壳聚糖纳米颗粒包裹猪白细胞介素2和融合白细胞介素4/6基因对猪圆环病毒2型疫苗免疫的强化

目的探讨猪Sus scrofa domesticus白细胞介素2(IL-2)和融合白细胞介素4/6(IL-4/6)基因的共表达对仔猪免疫应答的影响,为进一步研制新型免疫调节剂来加强仔猪抵御断奶后多系统衰竭综合征奠定基础。方法将猪IL-2和IL-4/6融合基因的共表达重组质粒,用壳聚糖材料包裹制成纳米颗粒,记作VRIL-4/6-2-CS。将仔猪分为2组,分别肌肉注射VRIL-4/6-2-CS(实验组)和生理盐水(对照组),2组均同时接种猪圆环病毒2型(PCV-2)疫苗,在接种后的第0、7、14、28天采集血样并分析免疫变化。结果实验组仔猪血清中的IgG2a抗体数量和血液中CD4~+、CD8+~T淋巴细胞数量均显著高于对照组(P0.05);同时,实验组仔猪的IL-2、IL-4、IL-6、TNF-α、TLRs(TLR-2,7)、STATs(STAT-1,2,3)基因表达水平也显著高于对照组(P0.05)。尽管2组之间PCV-2特异性抗体的含量差异无统计学意义,但是实验组仔猪的生长速率显著高于对照组(P0.05)。结论 VRIL-4/6-2-CS能促进仔猪对PCV-2疫苗的免疫应答,是一种安全有效的免疫佐剂。     

猪白细胞介素4,6融合基因和CpG基序对猪蓝耳病疫苗免疫的强化效应

为探索新型高效安全的猪蓝耳病疫苗免疫佐剂,本实验用离子交联法制备壳聚糖纳米颗粒包裹猪融合基因PIL-46和CpG基序的真核表达质粒(CNP-(VRIL-46 VR1C)),将CNP-(VRIL-46 VR1C)肌注接种过猪蓝耳病灭活苗的长白川白杂交猪,接种后1、2、4、6和8周采取实验猪静脉血,用Sandwich ELISA检测血清中白细胞介素2(IL-2)、IL-4、IL-6和IgG、IgA、IgM及特异抗体的含量,同时检测外周血液免疫细胞数量的变化。结果发现实验猪接种CNP-(VRIL-46 VR1C)后1～8周内,其血清中IL-2、IL-4、IL-6和IgG、IgA、IgM及特异抗体的含量较对照组均显著增多(P<0.05),淋巴细胞和单核细胞数量也明显升高(P<0.05)。这些表明接种CNP-(VRIL-46 VR1C)的实验猪的特异体液和细胞免疫水平均显著多于对照组,提示PIL-46基因和CpG基序组合能显著增强猪的特异体液和细胞免疫机能,明显提高其免疫防御力,具有研制为高效安全的分子免疫增强剂的应用前景。     

猪白细胞介素4、6融合基因对小鼠免疫的影响

目的：研究猪白细胞介素IL4、IL6融合基因（PIL46）对小鼠免疫应答的反应。方法：以壳聚糖纳米颗粒包裹融合基因（PIL46）的真核表达质粒（VPIL46）接种昆明小鼠,免疫后28日以口服攻毒实验小鼠,观察其体液和细胞免疫水平指标的变化和病变情况。实验结果发现：CNP包裹VPIL46接种小鼠体液免疫和细胞免疫指标不同程度地增多,均显著高于对照组（P〈0．05）;与CNP包裹VPIL4＋6免疫效果相近。免疫后28日以口服攻毒实验小鼠,检测结果发现：CNP包裹VPIL46组和CNP包裹VPIL4＋6组小鼠的上述免疫指标除中性粒细胞外均显著多于对照小鼠,免疫小鼠无症状和病变,健康存活;而对照小鼠均发病,消化道组织器官呈现明显出血病变。结论：PIL46基在具有显著增强小鼠体液和细胞免疫机能、提高对大肠杆菌感染抵抗力的免疫调节效应,可作为有效的抗感染免疫调节剂。     

