本发明涉及医学免疫学领域，特别是涉及疫苗佐剂及含有该佐剂的疫苗。新佐剂皮卡钙联合佐剂的组成：以皮卡钙佐剂(聚肌苷酸聚胞苷酸-卡那霉素-氯化钙英文缩写为：PICKCa)为核心联合其他免疫促进剂(皮卡钙联合佐剂)作为在制备免疫佐剂中的应用，较单独使用皮卡钙佐剂更进一步促进机体的非特异性免疫和特异性免疫的佐剂。皮卡钙联合佐剂不仅能进一步提高抗原的免疫原性，更具有进一步转变免疫类型的作用，使预防性疫苗转变成兼有治疗性和预防性疫苗，以在安全前提下进一步提高抗各种细胞内感染(病毒和细胞内感染的细菌、寄生虫等)、抗各种肿瘤的效果。皮卡钙佐剂中各种成分是通过氢键等化学键复合在一起，皮卡钙联合佐剂中皮卡钙佐剂与其它免疫促进剂不一定完全通过化学键结合在一起，而是重点在于通过不同机理联合刺激机体的免疫系统达到进一步提高免疫治愈疾病的效果。The invention relates to the field of medical immunology, in particular to a vaccine adjuvant and a vaccine containing the adjuvant. Composition of the new adjuvant PICKCa combined adjuvant: PICKCa adjuvant (polyinosinic acid polycytidylic acid-kanamycin-calcium chloride English abbreviation: PICKCa) combined with other immune enhancers (PICKCa) Combined adjuvant) as an application in the preparation of immune adjuvants, compared with the use of picocalcium adjuvant alone, it can further promote the non-specific immunity and specific immunity of the body. Pika calcium combined with adjuvant can not only further improve the immunogenicity of the antigen, but also have the effect of further changing the immune type, so that the preventive vaccine can be transformed into a therapeutic and preventive vaccine, so as to further improve the anti-cancer effect of various cells under the premise of safety. Intra-infection (viruses and intracellular bacteria, parasites, etc.), anti-tumor effects. The various components in Pika Calcium adjuvant are compounded together through chemical bonds such as hydrogen bonds. Stimulate the body's immune system to further improve immunity and cure diseases.
皮卡钙佐剂及含有该佐剂的疫苗我们自20世纪九十年代已先后获得三项专利(第一个是我单独发明的原专利《聚肌胞复合物免疫佐剂及含有该佐剂的疫苗》(ZL93 1 05862.7)以及其后与李烈涛联合申请的专利《包含基于聚肌苷酸-聚胞苷酸的佐剂免疫物质》(ZL 2006 80000776.7)、《含基于聚肌苷酸-聚胞苷酸的佐剂粘膜免疫物质》(ZL 2006 8 0001059.6)，30年来已在国内外取得了一系列重要进展(互联网林海祥)。这些专利中的佐剂物质是聚肌苷酸聚胞苷酸-卡那霉素-氯化钙英文缩写为PICKCa及含有该佐剂物质的各种抗原疫苗。Pika calcium adjuvant and the vaccine containing the adjuvant, we have obtained three patents since the 1990s (the first one is the original patent "polymyocyte complex immune adjuvant and the vaccine containing the adjuvant" invented by me alone) Vaccine" (ZL93 1 05862.7) and the subsequent patents jointly applied with Li Lietao "Containing Adjuvant Immune Substances Based on Polyinosinic Acid-Polycytidylic Acid" (ZL 2006 80000776.7), "Containing Polyinosinic Acid-Polycytidylic Acid-Based Adjuvant Mucosal Immune Substances of Glycylic Acids" (ZL 2006 8 0001059.6), a series of important progress has been made at home and abroad in the past 30 years (Internet Lin Haixiang). The adjuvant substances in these patents are polyinosinic acid polycytidylic acid - Kanamycin-Calcium Chloride English abbreviation is PICKCa and various antigen vaccines containing this adjuvant substance.
