丙型肝炎病毒E2蛋白对HepG2细胞MAPK／ERK的激活

人CD81是丙型肝炎病毒（hepatitis Cvirus,HCV）的细胞表面特异性受体,HCV包膜蛋白－2（E2）可与其结合。细胞个信号调节激酶（extracellular signal-regulated protein kinase,MAPK/ERK1,2）信号途径主要介导细胞增殖及分化。为探讨HCV E2蛋白与人CD81结合对MAPK／ERK活性变化的影响,以HCV E2蛋白刺激HepG2细胞,采用免疫印迹、免疫组化及免疫荧光等方法动态观察细胞内MAPK／ERK的激活情况,并以流式细胞术检测细胞表面CD81的表达。结果表明：HepG2细胞高表达人CD81;HCV E2蛋白可激活细胞内MAPK／ERK;MAPK／ERK的磷酸化反应与HCV E2蛋白浓度、作用时间呈依赖关系;HCV E2－CD81相互作用引发的细胞异常信号转导可能与HCV致病性相关。     

猪红细胞与人淋巴细胞形成玫瑰花环的影响因素

Lay等人曾发现人T淋巴细胞与猪红细胞形成花环,证实在人T淋巴细胞表面存在猪红细胞受体。为探讨该种受体的特性,我们观察了若干因素对人T淋巴细胞与猪红细胞花环形成的影响,现报告如下: 材料与方法猪红细胞悬液制备取抗凝猪血,用Hanks液洗涤3次,并以Hanks液配成0.5％猪红细胞悬液(约为8×10~7个细胞/毫     

蛋白激酶C抑制剂对U937细胞清道夫受体功能的影响

为了解细胞内蛋白质磷酸化水平对清道夫受体功能的影响,用蛋白激酶Ｃ抑掉剂形孢菌素（ｓｔａｕｒｏｓｐｏｒｉｎｅ,ＳＴＡ）处理人Ｕ９３７细胞,分别测定对照组和处理组细胞对碘标记的氧化低密度脂蛋白（＾１２５Ｉ）ｏｘ－ＬＤＬ的降解,结合,细胞表面受体复合物的内移以及细胞内脂质蓄积的程度,并利用放射自显影方法观察药物对细胞表面受体表达的影响,结果发现ＳＴＡ可以促进细胞结合（＾１２５Ｉ）ｏｘ－ＬＤＬ增加细胞表面     

酪氨酸蛋白激酶抑制剂genistein对PMA处理后A类清道夫受体表达的抑制

为研究清道夫受体与细胞内酷不氨酸蛋白激酶的关系,用酪氨酸蛋白激酶抑制剂genistein处理人U937细胞。分别测定对照组和处理组细胞对碘标记的氧化低密度脂蛋白[^125I]ox-LDL的结合、降解以及细胞内脂质蓄积的程度;并利用放射自显影的方法观察药物对细胞表面受体数目的影响,利用RT－PCR法进一步探讨药物作用的分子机制。结果发现genistein可以抑制细胞表达结合[^125I]ox-LDL,抑制细胞表面受全的表达以及细胞内降解[^125I]ox-LDL,抑制细胞内胆固醇脂的蓄积,并且抑制SR－A mRNA的转录,提示清道夫受体的活性可能与细胞内蛋白质酪氨酸磷酸化水平密切相关,genistein所引起的酪氨酸磷酸化水平下降可影响SR－A基因转录和翻译。     

与豚鼠红细胞形成自然花环的狗淋巴细胞类别的探讨

本文研究了豚鼠E花环形成狗淋巴细胞(E-RFC)的类别。狗淋巴细胞于37℃孵育后,可提高E花环形成率。E-RFC与总淋巴细胞的SIg阳性率相近。E-EAC(豚鼠红细胞—抗体和补体包被的绵羊红细胞)混合花环实验表明一部分狗淋巴细胞同时具有E和补体二种受体,总淋巴细胞与补体受体淋巴细胞的E花环阳性率无显著差异。这些结果说明E受体不仅表现于一部分T细胞上,而且见于某些B细胞上。因此,不宜用E花环试验作为检测狗T细胞的方法。     

