基因疗法,利用其对肿瘤细胞的杀伤效应及其旁观者效应,达到防治肿瘤的目的。单纯疱疹病毒胸苷激酶/丙氧鸟苷(herpes simplex virus thymidine kinase/ganciclovir, HSV-tk/GCV)系统是研究得最早、最成熟的、应用最广泛的自杀基因疗法。单纯疱疹病毒胸苷激酶(human herpes simplex virus-thymidine kinas, HSV-TK)基因能特异性地将无毒性的前体药物更昔洛韦(Gancivol, GCV)磷酸化为高毒性的三磷酸GCV,从而抑制DNA聚合酶的活性,诱导抗肿瘤效应。但是肿瘤细胞的多态性、高致瘤性以及低免疫原性依然是其难以攻克的重要原因。如何突破肿瘤的免疫耐受及肿瘤细胞的免疫逃逸机制,激活机体免疫系统识别并杀伤肿瘤细胞是肿瘤疫苗设计时需要解决的根本问题。而热休克蛋白(heat shock protein, HSP)可以结合细胞内的自我与非自我的全部异常的蛋白降解产物--多肽,形成带有该细胞的全部特征性指纹的HSP-肽复合物(HSP peptide complexes, HSP-PCs),因此HSP-PCs是多价的,这有利于抑制免疫逃逸从而提高机体的免疫作用。结核杆菌热休克蛋白70 (mycobacterium tuberculosis heat shock protein 70, mtHsp70)作为分子伴侣具有免疫激活作用,不仅可以作为抗原递呈载体将抗原运送至主要组织相容性抗原复合物(major histocompatibility complex, MHC)Ⅰ类和Ⅱ类分子,而且能够激活抗原递呈细胞树突状细胞(dendritic cells, DC),打破机体对肿瘤特异性抗原的免疫耐受。另外,mtHSP70可以促进Th细胞向Thl细胞转化,调整肿瘤病人机体的免疫状态,有利于控制肿瘤的发生。肿瘤基因治疗的另一障碍是不能靶向性将抗肿瘤基因传递到肿瘤组织内,这需要建立有效的向靶细胞转移目的基因的载体系统。近年来研究较多的病毒载体(如腺病毒、逆转录病毒、腺相关病毒等),存在的最主要的缺点就是多数实体肿瘤中存在的乏氧现象会大大削弱病毒载体的功能和目的基因的表达。而减毒沙门氏菌是肠道兼性厌氧菌,实体肿瘤乏氧现象恰恰为其提供了适合聚集生长和繁殖的场所；同时减毒沙门菌也是一种侵袭性胞内菌,主要通过M细胞进入肠上皮,被巨噬细胞和树突状细胞吞噬后携带至淋巴结、脾脏和其他组织。以上两种特性为减毒沙门氏菌用作口服型肿瘤基因的靶向载体提供了可能。同时它对肿瘤细胞具有侵袭性,在肿瘤细胞内有限复制,且易于以抗生素清除,具有良好的生物安全性。目前,肿瘤基因治疗的研究越来越深入,单基因治疗暴露出许多的不足,最主要的是易引起药物耐受。为解决这一问题,许多学者采用联合基因疗法,并已取得了相当可喜的成绩。但目前大都以融合基因的形式,自身存在许多缺点,最主要的缺点是融合蛋白的不稳定性,它容易发生降解,其次融合蛋白不易正确折叠,难以保证各组分在融合之后仍保持原来的活性。利用减毒沙门菌作为基因疫苗载体,它可分别将单个基因转入沙门菌中,避免了融合基因中两个基因表达相互干扰的问题；同时也使两类基因产生协同作用,突破了对肿瘤细胞类型的依赖性,尽可能地消除了瘤细胞对药物的耐受性。结合以上几种原因,本课题以减毒鼠伤寒沙门氏菌SL7207为载体,利用mtHsp70的免疫佐剂作用和HSV-TK对肿瘤细胞杀伤效应以及其旁观者效应,制备重组减毒沙门菌：SL7207/mtHSP70/HSV-TK双基因真核共表达的DNA疫苗口服液,建立小鼠黑色素瘤B16动物模型,拟制备的重组沙门菌口服液治疗荷B16黑色素瘤小鼠,观察减毒沙门菌对肿瘤的靶向性,以及重组菌的抑瘤效应及其可能机制。本研究共分为两章：第一章口服携带EGFP的重组沙门菌对肿瘤的靶向性；第二章口服mtHSP70/HSV-tk重组沙门菌对小鼠黑色素瘤的抑制作用。第一章口服携带EGFP的重组沙门菌对肿瘤的靶向性1.目的：研究减毒的沙门氏菌SL7207对于肿瘤组织的靶向性。2.方法：将本课题组前期已制备并通过鉴定的含绿色荧光蛋白(enhanced green fluorescent protein, EGFP)的重组沙门菌复苏,制备以EGFP为报告基因的重组沙门菌(SL7207/PCMV-IRES-EGFP)口服液(浓度为109 CFU/ml),以100μl灌胃饲服荷B16黑色素瘤小鼠,通过组织冰冻切片及组织匀浆菌落计数方法研究不同时间点细菌在肿瘤组织中和正常组织中的分布情况。3.结果：在口服重组沙门菌后第2天,肝脏、脾脏及肿瘤组织匀浆中均有少数菌落生长；而第4天,肝脏、脾脏及肿瘤组织匀浆中菌落数较口服后第2天明显增多,并且发现肿瘤组织中的菌落数增加更明显；在口服后第7天,肝脏、脾脏组织匀浆中菌落生长明显减少,而肿瘤组织中菌落大量生长；同时冰冻切片荧光显示同样的结果。说明了减毒沙门菌能特异性地聚集于肿瘤组织中。第二章口服mtHSP70/HSV-TK重组沙门菌对小鼠黑色素瘤的抑制作用1.目的：研究以减毒沙门菌SL7207为载体的结核杆菌热休克蛋白70 (mtHSP70)和人单纯疱疹病毒-胸苷激酶(HSV-TK)双基因共表达的DNA疫苗靶向治疗小鼠黑色素瘤的效应,以及该疫苗抗肿瘤的可能机制。2.方法：将本课题组前期已制备并通过鉴定的重组沙门菌：SL7207/PCMV-mtHSP70-IRES-TK、SL7207/PCMV-mtHSP70-IRES-EGFP SL7207/PCMV-TK-IRES-EGFP、SL7207/PCMV-IRES-EGFP复苏,制备成以减毒沙门菌SL7207为载体的口服DNA疫苗。将细胞浓度为107/ml的小鼠黑色素瘤细胞B16细胞0.1 ml接种于C57BL/6J小鼠右背皮下,建立小鼠黑色素瘤B16动物模型,当肿瘤体积达到100 mm3左右,将荷瘤小鼠C57BL/6J随机分成五组,每组10只,实验组及对照组口服(灌胃法)109 CFU/ml的重组沙门菌100μl,qd×2天,第一次口服48 h后,辅以HSV-TK基因前体药物GCV 50mg/Kg,腹腔注射,Bid×5天,此为一周期,共治疗三周期。同时空白对照组给予100μl PBS灌胃,同样给予GCV。治疗期间每三天测量荷B16小鼠瘤体大小,观察各组小鼠瘤体变化情况,并计算抑瘤率；最后一次治疗结束时每组随机处死5只,剩下的荷瘤鼠用于观察生存期。用HE染色、免疫荧光学检测肿瘤组织及其周围免疫细胞的变化；在无菌状态下取得免疫鼠脾脏,用LDH释放法检测CTL及NK的活性；制备脾细胞上清用Elisa法检测IFN-γ的含量。3.结果：五组B16黑色素瘤小鼠经重组沙门菌治疗三个周期后,①用重复测量方法对各组肿瘤体积进行统计分析,结果显示五组间差异有显著性(F=599.949,P=0.000),不同重组菌处理组与时间之间有交互作用(F=331.643,P=0.000),HT组分别与各对照组肿瘤体积差异有显著性(P=0.000)；H组及T组与Sa组、PBS组肿瘤体积差异有显著性(P=0.000),H组与T组肿瘤体积差异无显著性(P=0.398),Sa组与PBS组肿瘤体积差异有显著性(P=0.000)；相对PBS组,各重组沙门菌组在第三个疗程治疗结束的抑瘤率分别是：HT组抑瘤率为78.47%,H组抑瘤率为63.02%,T组抑瘤率为61.63%,Sa组抑瘤率为55.61%。②以Log-Rank法行生存分析,结果表明中位生存时间：HT组为35.60天,H组为31.17天,T组为31.50天,Sa组为29.73天,PBS组为27.53天；5组间生存期存在差异(x2=12.591,P=0.013),HT组生存期显著长于其他各组(P<0.05)。③LDH释放法检测结果表明：在不同的效靶比情况下5组间CTL杀伤活性差异有显著性意义(x2=11.867,P=0.018),HT组、H组、T组、Sa组、PBS组的秩次分别是：13.07、10.33、8.33、5.67、2.00,不同效靶比之间CTL杀伤活性差异无显著性意义(x2=1.820,P=0.403)；5组间NK杀伤活性差异无显著性意义(x2=7.121,P=0.130),不同效靶比之间差异有显著性(x2=6.316,P=0.043),效靶比为50：1、25：1和12.5：1时的杀伤活性的秩次分别为：11.60、7.90、4.50。④Elisa法检测治疗后各组荷瘤小鼠脾细胞上清IFN-r的含量结果显示：组间不符合方差齐性,故用welch方法检验：组间总体比较差异有显著性(F=54.441,P=0.000),进一步用Dunnett T3进行两两比较：HT组、H组、T组与PBS组之间差异有显著性(P<0.05),HT组、H组、T组、Sa组组间差异v没有显著性(P＞0.05)。⑤HE染色及免疫荧光法检测结果表明：通过重组菌治疗后获得免疫的荷瘤小鼠的肿瘤组织明显地坏死,肿瘤组织内及其周围炎症细胞、CD8+T淋巴细胞浸润明显。全文结论：(1)口服减毒沙门氏菌SL7207对肿瘤组织具有靶向性,并能在肿瘤细胞中表达目的基因,是肿瘤靶向基因治疗的理想载体。(2)口服SL7207/mtHSP70/HSV-TK重组沙门菌能够诱导CD8+T细胞产生特异性细胞免疫,激活CTL及NK细胞的活性,促进脾细胞分泌IFN-γ,抑制C57BL/6J小鼠B16黑色素瘤的生长,延长生存期。更多还原

