半番鸭肠道相关淋巴组织的M细胞来源

应用组织学技术对半番鸭肠道相关淋巴组织(GALT)M细胞的来源进行了研究;应用台盼蓝、辣根过氧化物酶(HRP)作为示踪物对GALT上皮细胞的功能进行了研究.结果表明:半番鸭GALT黏膜上皮最初为柱状上皮,柱状上皮逐渐脱离基底膜,基底膜部位的成纤维细胞转变为扁平上皮;鸭灌服台盼蓝后,在淋巴组织表面的扁平上皮细胞和肝脏的枯否氏细胞均出现了台盼蓝颗粒;肠腔内注入HRP后,HRP的阳性产物出现在扁平上皮和淋巴组织中;在柱状上皮内2种示踪物均未出现.可见,半番鸭GALT表面的M细胞来源于成纤维细胞.     

Release of multiple hormones by a direct action of interleukin-1 on pituitary cells

Exposure to bacterial endotoxins has long been known to stimulate the release of anterior pituitary hormones; administration of endotoxin was at one time a common clinical test of anterior pituitary function. Endotoxin is a potent stimulus for production of the endogenous pyrogenic protein, interleukin-1 (IL-1), by macrophages and monocytes. The possibility that IL-1 has a direct effect on the secretion of hormones by rat pituitary cells in a monolayer culture was investigated. Recombinant human IL-1 beta stimulated the secretion of adrenocorticotropic hormone, luteinizing hormone, growth hormone, and thyroid-stimulating hormone. Increased hormone secretion into culture supernatants was found with IL-1 concentrations ranging from 10(-9) M to 10(-12) M. Prolactin secretion by the monolayers was inhibited by similar doses. These concentrations of IL-1 are within the range reported for IL-1 in serum, suggesting that IL-1 generated peripherally by mononuclear immune cells may act directly on anterior pituitary cells to modulate hormone secretion in vivo. Incubation of IL-1 solutions with antibody to IL-1 neutralized these actions. These pituitary effects of IL-1 suggest that this monokine may be an important regulator of the metabolic adaptations to infectious stressors.     

11S通过NF-κB信号通路引起猪小肠上皮细胞损伤

通过体外培养猪小肠上皮细胞(IPEC-J2),研究大豆球蛋白(11S)对猪小肠上皮细胞的影响。将IPEC-J2细胞分为6组[A(对照组)、B、C、D、E和F],各组中分别添加0、1、5、10、5、5 mg·mL^-1的11S,并且在E组和F组分别添加1 μmol·L^-1吡咯烷二硫代氨基甲酸盐(PDTC)和1 μmol·mL^-1 Nω-硝基-L-精氨酸甲酯(L-NAME)。分别于8、16、24 h,用CCK-8法检测细胞存活率,用ELISA法检测细胞一氧化氮(NO)、二胺氧化酶(DAO)、5-羟色胺(5-HT)、白细胞介素6(IL-6)和白细胞介素10(IL-10)含量,用western blot法检测细胞p-NF-κB p65、iNOS、COX-2的蛋白表达量,用qPCR法检测细胞NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对表达量。结果表明:11S可以降低猪小肠上皮细胞存活率,添加PDTC和L-NAME可以提高小肠上皮细胞存活率;11S可促进NO、DAO、5-HT和IL-6的分泌,降低IL-10的分泌,增加p-NF-κB p65、iNOS、COX-2的蛋白表达量及NF-κB p65、IKKβ、iNOS、IKKα、COX-2 mRNA的相对表达量,添加PDTC和L-NAME可抑制11S的作用。综上,11S可引起猪小肠上皮细胞损伤,随着11S浓度的增大,损伤程度增加,PDTC和L-NAME可以降低11S对细胞的损伤。     

