睾酮诱导的小鼠骨髓巨噬细胞凋亡途径研究

用睾酮诱导,研究小鼠骨髓巨噬细胞(BMMs)凋亡过程中Fas/FasL途径上caspase-8表达的改变。以BMMs经L929条件培养基(LCM)诱导5d后,用流式细胞仪分选出F4/80阳性细胞,并将细胞分为两大组。第1组是空白对照组,睾酮(100nmol·L-1,下同)处理组,去除LCM组,去除LCM同时用睾酮处理组。第2组用FADD反义寡核苷酸(ASODN)转染BMMs后,重复第1组的4个处理组,并以FADD错义寡核苷酸(MSODN)转染后的睾酮处理组作为对照。处理12h后,用流式细胞仪检测巨噬细胞的凋亡情况,并通过RT-PCR、Real-timeRT-PCR和WesternBlot方法检测各组中caspase-8基因及蛋白的表达。结果显示,在缺少LCM或用睾酮处理时,睾酮可在体外诱导小鼠骨髓巨噬细胞凋亡,并伴随caspase-8的活化。FADD反义寡核苷酸能抑制睾酮诱导的小鼠骨髓巨噬细胞的凋亡,其下游的caspase-8表达也被抑制。     

Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells

Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles associated with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells.     

Phagocytosis of inhaled plutonium oxide-239pu particles by pulmonary macrophages

Pulmonary macrophages and plutonium particles were removed by washing the lungs of rats that had inhaled plutonium oxide-(239)Pu. A significant amount of plutonium was found in multiple washings of the same lung. The removal of toxic particles by washing is of potential therapeutic value. Particles were phagocytized by macrophages during the first 3 hours and retained within these cells for up to 25 days. Nearly all particles in washings were found in macrophages after the second day. The percent of macrophages with engulfed particles increased with increasing amounts of plutonium deposited in the lungs. The ability of pulmonary macrophages to rapidly phagocytize and retain plutonium particles deposited in the lungs has been shown.     

Cell corpse engulfment mediated by C. elegans phosphatidylserine receptor through CED-5 and CED-12

During apoptosis, phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and has been suggested to act as an "eat-me" signal to trigger phagocytosis. It is unclear how phagocytes recognize phosphatidylserine. Recently, a putative phosphatidylserine receptor (PSR) was identified and proposed to mediate recognition of phosphatidylserine and phagocytosis. We report that psr-1, the Caenorhabditis elegans homolog of PSR, is important for cell corpse engulfment. In vitro PSR-1 binds preferentially phosphatidylserine or cells with exposed phosphatidylserine. In C. elegans, PSR-1 acts in the same cell corpse engulfment pathway mediated by intracellular signaling molecules CED-2 (homologous to the human CrkII protein), CED-5 (DOCK180), CED-10 (Rac GTPase), and CED-12 (ELMO), possibly through direct interaction with CED-5 and CED-12. Our findings suggest that PSR-1 is likely an upstream receptor for the signaling pathway containing CED-2, CED-5, CED-10, and CED-12 proteins and plays an important role in recognizing phosphatidylserine during phagocytosis.     

白介素21在42日龄中国黄羽鹌鹑盲肠扁桃体中的定位

为探明白介素21(IL-21)在鹌鹑机体免疫调节的作用机制,观察IL-21的分布特点,取42日龄中国黄羽鹌鹑盲肠扁桃体,运用免疫组织化学法对白介素21进行组织学定位,观察阳性细胞的形态及分布情况。结果显示,IL-21阳性反应细胞多呈圆形或椭圆形,少数呈梭形,有的呈团块状,阳性反应细胞主要分布于黏膜上皮下固有层中,少数位于肠腺周围,生发中心周围也有少量阳性细胞分布。证实IL-21在鹌鹑盲肠扁桃体中广泛存在,分布数量及反应强度与局部免疫细胞及机体免疫状态有关。     