PHA刺激对淋巴细胞修复DNA损伤的影响

本文报道PHA刺激对淋巴细胞DNA修复的影响的实验结果。以254nm波长的UV照射细胞(30J/m~2)引起DNA损伤,以[~3H]-TdR掺入实验测定非程序DNA合成,用超微量法测定细胞的NAD~+含量,并以[~(35)S]-蛋氨酸掺入,聚丙烯酰胺凝胶电泳及放射自显影术测定蛋白质生物合成,其结果如下: (1)在被PHA转化的淋巴细胞内非程序DNA合成,随PHA刺激的时间加长而增高;PHA处理淋巴细胞42小时,合成的速率约增加4倍;(2)在转化的淋巴细胞内,非程序DNA合成及程序DNA合成都被N-乙基马来酰亚胺(一种DNA聚合酶α的抑制剂)抑制,表明在DNA修复过程中DNA聚合酶α可代替DNA聚合酶β发挥作用; (3)UV照射后,被PHA刺激的淋巴细胞内NAD~+含量大约减少43.2％,而对照淋巴细胞内NAD~+的含量只减少25％,似乎说明PHA刺激能促进淋巴细胞内的P-ADP-核糖化作用;(4)在受PHA刺激72小时的淋巴细胞内有多种蛋白质合成,这些细胞在UV照射后以含10μg/ml嘌呤霉素的培养基培养,则非程序DNA合成被明显抑制(P<0.01),这提示DNA修复是一需要蛋白质合成的过程。此外,在受UV照射后10-45小时的淋巴细胞内,诱导产生一种分子量大约34000道尔顿的蛋白质。 上述结果表明,当PHA使淋巴细胞从静止状态转化为增殖状态时,有多种酶被诱导。由于这些酶,如DNA聚合酶α及P-ADP-核糖聚合     

不同浓度A2E对离体猪视网膜色素上皮细胞的影响

目的评价体外合成的A2E对猪视网膜色素上皮（RPE）细胞的细胞活力和生物学特性影响,为进一步研究A2E在RPE细胞相关疾病中的作用提供细胞模型。方法利用全反式视黄醛和乙醇胺体外合成脂褐质荧光基团A2E。不同浓度的A2E（0,50,75,100μmol／L）作用第3代体外培养的猪RPE细胞30,45,60,90min,换10％FBS DMEM-F12培养液孵育24h后,倒置荧光显微镜观察荧光强度,IPP6．0软件灰度扫描定量荧光强度。采用MTT法检测A2E作用细胞各个时间段的吸光度值,应用SPSS11．0软件包对数据进行统计学分析,评价A2E的细胞毒性及RPE细胞活性。结果A2E被RPE细胞摄取后主要分布于细胞核周围,具有自发荧光。MTT实验及荧光灰度扫描结果显示,不同浓度的A2E被细胞摄取后细胞活力和荧光灰度扫描结果不同,以50μmol／L浓度A2E作用RPE细胞60min时,细胞内荧光强度高同时细胞活力强。结论体外培养的猪RPE细胞摄取体外合成的50μmol／L A2E 60min后细胞对A2E的摄取较多,A2E对细胞的毒性相对较低,该条件下进行A2E对离体猪RPE细胞的研究较好。     

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

T淋巴细胞表面的TRBC受体不同介导E花结形成的E受体(CD2)和E2分子。CD2的配体,人红细胞表面的CD58(LFA-3)和绵羊红细胞表面的T11 TS,S42,S14及S110-220,与TRBC受体的配体无关,TRBC玫瑰花结的形成是通过不同于E花结和人自身玫瑰花结的受体-配体相互作用来实现的,进一步表明,人和猴T淋巴细胞表面和TRBC表面,可能都有独特的蛋白质分子介导TRBC玫瑰花结的形成。     

肽类激素在受体介导下进入细胞的机制

许多肽类激素与靶细胞膜上的特异受体结合后形成的激素-受体复合物(H-R)在细胞膜内横向移动,聚集在细胞膜的特化结构区域。H-R进入细胞经高尔基氏区转运至溶酶体,激素在溶酶体内降解,受体也在细胞内降解。H-R的内移使细胞表面受体数目减少,这是激素对受体数目进行减数调节的原因。以入胞速率常数(Ke)为参数,可定量分析H-R内移过程。     

