【作者】 陆叶；

【导师】 贡成良； 沈卫德；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2010， 博士

【摘要】 IL-28A是Sheppard等在2003年报道的一组新型白细胞介素之一,即干扰素λ2（IFN-λ2）。这种干扰素属于新型的干扰素IFN-λs,也被称为Ⅲ型干扰素。它和Ⅰ型干扰素的功能类似,具有抗病毒、抗增殖及免疫调节等生物活性。国内外对其抗病毒活性的研究较多,常用的表达系统为大肠杆菌和哺乳动物的细胞等。对于用家蚕生物反应器来表达人的IL-28A及其表达产物的体内、体外抗肿瘤活性研究,未见相关报道。用家蚕生物反应器表达外源蛋白是高效生产生物制品的一种有效途径,家蚕生物反应器具有表达水平高、表达产物稳定、后加工较完善、安全性好、易产业化生产和形成我国的自主产业等优点,是蛋白质类药物口服化研究最有效的系统之一。为了进一步研究人的IL-28A（hIL-28A）在体内及体外的抗增殖活性,探讨口服重组hIL-28A的抗肿瘤效果,我们通过非转座子载体介导以及Bac-To-Bac系统分别在BmN细胞和家蚕蛹中成功表达了hIL-28A,并研究了口服杆状病毒表达的重组hIL-28A对裸鼠抗肿瘤能力的影响,主要从以下几个方面进行了研究。1、hIL-28A在BmN细胞中的表达及其抗增殖活性构建了重组表达载体pIZT/V5-His-hIL-28A并转染BmN细胞。使用终浓度为300-400μg/mL Zeocin（博来霉素）经过两个月的筛选获得了稳定转化的BmN细胞。SDS-PAGE和Western blotting检测结果显示,转化细胞表达的重组hIL-28A约为26 kDa;ELISA检测结果显示,hIL-28A的表达水平约为20.35 ng / 106个细胞。用MTT法测定hIL-28A的抗增殖活性,稳定转化细胞表达的hIL-28A具有抗增殖活性,hIL-28A对A549（肺癌细胞）,HL60细胞（急性早幼粒细胞白血病细胞）,BEL-7402（肝癌细胞）和M231（乳腺癌细胞）的50%的抑制浓度（IC50）分别为3.21,2.84,6.29和9.32 ng /ml。2、用Bac-to-Bac表达系统在BmN细胞及蛹中表达hIL-28A将hIL-28A基因克隆进pFastBac^TMDual载体中的杆状病毒多角体启动子下游,利用Bac-to-Bac系统构建重组病毒Bacmid-EGFP-hIL-28A;并在BmN细胞及家蚕蛹中表达hIL-28A。用ELISA试剂盒检测重组病毒感染24-144 h后BmN培养细胞及蚕蛹中hIL-28A蛋白的表达情况,结果显示BmN细胞、蚕蛹分别在重组病毒感染96、120h时hIL-28A表达水平最高,分别为11.4μg/10⁶细胞、33.2μg/ml蛹血淋巴,感染重组病毒120h的冻干蛹粉中hIL-28A的表达水平为8.49mg/g。同时,收集感染96h的BmN细胞和120 h的蚕蛹的血淋巴进行SDS-PAGE和Western blotting分析,结果显示BmN细胞和蚕蛹表达的重组hIL-28A的分子量约为26KD。3、口服杆状病毒表达的重组hIL-28A对裸鼠抗肿瘤能力的影响通过免疫组化的方法研究含有hIL-28A的蚕蛹冻干粉作为药物组对裸鼠A549和HL60移植瘤的抑制作用。与不含有hIL-28A的蚕蛹冻干粉作用的对照组相比,药物组移植瘤的大小及瘤重明显下降（P<0.05）,裸鼠的脾重和体重的比值明显高于对照组（P<0.01）。肿瘤细胞中PCNA和VEGF的阳性表达水平也明显下调（P<0.05）。观察肿瘤组织的病理变化,药物组的肿瘤组织细胞坏死程度明显增加,TUNEL（原位细胞凋亡）检测表明,药物组的肿瘤组织中细胞凋亡数量明显增加（P<0.05）。表明含hIL-28A蚕蛹冻干粉可以通过口服抑制裸鼠移植瘤的生长速度和大小。并提示其抗肿瘤作用可能和下调A549、HL60移植瘤中PCNA和VEGF的阳性表达水平,诱导肿瘤细胞凋亡以及提高裸鼠免疫力等有关。研究结果为hIL-28口服药物的研发奠定了基础,为家蚕制药提供了新的途径。更多还原

