第一部分 肿瘤细胞裂解物联合IL-2通过激活机体免疫预防肿瘤发生的初步研究

中文摘要

背景：随着对免疫学及肿瘤发病机制的逐步深入探讨，肿瘤免疫治疗已转变成为当前重要的研究手段之一。肿瘤疫苗属于免疫治疗的范畴，近年来在临床前研究和临床研究领域均获得诸多优秀的成果。本研究采用肿瘤细胞裂解物(Tumor cell lysates，TCL)联和佐剂建立肿瘤疫苗，拟探讨其在黑色素瘤中发挥出的潜在免疫激活及抑制肿瘤的作用，并为后续研究做好理论铺垫。

材料与方法：我们采用超声破碎仪制备B16F10黑色素瘤TCL，并使用台盼蓝染色明确其活性。24只C57BL/6小鼠随机分为四组，分别予PBS、IL-2、TCL及TCL+IL-2皮下注射，每周1次，共3周，第4周对侧植瘤，观察小鼠的出瘤时间及肿瘤大小。并连续采集小鼠外周血行CD4⁺T及CD8⁺T细胞的流式动态分析，并采用流式及免疫组化的方法检测脾脏及肿瘤组织中CD4⁺T及CD8⁺T细胞的表达。

结果：TCL+IL-2组小鼠经预防性免疫后，出瘤时间明显延迟(p=0.034)，肿瘤体积(p =0.023)及瘤重(p=0.0015)也显著小于对照组。流式检测结果提示TCL+IL-2组及单用TCL组小鼠，外周血CD8⁺T细胞较对照组上(p=0.0016, p=0.012)。TCL+IL-2组的CD4⁺T细胞在植瘤后较对照组有所下降(p=0.0089)。脾脏及肿瘤组织中CD4⁺T及CD8⁺T细胞与外周血中的表达趋势相同。

结论：TCL佐以IL-2可以有效激活机体免疫，预防小鼠黑色素瘤发生并遏抑肿瘤的生长。

第二部分 超声联合纳米粒的物理治疗方式在乳腺癌中的应用及免疫机制探索

中文摘要

背景：近年来，新材料技术和医学影像学迅猛发展，新型物理治疗方式成为肿瘤治疗的又一重要策略。超声微气泡物理治疗技术是一种无创的物理治疗手段。相变纳米粒在外界超声干预下，发生液气相变从而导致细胞原位损伤。本研究拟探讨超声联合相变纳米粒的物理治疗方式在乳腺癌中的作用及激活免疫的相关机制研究。

材料与方法：制备氟碳脂质纳米粒(Lipid Fluorocarbon Nanoparticles，LIP-PFH纳米粒)，使用马尔文粒径及电位检测设备测试其理化特性，并使用共聚焦拍摄手段明确4T1乳腺癌细胞对其吞噬作用。通过CCK-8实验，凋亡及周期实验分析低强度聚焦超声(Low-intensity focused ultrasound, LIFU)联合纳米粒的物理治疗技术在体外的抑瘤作用。建立Balb/c乳腺癌小鼠移植瘤模型，进一步证实该种物理治疗的体内抑瘤作用，并通过肺转移灶的H&E染色，初步提示对远处转移的作用。流式细胞术监测小鼠外周血、脾脏及肿瘤组织中CD4⁺T，CD8⁺T细胞的改变，明确治疗后对机体免疫的影响。进行免疫原性细胞死亡标记物的检测，及DC细胞成熟度的流式分析，以研究潜在的免疫机制，并用缺乏免疫功能的裸鼠实验再次证实。

结果：LIFU联合LIP-PFH纳米粒的物理治疗手段，在体外和体内实验中均看到有效抑制4T1细胞的增殖，并促进其凋亡，并且在体内动物实验中初步看到对远处肺转移也具有抑制作用。外周血，脾脏和肿瘤组织中CD4⁺T和CD8⁺T细胞亚群比例在该种物理治疗后显著增加，表明该种治疗技术有效增强了乳腺癌细胞中的抗肿瘤免疫应答。免疫原性细胞死亡标记物(ATP，高迁移率族蛋白B和钙网蛋白)以及DC细胞成熟度的流式分析表明，超声联合纳米粒的物理治疗技术可通过诱导4T1乳腺癌细胞发生免疫原性细胞死亡，介导DC成熟明显增加，以进一步促进细胞毒性T细胞比例的增多，增加肿瘤组织中T淋巴细胞的浸润。裸鼠实验进一步验证了该种作用机制。

结论：超声联合相变纳米粒的物理治疗可以有效增强乳腺癌细胞的抗肿瘤免疫反应，改善其免疫原性，为未来提高乳腺癌免疫治疗疗效提供潜在的研究方向。

The Part I

Abstract

Background: With the gradual and in-depth discussion of immunology and tumor pathogenesis, tumor immunotherapy has become one of the most anticipated research fields. As one of the important components of immunotherapy, tumor vaccines have grown rapidly in recent years and have obtained many excellent results in the fields of basic research and clinical research. This study uses tumor cell lysates (TCL) combined with adjuvant to prepare tumor vaccine, and intends to explore its potential immune activation and tumor suppression effects in melanoma, and to pave the way for follow-up research.