猪胸腺激素对淋巴细胞DNA复制和转录的刺激作用

胸腺激素可促进T细胞成熟分化,调节免疫细胞功能,因而在免疫中起重要作用。T细胞在成熟过程中,将出现Lyt 1,2,3及绵羊红血球受体等表面标志和TDT。本研究以~3H-TdR和~3H-Leu掺入试验观察猪胸腺激素PTH和DNA复制及蛋白质合成的关系,以玫瑰花结及TDT为标志研究PTH在T细胞成熟中的作用。     

Polyadnylic acid sequences in RNA able to transfer specific delayed type hypersensitivity in vitro.

Antibody-depedent cell-mediated cytotoxicity (ADCC) could be initiated without protein synthesis [human peripheral blood lymphocytes as effector cells incubated with 10^?3M cycloheximide, (Cy)], although the reaction did not achieve its full lytic ability. This partial inhibition of ADCC was dependent on the dose of Cy. Both ADCC and protein synthesis returned to normal values after removal of the inhibitor. The kinetics of the reaction carried out by Cy-treated effector cells for short periods was similar to that of controls. After this time, the percentage of lysed target cells increased continuously in controls, while the cytotoxiciy of Cy-treated effector cells reached a plateau. When effector cells carried out ADCC in the presence of Cy, their lytic mechanism was “wasted,” and it could be recovered only by removal of the inhibitor. Our results indicate that effector cells have a preformed lytic mechanism operative in ADCC. This lytic mechanism is consumed during the reaction and its recovery requires protein synthesis.     

Oxidative stress stimulates the synthesis of the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid by inflammatory cells

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that acts through a selective G-protein coupled receptor. It is formed by oxidation of the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Although leukocytes and platelets display high microsomal 5-HEDH activity, unstimulated intact cells do not convert 5-HETE to appreciable amounts of 5-oxo-ETE. To attempt to resolve this dilemma we explored the possibility that 5-oxo-ETE synthesis could be enhanced by oxidative stress. We found that hydrogen peroxide and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U937 monocytic cells. This was dependent on the GSH redox cycle, as it was blocked by depletion of GSH or inhibition of glutathione reductase and mimicked by oxidation of GSH to GSSG by diamide. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, as its effect was reversed by the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-butyl hydroperoxide. Oxidative stress could act by depleting NADPH, resulting in the formation NADP+, the cofactor for 5-HEDH. This is opposed by the pentose phosphate pathway, which converts NADP+ back to NADPH. Oxidative stress could be an important mechanism for stimulating 5-oxo-ETE production in inflammation, promoting further infiltration of granulocytes into inflammatory sites.     

Interaction of Bacillus Calmette--Guérin-activated macrophages and neoplastic cells in vitro II. The relationship of selective binding to cytolysis

5′-Methylthioadenosine (MTA), a degradation product of S-adenosylmethionine, inhibits DNA and protein synthesis as well as cellular proliferation in human lymphocyte cultures stimulated with mitogens, antigens, or allogeneic cells. MTA (10^?3M) inhibited [³H]Tdy uptake in PHA- or Con A-induced transformation greater than 95%, and inhibited the uptake of both [³H]Tdy and [¹⁴C]Leu to the same degree in lymphocytes stimulated with PPD or allogeneic lymphocytes. MTA inhibition was dose dependent, inhibition being lost when exogenous levels reached approximately 10^?5M. The inhibitory effects of MTA were not produced by cytotoxicity since MTA-inhibited cells washed free of this compound could be stimulated at least as well as uninhibited cells. Understanding the mode of action of MTA and the mechanisms controlling its metabolism may lead to new approaches for regulating cellular proliferation.     