Multiple sclerosis: distribution of T cell subsets within active chronic lesions

The distribution of T cells and T cell subsets was examined within the human central nervous system in active lesions from seven patients with chronic multiple sclerosis. The monoclonal antibodies anti-T11, anti-T4, and anti-T8 were used to detect total (whole) T cells, helper T cells, and suppressor-cytotoxic T cells, respectively, and a monoclonal antibody against human Ia was used for macrophages and B cells. Lesion progression was associated with large numbers of T4+ cells at the lesion margin and these extended great distances into the adjacent normal-appearing white matter. T8+ cells were most commonly concentrated around the lesion margin and displayed a preferential perivascular distribution. Within the lesion center, only a few T cells were found. Ia+ macrophages were most numerous within the centers of active lesions and were always present in the adjacent normal white matter. The monoclonal antibodies to T cells did not cross-react with glial cells including oligodendrocytes. These results indicate that T4+ cells are actively involved in lesion extension and Ia+ cells, in demyelination.     

斑蝥素对昆虫细胞Spex-Ⅶ和Sf9的作用效果

以昆虫细胞Spex-Ⅶ和Sf9为试材研究斑蝥素对昆虫的杀虫机理。采用MTT法,结果表明,斑蝥素对这两种细胞凋亡的增殖有剂量和时间依赖性抑制作用,且对Sf9作用更为敏感:处理2,3,4 d后,斑蝥素对Spex-Ⅶ细胞的抑制中浓度(IC50)分别为17.623 8,13.225 1,10.707 8μg/mL;对Sf9细胞的IC50为5.433 8,1.228 9,1.222 6μg/mL;采用瑞氏-吉姆萨细胞染色和Hoechst33258荧光细胞染色法,结果表明12.5μg/mL斑蝥素处理Sf9细胞,25μg/mL和50μg/mL斑蝥素处理Spex-Ⅶ细胞,2 d后,斑蝥素对两种细胞有诱导凋亡的作用。可以观察到凋亡形态特征:胞膜完整,染色质固缩,核碎裂,凋亡小体形成。     

Phosphatidylserine receptor is required for clearance of apoptotic cells

Cells undergoing apoptosis during development are removed by phagocytes, but the underlying mechanisms of this process are not fully understood. Phagocytes lacking the phosphatidylserine receptor (PSR) were defective in removing apoptotic cells. Consequently, in PSR-deficient mice, dead cells accumulated in the lung and brain, causing abnormal development and leading to neonatal lethality. A fraction of PSR knockout mice manifested a hyperplasic brain phenotype resembling that of mice deficient in the cell death-associated genes encoding Apaf-1, caspase-3, and caspase-9, which suggests that phagocytes may also be involved in promoting apoptosis. These data demonstrate a critical role for PSR in early stages of mammalian organogenesis and suggest that this receptor may be involved in respiratory distress syndromes and congenital brain malformations.     

Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry

The asymmetrical distribution of phospholipids on the plasma membrane is critical for maintaining cell integrity and physiology and for regulating intracellular signaling and important cellular events such as clearance of apoptotic cells. How phospholipid asymmetry is established and maintained is not fully understood. We report that the Caenorhabditis elegans P-type adenosine triphosphatase homolog, TAT-1, is critical for maintaining cell surface asymmetry of phosphatidylserine (PS). In animals deficient in tat-1, PS is abnormally exposed on the cell surface, and normally living cells are randomly lost through a mechanism dependent on PSR-1, a PS-recognizing phagocyte receptor, and CED-1, which contributes to recognition and engulfment of apoptotic cells. Thus, tat-1 appears to function in preventing appearance of PS in the outer leaflet of plasma membrane, and ectopic exposure of PS on the cell surface may result in removal of living cells by neighboring phagocytes.     

Restoration of gamma globulin production in agammaglobulinemic chickens

Chickens irradiated and bursectomized in the newly hatched period consistently develop agammaglobulinemia and form no circulating antibodies; if the birds are treated immediately after operations by intra-abdominal injection of unirradiated autologous bursa cells, immunoglobulin production, lymphoid germinal centers, and plasma cells are restored; however, the birds fail to produce antibody to specific antigenic challenge.     