Materials and Methods: We used an ultrasonic disruptor to prepare B16F10 melanoma TCL and used trypan blue staining to confirm its activity. Twenty-four C57BL/6 mice were randomly divided into four groups. PBS, IL-2, TCL and TCL+IL-2 were injected subcutaneously respectively, once a week for 3 weeks. In the 4th week, the contralateral tumor was implanted, and the tumor emergence time and tumor size were observed. The peripheral blood of mice was continuously collected for CD4⁺T and CD8⁺T flow dynamic analysis. The expression of CD4⁺T and CD8⁺T in the spleen and tumor tissues were also detected by flow cytometry and immunohistochemistry.

Results: The tumor emergence time was significantly delayed after prophylactic immunization in the TCL+IL-2 group(p=0.034), and the tumor volume(p=0.023) and tumor weight(p=0.0015) were also significantly smaller than those in the control group. The results of flow cytometry showed that the proportion of CD8⁺T cells in the peripheral blood of the TCL+IL-2 group and the TCL group alone was significantly higher than that of the control group (p=0.0016, p=0.012). The CD4⁺T cells in the TCL+IL-2 group decreased after tumor implantation compared with the control group(p=0.0089). The proportions of CD4⁺T and CD8⁺T cells in the spleen and tumor tissues were the same as the expression trends in peripheral blood.

                Conclusions: TCL combined with IL-2 can effectively  activate the body's immunity, prevent the occurrence of melanoma in mice and inhibit the growth of tumors.

  The Part II

Abstract

Backgrounds: With the rapid development of material technology and medical imaging technology, new physical therapy methods have become another important strategy for tumor treatment in recent years. Ultrasound microbubble physical therapy technology is a non-invasive physical therapy method. Phase change nanoparticles undergo a liquid-to-gas phase change under the intervention of external ultrasound, which leads to cell injury in situ. This study intends to investigate the role of a physical therapy consisited of ultrasound combined and phase-change nanoparticles in breast cancer and the related immune activation mechanisms. 

Materials and Methods: Preparing lipid fluorocarbon nanoparticles (LIP-PFH nanoparticles), use Malvern particle size and potential detection equipment to test their physical and chemical properties, and use confocal methods to determine the phagocytosis of 4T1 breast cancer cells. Through CCK-8 experiment, apoptosis and cycle experiment, the anti-tumor effect of low-frequency ultrasound combined with nano-particle physical therapy technology in vitro is analyzed. The establishment of Balb/c breast cancer mouse xenograft model to further confirm the anti-tumor effect of the physical therapy, and through the HE staining of lung metastases, preliminary suggestion of the effect on distant metastasis. Flow cytometry was used to monitor the changes of CD4⁺T, CD8⁺T cells in peripheral blood, spleen and tumor tissues of mice. Detection of immunogenic cell death markers to study the underlying immune mechanism, and use nude mice to confirm again.

Results: Low-intensity focused ultrasound combined with LIP-PFH nanoparticles can effectively inhibit the proliferation of 4T1 cells and promote their apoptosis both in vitro and in vivo. An inhibitory effect on distant metastasis also have been preliminarily showed in vivo animal experiments. The proportion of CD4⁺T and CD8⁺T cell subsets in peripheral blood, spleen and tumor tissues increased significantly, indicating that this physical therapy technique effectively increases the anti-tumor immune response of breast cancer cells. Flow cytometric analysis of immunogenic cell death markers (ATP, high mobility group box B1 and calreticulin) and DC cell maturity showed that the physical therapy technology can induce immunogenic cell death in 4T1 breast cancer cells and mediates DC maturation to increase the proportion of cytotoxic T cells. Nude mouse experiments further verified this mechanism.

Conclusion: The new physical therapy combined with ultrasound and nanoparticles can effectively enhance the anti-tumor immunity of breast cancer cells and improve their immunogenicity, providing potential research directions for improving the efficacy of breast cancer immunotherapy in the future