改组猪IL-2和重组IL-6C基因增强猪口蹄疫疫苗的免疫应答

以离子交联法制备壳聚糖纳米颗粒,包裹重组改组的猪IL-2基因表达质粒(VRIL2S)、重组IL-6和CpG(VPIL6C)质粒(CNP-VRIL2S-VPIL6C-CNP), 按0.5 mg/头肌肉注射壳聚糖包装质粒接种21日龄健康三元杂交猪,同时肌肉注射免疫灭活口蹄疫疫苗;在免疫后7、14、28、42和56 d,采血检测实验猪的免疫球蛋白、特异抗体和免疫细胞的动态变化.结果发现:接种后56 d,改组猪IL-2和重组IL-6C质粒接种猪血液中的特异性抗口蹄疫抗体、IgG、IgA和IgM含量均较对照空白疫苗免疫猪显著升高(P<0.05),其IL-2、IL-6水平和淋巴细胞、单核细胞的数量也相应较灭活疫苗对照组明显增加(P<0.05).这些证明猪IL-2和IL-6基因与CpG有良好的免疫协同增强效应,能有效地增强疫苗的特异体液和细胞免疫应答反应,可作为安全高效的新型免疫基因分子佐剂,促进猪对口蹄疫疫苗的免疫防御抗病力,抵御口蹄疫的感染.     

白细胞介素6重组逆转录病毒载体的构建及鉴定

目的 构建特异性过表达大鼠IL-6基因的重组逆转录病毒载体,并在大鼠嗜铬细胞瘤PC12细胞和人胚肾HEK293细胞中检测IL-6的表达。方法以大鼠骨髓间充质干细胞mRNA为模板,经PCR获得目的基因IL-6,将其定向克隆到逆转录病毒载体pSEB-3H中,构建重组逆转录病毒质粒pSEB—IL-6,经脂质体分别转染到PC12细胞和HEK293细胞中,应用Real—timePCR和ELISA的方法在mRNA和蛋白质水平检测IL-6的表达变化。继而用HEK293细胞中包装获得的含有pSEB—IL-6的病毒颗粒进一步感染PC12细胞,Real—timePCR检测,IL-6mRNA的表达水平变化。结果PCR电泳及酶切鉴定证实目的基因正确克隆至逆转录病毒载体中,其基因序列与Genbank报道一致;Real—timePCR和ELISA结果均显示,逆转录病毒质粒pSEB—IL-6转染PC12细胞和HEK293细胞后,IL-6的表达水平较对照组显著上调;经pSEB—IL-6逆转录病毒颗粒感染的PC12细胞中,IL-6mRNA表达水平较对照组提高4倍。结论成功构建了特异性表达大鼠儿-6基因的重组逆转录病毒载体pSEB—IL-6,并获得了具有感染能力的逆转录病毒颗粒,感染真核细胞后可高表达IL-6,为进一步研究IL-6的功能及其在多种疾病中的免疫调节机制提供重要的分子手段。     

共表达猪瘟病毒E2基因和猪白细胞介素2基因的重组腺病毒的构建及其免疫原性研究

为研制猪瘟活载体疫苗,构建共表达猪瘟病毒E2基因和猪白细胞介素2基因的重组腺病毒并研究其免疫原性。应用PCR方法从pMD19-T-E2质粒和pMD19-T-pIL2质粒中分别扩增E2基因和pIL-2基因,将扩增的全长E2基因和pIL-2的编码区基因序列通过柔性接头序列(5个甘氨酸密码子)串联,插入腺病毒穿梭载体AdTrack中,在受体菌中与骨架载体AdEasy同源重组。重组质粒AdEasy-E2-pIL-2转染HEK293细胞,包装出重组腺病毒rAd-E2-pIL-2。用rAd-E2-pIL-2免疫家兔,经兔体交互免疫试验评定其免疫效果。结果显示,经RT-PCR和West-ern blot检测,成功构建了rAd-E2-pIL-2,重组病毒滴度达108.12PFU/mL。rAd-E2-pIL-2接种家兔后刺激接种兔产生猪瘟病毒特异性抗体,淋巴细胞转化试验结果显示猪瘟病毒诱导兔淋巴细胞特异性增殖,攻毒后rAd-E2-pIL-2接种兔和猪瘟病毒C株接种兔均未出现定型热反应。研究结果表明,rAd-E2-pIL-2免疫兔可以预防猪瘟病毒C株接种引发的体温反应,rAd-E2-pIL-2可望成为猪瘟候选疫苗。     