PEST domain-enriched tyrosine phosphatase (PEP) regulation of effector/memory T cells

Protein tyrosine kinases and phosphatases cooperate to regulate normal immune cell function. We examined the role of PEST domain-enriched tyrosine phosphatase (PEP) in regulating T cell antigen-receptor function during thymocyte development and peripheral T cell differentiation. Although normal na?ve T cell functions were retained in pep-deficient mice, effector/memory T cells demonstrated enhanced activation of Lck. In turn, this resulted in increased expansion and function of the effector/memory T cell pool, which was also associated with spontaneous development of germinal centers and elevated serum antibody levels. These results revealed a central role for PEP in negatively regulating specific aspects of T cell development and function.     

Virus Location and Lymphocyte Apoptosis in Lymph Nodes of Piglets Infected with PCV-2

The present study has been performed to understand the location of the virus, type of apoptotic cells, and their relation to lymph nodes of piglets infected with porcine circovirus type Ⅱ （PCV-2）. Nine 32-day-old conventional piglets free of infection with PCV-2 were used, and distributed into three groups： control group （n = 3）, piglets inoculated with PCV-2 alone （PCV-2, n = 3）, and PCV-2 inoculated and KLH immunostimulated group （PCV-2 ＋ KLH, n = 3）. Superficial inguinal lymph nodes from all piglets were collected for histological examination after 32 days postinoculation, and immunohistochemistry for PCV-2 detection. Location of apoptotic cells was detected with TdT-mediated dUTP nick end labeling （TUNEL） and cell cycle, and the apoptotic rates were measured by flow cytometry. The characteristic histopathological lesions of the piglets in PCV-2 and PCV-2 ＋ KLH were lymphocyte depletions in the cortex and paracortex of the lymph nodes, epithelioid-like macrophage infiltration, and intracytoplasmic inclusion bodies presented in epithelioid-like macrophages. PCV-2 was mainly found in epithelioid-like macrophages by immunohistochemistry. In the lymph nodes, lymphocytes presented higher apoptotic rates in the cortex by TUNEL, special B-cell areas, and similar apoptotic cells were found in this compartment in the control. The apoptotic rates of the lymph nodes were 0.41, 3.34, and 4.88% in the control, PCV-2, and PCV-2 ＋ KLH groups by flow cytometry, respectively. The apoptotic rates of lymph nodes for PCV-2 and PCV-2 ＋ KLH piglets were significantly higher than those for the control group （P〈0.05 and P〈0.01）. The proliferation index （PI） was 0.17_＋0.01, 0.12_＋0.01 and 0.12_＋0.04 in the control, PCV-2, and PCV-2 ＋ KLH group, the PI of the control group was higher than that of the other groups, but without the statistical difference. PCV-2 can induce lymphocyte depletion in lymph nodes of piglets by blocking cell proliferation and promoting apoptosis. This is one o     

固始鸡脾脏内淋巴细胞自然凋亡的超微结构研究

为了探讨固始鸡脾脏内淋巴细胞自然凋亡的过程和超微结构的变化。应用透射电子显微镜技术,对固始鸡17日龄胚胎和4,19周龄固始鸡3组正常发育的脾脏内淋巴细胞自然凋亡的超微结构进行了研究。结果表明,脾脏内自然凋亡的淋巴细胞主要表现为细胞核的变化,其次是线粒体增多。核质凝集为新月形、C字形、圆环形、花瓣状等多种形态,凋亡细胞裂解为凋亡小体,最后被巨噬细胞吞噬;固始鸡胚胎和4,19周龄固始鸡脾脏内自然凋亡细胞有逐渐增加的趋势。由此可见,固始鸡脾脏内淋巴细胞存在着自然凋亡现象,凋亡细胞主要表现为细胞核的变化,其次是线粒体增多。     

Receptor activation alters inner surface potential during phagocytosis

The surface potential of biological membranes varies according to their lipid composition. We devised genetically encoded probes to assess surface potential in intact cells. These probes revealed marked, localized alterations in the charge of the inner surface of the plasma membrane of macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement of phosphatidylserine accounted for the change in surface potential at the phagosomal cup. Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by electrostatic interactions were rapidly released from membrane subdomains where the surface charge was altered by lipid remodeling during phagocytosis.     