本发明是对皮卡钙佐剂的进一步发展，即皮卡钙佐剂联合其他免疫促进剂在安全基础上使佐剂效果进一步提高。其他免疫促进剂包括各种Toll样受体激动剂、矿物盐如铝佐剂、悬浮微粒清洁剂如MF59、ASO3、AFO3、乳化剂，皂化剂QS21、ISCOM，、GLA-SE，颗粒释放剂如nanoparticles、PLGA、VLP、Liposomes，Virosomes，人细胞因子，趋化因子，活化配体，DC共刺激分子，IL-12，IL-15，GM-CSF，MCP-1，CD40L，DEC-205，细菌外毒素CT，LT(modified)，脂质体尤其是阳离子脂质体如DOTIM、DC-Chol、CCS、diC14咪、DOTAP、DDA，PD1/PD-L1、TIM-3阻断性抗体，激活T细胞的CTLA4，促进细胞转染制剂如PEI、PEG以及除钙离子以外的金属离子如锌、镁等；皮卡钙联合佐剂的疫苗是指含有皮卡钙联合佐剂的疫苗包括预防用或治疗用，人用或兽用疫苗而具有进一步提高抗各种细胞内感染(病毒和细胞内感染的细菌、寄生虫等)抗各种肿瘤的效果。皮卡钙联合佐剂本身即具有免疫促进作用能够非特异性地抗各种细胞内感染(病毒和细胞内感染的细菌、寄生虫等)和抗各种肿瘤的效果，能单独制备成各种抗细胞内感染和抗肿瘤的针剂、喷剂、膏剂等。皮卡钙联合佐剂是国内外都没有的一类新药，本发明皮卡钙联合佐剂及其针剂、喷剂、膏剂等也属于国内外都没有的一类新药。The present invention is a further development of the pica-calcium adjuvant, that is, the pica-calcium adjuvant is combined with other immune enhancers to further improve the adjuvant effect on the basis of safety. Other immune boosters include various Toll-like receptor agonists, mineral salts such as aluminum adjuvants, aerosol detergents such as MF59, ASO3, AFO3, emulsifiers, saponifiers QS21, ISCOM, GLA-SE, particle release agents such as Nanoparticles, PLGA, VLP, Liposomes, Virosomes, human cytokines, chemokines, activating ligands, DC co-stimulatory molecules, IL-12, IL-15, GM-CSF, MCP-1, CD40L, DEC-205, bacteria Exotoxin CT, LT (modified), liposomes, especially cationic liposomes such as DOTIM, DC-Chol, CCS, diC14 imid, DOTAP, DDA, PD1/PD-L1, TIM-3 blocking antibodies, activated T CTLA4 of cells, promoting cell transfection agents such as PEI, PEG, and metal ions other than calcium ions such as zinc, magnesium, etc.; vaccines with pica calcium combined with adjuvants refer to vaccines containing pica calcium combined with adjuvants, including preventive or therapeutic use , Human or veterinary vaccines can further improve the effect of resisting various intracellular infections (viruses and intracellular infected bacteria, parasites, etc.) against various tumors. Pika Calcium combined with adjuvant itself has immune-promoting effect, can non-specifically resist various intracellular infections (viruses and intracellular infected bacteria, parasites, etc.) and anti-tumor effects, and can be prepared separately into various anti-cellular Internal infection and anti-tumor injections, sprays, ointments, etc. The Pika calcium combined adjuvant is a class of new drugs that do not exist at home and abroad, and the Pika calcium combined adjuvant and its injections, sprays, ointments, etc. of the present invention also belong to a class of new drugs that do not exist at home and abroad.
为了获得理想的免疫应答联合佐剂应用的例子是很多的，如完全弗氏佐剂(FCA)就是将免疫调节剂结核杆菌与W/O乳剂联合应用导致Th1和Th2应答。鲨烯和MOD联合应用成功；脂质体可以和各种免疫调节分子MPL、MDP、QuiA联合；MPL和QS21联合。CPG和铝佐剂联合应用具有协同作用，促进重组丙型肝炎抗原的体液免疫，铝佐剂作为CPG的运载系统使CPG的高效免疫效应充分发挥。There are many examples of combined adjuvant application in order to obtain an ideal immune response, such as complete Freund's adjuvant (FCA), which combines the immunomodulator Mycobacterium tuberculosis with W/O emulsion to induce Th1 and Th2 responses. The combined application of squalene and MOD is successful; liposomes can be combined with various immune regulatory molecules MPL, MDP, QuiA; MPL and QS21 can be combined. The combined application of CPG and aluminum adjuvant has a synergistic effect, promoting the humoral immunity of the recombinant hepatitis C antigen, and the aluminum adjuvant is used as the delivery system of CPG to fully exert the high-efficiency immune effect of CPG.