【Abstract】 Sheppard has reported interlukin-28A (IL-28) in 2003, as one of novel interleukins, as known as interferonλ2 (IFN-λ2), which belongs to a new type of IFN-λs, also known as typeⅢinterferon. It contains the similar functions with typeⅠinterferon, including anti-virus, anti-proliferation immune regulation and other biological activities. The antiviral activity has been investigated both at home and abroad, using E. coli and mammalian cell expression systems usually. There are no relevant reports over the IL-28A expression based on Silkworm bioreactor and its impact on antiproliferation.The expression of exogenous proteins in Silkworm Bioreactor is an effective way to produce highly efficient biological products, which possesses many advantages, such as high expression level, stable expression product, better post processing, better security, easy to industrial production and the formation of our country’s independent industry. Therefore, it is one of the most effective systems for the study on oral administration protein drugs. To further study anti-tumor effect of oral administration of recombinant human IL-28A (hIL-28A) in vivo and in vitro, and based on the non-transposon vector-mediated and the Bac-to-Bac system, respectively.we successfully constructed the recombinant expression vector which can express hIL-28A in BmN cells and silkworm pupae to investigate whether the oral administration of recombinant baculovirus expressing hIL-28A could improve the anti-tumor ability on nude mice. The following aspects are the detail research contents.1. The expression of hIL-28A in stable transformed BmN cell and the Anti-proliferation activity detectionThe recombinant expression vector pIZT/V5-His-hIL-28A was constructed to transfect the BmN cells, aiming to obtain the stable transformed BmN cell line which can continuously express hIL-28A gene, and investigate its anti-proliferation activity. After screening with Zeocin at a terminal concentration 300-400μg/mL for two months, we obtained the stable transformed BmN cells. The SDS-PAGE and Western blotting results illustrated that the transformed cells could express the recombinant hIL-28A protein of 26 kDa molecular weight. And ELISA result demonstrated the expression level of hIL-28A at 20.35 ng in 106 cells. Meanwhile, the anti-proliferation activity of hIL-28A was determined by MTT assay, the consequence showed that the 50% inhibitory concentrations (IC50) of the recombinant hIL-28A on A549 (Lung Cancer Cells), HL60 (Acute Promyelocytic Leukemia Cells), BEL-7402(Liver Cancer Cells) and M231 cells (Breast Cancer Cells) were approximately 3.21, 2.84, 6.29 and 9.32 ng/ml, respectively. In summary, hIL-28A can be stable expressed in the transformed BmN cell line, and the expressed recombinant hIL-28A harbors the anti-proliferation activity to tumor cells in vitro.2. The expression of hIL-28A gene in BmN cell and silkworm pupa using Bac-to-Bac systemThe hIL-28A was cloned into the downstream of polyhedron promoter of the pFastBacTMDual vector, and the recombinant virus Bacmid-EGFP-hIL-28A vector was finished using Silkworm/ Baculovirus expression vector system (Bac-to-Bac), to express the hIL-28A in BmN cells and silkworm pupae.The expression level of hIL-28A in BmN cell (harvested at 24 h to 144h post infection) was detected by ELISA. ELISA result showed that the amount of expressed hIL-28A protein in BmN cells at 96h post infected and blood lymph in silkworm pupae at 120h post infected reached highest level, 11.4μg/106 cells and 32.3μg/ml, respectively. The amount of expressed hIL-28A protein in silkworm pupae powder was 8.49mg/g. At the same time, BmN cells (96h post infection) and blood lymph of silkworm pupae (120h post infection) were detected by SDS-PAGE and western blotting. The results indicated that the molecular weight of expressed recombinant hIL-28A protein was about 26 kD.3. Effect of oral administration recombinant hIL-28A on anti-tumor ability in nude miceThe effects of hIL-28A contained silkworm pupae freeze-dried powder on the inhibitory of A549 (Lung Cancer Cells) and HL60 (Acute Promyelocytic Leukemia Cells) tumor cells in vivo were investigated by immunohistochemical methods. Compared with the control group, the size and weight of transplanted tumor in treated group have significantly decreased(p<0.05), and the ratio of spleen weight and body weight has increased significantly (p < 0.01).The expression level of PCNA(proliferating cell nuclear antigen) and VEGF(vascular endothelial growth factor) of transplanted tumor was decreased in treated group, compared with the control(p<0.05).The pathological changes were observed. In treated group, the number of cellular necrosis increased markedly compared with control group, and cell apoptosis can be observed. TUNEL (TdT-mediated dUTP-biotin nick end labeling) analysis showed that the number of apoptotic cells increased significantly in the treated group(p<0.05). These results illustrated that oral administration silkworm freeze-dried powder containing hIL-28A could inhibit tumor growth rate and size in nude mice, suggesting its anti-tumor effect may be related to reducing the level of positive expression of PCNA and VEGF in transplanted A549 and HL60 tumor cells, inducing apoptosis of tumor cells, as well as improving their own immunity in nude mice.The research results laid a foundation on the oral drug research and development of hIL-28A, and provided a new way for the pharmaceutical silkworm.更多还原