印楝素对小菜蛾胚胎细胞增殖和凋亡的影响

【目的】探讨印楝素对小菜蛾Plutella xyllostella胚胎细胞增殖和凋亡的影响及可能的机制。【方法】将体外培养的小菜蛾胚胎细胞分为印楝素处理组和未处理组,采用CCK-8试剂盒检测细胞增殖的抑制率,激光共聚焦镜检PI染色后的细胞死亡和DAPI染色后的凋亡小体,通过Western-blotting检测各组细胞Caspase-3的表达情况以及通路蛋白Akt的磷酸化水平。【结果】印楝素对小菜蛾胚胎细胞有明显的增殖抑制作用,且呈现浓度依赖性,24 h的IC_(50)为4.4μg·mL~(-1)。印楝素处理后的细胞经PI染色后能明显观察到死亡细胞,DAPI染色后可见凋亡小体;Caspase-3蛋白发生剪切,并且抑制Akt的磷酸化水平。【结论】印楝素对小菜蛾胚胎细胞的增殖有明显的抑制作用,并通过抑制Akt信号通路的活化,诱导细胞产生依赖于Caspase-3的Ⅰ型凋亡。     

鸡包涵体肝炎的病理学研究

介绍了河北省中南部地区１９９４－－１９９５年间,８７个鸡场,３２００万只鸡发生鸡包涵体肝炎的系统病理学研究。结果表明,病鸡发生以肝脏严重变性、坏死,并在肝细胞核内形成核内包涵体的特征性病变。同时,机体免疫器官发生广泛损伤,产蛋鸡生殖器官发迟缓或停滞。     

代乳粉添加单宁酸对7—28日龄湖羊羔羊胃肠道发育的影响

目的 观察代乳粉中添加单宁酸对7—28日龄湖羊羔羊胃肠道生长发育的影响。方法 选择同质性好的7日龄湖羊公羔（双羔）30只,随机分为2组,每组15只,每只1个重复,分别饲喂对照代乳粉或单宁酸含量为0.2%的代乳粉,试验期21 d。羔羊28日龄时饲养试验结束,每组随机选择8只羔羊进行屠宰,分离胃室和肠段,分别称量各胃室和各肠段净质量以及含内容物质量,并量取各肠段的长度,计算各胃室和各肠段的相对质量和相对长度及内容物分布。肠道组织形态学和上皮细胞凋亡率用皱胃胃底腺区及十二指肠、空肠和回肠中段的组织样品测定。肠道屏障功能相关的闭锁蛋白（occludin）、闭锁小带1（zonula occludens-1,ZO-1）和紧密连接蛋白1（claudin 1）mRNA表达量用采集的十二指肠、空肠和回肠黏膜测定。结果 除十二指肠指数（%活体质量,P=0.012）和相对质量（%全肠质量,P=0.034;%全胃肠质量,P=0.017）、空肠和结肠相对长度（%全肠长度,P=0.030,P=0.004）及结肠内容物分布（%活体质量,P=0.039）外,单宁酸对羔羊胃肠指数（%活体质量）、胃肠相对质量（%全胃质量、%全肠质量和%全胃肠质量）、肠道相对长度（%全肠长度）、内容物相对质量（%活体质量）、胃肠内容物相对总胃/肠内容物及总胃肠内容物相对质量（%总胃内容物、总肠内容物、总胃肠内容物）以及小肠claudin 1蛋白mRNA表达量均没有产生显著影响（P＞0.05）,但显著提高羔羊十二指肠肌层厚度并降低绒毛宽度（P=0.013,P=0.001）,显著上调空肠ZO-1蛋白mRNA表达量（P=0.003）,此外,单宁酸有上调羔羊空肠occludin蛋白mRNA表达量并降低空肠绒毛宽度和隐窝深度以及空肠和回肠上皮细胞凋亡率的趋势（P=0.077,P=0.073,P=0.062,P=0.097,P=0.052）。结论 单宁酸显著降低7—28日龄湖羊羔羊十二指肠相对质量和空肠相对长度,但可通过提高十二指肠肌层厚度、上调空肠ZO-1蛋白和occludin蛋白mRNA表达量并降低空肠隐窝深度及空肠和回肠上皮细胞凋亡率来改善肠屏障功能。     

Evolution of autoantibody responses via somatic hypermutation outside of germinal centers

Somatically mutated high-affinity autoantibodies are a hallmark of some autoimmune diseases, including systemic lupus erythematosus. It has long been presumed that germinal centers (GCs) are critical in autoantibody production, because they are the only sites currently believed to sustain a high rate of somatic hypermutation. Contrary to this idea, we found that splenic autoreactive B cells in autoimmune MRL.Fas(lpr) mice proliferated and underwent active somatic hypermutation at the T zone-red pulp border rather than in GCs. Our results implicate this region as an important site for hypermutation and the loss of B cell self-tolerance.     