【Abstract】 Malignant melanoma is a malignant tumor that derived from malignant melanoma cells and neural crest melanocytes; it can occur in the skin, mucous membranes, and malignant melanoma occurred in the oral mucosa is the highest level of oral malignant tumor, accounting for 1% to 2% in systemic malignancy. And In China and Southeast Asia occurred in oral mucosa were more inversely proportional to the facial skin, accounting about 80% of total. In recent years, many surveys indicate that the morbidity of malignant melanoma constantly increase, and about 70% early metastasis to regional lymph nodes, also by the blood flow distant metastasis to lung, liver, bone, brain and other organs, the distant metastasis rate up to 40%. Though, the traditional method of treating cancer (including:surgery, radiotherapy, chemotherapy, and adjuvant therapy) has achieved right effect, but the malignant melanoma cells is relatively insensitive to chemotherapeutic drugs, occur certain resistance to the radiation, besides the B16 melanoma derived from the melanoma tumor cells with high oncogenicity and low immunogenicity, so finding a safe and effective method of preventing and treating malignant melanoma is quite important.With the immunology and molecular biology are in-depth understanded, targeted therapy of cancer gene immune has become a research focus of tumor therapy. From 90 years of the 20th century, this concept of DNA vaccine was produced, DNA vaccines have entered a number of areas and have made great progress, and on the prevention and treatment of cancer genes, the types of DNA vaccines are numerous, of which suicide gene therapy is considered that one of the most promising cancer gene therapy at the present time. Suicide gene therapy of tumor (suicide gene therapy), also known as gene directed-enzyme prodrug therapy (GDEPT), is a new development gene therapy for solid tumors in recent years, exploit its killing effect on tumor cells and bystander effect to control the tumor. Herpes simplex virus type I thymidine kinase/ganciclovir (HSV-tk/GCV) system is a suicide gene therapy is the earliest studied, most mature, and most widely used. HSV-TK gene can specifically triphosphate non-toxic phosphate prodrug GCV into the high toxicity GCV, thereby inhibiting the activity of DNA polymerase, inducting anti-tumor effect.However, because the polymorphism, high cause tumor and low immunogenicity of tumor cells remains be an important reason which was difficult to overcome. How to break immune tolerance of tumor and immune escape mechanism of tumor cells, and activate the body’s immune system to recognize and kill tumor cells, these which are the design of tumor vaccines need to address the fundamental problem. The heat shock protein (HSP) can combine with all non-self and self abnormal protein degradation products of cells, and form with the HSP peptide complexes (HSP-PCs) with all the characteristic fingerprint of the cells, so HSP-PCs are more much price, which is conducive to