通草提取物对奶牛乳腺上皮细胞乳糖合成及相关基因表达的影响

为探讨通草对奶牛乳腺上皮细胞乳糖合成及相关基因表达的影响,采用不同剂量通草提取物处理奶牛乳腺上皮细胞,利用四甲基偶氮唑盐(MTT)法检测乳腺上皮细胞增殖能力,乳糖/半乳糖检测试剂盒(Lactose/D-Galactose (Rapid) Assay Kit)检测乳腺上皮细胞培养液中乳糖含量,实时荧光定量PCR技术检测乳腺上皮细胞乳糖合成相关基因葡萄糖转运蛋白1(GLUT1)、葡萄糖转运蛋白4(GLUT4)、葡萄糖转运蛋白8(GLUT8)、葡萄糖转运蛋白12(GLUT12)、己糖激酶Ⅰ(HKⅠ)、己糖激酶Ⅱ(HKⅡ)、β-1,4-半乳糖基转移酶-1(β-4GALT1)和α-乳清白蛋白(α-LA)基因mRNA表达水平。结果表明,200、400、600μg(生药)·mL-1通草提取物提高奶牛乳腺上皮细胞增殖能力(P<0.05),促进奶牛乳腺上皮细胞合成分泌乳糖(P<0.05),并显著上调细胞GLUT1、GLUT8、HKⅡ、β-4GALT1及α-LA mRNA表达水平(P<0.05),但对GLUT4、GLUT12和HKⅠmRNA表达水平无显著影响(P>0.05)。研究表明,适当浓度通草提取物可提高奶牛乳腺上皮细胞增殖能力,并通过上调乳腺上皮细胞GLUT1、GLUT8、HKⅡ、β-4GALT1和α-LA的mRNA表达,促进乳腺上皮细胞合成乳糖。     

猪伪狂犬病毒体外诱导RAW264.7细胞炎症反应模型的建立

【目的】探讨猪伪狂犬病毒（Pseudorabies virus,PRV）感染对小鼠单核巨噬细胞（RAW264.7）炎症反应的影响,确定PRV的最佳感染剂量和感染时间,为建立RAW264.7细胞体外病毒感染炎症反应模型打下基础。【方法】PRV按10倍递增稀释成10-5~10-1 PRV稀释液,感染RAW264.7细胞并孵育1.5 h,弃病毒液后加入含5%胎牛血清的DMEM维持培养液继续培养,分别于继续培养2、4、8、12、24和48 h时收集细胞上清液,采用ELISA测定IL-6、IL-10、IL-1β、TNF-α、MCP-1和IFN-γ分泌水平及环氧合酶（COX-1和COX-2）活性,并以CCK-8法测定细胞活性。【结果】以PRV感染RAW264.7细胞4~48 h后均能通过PCR扩增获得PRV核酸的特异性条带,故选择4~48 h作为后续研究的PRV感染时间范围;10-2 PRV~10-1 PRV感染可显著降低RAW264.7细胞活性（P<0.05,下同）,10-3 PRV组仅在培养48 h时出现下降趋势,而10-5 PRV~10-4 PRV感染对RAW264.7细胞活性无显著影响。PRV感染RAW264.7细胞后,其胞内炎症因子IL-6、IFN-γ、TNF-α、IL-1β和MCP-1的分泌水平整体上呈升高趋势,其中10-3 PRV感染RAW264.7细胞12 h能显著或极显著（P<0.01）提高IL-6、IFN-γ、TNF-α、IL-1β和MCP-1的分泌水平;10-4 PRV~10-1 PRV感染组的IL-10分泌水平均呈升高趋势,而10-5 PRV感染组在感染8和24 h时IL-10分泌水平明显低于空白对照组,至感染48 h所有病毒感染组的IL-10分泌水平均降低;10-3 PRV~10-1 PRV感染8~24 h能有效提高RAW264.7细胞的COX-2活性,但对COX-1活性的影响不明显。【结论】PRV感染能诱导RAW264.7细胞发生炎症反应,其中10-3 PRV体外感染RAW264.7细胞8~12 h是建立RAW264.7细胞炎症反应模型的最佳条件。该模型可应用于PRV感染与RAW264.7细胞炎症反应相关干预药物的研究,为进一步揭示PRV感染机理及开发抗病毒感染药物提供理论依据。     