非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位

【目的】制备非洲猪瘟病毒（African swine fever virus,ASFV）p30蛋白的单克隆抗体（monoclonal antibodies,MAbs）并初步分析其所识别的线性抗原表位,为ASFV及其抗体检测方法的建立及p30蛋白结构和功能的研究奠定基础。【方法】将原核表达并纯化的p30重组蛋白作为免疫原,免疫6—8周龄BALB/c雌鼠,每两周免疫1次,共免疫3次,首次免疫是抗原与等体积的弗氏完全佐剂乳化后免疫,第二次和第三次免疫与等体积的弗氏不完全佐剂乳化,3次免疫后1 w断尾采血,间接酶联免疫吸附试验（ELISA）检测血清抗体效价,选择血清效价最高的小鼠进行加强免疫,3 d后取小鼠脾淋巴细胞与SP2/0骨髓瘤细胞按照4﹕1的比例使用PEG进行常规细胞融合。利用重组p30蛋白作为包被抗原,间接ELISA筛选阳性杂交瘤细胞,有限稀释法进行克隆纯化,直至筛出能够稳定分泌抗体的MAbs。将ASFV接种于猪肺泡巨噬细胞,以筛选的MAbs为一抗、兔抗鼠HRP-IgG为二抗,进行间接免疫荧光试验（IFA）。将感染和未感染ASFV的细胞沉淀处理后进行 SDS-PAGE并转印至硝酸纤维素膜,分别以IFA鉴定为阳性的MAbs上清为一抗、兔抗鼠HRP-IgG为二抗,进行Western blotting分析,筛选获得p30 MAbs。根据已知序列设计引物扩增p30ab与p30bc两段截短基因,其中p30ab代表由第86—153位氨基酸残基的截短体,p30bc代表由第120—187位氨基酸残基的截短体,原核表达部分重叠的截短p30蛋白,最终获得重组蛋白GST-p30ab与重组蛋白GST-p30bc。分别以GST-p30ab和GST-p30bc融合蛋白为包被抗原,以5株MAbs为一抗,以兔抗鼠HRP-IgG为二抗, 通过间接ELISA方法初步定位p30蛋白的抗原表位。【结果】以纯化的重组蛋白为包被抗原,经间接ELISA试验筛选出25株可分泌抗重组 p30蛋白的杂交瘤细胞株。IFA结果显示,5株MAbs（8F4、1D3、1H2、6C3和8E11）与ASFV感染的猪肺泡巨噬细胞IFA 试验呈阳性;Western blotting结果显示,5株MAbs均能够与ASFV感染的细胞呈阳性反应,与未感染病毒的细胞呈阴性反应。试验构建的p30截短体重组蛋白GST-p30ab以可溶和包涵体两种形式表达,而GST-p30bc仅以包涵体形式表达,以两组截短体融合蛋白为包被抗原,通过间接ELISA检测出MAbs 8F4、1H2和6C3与两个重组蛋白均能有效结合,证明MAbs 8F4、1H2和6C3抗原识别区域为两组截短蛋白重叠区域,即第120—153位氨基酸;MAbs 8E11与1D3则只能与GST-p30ab蛋白结合, 证明MAbs 8E11与1D3抗原识别区域为两个重组蛋白的非重叠区域,即第86—119位氨基酸。【结论】本研究可溶性地表达了p30蛋白的第86—153位氨基酸截短体重组蛋白,制备了5株p30 MAbs,定位到2个p30蛋白抗原表位。结合ELISA和IFA,可建立十分可靠的ASFV及其抗体的检测手段。