Fc receptors and human T lymphocytes in frozen-thawed peripheral blood cells

The present study was carried out to investigate the influence of cryopreservation on human T-cell subsets defined by their membrane receptors for Fc IgM (TM) and Fc IgG (TG) and by their membrane antigens. For this purpose isolated T cells, obtained by neuraminidase-treated sheep erythrocyte (E-N) rosetting, and enriched mononuclear cells were cryopreserved using a programmed freezing procedure. A significant decrease of the TM and TG cells was found whereas the proportion of T cells and their subsets determined by monoclonal antibodies seemed not to be influenced. The effectiveness of T-cell separation by E-N rosetting of frozen lymphocytes demonstrated no impairment of the E-receptor binding capacity of T cells. The PHA reactivity of separated T cells was maintained after cryopreservation; however, the spontaneous blastogenesis was reduced significantly. The selective loss of the TM and TG cells seemed to be dependent on the length of the phase transition time; over 90 sec the capacity of the expression of Fc receptors was profoundly affected. Neither an additional 20 hr incubation after hypotonic shock prior to cryopreservation nor incubation after thawing could repair this function of T cells. The data suggest irreversible damage of the Fc receptor expression capacity on the cell membrane as a result of a disturbance of metabolic pathways rather than a preferentially greater sensitivity of these cells to cryopreservation.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP: I. Antigen-induced uptake of nucleotides and nucleosides by lymphocytes

Addition of N⁶, O²′-dibutyryl cAMP (DbcAMP) to keyhole limpet hemocyanin (KLH)-primed rabbit lymph node cells for 1 hr, followed by its removal and the addition of KLH, had no effect on the subsequent antibody response, whereas addition of KLH for 1 hr followed by DbcAMP resulted in a 100% enhancement of antibody synthesis. Addition of cholera enterotoxin (CT), which rapidly and irreversibly binds to lymphocytes and activates adenylate cyclase, either before or after the addition of antigen, elevated the antibody response by 100%. These results suggested that some antigen-induced event(s) may be required for DbcAMP to exert its enhancing effects on the antibody response. The effect of KLH on the uptake of DbcAMP by KLH-primed lymph node cells was investigated. One and one hundred micrograms of KLH, which induce optimal and supraoptimal antibody synthesis, respectively, promoted maximal uptake of DbcAMP. This induced uptake was first detectable about 12 hr after addition of KLH, and it peaked during 24–48 hr of culture. DbcAMP uptake induced by a brief exposure of KLH (0–1 hr) was equivalent to that observed with long-term KLH addition (0–24 hr). KLH-induced DbcAMP uptake required KLH-reactive lymphocytes and represented active transport. Antibody to rabbit T lymphocytes inhibited this antigen-induced uptake. The mitogens concanavalin A (Con A) (T cells) and goat anti-rabbit Fab' (anti-Fab') (B cells) also stimulated DbcAMP uptake, as did human serum albumin (HSA) and myoglobulin (Mb) when added to homologously primed cells, indicating the generality of the phenomenon. [³H]DbcAMP entered the cells as di- or monobutyryl cAMP with about 40% metabolized to 5′AMP. This uptake could be competitively inhibited by other adenine or guanine nucleotides and nucleosides.     

Immune modulation of connective tissue functions: studies on the production of collagen synthesis inhibitory factor by populations of human peripheral blood mononuclear cells

It has been shown previously that a soluble factor(s) from human peripheral blood mononuclear cells was capable of specifically suppressing collagen synthesis by normal human dermal fibroblasts (S. A. Jimenez, W. McArthur and J. Rosenbloom, J. Exp. Med.150, 1421, 1979). In this communication, the cell sources and the conditions for synthesis of this collagen synthesis inhibitory factor (CSIF) are identified. CSIF production by mononuclear cells was directly related to the number of cells in culture and was significantly enhanced by a variety of mitogens and by antigens. Homologous serum or bovine serum albumin was required for CSIF production and maximal levels were reached 48 hr after stimulation. Thymus-derived lymphocytes appeared to be the main cells responsible for CSIF synthesis but B lymphocytes also produced the factor in response to proper B-cell mitogens. Preparations of plastic-adherent mononuclear cells were also found to produce increased CSIF but it was not possible to exclude completely the presence of T lymphocytes in these preparations and therefore, the cell source of CSIF in these preparations was not clearly established. Through the use of metabolic inhibitors it was shown that CSIF production required de novo protein synthesis but not cell division. Indo-methacin had no effect on either the production of CSIF or on CSIF-mediated inhibition of collagen synthesis. The results indicate that CSIF has the classic characteristics of a lymphokine and suggest a mechanism by which the immune response could modulate connective tissue function.     

