鸡白细胞介素2(IL-2)对鸡免疫功能的影响

为研究鸡IL-2对雏鸡免疫功能的影响，进行了3个方面的试验。①对体液免疫及细胞免疫的影响。结果表明，鸡IL-2能明显提高鸡新城疫疫苗免疫后HI水平、T淋巴细胞增殖反应及ANAE^＋细胞百分数；②对雏鸡腹腔巨噬细胞吞噬功能的影响。结果表明使用IL-2与否对巨噬细胞的吞噬功能没有明显的影响；⑧人工感染法氏囊病的防治试验。在攻毒前后注射IL-2，均可明显降低发病率和死亡率。本研究结果表明，鸡IL-2可明显提高特异性免疫应答能力，与疫苗共同注射可作为佐剂提高疫苗的免疫原性，对法氏囊等病毒性疾病具有一定的防治作用。     

稳定表达CD163蛋白的猪肺泡巨噬细胞系的建立

以RT-PCR方法从原代猪肺泡巨噬细胞中获得CD163 cDNA序列,然后测序鉴定,对突变碱基进行修正,将修正后碱基序列克隆入真核表达载体pcDNA4.0中,构建重组真核表达质粒pcDNA4.0-CD163,通过BamHⅠ/NotⅠ双酶切及测序鉴定其正确性。使用脂质体2000将pcDNA4.0-CD163转染猪肺泡巨噬细胞系3D4/21(CRL-2843),通过Zeocin抗性筛选,建立稳定表达CD163的猪肺泡巨噬细胞系。RT-PCR、间接免疫荧光(IFA)检测结果表明CD163在mRNA和蛋白质水平均已表达。通过Zeocin抗性筛选,共获得8株表达CD163的重组细胞株,IFA检测结果表明表达的CD163定位于重组细胞的细胞膜表面。去除筛选抗性后连续传代10代,间接免疫荧光检测结果确定重组细胞株在10代内性状稳定,说明稳定表达CD163的猪肺泡巨噬细胞系构建成功。     

Bisphosphonates significantly increase the activity of doxorubicin or vincristine against canine malignant histiocytosis cells

Canine malignant histiocytosis (MH) is an aggressive neoplasm of macrophages and dendritic cells. It carries a poor prognosis because of the development of widespread metastasis and poor sensitivity to chemotherapy. Thus, there is a large need for new treatments for MH. We hypothesized that bisphosphonates might be useful to increase the effectiveness of cytotoxic chemotherapy against MH. To address this question, we conducted in vitro screening studies using MH cell lines and a panel of 6 chemotherapy and 5 bisphosphonate drugs. The combination of clodronate with vincristine was found to elicit synergistic killing which was associated with a significant increase in cell cycle arrest. Second, zoledronate combined with doxorubicin also significantly increased cell killing. Zoledronate significantly increased the uptake of doxorubicin by MH cells. On the basis of these findings, we conclude that certain bisphosphonate drugs may increase the overall effectiveness of chemotherapy for MH in dogs.     

亚洲棉、雷蒙德棉和陆地棉NRAMP基因家族的鉴定和进化分析

自然抗性相关巨噬细胞蛋白(NRAMP)家族是跨膜转运蛋白家族,参与多种金属离子的转运过程。为了更好了解棉花NRAMP基因的成员和特征,二倍体和四倍体棉花之间的进化关系,本研究利用二倍体亚洲棉、雷蒙德棉和陆地棉基因组的氨基酸序列对三个棉种中的NRAMP家族成员进行鉴定,分析了NRAMP家族的种类、理化性质、进化关系、基因结构、蛋白基序、染色体定位、共线性关系和选择压力。结果显示,在二倍体亚洲棉基因组(A)中确定12个成员,在二倍体雷蒙德棉基因组(D)中确定15个成员,在四倍体陆地棉基因组(At, Dt)中确定23个成员。按照进化关系的远近将这50个成员分为3个亚类,进化关系较近的成员之间具有更为相似的理化性质、基因结构和蛋白质基序;通过和其他植物的进化关系分析发现,NRAMP基因在单子叶和双子叶植物分离前已经产生;染色体定位发现NRAMP在染色体上的分布并不规律;共线性分析和选择压力分析表明,片段复制在棉花NRAMP基因的遗传进化过程中发挥主导作用,同源基因对在进化过程中处于纯化选择。     

当归多糖对免疫系统作用的研究

当归多糖(AP)对动物机体具有明显的免疫作用。研究表明,AP通过活化巨噬细胞作用于各种免疫效应细胞,产生相应的细胞因子从而实现其免疫作用。AP对淋巴细胞也有较强的活化作用,能提高IL-2和免疫球蛋白IgG、IgM、补体C5的水平。     

安吉白茶多糖抗肿瘤及免疫调节研究

选用MTT法研究了安吉白茶多糖的体外抗肿瘤活性;采用建立小鼠移植瘤模型,通过测定安吉白茶多糖对荷瘤小鼠抑瘤率和腹腔巨噬细胞吞噬能力,研究其体内抗肿瘤活性和免疫调节作用。结果表明,安吉白茶多糖体外能显著抑制S180细胞的生长,且具有剂量依赖关系。体内试验表明,与模型对照组相比,安吉白茶多糖对小鼠S180实体瘤和H22腹水型肝癌移植瘤的生长均有显著抑制作用(P<0.05),对荷瘤小鼠脾指数和胸腺指数也有较强增高作用。体内免疫调节试验结果显示,安吉白茶多糖可使荷S180实体瘤小鼠腹腔巨噬细胞吞噬能力显著升高(P<0.05)。     