小鼠白细胞介素IL-35基因慢病毒表达载体的构建及鉴定

[目的]构建小鼠白细胞介素IL-35基因慢病毒表达载体并鉴定。[方法]通过PCR法扩增出小鼠IL-35基因片段,将其与经XhoⅠ和XbaⅠ双酶切的慢病毒载体pLVX-IRES-ZsGreen1连接,构建慢病毒重组质粒pLVX-IL-35-IRES-ZsGreen1。用无内毒素试剂盒提取重组质粒后,将其与病毒包装所需的3个辅助质粒共转染到人肾胚(HEK)293T细胞中包装能表达IL-35的慢病毒颗粒。该病毒培养上清经浓缩后,梯度稀释法检测其滴度。用浓缩后的病毒感染HEK293T细胞,然后通过荧光显微镜观察荧光蛋白的表达并结合RT-PCR法检测IL-35基因在该细胞中的表达。[结果]IL-35慢病毒表达载体构建成功,包装的慢病毒滴度为1×109TU/m L,通过荧光显微镜和RTPCR检测发现IL-35在HEK293T细胞中表达。[结论]成功构建IL-35慢病毒表达载体且IL-35蛋白能在HEK293T细胞中过表达。     

融合蛋白pIL6-IL2在E.coli中的表达及活性鉴定

白细胞介素 2 (interleukin 2 ,IL-2 )与白细胞介素 6(interleukin 6,IL-6)能分别刺激T淋巴细胞增殖与B淋巴细胞分泌免疫球蛋白 ,从而促进动物机体的细胞免疫与体液免疫 ;另外IL2与IL6在发挥生物学活性时还有相互协同作用。因此 ,将去除信号肽的猪白细胞介素 6(pIL-6)与猪白细胞介素 2 (pIL-2 )cDNAs序列通过一段Linker相连 ,克隆到E .coli表达载体pPET-2 8a中。该融合蛋白IL6-IL2在E .coli表达菌BL2 1 (DE3)中获得成功表达 ,SDS-PAGE分析分子量约为40kDa ,表达量达到总菌体的 66.26%。用IL6依赖的B9细胞与IL2依赖的CTLL细胞增殖试验进行融合蛋白IL6 IL2的生物活性检测 ,其活性可分别达到 0.8× 10³U /mg和 6.4× 10³ U/mg。     

真核表达猪白细胞介素17及其生物活性研究

目的:为真核表达猪白细胞介素17(IL-17),研究产物在细胞培养下的免疫生物活性。方法:通过PCR扩增出猪IL-17基因并插入到真核表达载体p VAX1,然后转染到IPEC-J2细胞、Ha Ca T细胞和L02细胞中。在转染后第24、48和72h收集细胞,第48h收集上清液。收集细胞通过实时荧光定量PCR检测相关免疫基因的表达水平,收集上清液通过抑菌试验检测相关抗菌肽的生物活性。结果:采用p VAX1载体构建了表达猪IL-17的重组质粒,转染到细胞中。证实IL-17基因能诱导抗菌肽基因(RegⅢ、S100A8和BD2)的表达,显著上调JAK-STAT信号通路基因(JAK1、STAT1和STAT3)和细胞因子基因(IL-6、IL-12和TNF-α)的表达。此外,细胞上清液能够在不同程度上抑制大肠杆菌及金黄色葡萄球菌的增殖。结论:成功将猪IL-17基因真核表达,其表达产物能诱导效应细胞表达多种细胞因子,产生多种抗菌肽,具有抑菌能力;这为进一步研发猪IL-17作为抗菌免疫分子制剂奠定了初步基础。     

新型纳米转染试剂转染PNP自杀基因体外杀伤实验

将壳聚糖纳米粒包裹的报告基因pEGFP-N1质粒转染至HEK293细胞,并在HEK293细胞中成功表达荧光蛋白的基础上,进一步将本室自行构建的PNP基因的真核高效表达载体质粒pcDNA3-PNP转染至HEK293细胞。转染72h后,对转染的HEK293细胞给予前体药6-MPDR至终浓度40μg/ml,一天后,采用MTT比色法测定药物对细胞增值的影响,并进行统计学处理。实验结果表明采用壳聚糖纳米粒转染试剂转染并给予前体药6-MPDR的实验组活细胞数,与用壳聚糖转染但不给前体药6-MPDR的对照组活细胞数相比,有显著差异(P<0.05),说明新筛选出的壳聚糖纳米粒转染试剂可以将PNP自杀基因递送至靶细胞中,并在细胞中进行表达,从而使PNP/6-MPDR自杀基因系统发挥杀伤细胞的作用。分别采用相同工作浓度的脂质体与壳聚糖纳米粒转染试剂转染相同浓度的基因质粒,壳聚糖纳米粒对靶细胞生长数量影响很小,说明的壳聚糖纳米粒细胞毒性大大低于阳离子脂质体的细胞毒性。     

Interleukin-6

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.     