佐剂的配方应该是将佐剂与免疫调节剂和运载体联合使用，使免疫活性达到最优，并根据疾病的发生机理和佐剂的作用机理选择不同的免疫促进剂联合应用以增强机体所需的免疫应答。免疫促进剂包含多种，通过不同机理和渠道促进机体的免疫反应，适当的联合应用使佐剂效果更加明显。大量的临床前试验和临床试验数据表明TLR受体激动剂是强效的疫苗佐剂，Toll样受体在小白鼠中发现13种，在人体中发现11种。人Toll样受体1、2、4、5、6识别细胞外和表达在细胞表面的微生物成分，如：Toll样受体2、4识别细菌的脂蛋白和脂多糖。Toll样受体3、7、8、9识别病毒或细菌的核酸并在核内体和溶酶体。Toll样受体可以识别各种不同微生物成分，经过其重要的信号传导作用，首先引起非特异性免疫反应，继而发动特异性免疫反应，产生细胞因子和趋化因子使初始T细胞分化。Toll样受体与致病因子结合也产生炎性组织使B细胞核内因子kB(NF-kB)和干扰素调节因子活化，诱发免疫、炎性基因释放炎性趋化因子和1型干扰素(IFN1)。Toll样受体的信号传导通过依赖和非依赖髓样分化因子(MyD88)两种通路。TLR1、2、4、5、6、7、8、9是依赖髓样分化因子(MyD88)通路；TLR3、4是非依赖髓样分化因子(MyD88)通路或者TRIF(含有诱生干扰素-β接头的Toll样受体区域)通路。含有接头蛋白的TIR(Toll/白细胞介素-1受体)域，如：MyD88、TRIF、TIRAP(含有接头蛋白的Toll/白细胞介素-1受体)、TRAM(有关TRIF接头分子)的下游信号传导对诱生特异性免疫起着主导作用。The formulation of the adjuvant should be used in combination with the adjuvant, the immunomodulator and the carrier to optimize the immune activity, and select different immunopromoters for combined application according to the pathogenesis of the disease and the mechanism of action of the adjuvant to enhance the body's required immune response. immune response. There are many kinds of immune enhancers, which can promote the immune response of the body through different mechanisms and channels. Appropriate joint application can make the adjuvant effect more obvious. A large number of preclinical and clinical test data show that TLR receptor agonists are powerful vaccine adjuvants. Toll-like receptors have been found in 13 types in mice and 11 types in humans. Human Toll-like receptors 1, 2, 4, 5, and 6 recognize microbial components that are extracellular and expressed on the cell surface, such as: Toll-like receptors 2 and 4 recognize bacterial lipoproteins and lipopolysaccharides. Toll-like receptors 3, 7, 8, 9 recognize viral or bacterial nucleic acids and are present in endosomes and lysosomes. Toll-like receptors can recognize various microbial components. Through their important signal transduction, they first cause non-specific immune responses, and then initiate specific immune responses, producing cytokines and chemokines to differentiate initial T cells. The combination of Toll-like receptors and pathogenic factors also produces inflammatory tissues, activates B cell nuclear factor kB (NF-kB) and interferon regulatory factors, induces immunity, and inflammatory genes release inflammatory chemokines and type 1 interferon ( IFN1). Toll-like receptors signal through two pathways: dependent and independent of myeloid differentiation factor (MyD88). TLR1, 2, 4, 5, 6, 7, 8, 9 are dependent on myeloid differentiation factor (MyD88) pathway; TLR3, 4 are independent of myeloid differentiation factor (MyD88) pathway or TRIF (containing inducible interferon-β linker Toll-like receptor region) pathway. TIR (Toll/Interleukin-1 receptor) domain containing adapter proteins, such as: downstream of MyD88, TRIF, TIRAP (Toll/Interleukin-1 receptor containing adapter proteins), TRAM (related to TRIF adapter molecules) Signal transduction plays a leading role in the induction of specific immunity.