Impairment of alternate pathway (CD2) of T cell activation in leprosy

Recent studies in basic immunology have been directed towards the understanding of the mechanism of T cell activation. T cells can be activated to proliferatevia the classical pathway through the antigen receptor (CD3-Ti) orvia the alternate pathway through the CD2 receptor. Since immunologic unresponsiveness in lepromatous leprosy is considered to be due to the inability of T cells to proliferate upon stimulation, we have been interested in the nature of these receptors and the activation pathways in lymphocytes of leprosy patients. In the present investigation we demonstrate: (i) CD2 receptor (E-receptor) is downregulated in bacterial index positive lepromatous leprosy patients. (ii) The alternate pathway of T cell activation is impaired in lepromatous patients as revealed by the inability of their lymphocytes to proliferate in response to a pair of mitogenic anti-CD2 monoclonals. (iii) The addition of recombinant interleukin 2in vitro restores the ability of lymphocytes from lepromatous patients to proliferate in response to anti-CD2 antibodies. (iv) Interestingly, CD2 modulation and the associated functional impairment could be brought about in peripheral blood lymphocytes from normal subjects by prior treatment withMycobacterium leprae in vitro. This approach would be useful in understanding the molecular events leading to the defective T cell functions in leprosy.     

关于鸡法氏囊淋巴滤泡皮质部形成的研究

初生雏鸡孵出后立即结扎法氏囊管,使外界抗原进入法氏囊腔通路受阻,法氏囊髓质部细胞未见增殖分化,从而没有淋巴细胞穿过基膜形成皮质部。结扎法氏囊管后喂养半个月的雏鸡,再拆除结扎线,恢复泄殖腔与法氏囊的通道,外界抗原又可进入法氏囊腔,刺激滤泡髓部细胞分裂增殖,并穿过基膜形成皮质部。但由于曾结扎半月,所以迁移到皮质部的细胞与对照组比较相对减少。结扎法氏囊管后同时注射枯草杆菌(Bs)和四球菌(Mt),法氏囊滤泡皮质部与正常对照组相似,有的甚至比对照组更为发达。电镜观察皮质部具有不同成熟度的浆细胞。孵出的雏鸡用睾酮(TP)处理后法氏囊滤泡虽有皮质部,但不是正常的皮质部淋巴细胞。实验结果表明滤泡皮质部的形成与孵化后外界抗原的刺激有关,法氏囊作为鸟类特有的体液免疫的中枢淋巴器官,可能仅指胚胎时期发育的淋巴滤泡髓质部,而法氏囊皮质部则可能相当于外周淋巴器官。它的形成必须依赖于外界抗原的刺激。     

Characterization of a monoclonal antibody to guinea pig T cells that inhibits rosette formation of the cells with rabbit erythrocytes: similarity of the antigen to E-receptor on human T cells

A monoclonal antibody, GPT-1, was prepared by fusion of the splenic cells of mice immunized with guinea pig thymocytes with a mouse myeloma cell line. GPT-1 completely inhibited spontaneous rosette formation of T cells with papain-treated rabbit erythrocytes. GPT-1 reacted with 90% of thymocytes, 70% of peripheral blood lymphocytes, and 45% of splenic lymphocytes, but not with B cells. These results indicate that GPT-1 has pan-T reactivity. The antibody specifically bound to a single polypeptide chain with a molecular size of 50-65 kD. The surface density of the antigen was higher on thymocytes than on peripheral T cells, suggesting that the antigen is a certain differentiation antigen on T cells. Phytohemagglutinin-activated T cells expressed more antigen molecules than resting T cells. In addition, GPT-1 suppressed the proliferation of T cells induced by the mitogen, indicating that GPT-1 recognizes a T cell-specific surface antigen which is associated with T cell activation. Based on these results, it was concluded that GPT-1 reacts with a guinea pig T cell surface antigen which is similar to the E-receptor protein on human T cells (CD2 molecule).