Interleukin-3

Interleukin-3 (IL-3) is a hemopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hemopoietic cells. In five mammalian species, including man, the gene encoding IL-3 has been isolated and expressed to yield the mature recombinant proteins. The human IL-3 gene encodes a protein of 133 amino acids with two conserved cysteine residues and 2 potential N-linked glycosylation sites; human native IL-3 has not been characterized. Comparison of the IL-3 genes revealed a more rapid evolutionary divergence than has been observed for other hemopoietic growth factors, and, hence, a more pronounced species specificity of the functional proteins was found. In agreement with its stimulatory action on immature multipotent cells, thein vivo actions of homologous recombinant IL-3 in nonhuman primates include a highly increased production of blood cells along the neutrophilic, eosinophilic and basophilic granulocyte as well as the monocyte, red cell and platelet lineages.     

Enhancing Interleukin-6 and Interleukin-11 receptor cleavage

Proteolytic cleavage of the membrane-bound Interleukin-6 receptor (IL-6R) by the metalloprotease ADAM17 releases an agonistic soluble form of the IL-6R (sIL-6R), which is responsible for the pro-inflammatory trans-signaling branch of the cytokine's activities. This proteolytic step, which is also called ectodomain shedding, is critically regulated by the cleavage site within the IL-6R stalk, because mutations or small deletions within this region are known to render the IL-6R irresponsive towards proteolysis. In the present study, we employed cleavage site profiling data of ADAM17 to generate an IL-6R with increased cleavage susceptibility. Using site-directed mutagenesis, we showed that the non-prime sites P3 and P2 and the prime site P1′ were critical for this increase in proteolysis, whereas other positions within the cleavage site were of minor importance. Insertion of this optimized cleavage site into the stalk of the Interleukin-11 receptor (IL-11R) was not sufficient to enable ADAM17-mediated proteolysis, but transfer of different parts of the IL-6R stalk enabled shedding by ADAM17. These findings shed light on the cleavage site specificities of ADAM17 using a native substrate and reveal further differences in the proteolysis of IL-6R and IL-11R.     

Interleukin-2

Between October 1987 and October 1989 we have treated 110 patients with advanced solid tumors with recombinant interleukin-2 (rIL2) based immunotherapy. In renal cell cancer we have studied rIL2 alone, rIL2 combined with rIL2 activated lymphocytes (LAK), and in an ongoing study rIL2 and LAK and alpha-interferon (alpha IFN). There is suggestive evidence of increasingly good results in these consecutive studies. In melanoma the combination of rIL2 and chemotherapy was investigated, followed by an ongoing study of rIL2 and alpha IFN. In these studies rIL2 has been administered as a continuous intravenous infusion of 18 x 10(6) International Units/m2/day for 5 days (18 x 10(6) IU = 3 x 10(6) Cetus Units = 6.9 Biological Response Modifiers Program (BRMP) Units). Patients with non-small cell lung cancer are entered in a phase I-II study of rIL2 and alpha IFN. The rIL2 administration differs from the above mentioned schedule in that rIL2 is given at a maximum dose of 6 x 10(6) IU/m2/day for 28 days on an outpatient basis. In a phase I study we have searched for the maximum tolerated dose of a daily time 4 schedule of rIL2. In the second part of this study a daily time 4 schedule, every week for 4 weeks is being investigated. Finally, we are investigating the safety and efficacy of local regional administration of rIL2 in patients with head and neck cancer, mesothelioma, and liver metastasis of colorectal cancer.     