In infectious pancreatic necrosis virus carrier Atlantic salmon, Salmo salar L., post-smolts, almost all kidney macrophages ex vivo contain a low level of non-replicating virus

The level of infection by infectious pancreatic necrosis virus (IPNV) of kidney macrophages from 12 asymptomatic carrier Atlantic salmon post-smolts was studied. Kidney leucocytes were fractionated on 34/51% Percoll gradients, allowed to adhere to plastic wells overnight, washed to remove non-adherent cells and cultured for up to 7 days with or without renewal of medium on day 3. On day 1, supernatants were harvested, macrophages were counted, lysed and IPNV in the supernatants and lysates was titred in chinook salmon embryo (CHSE-214) cells. The multiplicity of infection ranged between 1:2.2 and 1:7.4 (virus:macrophages). On day 3, the titres of IPNV in macrophage lysates decreased and in wells where the medium was renewed on day 3, IPNV was no longer detectable on day 7. In the supernatants, one fish was positive for IPNV on day 1, four fish on day 3 but none were detectably positive on day 7. In parallel wells in which the medium was not renewed, on day 7 IPNV was detected in macrophage lysates of three fish and the supernatants were also IPNV positive in two of these fish. This suggests that virus might be shed from infected macrophages and then reinfect other macrophages. When macrophages were serially diluted in wells and cultured for 24 h, IPNV could be cultured from macrophage lysates of wells containing between two and 70 macrophages. These results indicate that a very high proportion of the adherent kidney macrophages must be infected with very few non-replicating virions.     

秦川牛天然免疫力相关巨噬细胞蛋白1基因(Nramp1)N端序列的原核表达及纯化

为了研究牛天然免疫力相关巨噬细胞蛋白1(Nramp1)的功能,探求转基因抗胞内感染菌牛新材料创制的功能基因,本实验应用RT-PCR方法从秦川牛(Bos taurus)外周血中扩增出Nramp1编码基因N端序列,并将其克隆到pMD18-T simple载体,酶切后连接于pGEX-4T-1表达载体,命名为pGEX-N2。将重组质粒pGEX-N2转化大肠杆菌(Escherichia coli)BL21(DE3),经IPTG诱导,SDS-PAGE电泳检测GST-Nramp1-N融合蛋白表达,并筛选最佳诱导条件,用谷胱甘肽Sepharose4B介质分离纯化该融合蛋白。结果表明,牛Nramp1N端编码序列长186bp,与GenBank中的相应的牛基因序列(U12862.1)对比存在一个碱基差异,致使其编码的第49位氨基酸为丙氨酸。构建的牛Nramp1N端表达重组质粒pGEX-N2,于35℃、0.1mmol/LIPTG、诱导10h,在大肠杆菌中高效表达,表达产物的分子量为33kD。结果为进一步研究牛Nramp1在机体抵抗胞内菌感染中的作用并为利用该基因进行转基因抗病牛的培育提供了实验依据和实验材料。     

The TLR Expression Pattern on Monocyte-Derived Macrophages for Lipopolysaccharid Stimulation of Calves

In this paper, toll-like receptor expression pattern in monocytes-derived macrophages by lipopolysaccharid （LPS） stimulation was examined. Jugular venous blood samples from 4 Japanese calves were obtained and the peripheral blood mononuclear cells （PBMC） were isolated. The PBMC were cultured for 7 d so as to collect monocytes-derived macrophages in Repcell. The PBMC were stimulated by LPS for 24 h and the mRNA expression pattern of TLR and cytokines in monocytes-derived macrophages （Mod-Mφ） was analyzed. Results showed that LPS stimulation of Mod-Mφ could increase the mRNA levels of the genes of TNF-α, IL-6, and IL-8. In addition, the mRNA levels of the genes of TNF-α and IL-6 in the group of LPS stimulation were most significantly （P 〈 0.01） higher than those in control group and the mRNA levels of TLR1, 3, 5, 8, and 10 were significantly （P 〈 0.05） decreased after LPS stimulation. There was no difference in the mRNA expressions of TLR2, 4, 6, and 7 between the groups of the control and LPS stimulation. Besides, expression of TLR9 was not found. It suggested that monocytes-derived macrophages could respond to LPS and they might take an important role in the innate immunity. The important function of the cells might contribute to better disease treatment.     

具核梭杆菌体外对小鼠巨噬细胞超微形态和机能的影响

本研究对具核梭杆菌(F.nucleatum)感染鼠巨噬细胞进行共同培养之后,分别在10个时段采取上清液和巨噬细胞,用ELISA、qRT-PCR法和扫描电镜检测TNF-α、 IL-1α、 IL-6和IL-8细胞因子,并观察巨噬细胞的超微形态变化。结果显示,4种细胞因子均有高水平表达;IL-8在共培养6～72 h呈高水平表达;IL-6和TNF-α表达水平也较高;IL-1从3 h表达,12 h达最高值,48 h后无表达。qRT-PCR检测结果显示,TNF-α的RNA表达呈波浪式,分别于6,60 h各出现1个高峰值;IL-1α和IL-6呈现正态表达,从3 h开始明显表达至60 h达峰值,此后缓慢下降;IL-8从6 h逐渐上升,至72 h仍呈高水平表达。超微形态观察显示,在F.nucleatum感染的前24 h,巨噬细胞呈收缩状,其表面和周围有大量发育良好F.nucleatum,并呈团块状黏附,巨噬细胞逐渐伸出伪足。30 h后F.nucleatum逐渐发育不良,小而细,而巨噬细胞表面粗糙,有许多大小不一的结节,伪足变粗大。48 h巨噬细胞表面出现胞吐孔,少量细小的F.nucleatum仍然黏附于...