鼻咽癌患者与不同IgA/VCA抗体滴度者外周血单个核细胞对EB病毒感染的早期免疫反应

本文用本室建立的EB病毒(EBV)体外感染外周血单个核细胞(PBMC)早期反应综合检测观察系统,测定了鼻咽癌(NPC)患者、IgA/VCA高滴度者、低滴度和阴性者3组人的PBMC体外感染EBV的早期免疫反应。发现在EBV感染PBMC早期,NPC组的EBNA阳性细胞数、~3H-TdR掺入量和免疫球蛋白(IgG、IgG、IgG)分泌量最低,IgA/VCA低滴度和阴性组最高,两组间所有指标差异均有显著或非常显著性,而IgA/VCA高滴度组的多数指标与NPC组接近.结果表明,在EBV体外感染PBMC所引起的早期免疫反应中,NPC患体者和IgA/VCA高滴度者EBV活化的B细胞功能较IgA/VCA低滴度和阴性者降低.这对了解NPC发生发展过程中EBV感染活化B细胞的功能在体内外变化的关系方面具有一定意义.     

壳寡糖对大熊猫轮状病毒VP6-VP7亚单位疫苗免疫作用的研究

为研究壳寡糖(COS)对大熊猫轮状病毒(GPRV)重组蛋白VP6-VP7的免疫增强作用,以原核表达的重组VP6-VP7蛋白为抗原,添加浓度为2.0 mg·mL^-1 COS作为免疫佐剂制成GPRV VP6-VP7-COS亚单位疫苗。选取SPF级昆明小鼠78只随机分为4组。PC组免疫纯化的重组VP6-VP7蛋白,A组免疫VP6-VP7-COS疫苗,B组免疫VP6-VP7氢氧化铝胶佐剂疫苗,NC组注射PBS(对照组)。在0、15、30 d进行免疫接种,每次接种完成后于每周每组随机挑选6只小鼠采血分离血清,分别检测小鼠血清IgG、IgA抗体效价、T淋巴细胞转化率、细胞因子的含量,评估COS对GPRV重组VP6-VP7蛋白诱导的免疫应答反应的调节作用。试验结束后,取各组小鼠注射部位的肌肉组织进行病理组织学分析,以评价COS作为免疫佐剂的安全性。结果表明,VP6-VP7-氢氧化铝胶佐剂组、VP6-VP7-COS组免疫小鼠血清中所产生的IgG均显著(P<0.05)高于其他各组。VP6-VP7-COS组小鼠产生的IgA抗体含量显著(P<0.05)高于其他各组,VP6-VP7-氢氧化铝胶佐剂组和VP6-VP7-COS物理混合组诱导T淋巴细胞增殖的能力显著(P<0.05)高于VP6-VP7蛋白组。VP6-VP7-COS组、VP6-VP7-氢氧化铝胶佐剂组中IL-2、IL-4、IL-5及IFN-γ的含量均显著(P<0.05)高于其他组,组织病理学分析结果表明,以COS作为佐剂的VP6-VP7亚单位疫苗免疫小鼠后注射部位的肌肉组织未出现病理变化。本研究表明,GPRV重组蛋白VP6-VP7能够显著诱导动物机体的免疫应答,壳寡糖对GPRV VP6-VP7蛋白呈现较好的免疫增强效果。本研究为研制安全、无副作用和高免疫保护力的GPRV 亚单位疫苗提供了参考。     

Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA

Both interleukin-1 (IL-1) and platelet-derived growth factor (PDGF) induce proliferation of cultured fibroblasts and smooth muscle cells. These polypeptide mediators are released by activated macrophages and other cell types in response to injury and are thought to have a role in tissue remodeling and a number of pathologic processes. Analysis of the kinetics of [3H]thymidine incorporation by cultured fibroblasts demonstrated that the response to IL-1 is delayed approximately 8 hours relative to their response to PDGF. IL-1 transiently stimulated expression of the PDGF A-chain gene, with maximum induction after approximately 2 hours. Subsequent synthesis and release of PDGF activity into the medium was detected as early as 4 hours after IL-1 stimulation, and downregulation of the binding site for the PDGF-AA isoform of PDGF followed PDGF-AA secretion. Antibodies to PDGF completely block the mitogenic response to IL-1. Therefore, the mitogenic activity of IL-1 for fibroblasts and smooth muscle cells appears to be indirect and mediated by induction of the PDGF A-chain gene.     