suppress the immune escape machinery, so as to enhance the immune function. Mycobacterium tuberculosis heat shock protein70 (mtHsp70) as a molecular chaperone process immune activational effect, mtHsp70 was not only as a carrier of antigen to antigen delivery to major histocompatibility complex (MHC) I and II molecules, but also can activate antigen presenting cells (dendritic cells DC), breaking the immune tolerance of tumor specific antigen. In addition, HSP70 can promote the transformation of Th cells to Th1 cells, adjust the immune status of cancer patients, and help control occur of the cancer.Another hindrance of cancer gene therapy is that it can not specifically transmit on anti-tumor gene to tumor tissues, which requires the establishment of an effective carrier system of gene that can divert the purposed gene to targeted cells. Viral carrier which is recently considerably much studies (eg adenovirus, retrovirus, adeno-associated virus, etc.), subsistent main drawback is that the most solid tumors exist the phenomenon of hypoxia will significantly weaken function of the virus carrier and expression of the purposed gene. The attenuated Salmonella are intestinal facultative anaerobes, the hypoxia phenomenon of solid tumor just provide a suitable place for growth and reproduction of the attenuated Salmonella; as the same time, The attenuated Salmonella is a kind of intracellular bacteria of invasion, primarily through M cells into the intestinal epithelium, that is swallowed by macrophages and dendritic cells into the lymph nodes, spleen and other organizations. These two characteristics provide possibility for the attenuated Salmonella as targeting carrier of oral tumor gene. And then it also has attacking to tumor cells, limited replication in tumor cells, and is easily cleared by antibiotics, with good biological safety.With more and more in-depth study of tumor gene therapy, single-gene therapy exposed the more and more deficiencies, the most important element is easy to cause drug resistance. To solve this problem, many scholars using combined gene therapy and have achieved fairly good results. But the majority are the form of the fusion gene, which exist many shortcomings, the uppermost defect is the instability of the fusion protein, it is prone to degradation, secondly fusion protein is not easy to by fold correctly, so it is difficult to ensure that all components remain maintain the original activity after the integration. Using attenuated Salmonella as a genetic vaccine vector, it can transfer the single gene into Salmonella, avoiding the mutual interference of the fusion gene when express the two types of genes; and it also make that the two types of gene product synergies, eliminating drugs tolerance of the tumor cells.To combine the above reasons, the subject use attenuated Salmonella typhimurium as carrier, immuno-adjuvant effect of Mycobacterium tuberculosis Hsp70 on the antigen and the killing effect of the suicide gene on tumor cellt and bystander effect, preparating DNA vaccines Oral of recombinant attenuated Salmonella:SL7207/mtHSP70/HSV-TK dual-gene co-expression, seting up the animal model of B16 mouse melanoma, exploit the prepared recombinant Salmonella Oral to treat bearing B16 mice, and observing the targeting to tumor, the inhibited effect on tumor and its interrelated mechanismThe study is divided into two chapters:the first chapter:the oral recombinant Salmonella carrying EGFP on tumor; the second Chapter:Inhibited effect of oral recombinant Salmonella carrying mtHSP70/HSV-TK to mouse melanoma.Chapter One:The targeted effect Oral recombinant Salmonella carrying EGFP on tumor1. Objective:To Study targeting of attenuated Salmonella carrying EGFP (SL7207/PIRES-EGFP) to tumor-tissue. 