【作者】 陈艳；

【导师】 龚卫娟；

【作者基本信息】 扬州大学 ， 免疫学， 2015， 硕士

【摘要】 DsNKG2D-IL-15为2个NKG2D分子的胞外区和IL-15的融合蛋白,基于NKG2D分子识别高表达于大部分肿瘤细胞表面的MICA等配体分子,并通过IL-15发挥激活NK和CD8+T细胞的功能,前期研究已证实,该融合蛋白具有明显的抗肿瘤效应。壳聚糖纳米材料是一种新兴的药物载体,在基因疫苗以及各类抗肿瘤药物的运输过程中扮演越来越重要的角色。壳聚糖具有生物相容性高,毒性较低,且具有生物可降解性等优势,可作为载体来制备抗肿瘤纳米颗粒。本课题拟将前期构建的pcDNA3.1-dsNKG2D-IL-15重组质粒、以及自行制备的dsNKG2D-IL-15融合蛋白分别装载入HTCC(2-羟丙基三甲基氯化铵)壳聚糖纳米颗粒中,形成一定大小的纳米凝胶颗粒,体内外分别评估其激活淋巴细胞的效应以及抗肿瘤的效果。研究内容分为2个部分：1.装载pcDNA3.1-dsNKG2D-IL-15重组基因的纳米颗粒的制备及其抗肿瘤活性目的：利用化学修饰过的壳聚糖(HTCC)以及前期工作中构建好的重组基因(pcDNA3.1-dsNKG2D-IL-15)来制备抗肿瘤纳米颗粒。检测该纳米颗粒的理化性质,并观察其能否将重组基因转导入细胞,且在细胞内表达NKG2D和IL-15的融合蛋白。体外观察肿瘤细胞受该纳米基因颗粒转染后,对NK与CD8+T细胞活性的影响,并且体内观察该纳米基因颗粒拮抗B16BL6-MICA黑色素移植瘤体内生长的影响。方法：首先将HTCC与重组质粒分别按照质量比1：2,2：1,1：1混合,震荡装载后,离心取上清,用分光光度计检测其中DNA浓度,按照(DNA总量—游离DNA)/DNA总量的公式来计算包封率。取制备好的纳米颗粒,用适量的超纯水或PBS缓冲液稀释后,检测纳米颗粒的粒径大小及其表面电荷。其次使用MTS/PMS试剂盒检测纳米颗粒对RAW264.7以及B16BL6等细胞的毒性大小。然后,将脂质体以及单体蛋白作为对照,使用纳米颗粒转染CT-26等细胞,72h后通过FCM检测NKG2D的表达；使用检测IL-15的ELISA试剂盒,分析受纳米基因颗粒转染后,肿瘤细胞培养上清中IL-15的浓度。并且将转染后的细胞与小鼠脾脏细胞共孵育,次日检测其NK细胞和CD8+T细胞频率的改变,以及其活化的情况。最后,给C57BL6小鼠背部皮下接种黑色瘤细胞B16BL6-MICA,成瘤5日后,分别注射PBS,壳聚糖,肌肉注射,瘤内注射,持续三周,每天记录荷瘤小鼠肿瘤生长情况,以及小鼠的生存状况。处死小鼠后,通过FCM检测并分析小鼠脾脏NK细胞和CD8+T细胞的频率以及CD69、NKG2D和CD44的表达。结果：该纳米材料能够装载入pcDNA3.1-dsNKG2D-IL-15重组基因,并且包封率可达到92.8+0.37%。该纳米基因颗粒在较高浓度时,对肿瘤细胞有一定的毒性。该纳米颗粒具备转运重组基因dsNKG2D-IL-15至细胞的能力,且FCM以及ELISA检测证实,肿瘤细胞转染该纳米颗粒后,胞浆内NKG2D与IL-15均表达。该纳米颗粒转染细胞后,可促进NK细胞表达CD69,并上调CD8+T细胞表面NKG2D和CD44的表达。另外,荷瘤小鼠经该纳米基因颗粒处理后,明显抑制移植瘤的生长、且延长小鼠的生存期。结论：装载pcDNA3.1-dsNKG2D-IL-15重组基因的纳米颗粒可被细胞吞噬并表达dsNKG2D-IL-15融合蛋白。该纳米基因颗粒体内使用后,可通过激活NK和CD8-T细胞的功能而发挥抗肿瘤活性。2.装载dsNKG2D-IL-15重组蛋白的纳米颗粒制备及其抗肿瘤活性目的：通过HTCC修饰的壳聚糖以及前期制备的dsNKG2D-IL-15融合蛋白,制备装载待重组蛋白的纳米颗粒。体外检测该纳米颗粒的理化性质和细胞毒性,体内注射荷瘤小鼠,观察该纳米颗粒的抗肿瘤效果。方法：利用配制好的HTCC壳聚糖,以及课题组前期制备的dsNKG2D-IL-15重组蛋白,制备负载融合蛋白的抗肿瘤纳米颗粒。取装载混合物上清,使用分光光度计检测游离蛋白浓度,按照下列公式计算包封率：(总蛋白量-游离蛋白量)／总蛋白量×100%。用动态光散射仪检测纳米颗粒的粒径,以及纳米颗粒表面的Zeta电位。使用MTS/PMS试剂盒检测该纳米颗粒对细胞的毒性作用,并通过透析和蛋白凝胶电泳检测纳米颗粒中蛋白的释放。最后,给C57BL/6小鼠背部皮下接种B16BL6-MICA黑色素瘤细胞,成瘤5天后,分别给每组小鼠注射生理盐水(PBS),壳聚糖(HTCC)溶液,制备好的纳米颗粒以及单体重组蛋白。持续3周,每天记录小鼠生存状况,以及肿瘤生长的情况。处死小鼠后,分析荷瘤小鼠脾脏NK和CD8+T细胞频率的改变和活化状态。结果：HTCC壳聚糖溶液可包裹溶液中的dsNKG2D-IL-15重组蛋白,且质量比为1：1时最高,达到95.3%。粒径大小在200 nm左右,表面带5.3 mV正电荷。该纳米凝胶对B16BL6等肿瘤细胞有一定毒性,而对小鼠巨噬细胞RAW264.7毒性相对较小。在体外释放实验中,能够在24小时有效释放重组蛋白。在体内实验中,该纳米颗粒治疗组与PBS以及壳聚糖对照组相比,在C56BL/6小鼠体内能够有效抑制B16BL6-MICA黑色素移植瘤的生长,并延长荷瘤小鼠的生存期。荷瘤小鼠脾脏中NK以及CD8+T细胞的频率增多,其表面NKG2D、CD44的表达上调。结论：装载dsNKG2D-IL-15重组蛋白的纳米颗粒体外有效释放重组蛋白的能力,体内使用具有明显抑制肿瘤的活性,该活性与促进NK以及CD8+T细胞的活化有关。更多还原

【Abstract】 DsNKG2D-IL-15 fusion protein composed by two of NKG2D extracellular domains and a IL-15 domain. Previous research has confirmed that NKG2D highly expresses on the surface of most tumor cells which can recognize MICA and other ligands, and meanwhile IL-15 has the function of stimulating NK and CD8+T cells, the fusion protein has dramatic anti-tumor effects. The nanometer material of Chitosan is the emerging advantage drug carrier which plays a more and more important role in the transfer process of gene vaccine and a variety of antitumor drugs. With the advantage of high biocompatibility, low toxicity and