重组捻转血矛线虫半乳糖结合凝集素免疫山羊皱胃细胞因子表达定位

【目的】检测重组雌、雄捻转血矛线虫半乳糖结合凝集素(rHco-GAL-f/m)免疫山羊皱胃转录细胞因子。【方法】用rHco-GAL-f/m免疫山羊,通过原位杂交法对阴性对照组(A)、攻虫对照组(B)、免疫攻虫组(C)和免疫不攻虫组(D)山羊皱胃中白介素-1β(IL-1β)、白介素-2(IL-2)、白介素-4(IL-4)、白介素-6(IL-6)、干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)mRNA转录进行定位,并对上述细胞因子阳性反应细胞数量进行分析。【结果】A组山羊皱胃组织中没有检测到细胞因子,其它3组仅在皱胃黏膜下层中检测到了上述细胞因子。B、C和D组间IL-4和IL-6阳性反应细胞数差异显著(P0.05),其中C组明显高于其它两组;B、C和D组间IL-1?、IL-2和TNF-α阳性反应细胞数差异显著(P0.05),其中D组明显高于其它两组;IFN-γ阳性反应细胞数在B组和C组之间差异不显著(P0.05),D组与B组和C组之间差异显著(P0.05),且明显高于其它两组。【结论】重组捻转血矛线虫半乳糖结合凝集素能够诱导山羊皱胃局部免疫应答。     

Rheumatoid factor secretion from human Leu-1+ B cells

A human B cell subpopulation identifiable by the expression of the cell surface antigen Leu-1 (CD5) is responsible for most of the immunoglobulin M rheumatoid factor secreted in vitro after the cells are stimulated with Staphylococcus aureus. The ability of B cells bearing the Leu-1 marker (Leu-1+) to secrete rheumatoid factor is present early in development and extends to adulthood, since Leu-1+ B cells from cord blood and from peripheral blood lymphocytes of both normal adults and patients with certain autoimmune conditions secrete rheumatoid factor in comparable amounts. The neonatal enrichment of Leu-1+ B cells, the presence of Leu-1+ B cells in increased frequencies in patients with autoimmune disease, and the involvement of Leu-1+ B cells in autoantibody secretion suggest both developmental and functional homologies between this human B cell subpopulation and the murine Ly-1 B cell subpopulation.     

维生素C对β-伴大豆球蛋白诱导的仔猪肠上皮细胞炎性损伤的保护作用

为分析不同浓度维生素C(vitamin C,V_C)对β-伴大豆球蛋白(7S)诱导的仔猪肠上皮细胞(IPEC-J2)炎性损伤的保护作用,取对数生长期的IPEC-J2用于试验,随机分为对照组、7S模型组(5 mg·mL^-1 7S)和V_C(25、50、100、200、400、600、800、1 000 μmol·L^-1)保护组。细胞培养24 h后采用CCK-8法检测细胞存活率,倒置显微镜观察细胞形态学变化,ELISA法检测细胞上清液中LDH、ALP、DAO、IFABP1含量和IL-1β、IL-6、TNF-α、IL-4、IL-10的分泌水平,用qRT-PCR法检测IL-1β、IL-6、TNF-α、IL-4和IL-10 mRNA相对表达量。结果显示:7S可显著(P<0.01)降低IPEC-J2活力、破坏细胞膜完整性,上调细胞促炎性因子和下调抗炎因子的产生;与7S模型组相比,同时添加7S和V_C的试验组细胞活力增加,细胞上清液中LDH、ALP、DAO、IFABP1含量显著(P<0.01)降低,促炎因子IL-1β、IL-6、TNF-α分泌水平降低,抗炎因子IL-4和IL-10的分泌水平升高。因此,不同浓度V_C均可保护由7S诱导引起的仔猪肠上皮细胞损伤,100 μmol·L^-1 V_C的修复和保护作用最佳。     