部分TLR及其配体Some TLRs and their ligands
对一些疫苗只使用单一靶点的佐剂或免疫促进剂并不能诱导产生有效的保护性免疫反应。除上述皮卡钙联合佐剂内容外，还可以根据疾病机理和皮卡钙联合佐剂作用机理，加入如PD-1/PD-L1抗体去除对T细胞的耗竭和对肿瘤的免疫抑制，加入如激活T细胞的CTLA4以及促进细胞转染的促进剂如PEI、PEG等等成为联合佐剂成分，更能达到目标目的。为此本发明提供的技术方案是：For some vaccines, the use of single-target adjuvants or immune boosters cannot induce effective protective immune responses. In addition to the above-mentioned content of Pika calcium combined with adjuvant, according to the disease mechanism and the mechanism of Pika calcium combined with adjuvant, it is also possible to add antibodies such as PD-1/PD-L1 to remove the depletion of T cells and immunosuppression of tumors, and add such as activation CTLA4 of T cells and promoters such as PEI, PEG, etc., which promote cell transfection, become combined adjuvant components, which can better achieve the target purpose. The technical scheme provided by the present invention is for this reason:
1.一种以含卡那霉素和钙离子的聚肌苷酸聚胞苷酸(皮卡钙佐剂)为核心联合其他免疫促进剂协同增强机体免疫刺激物质——皮卡钙联合佐剂作为在制备免疫佐剂中的应用。1. A kind of polyinosinic acid polycytidylic acid (pika calcium adjuvant) containing kanamycin and calcium ions as the core combined with other immune enhancers to synergistically enhance the body’s immune stimulating substance—pika calcium combined adjuvant as an adjuvant in the Application in the preparation of immune adjuvants.
2.根据权利1所述的一种含有皮卡钙联合佐剂的疫苗。其特征在于所述的佐剂是含有皮卡钙联合佐剂，所述的疫苗包括各种抗细胞内感染(病毒和细胞内感染的细菌、寄生虫等)疫苗和各种抗肿瘤疫苗。用于体内免疫、黏膜免疫、局部免疫。2. a kind of vaccine containing pica calcium combined adjuvant according to right 1. It is characterized in that the adjuvant is a combined adjuvant containing pica calcium, and the vaccine includes various anti-intracellular infection (viruses and intracellular infected bacteria, parasites, etc.) vaccines and various anti-tumor vaccines. For in vivo immunity, mucosal immunity, local immunity.
3.一种含有皮卡钙联合佐剂在制备用于哺乳动物或人的预防性和治疗性疫苗中的应用，包括体内免疫、黏膜免疫、局部免疫。3. The application of a combined adjuvant containing pica calcium in the preparation of preventive and therapeutic vaccines for mammals or humans, including in vivo immunity, mucosal immunity and local immunity.
4.根据权利1所述用含有皮卡钙联合佐剂的免疫刺激物体外刺激免疫干细胞，如体外刺激DC-CIK细胞、NK细胞、TCL/TCR-T/CAR-T细胞使扩增、成熟并荷载肿瘤或感染因子抗原回输体内以达到增强抗肿瘤、抗细胞内感染的免疫效果。其特征在于所述的免疫刺激物含有皮氨钙联合佐剂。4. According to right 1, stimulate immune stem cells in vitro with an immunostimulatory body containing pica calcium combined with adjuvant, such as in vitro stimulation of DC-CIK cells, NK cells, TCL/TCR-T/CAR-T cells to expand, mature and Carry tumor or infection factor antigen back into the body to achieve enhanced anti-tumor and anti-intracellular infection immune effects. It is characterized in that the immune stimulant contains calcium cortex combined with adjuvant.
5.根据权利1所述用皮卡钙联合佐剂本身作为非特异性促进机体免疫力的应用，包括制备成水针剂和各种膏剂的应用。其特征在于所述的免疫增强剂或免疫促进剂含有皮氨钙联合佐剂。5. According to claim 1, the application of calcium pica combined with the adjuvant itself as a non-specific promotion of body immunity includes the application of preparing water injections and various ointments. It is characterized in that the immune enhancer or immune enhancer contains calcium dermamine combined with an adjuvant.