biodegradability, chitosan is the good choice for us to use as the material to prepare nanoparticles. In our study, the recombinant plasmid of pcDNA3.1-dsNKG2D-IL-15 and the fusion protein of dsNKG2D-IL-15 both had been already prepared. Besides HTCC(2-Hydroxy-N,N,N-trimethyl-l-propanaminium chloride) chitosan is the material we use to load the agencies. And then we aimed to evaluate the anti-tumor effection of the nanoparticles through experiments in vitro and in vivo.Our study can be divided into two parts.Part I. Preparation of the nanoparticle loading with recombinant plasmid pcDNA3.1-dsNKG2D-IL-15 and its antitumor activity.Objective:To make the antineoplastic nanoparticle with the chemically modified HTCC chitosan and the recombinant gene (pcDNA3.1-dsNKG2D-IL-15) prepared in our early work. To test part of the physical and chemical properties of the nanoparticle and observe whether the recombinant gene can be transferred into the cells and express NKG2D, IL-15. To find out its influence on lymphocyte especially NK and CD8+T cells and the influence on B16BL6-MICA melanoma in the C57BL6 mice.Methods:Firstly, mix HTCC with recombinant plasmid in the mass ratio of 1:2,2:1,1:1. After shaking and loading, centrifuge the mixture and get the supernatant. Detecting the concentration of DNA in the supernatant with spectrophotometry and calculating the encapsulation efficiency by the following formula (total DNA-free DNA)/total DNA* 100%. Take the prepared nanoparticles and test its diameter and electric potential after dilution. Then test its toxicity to RAW264.7 and B16BL6 cells with MTS/PMS kits. Compared with lipidosome and monomeric protein, transfer the nanoparticles into the CT-26 cells, test the express of NKG2D by FCM after 72 hours. In addition, analyse the concentration of IL-15 in the supernatant of the transferred cells. Use the transferred cells co-incubation with the spleen cells from mice, analyse the frequency and activity of NK and CD8+T cells morrow. Finally, the in vivo experiment was performed by subcutaneous injection of B16BL6-MICA transplantation tumor into C57BL/6 mice. Inject each 4 group with PBS, HTCC, nanoparticle intramuscularly and intraperitoneally respectively. Record the subsistence of tumor-bearing mice and the growth of tumor everyday for three weeks. Eventually, execute the mice and analyse the change in frequency and activation of NK and CD8+T cells in the spleen of tumor-bearing mice. Detect the express of CD69, NKG2D, CD44 by FCM.Results:The HTCC material can load with the pcDNA3.1-dsNKG2D-IL-15 recombinant gene and the loading efficiency can reach 92.8±0.37%. It has certain cytotoxicity to tumor cells in a high concentration. Besides, the nanoparticle can transfer the recombinant gene to CT-26 and RAW 264.7 cells. The expressed NKG2D and IL-15 can be detected by FCM and ELISA respectively. The frequency of the NK and CD8+T cells in the spleen of tumor-bearing mice also increased. And so does the NKG2D, CD44 enhanced. The life