密蒙花滴眼液对去势雄兔干眼泪腺组织中ICAM-1、IL-6、IL-17表达的影响

目的 观察不同浓度密蒙花滴眼液对雄兔干眼泪腺组织中炎性细胞因子：细胞间黏附因子-1（intercellular cell adhesion molecule-1,ICAM-1）、白细胞介素-6（interleukin-6,IL-6）、白细胞介素-17（interleukin-17,IL-17）表达的影响,从而探讨其疗效。方法 将36只成年新西兰雄兔随机分为空白组（A）,模型组（B）,密蒙花滴眼液低（C）、中（D）、高（E）浓度组,睾酮组（F）组,每组6只。除A组外,其余各组均切除双侧睾丸及附睾,造模成功后,C、D、E组开始双眼滴用滴眼液,F组注射丙酸睾酮注射液。术前及治疗后第4周各组雄兔均测定1次泪膜破裂时间（tear break-up time,BUT）和泪液分泌功能（Schirmer Ⅰ test,SIT）。治疗后第4周处死实验用兔,并摘取其双侧泪腺,采用免疫组化的方法检测ICAM-1、IL-6与IL-17的表达。结果 （1） SIT和BUT：B组自身前后对比,治疗后第4周：B组与C、D、E、F组相比,E组与C、D组相比,F组与C、D、E组相比,差异均有统计学意义（P<0.05或P<0.01）;（2） ICAM-1、IL-6与IL-17电镜下表达的结果：A、F组未见明显表达;B组可以见到大量棕黄色颗粒存在细胞浆与细胞膜中;C、D、E组可见散在的表达;（3）治疗后第4周,组间ICAM-1、IL-6与IL-17的表达相比较：B组与其余各组相比,E组与C、D组相比,差异均有统计学意义（P<0.05或P<0.01）。结论 密蒙花滴眼液具有拟雄激素的作用,可以抑制炎性细胞因子ICAM-1、IL-6与IL-17的表达,疗效弱于雄激素。不同浓度的密蒙花滴眼液中,高浓度的疗效最佳。     

Lymphotoxin is an important T cell-derived growth factor for human B cells

Two different assays for B cell growth factors (BCGF) and an antibody against lymphotoxin were used to show that the presence of lymphotoxin in conditioned media derived from normal activated T cells and in a partially purified BCGF accounts for a substantial portion of their B cell growth-promoting activity. A competitive binding assay confirmed the presence of significant amounts of lymphotoxin in the partially purified BCGF. Recombinant lymphotoxin enhanced the proliferation of activated B cells and augmented B cell proliferation and immunoglobulin secretion induced by interleukin-2.     

Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males.     

神经干细胞定向诱导分化条件研究

通过模拟体内分化诱导条件,在体外对神经干细胞(NSCs)进行定向诱导.结果表明,由添加2% B27 100 pg·mL-1 IL-1 0.05 g·L-1 Vc 5 u·mL-1 EPO的DMEM/F12的诱导液A和1份含2% B27的DMEM/F12 1份纹状体提取液混合组成的诱导液B均可以将中脑和大脑皮质的NSCs诱导分化为多巴胺能神经元,TH阳性细胞表达率随诱导时间的增加而提高,中脑来源的NSCs的TH阳性细胞诱导率极显著地高于大脑皮质来源的NSCs,诱导液A的诱导效果优于诱导液B.     

Study on the production of IL-12, IL-10 in early stage after infection with Babesia microti and Babesia rodhaini in mice

The serum levels of IL-12 and IL-10 in mice after infected with Babesia microti(B. microti) and Babesia rodhaini (B. rodhaini) were examined. Collected the mice serum and examined the concentration of IL-12 and IL-10 by using ELISA after infection with B. microti and B. rodhaini at 0, 3, 6, 9, 12, 18, 24, 36, 72, 96 h in mice. The results showed that B. microti infection resulted in IL-12 increasing, which peaked at 3 and 24 h after the infection, while same infection did not induce a significant change in IL-10 compared to uninfected mice. When mice were infected with B. rodhaini, any significant changes were not decteted both in IL-12 and IL-10 in comparison with uninfected animals during the period of 3-72 h after infection. Instead, a significant decline in IL-12 and IL-10 was found compared to uninfected mice 96 h after infection with B. rodhaini. It indicates that the mutagenetic cytokine is IL-12 in the serum of mice after infection with B. microti, and no any significant changes were detected in both IL-12 and IL-10 from 0 to 72 h after infected with B. rodhaini.     