2. Methods:Using recombinant Salmonella with enhanced green fluorescent protein (EGFP) that has been prepared and identified by our discussion group to recovery, preparating the recombinant Salmonella (SL7207PCMV-/PIRES-EGFP) Oral that using EGFP as reporter gene.100μl of 109 CFU/mL recombinant salmonella typhimurium was given tomice bearing B16 melanoma mice by oral (gavage method). Through the frozen sections of tissue and tissue spare pulp count method of bacteria colony to observe bacterial distribution in tumor tissue and normal tissue at different time points.3. Result:Two days after the oral, there is a small number of colony growth in liver, spleen and tumor tissue’s spare pulp. And four days after the oral, the number of colony growth of liver and spleen tissue’s spare pulp increased more significantly than after two days; and tumor tissue were more significantly increased in colonies as liver and spleen tissue. However, to the seventh day, the number of colony growth of liver and spleen tissue’s spare pulp significantly reduced; but bacterial colony of tumor tissue group in a large of numbers. At the same time, Frozen sections by fluorescence show the same results as tissue’s spare pulp.Chapter Two:Inhibited effect of oral recombinant Salmonella carrying mtHSP70/HSV-TK to mouse melanoma. 1. Objective:To study the targeted effect of oral attenuated Salmonella typhimurium as carrier of Mycobacterium tuberculosis heat shock protein 70 (mtHSP70) and human herpes simplex virus thymidine kinase (HSV-TK) DNA vaccine of double-gene coexpression on mouse melanoma and the possible mechanism of the anti-tumor vaccines.2. Methods:Using identified recombinant Salmonella that our discussion group has had prepared earlier stage:SL7207/PCMV-mtHSP70-IRES-TK、SL7207/PCMV-mtHSP70-IRES-EGFP、SL7207/PCMV-TK-IRES-EGFP、SL7207/PIRES-EGFP, to recovery, and were prepared with attenuated Salmonella typhimurium SL7207 as a carrier for oral DNA vaccine. First of all, 0.1mL mouse melanoma B16 cells(cell concentration 107/mL) were subcutaneously inoculated into the back on the right side of C57BL/6J mice, so as to established animal model of mouse melanoma. When tumor volume reached about 100mm3, the C57BL/6J mice were randomly divided into five groups and each group hanve ten mices. for the experiment, with 10 tumor bearing mice for each group. For experimental group and control group, 100μL of 109CFU/mL recombinant salmonella typhimurium was given by oral (gavage method). The blank control group oral 100μL PBS. qd×2day.48h after the first oral, GCV 50mg/Kg in 0.5mL PBS was intraperitoneally injected, twice a day for five consecutive day.Each treatment course lasted for seven days. In total three successive treatment course were given. During treatment, each group was measured with vernier caliper once every three day. Calculating inhibitory rate based on measured tumor volume. At the end of the last treatment, five B16 bearing mice were sacrificed randomly from each group, The remaining tumor-bearing mice survival time for observation. Using HE staining, immunofluorescence measuring tumor tissue and surrounding changes in immune cells; under