6.根据权利要求1至5项，无论皮卡钙联合佐剂还是含有皮卡钙联合佐剂的疫苗通过任何途径(呼吸道、肠道、肌肉、皮下、外用等)或以任何免疫剂型(水针剂、冻干制剂、乳剂、膏剂、吸入剂、胶囊、灌肠剂等)对机体免疫或直接杀伤以达到抗肿瘤抗细胞内感染目的均为本发明范围。6. According to claims 1 to 5, no matter whether the Pika calcium combined adjuvant or the vaccine containing the Pika calcium combined adjuvant is passed through any route (respiratory tract, intestinal tract, muscle, subcutaneous, external use, etc.) or in any immune dosage form (water injection, Freeze-dried preparations, emulsions, ointments, inhalants, capsules, enemas, etc.) are within the scope of the present invention to immunize or directly kill the body to achieve the purpose of anti-tumor and anti-intracellular infection.
下面结合实例阐述本发明必要性，即作用机理和较单独使用皮氨钙佐剂及其疫苗的进一步的免疫效果。本发明并非仅限定这些举例的皮卡钙联合佐剂及其疫苗而在帮助理解。The necessity of the present invention, that is, the mechanism of action and the further immune effect of using the calcium dermamine adjuvant and its vaccine alone are illustrated below in conjunction with examples. The present invention is not limited to these exemplified pica-calcium combined adjuvants and vaccines thereof but to help understanding.
一.皮卡钙佐剂与TLR9配体CPG联合应用：皮卡钙佐剂属于双链RNA如PIC佐剂，是TLR3受体的配体。CPG是CPG寡核苷酸，是TLR9的配体，根据核苷酸序列和长度分为A、B、C类，A和C类活化B细胞(溶酶体部分)及浆细胞样DC，产生IFN-α。B类活化B细胞(内体部分)并使DC成熟。与皮卡钙佐剂联合应用就具有进一步提高免疫的效果。用合成蛋白MUC作为肿瘤疫苗联合应用CPG2395和PIC，较单独使用更能有效地增强机体抗肿瘤免疫应答，对CD4+T细胞和CD8+T细胞都较单独使用高，对肺癌小白鼠的预防和转移灶的减少联合应用也比单独使用好。1. Combined application of PICA calcium adjuvant and TLR9 ligand CPG: PICA calcium adjuvant belongs to double-stranded RNA such as PIC adjuvant, and is a ligand of TLR3 receptor. CPG is a CPG oligonucleotide, which is the ligand of TLR9. It is divided into A, B, and C types according to the nucleotide sequence and length. A and C types activate B cells (lysosomes) and plasma cell-like DCs to produce IFN-alpha. Class B activates B cells (endosomal fraction) and matures DCs. Combined application with Pika calcium adjuvant has the effect of further improving immunity. Using the synthetic protein MUC as a tumor vaccine in combination with CPG2395 and PIC can enhance the body’s anti-tumor immune response more effectively than using alone, and the effect on CD4+ T cells and CD8+ T cells is higher than that of using alone. Metastases reduction was also better in combination than in either alone.
在体外，用CPG2395和PIC单独或联合刺激脾细胞，进行细胞因子分泌情况以及细胞增殖能力的检测；在体内CPG2395和PIC单独或者联合作为MUCI多肽抗原的佐剂免疫小鼠，通过ELISA方法检测免疫血清中的抗体滴度、CTL杀伤实验检测杀伤性T细胞的细胞毒作用、流式细胞技术分析免疫前后各细胞亚群的变化；在肿瘤预防实验中，通过抑瘤率以及肺转移结节数等指标评价二者作为佐剂对MUCI免疫小鼠肿瘤预防效果的影响。In vitro, splenocytes were stimulated with CPG2395 and PIC alone or in combination, and the secretion of cytokines and cell proliferation were detected; in vivo, CPG2395 and PIC were used alone or in combination as an adjuvant for MUCI polypeptide antigens to immunize mice, and the immune response was detected by ELISA. Antibody titer in serum, CTL killing assay to detect the cytotoxicity of killer T cells, flow cytometry to analyze the changes of cell subpopulations before and after immunization; Evaluate the effects of the two as adjuvants on tumor prevention in MUCI-immunized mice.