β-伴大豆球蛋白通过p38/JNK MAPK信号通路引起IPEC-J2细胞损伤

采用细胞体外培养技术,研究不同浓度7S(β-伴大豆球蛋白)对IPEC-J2(猪小肠上皮细胞)的影响。实验分为6组,A(对照组)、B(1 mg·mL^-1 7S)、C(5 mg·mL^-1 7S)、D(10 mg·mL^-1 7S)、E(5 mg·mL^-1 7S+1 μmol·L^-1 SP600125)、F(5 mg·mL^-1 7S+1 μmol·L^-1 SB202190),24 h后,用CCK-8法检测细胞存活率,收集细胞用ELISA法检测NO、5-HT、IL-6和IL-10含量,用Western blot法检测p-JNK、p-p38、Bcl-2蛋白表达量,用PCR法检测Bad、Bax、Bcl-2、Bcl-x_L和Caspase-3 mRNA相对表达量。检测结果表明:C组和D组的IPEC-J2细胞活性极显著降低(P<0.01),E组和F组的细胞活性显著高于C组(P<0.05),说明7S促进炎性细胞因子NO、5-HT和IL-6的分泌,降低IL-10的分泌,添加抑制剂使细胞因子NO、5-HT和IL-6分泌减少,IL-10的分泌增多;同时7S促进p-JNK、p-p38蛋白表达,降低Bcl-2蛋白表达,添加抑制剂可抑制p-JNK、p-p38蛋白表达,使Bcl-2蛋白表达增高。Bcl-2/Bax mRNA比值随7S浓度增加而降低,Bax/Bcl-x_l比值随7S浓度增加而升高,Caspase-3 mRNA相对表达量随7S浓度增加而升高。添加抑制剂后Bcl-2/Bax比值增高,Bax/Bcl-x_l比值降低,Caspase-3 mRNA降低。结果表明,7S通过p38/JNK MAPK信号通路引起仔猪肠上皮细胞损伤。     

马尾藻多糖对猪脾细胞免疫活性及其抗病毒活性的影响

采用MTT法测定不同浓度马尾藻多糖(Sargassum polysaccharide,SP)对正常猪脾淋巴细胞体外增殖及其感染猪繁殖与呼吸障碍综合征病毒(PRRSV)后培养活性的影响,并用Griess和ELISA法分别检测细胞培养上清液中一氧化氮(NO)、白细胞介素2(IL-2)和γ干扰素(IFN-γ)的水平。试验结果表明:25～400μg.mL-1的SP能协同伴刀豆球蛋白(ConA)显著促进T淋巴细胞体外增殖,400μg.mL-1的SP显著促进脂多糖(LPS)刺激的B淋巴细胞体外增殖活性。SP提高正常猪脾细胞体外培养不同时间段的NO分泌量,与对照组比较,差异显著(P<0.05);不同浓度SP提高PRRSV感染的猪脾细胞体外培养分泌NO量,与病毒对照组比较,培养8 h时200μg.mL-1SP、12 h时100～400μg.mL-1SP、24 h时100μg.mL-1和400μg.mL-1SP均能显著地促进NO分泌(P<0.05)。400μg.mL-1SP极显著促进体外培养的猪脾细胞分泌IL-2(P<0.01),100μg.mL-1和400μg.mL-1SP显著促进PRRSV感染猪脾细胞IL-2和IFN-γ的分泌。结论:马尾藻多糖通过促进猪脾细胞增殖和分泌NO、IL-2和IFN-γ来调节免疫细胞活性和抗病毒能力。