aseptic state getting immunized mice spleen preparated spleen cell suspension, LDH release assay checking CTL and NK activity; detecting IFN-γlevels of spleen cell supernatants.3. Result:Five groups of B16 melanoma in mice by recombinant Salmonella treatment three cycles, the tumor volume in each group were analyzed by Statistics, results showed that differences between the five groups possess significance (F=599.949, P=0.000), treatment group with different recombinant exist interaction between time (F=331.643, P=0.000); significant differences in tumor volume when comparing HT group to every control groups (P=0.000); H group and T group exist significant difference in tumor volume (P=0.000); H group and T group tumor volume difference was not statistically significant between Sa group and PBS group(P=0.398); significant differences in tumor volume when comparing Sa group to PBS group(P=0.000); In relation to PBS group, all recombinant Salmonella groups in the end of the third course of treatment, the inhibitory rates were:HT tumor inhibition rate was 78.47%, H tumor inhibition rate was 63.02%, T tumor inhibition rate was 61.63%, Sa tumor inhibition rate was 55.61%; with Log-Rank method proceed survival analysis showed that the median survival time:HT group was 35.60 days, H group was 31.17 days, T group was 31.50 days, Sa group was 29.73 days, PBS group was 27.53 days; survival rates between five groups exist differences (x2=12.591, P=0.013), HT group survival was significantly longer than the other groups (P＜0.05). LDH release assay results showed that:in a different effective target, the difference of CTL killing activity between five groups was significant (x2=11.867, P=0.018), the order of HT group, H group, T group, Sa group, PBS group in turn are:13.07,10.33, 8.33,5.67,2.00, the difference of CTL killing activity between different effector to target ratios was not significant (x2=1.820, P=0.403); the difference of NK killing activity between five groups was not significant (x2=7.121, P=0.130), the differences between different effector to target ratio was significant (x2=6.316, P=0.043), effector to target ratio of 50:1,25:1,12.5:1, the order in turn are:11.60,7.90,4.50. Elisa assay was performed to detect IFN-r concentration in tumor tissue of bearing mice after treatment of recombinant vaccine, the resuls ware as follow:all groups does not meet homogeneity of variance, so, with welch way:the overall difference between groups was significant (F=54.441, P=0.000), further using Dunnett T3 compared to two groups:difference in HT group, H group, T group between PBS group was significant (P＜0.05), HT group, H group, T group, Sa group group There was no significant difference (P＞0.05). HE staining and immunofluorescence results showed that:the tumor tissue of tumor-bearing mice that obtain immune by recombinant treatment was clearly necrotic, in tumor tissue and it’s circumambience which there are a lots of inflammatory cells, CD8+T lymphocyte infiltrated into.Conclusion:(1) Oral attenuated Salmonella typhimurium SL7207 is targeting to tumor tissue and can express the target gene in tumor cells, is an ideal tumor targeting gene therapy vectors.(2) Oral recombinant Salmonella (SL7207/mtHSP70/HSV-TK) can induce CD8+ T cells to produce specific cellular immunity, activated CTL and NK cell activity, promot spleen cells to perspire IFN-y, inhibit C57BL/6J mice B16 tumor growth and prolong B16 mouse survival.更多还原