Interleukin-1

Interleukin-I (IL-1) is the prototypic pro-inflammatory cytokine. There are two forms of IL-1, IL-1 and IL-Iβ and in most studies, their biological activities are indistinguishable. IL-1 affects nearly every cell type, often in concert with another pro-inflammatory cytokine, tumor necrosis factor (TNF). Although IL-1 can upregulate host defenses and function as an immunoadjuvant, IL-1 is a highly inflammatory cytokine. The margin between clinical benefit and unacceptable toxicity in humans is exceedingly narrow. In contrast, agents that reduce the production and/or activity of IL-1 are likely to have an impact on clinical medicine. The synthesis, processing, secretion and activity of IL-1, particularly IL-Iβ, are tightly regulated events. A unique aspect of cytokine biology is the naturally occurring IL-1 receptor antagonist (IL-1Ra). IL-IRa is structurally similar to IL-Iβ but lacking agonist activity is used in clinical trials to reduce disease severity. In addition, regulation of IL-1 activity extends to low numbers of surface receptors, circulating soluble receptors and a cell surface “decoy” receptor to down-regulate responses to IL-Iβ. This review updates the current knowledge on IL-1.     

白介素22

近两年 ,发现了多种白介素 10家族新成员 :黑素瘤分化相关基因 7产物 (ｍｅｌａｎｏｍａｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ａｓｓｏｃｉａｔｅｄｇｅｎｅ 7,ＭＤＡ 7)、单纯疱疹病毒转化的Ｔ淋巴细胞产生的ＡＫＫ 15 5蛋白、Ｚｃｙｔｏ 10、巨细胞病毒编码ＩＬ 10 (ｃｍｖＩＬ 10 )、白介素 19(ＩＬ 19)、ＩＬ 2 0和ＩＬ 2 2。本文重点简述ＩＬ 2 2及其受体的研究进展。ＩＬ 2 2也称为ＩＬ 10相关Ｔ细胞衍生的可诱导因子 (ＩＬ 10 ｒｅｌａｔｅｄＴｃｅｌｌ ｄｅｒｉｖｅｄｉｎｄｕｃｉｂｅｆａｃｔｏｒ,ＩＬ ＴＩＦ)。比利时Ｌｕｄｗｉｇ癌…     

IL-18、IL-10及其比值与冠状动脉粥样硬化斑块易损性研究进展

急性冠脉综合征主要是由于具有易损性的冠状动脉粥样硬化斑块发生破裂或蚀损,继发血栓形成,并引起具有严重危害的急性冠状动脉事件.炎症反应是影响冠状动脉粥样硬化斑块易损性的主要因素,参与反应的炎症因子是近年来研究的热点.有研究发现白细胞介素-18(IL-18)作为一个促炎症因子,会增加斑块的易损性,而白细胞介素-10(IL-10)作为主要的抗炎症因子,则具有抗动脉硬化及稳定粥样斑块的作用.IL-18/IL-10的比值代表了机体促炎性与抗炎性动态平衡的状态,其比值失衡可能是影响斑块易损性的重要因素.近来有研究认为,IL-18/IL-10的比值可作为急性冠脉综合征患者近期冠脉事件的预测因子.文章就近几年来有关IL-10、IL-18及其比值与冠状动脉粥样硬化斑块易损性的研究进展作一综述,以探讨其可能的临床意义.     

Interleukin-25 Mediated Induction of Angiogenin-4 Is Interleukin-13 Dependent

The intestinal surface is directly exposed to both commensal microorganisms as well as pathogens with a single layer of epithelium separating luminal microorganisms from internal tissues. Antimicrobial peptides play a crucial role in allowing epithelial cells to contain in the lumen beneficial and pathogenic microorganisms. The commensal dependent, epithelial produced, Th2 cytokine IL-25 can induce IL-13 and potentially the antimicrobial peptide angiogenin-4. Here we show that IL-13 downstream of IL-25 is required to induce angiogenin-4. IL-25 mediated induction of angiogenin-4 is furthermore not dependent on IL-22 or IL-17.     

Interleukin-2 self-association

The self-association of human recombinant interleukin-2 (IL-2) from E. coli was explored. Self-association, with an apparent Kd of 0.6 micromolar, has pronounced effects on (1) the surface exposure of Trp-121, deduced from quenching studies employing potassium iodide and acrylamide, (2) the apparent quantum yield of Trp-121, the fluorescence of Trp-121 in IL-2 aggregates is 4-fold lower than in IL-2 "monomers", and (3) IL-2-mediated phospholipid vesicle fusion/aggregation.