结果表明体外用CPG2395、PIC以及二者联合与正常小鼠的脾细胞混合培养24小时后收集细胞培养上清，检测到CPG2395与PIC联合与单独应用相比能更好地诱导IL-12、TNF-a以及IFN-Y的产生。对免疫后小鼠脾细胞进行流式细胞术分型，联合组小鼠CD4+T细胞及CD8+细胞数量均较单用组升高。CPG2395与PIC联合作为佐剂与MUCI联合免疫C57BL/6小鼠后接种Lewis肺癌后，对肺癌的预防效果优于CPG2395或PolyIC单独作为佐剂的平行组。而且肺转移灶的数量也明显较少。具体结果是不同佐剂分别与MUCI疫苗复合后接种荷瘤小白鼠后33天处死，剥离瘤体称重，解剖取肺脏，观察并记录肺部转移灶的数量与大小。统计并计算各组肿瘤的平均重量。结果表明对照组中平均瘤重达到6.14g士1.749，CPG2395佐剂组平均瘤重为3.79g士2.789，P＝0.282。PI：C佐剂组平均瘤重为3.19g士2.199，P＝0.141，联合组肿瘤最小，平均瘤重仅为2.5g士2.479，P＝0.016。表明CPG2395、PIC与MUCI联合免疫的佐剂效果优于单用组，能够更好的激活机体的免疫应答，更好地抑制肿瘤的发生发展。The results showed that the combination of CPG2395, PIC and splenocytes from normal mice were cultured in vitro for 24 hours and then the cell culture supernatant was collected. It was detected that the combination of CPG2395 and PIC could induce IL-12 and TNF better than single application. -a and the production of IFN-Y. The splenocytes of the immunized mice were classified by flow cytometry, and the numbers of CD4+ T cells and CD8+ cells in the combination group were higher than those in the single use group. CPG2395 combined with PIC as an adjuvant and MUCI combined with immunization of C57BL/6 mice and then inoculated with Lewis lung cancer had better preventive effect on lung cancer than CPG2395 or PolyIC alone as an adjuvant parallel group. And the number of lung metastases was also significantly lower. The specific result was that different adjuvants were compounded with MUCI vaccine respectively and the tumor-bearing mice were inoculated 33 days after inoculation and sacrificed, the tumors were stripped off and weighed, the lungs were dissected, and the number and size of lung metastases were observed and recorded. The average weight of tumors in each group was counted and calculated. The results showed that the average tumor weight in the control group reached 6.14g±1.749, and the average tumor weight in the CPG2395 adjuvant group was 3.79g±2.789, P=0.282. PI: The average tumor weight in the C adjuvant group was 3.19g±2.199, P=0.141, and the tumor in the combination group was the smallest, with an average tumor weight of only 2.5g±2.479, P=0.016. It shows that the adjuvant effect of combined immunization with CPG2395, PIC and MUCI is better than that of the single-use group, which can better activate the body's immune response and better inhibit the occurrence and development of tumors.
二.TLR3受体的配体PIC和TLR7/8受体的配体R848联合应用并结合癌胚抗原CEA526-533肽段后促进树突状细胞(DC)成熟，与单独使用PIC比较能显著增强抗癌效果：2. The combination of TLR3 receptor ligand PIC and TLR7/8 receptor ligand R848 combined with carcinoembryonic antigen CEA526-533 peptide promotes dendritic cell (DC) maturation, which can be significantly enhanced compared with PIC alone Anticancer effect:
Toll样受体(TLR)2、3、4、7、8、9的配体选择性地联合应用可以进一步诱导树突状细胞(DC)成熟，产生一系列肿瘤免疫所期望的正向效应，如上调CCR7、CD40、CD80、CD86的表达，上调Th1，，下调Th2反应，介导IFN-Y、IL-12、TNF、IL-6等的分泌，以及在一定程度上阻断Treg的负向调节作用。The selective combination of ligands of Toll-like receptors (TLR) 2, 3, 4, 7, 8, and 9 can further induce the maturation of dendritic cells (DCs) and produce a series of positive effects expected by tumor immunity. Such as up-regulating the expression of CCR7, CD40, CD80, CD86, up-regulating Th1, down-regulating Th2 response, mediating the secretion of IFN-Y, IL-12, TNF, IL-6, etc., and blocking the negative activity of Treg to a certain extent Regulatory effect.
1、DC分组及具体操作方法：1. DC grouping and specific operation methods:
A.联合TLR组DC(TLR-DC)：在培养的最后两天加入20ug/ml PIC和1.5ug/ml R848。A. Combined TLR group DC (TLR-DC): 20ug/ml PIC and 1.5ug/ml R848 were added in the last two days of culture.