of tumor-bearing mice treated by the nanoparticle is extended, and the growth of transplanted tumor can be inhibited.Conclusions:The nanoparticle loading with pcDNA3.1-dsNKG2D-IL-15 recombinant gene can be endocytosed by tumor cells. In vivo nanoparticle can stimulate NK and CD8+T cells function andPart Ⅱ. Preparation of the nanoparticle loading with dsNKG2D-IL-15 fusion protein and its antitumor activity.Objective:To prepare the nanoparticle loading with dsNKG2D-IL-15 fusion protein and test part of its physicochemical property and toxicity. Observe its anti-tumor effect in vivo and the influence on tumor-bearing mice. To observe the influence about frequency and phenotype of lymphocyte such as NK, CD8+T cells.Methods:Mix HTCC solution and dsNKG2D-IL-15 fusion protein to prepare nanopartcile. Test the concentration of free protein in the supernatant of mixture with spectrophotometry and calculate the encapsulation efficiency by the following formula:(total protein-free protein)/total protein* 100%. Besides, the diameter of the nanoparticle is measured by the dynamic light scattering and the electric potential variation on the surface of nanometer materials can be observed by zeta potential meter. Test the toxicity of our nanoparticle with MTS/PMS kit. Then, the dialysis release experiment is used to test whether the nanoparticle could release the fusion proein, the result is checked by protein gel electrophoresis. After that, inject B16BL6-MICA transplantation tumor into C57BL/6 mice subcutaneously. Inject each 4 group with PBS, HTCC chitosan, nanoparticle and fusion protein respectively. Record the subsistence of tumor-bearing mice and the growth of tumor everyday for three weeks. Eventually, execute the mice and analyse the change in frequency and activation of NK and CD8+T cells in the spleen of tumor-bearing mice. Detect the express of CD69, NKG2D, CD44 by FCM.Results:The HTCC chitosan can load the recombinant protein in the solution. When the mass ratio is 1:1, it reaches its utmost load efficiency:95.7 ± 0.36%. The nanogel carrying dsNKG2D-IL-15 is poisonous to some tumor cells such as B16BL6 while less poisonous to mice macrophage RAW 264.7. The nanometer material of Chitosan releases recombinant preoteins effectively in 24 hours in vitro release test. In vivo test, the group treated by nanogel is in contrast to the group with PBS and chitosan which can inhibit the growth of B16BL6- MICA melanoma in the C56BL/6 mice and prolong the life of tumor-bearing mice to a certain extent. Our team finds the increase in the frequency of the NK and CD8+T cells in the spleen of tumor-bearing mice, and phenotypes such as NKG2D, CD44 enhanced.Conclusions:The nanoparticle loading with dsNKG2D-IL-15 fusion protein has the ability to release protein in vitro. Moreover in vivo with the treatment of nanoparticle the transplantation tumor can be inhibition. That mainly because with the expression of fusion gene, the NK and CD8+T cells are activated effectively.更多还原