B.联合TLR及荷载CEA526-533肽段组DC(TLR-DC+CEA)：在培养的最后两天加入20ug/ml PIC和1.5ug/ml R848，然后加入4oug/ml的肽段CEA526-533，置于细胞培养箱中。3-4小时后取出，洗去多余未结合的肤段，即得到联合TLR并荷载CEA的DC。B. Combining TLR and loading CEA526-533 peptide group DC (TLR-DC+CEA): add 20ug/ml PIC and 1.5ug/ml R848 in the last two days of culture, and then add 4oug/ml peptide CEA526-533 , placed in a cell culture incubator. Take it out after 3-4 hours, wash off the excess unbound peptide, and obtain the DC that combines TLR and loads CEA.
C.常规培养的荷载CEA526-533肽段组DC(常规DC+CEA)：加入4oug/ml的肽段CEA526-533，置于细胞培养箱中，3-4小时后取出，洗去多余未结合的肤段，即得到荷载CEA526-533的DC。C. Routinely cultured CEA526-533-loaded peptide group DC (conventional DC+CEA): add 4oug/ml peptide CEA526-533, put it in the cell culture incubator, take it out after 3-4 hours, and wash away the excess unbound Peptide segment, that is, to obtain the DC of the load CEA526-533.
D.常规培养DC组。D. Conventionally cultured DC group.
2、结果：2. Results:
(1).联合TLR组DC的IL-12分泌量显著高于常规组，分别为948.67士104.36pg和215.24士19.3spg，P＜0.01。(1). IL-12 secretion of DC in combined TLR group was significantly higher than that in conventional group, respectively 948.67±104.36pg and 215.24±19.3spg, P<0.01.
(2).与联合TLR组DC共培养后获得的效应细胞对MC38-CEA肠癌细胞在较高效应细胞∶靶细胞(E∶T)比值时具有一定的杀伤作用，显著优于常规组(P＜0.05)。(2). The effector cells obtained after co-culture with DCs in the combined TLR group had a certain killing effect on MC38-CEA intestinal cancer cells at a higher ratio of effector cells: target cells (E: T), which was significantly better than that of the conventional group ( P<0.05).
(3).对于MC38细胞与联合TLR组以及常规组DC共培养的效应细胞均无明显杀伤效应，两组无明显差异(P＞0.05)。(3). There was no obvious killing effect on the effector cells co-cultured with MC38 cells, combined TLR group and conventional group DC, and there was no significant difference between the two groups (P>0.05).
(4).MC38-CEA肠癌细胞建立的小鼠肿瘤模型中，联合TLR激活并且荷载CEA肽段的DC疫苗组的小鼠肿瘤平均体积明显小于其余各组(P＜0.01)，并且存活期明显优于其余各组(P＜0.01)。(4). In the mouse tumor model established by MC38-CEA intestinal cancer cells, the average tumor volume of mice in the DC vaccine group combined with TLR activation and loaded with CEA peptides was significantly smaller than that of the other groups (P<0.01), and the survival period Significantly better than the rest of the groups (P <0.01).
(5)MC38肠癌细胞建立的小鼠肿瘤模型中，各组肿瘤平均体积无明显差异(P＞0.05)，且各组小鼠存活期无明显差异(P＞0.05)。(5) In the mouse tumor model established by MC38 intestinal cancer cells, there was no significant difference in the average tumor volume of each group (P>0.05), and there was no significant difference in the survival period of the mice in each group (P>0.05).
3、结论3. Conclusion
(1).联合TLR组的DC疫苗较常规DC疫苗能分泌更多的IL一12。(1). DC vaccine combined with TLR can secrete more IL-12 than conventional DC vaccine.
(2).联合TLR组的DC疫苗诱导的抗原特异性CTLs能有效地产生抗小鼠MC38-CEA肠癌细胞的特异性免疫反应。(2). Antigen-specific CTLs induced by DC vaccine combined with TLR group can effectively produce specific immune response against mouse MC38-CEA intestinal cancer cells.
(3).16.3.21联合TLR组且荷载CEA的DC疫苗对结肠癌荷瘤小鼠具有显著的抑瘤效应并可以延长小鼠的生存期。(3).16.3.21 The DC vaccine combined with TLR group and loaded with CEA has a significant tumor inhibitory effect on colon cancer tumor-bearing mice and can prolong the survival period of mice.
三.双链RNA佐剂联合应用转染制剂作用：3. Effect of double-stranded RNA adjuvant in combination with transfection preparation:
1、显著抑制肿瘤细胞生长：双链RNA佐剂细胞外直接作用肺癌细胞株A549，对肺癌细胞抑制率低于30％，加入转染试剂PEI，在合适搭配比例下，通过MTT 570nm测定OD值，表明双链RNA佐剂细胞内作用明显抑制A549的生长P＜0.05。1. Significantly inhibit the growth of tumor cells: the double-stranded RNA adjuvant directly acts on the lung cancer cell line A549 extracellularly, and the inhibition rate on lung cancer cells is less than 30%. The transfection reagent PEI is added, and the OD value is measured by MTT 570nm at an appropriate ratio. , indicating that the double-stranded RNA adjuvant significantly inhibited the growth of A549 P<0.05.
2、显著促进肺癌细胞凋亡：采用Annexin V/FITC-PI双染方法显示双链RNA佐剂与转染试剂PEI在合适搭配比例下，作用24小时A549细胞出现明显凋亡，较对照组(PBS组)、单独双链RNA组、单独转染试剂PEI组均有显著差异P＜0.05。2. Significantly promote the apoptosis of lung cancer cells: the Annexin V/FITC-PI double staining method shows that the double-stranded RNA adjuvant and the transfection reagent PEI are in an appropriate ratio, and A549 cells undergo obvious apoptosis after 24 hours of treatment, compared with the control group ( PBS group), single double-stranded RNA group, and single transfection reagent PEI group had significant differences P<0.05.
3、上调细胞受体TLR3、RIG-1、MDA-5和IFN-β的表达：双链RNA是TLR3、RIG-1、MDA-5的配体。用双链RNA佐剂与转染试剂PEI在合适搭配比例下作用于A549细胞3、6小时后使用荧光定量PCR检测TLR3、RIG-1、MDA-5和IFN-βmRNA表达水平，结果显示双链RNA佐剂与转染试剂PEI组明显上调TLR3、RIG-1、MDA-5和IFN-βmRNA的表达，在6小时时，与其他对照组(PBS组)、单独双链RNA组、单独转染试剂PEI组均有显著差异P＜0.01。3. Up-regulate the expression of cell receptors TLR3, RIG-1, MDA-5 and IFN-β: double-stranded RNA is the ligand of TLR3, RIG-1 and MDA-5. The expression levels of TLR3, RIG-1, MDA-5 and IFN-βmRNA were detected by fluorescence quantitative PCR after the double-stranded RNA adjuvant and transfection reagent PEI were applied to A549 cells at an appropriate ratio for 3 and 6 hours, and the results showed that the double-stranded RNA The expression of TLR3, RIG-1, MDA-5 and IFN-β mRNA was significantly up-regulated in the RNA adjuvant and transfection reagent PEI group, and at 6 hours, compared with other control group (PBS group), single double-stranded RNA group, single transfection Reagent PEI group had significant difference P<0.01.
以上所述仅为本发明的相关实施例，这些例子并非因此限制本发明的专利范围，只是利用本发明举例说明和明确了皮卡钙联合佐剂和含有皮卡钙联合佐剂疫苗较单纯皮卡钙佐剂和含有皮卡钙佐剂疫苗有更加突出的促进免疫效果。凡是利用本发明举例说明皮卡钙联合佐剂配方、检定内容和皮卡钙联合佐剂疫苗配方、检定内容所作的数据或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。The above are only related examples of the present invention, and these examples are not therefore limiting the scope of the patent of the present invention, but use the present invention to exemplify and clarify that picocalcium combined adjuvant and vaccines containing picocalcium combined adjuvant are more effective than simple picocalcium adjuvant vaccines. Vaccines and vaccines containing Pika calcium adjuvants have more prominent immune-promoting effects. The same is true for any data or equivalent process transformation made by using the present invention to illustrate the formula and test content of Pika calcium combined adjuvant vaccine formula and test content, or directly or indirectly used in other related technical fields. included in the scope of patent protection of the present invention.
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