医用丝素蛋白皮肤再生膜的细胞相容性评价

评价医用丝素蛋白皮肤再生膜的细胞相容性。其方法是采用细胞增殖度试验和溶血试验,对医用丝素蛋白皮肤再生膜进行细胞毒性和溶血反应的实验研究。结果表明：该再生膜无明显细胞毒性存在,溶血率为1.15%。医用丝素蛋白皮肤再生膜具有良好的细胞相容性。     

医用丝素蛋白皮肤再生膜的生物相容性评价

BACKGROUND: Skin reproducing membrane is a biomaterial used directly in contact with skin defects, so its toxicity to the body must be taken into consideration.     

重型再生障碍性贫血患者骨髓T细胞克隆的初步研究

目的建立源于重型再生障碍性贫血(重型再障)患者骨髓的T淋巴细胞克隆,以便研究该病的发病机理.方法用免疫分选法分别从1名初诊重型再障患者及对照的骨髓中富集CD34+和CD3+细胞.将CD3+细胞作反应细胞、照射过CD34+细胞作刺激细胞,进行14d单向自身混合淋巴细胞培养.对活细胞作有限稀释和单个细胞分孔接种,与含IL-2和PHA-P的饲养细胞体系共培养.观察挑选细胞生长孔,计算接种率,用泊松(Poisson)分布初步判断单个细胞分离过程成功与否.而后将阳性孔细胞作转孔培养,定期用饲养抚育刺激、常规用IL-2支持增殖以扩大各克隆的数量.用SABC-P免疫细胞化学染色法,初步鉴定各克隆CD4/CD8抗原表型.结果自身混合淋巴细胞培养后的活细胞数,病例为4×103,对照为65.单细胞分离接种培养后,对照组的细胞生长孔数为0;而病例组的培养板上,共有11孔发生细胞增殖,相应的实际接种率是2.1%,未超出Poisson理论接种率值范围(＜26%),判明平均每个增生孔的细胞克隆前体数不大于1.这11个克隆中,9个呈CD4单阳性表型,阳性为98%;2个为CD8单阳性表型,阳性率为98%.结论从1例重型再障患者骨髓中分离出了11株T淋巴细胞克隆.进一步鉴定分析这些克隆的表型和特异性,以及探讨其Th1(Tc1)/(Th2)(Tc2)效应类型等功能表现,对研究重型再障的免疫学机理具有积极的意义.     

再生障碍性贫血患者T细胞CD69的表达

CD69又称活化诱导分子（AIM）、早期活化抗原1（EA-1），是淋巴细胞活化最早表达的膜表面分子。CD69由两个相同的单体经不同的糖基化后构成，属植物血凝素C超家族Ⅱ型膜贯通型糖蛋白，细胞外区C型凝集素（钙离子依赖性）有一个N结合型糖链附着部位。静止状态的T细胞不表达CD69，     

引导性组织再生膜材料的研究进展

引导性组织再生是人为地放置物理性隔膜，选择有利于组织再生的细胞及组织聚集生长。膜材料主要分为：非降解膜材料，其代表材料为ｅ－ＰＴＦＥ；可降解膜材料，其代表性材料主要为ＰＬＡ和胶原膜。这两大类膜材料有着各自的特性，在科研及临床上应用时又有着不同的优缺点。     

许旺细胞在不同孔径丝素蛋白支架上的生长

背景：细胞在生物支架上的生长行为受到支架表面形貌、润湿性、孔径及孔隙率等多种因素影响。 目的：观察许旺细胞在不同孔径丝素蛋白支架上的生长情况。 方法：制备大孔径50~60 µm、小孔径10~   20 µm两种多孔丝素材料。选用许旺细胞永生化细胞R3 [33-10ras3]为种子细胞,当细胞在培养瓶底形成致密单层时即可消化细胞并进行接种实验,将许旺细胞悬液种于不同形貌的多孔丝素材料表面。复合培养1周后,扫描电镜观察许旺细胞的生长形态及增殖等情况。 结果与结论：不同孔径丝素材料的表面可见许旺细胞生长情况不一。在10~20 µm孔径材料支架上,细胞浓度较低,细胞表现为特异的双极性形态,细胞与细胞之间或平行排列,或首尾相连成细胞链;细胞与细胞之间或平行排列,或首尾相连成细胞链;在50~ 60 µm孔径丝素材料支架上,细胞浓度较高,细胞多为球形,单个分散在多孔支架表面,或呈现成团成串葡萄样聚集在孔的底部,未延展成双极性形态,只有极少量生长在孔与孔之间嵴上的细胞呈双极样。说明多孔丝素蛋白支架的孔径对许旺细胞的黏附、生长有一定的影响,许旺细胞更适合生长在孔径略大于胞体直径的支架材料上。     

过敏性哮喘发病中T细胞激活的研究

为探讨T淋巴细胞在过敏性哮喘发病中的作用,本研究利用APAAP法观察了20例过敏性哮喘发作期患者外周血T细胞亚群及其活化状态改变,并与20例缓解期患者和20例健康对照者进行了比较.结果表明:CD4~+T细胞,CAD8~T细胞相对百分数和CD4/CD8比值在三组间无差异(P>0.05),而CD25~+T细胞和HLA-DR~+T细胞在哮喘发作组明显高于其它两组(P<0.01),且与血清总IgE水平呈正相关(r分别为0.3855,0.3554,P均<0.05),与FEV_1呈负相关(r分别为—0.6884,—O.5875,P均<0.01).上述结果提示讲:CD4~+T细胞活化在过敏性哮喘的发病中起重要作用.     

雷公藤对Ly—22.2^＋细胞（抑制性T淋巴细胞）的激活作用

本文用单克隆抗体观察了雷公藤对小鼠脾脏的Ｔｈｙ－１＾＋细胞，Ｌ３，ＬＴ＾＝细胞Ｌｙｔ－２．３％＋细胞和Ｌｙ－２２．２＾＋细胞的影响，发现Ｌｙ－２．３＾＋细胞增多的同时，Ｌｙ－２２．２＾＋细胞明显增加，Ｌ３Ｌ５＾＝细胞／Ｌｙｔ－２．３＾＋细胞明显下降。     

再生丝素蛋白膜作为人嗅鞘细胞移植支架的研究

探讨再生丝素蛋白膜对人嗅鞘细胞(hOECs)生物学特性的影响.人嗅鞘细胞接种在再生丝素蛋白膜上,取不同时相的细胞做下列实验.72 h固定,用免疫荧光染色技术观察人嗅鞘细胞的标志蛋白NT-3、S-100、GFAP、NGFRp75,用电子显微镜观察细胞在再生丝素蛋白膜上的生长状况;分别于48、72、96 h提取总蛋白,用免疫印迹法分析人嗅鞘细胞标志蛋白的表达情况;用MTF法分析细胞在再生丝素蛋白膜上的生长曲线.hOECs在再生丝素蛋白膜上能够很好地附着、铺展、生长,嗅鞘细胞的形态大多为梭形,嗅鞘细胞标志蛋白NT-3、NGFRp75、S-100及GFAP均呈免疫反应阳性,且免疫印迹结果与形态学结果一致;电镜结果显示,再生丝素蛋白膜的表面微纳结构适于细胞贴附、铺展;嗅鞘细胞在再生丝素蛋白膜上的生长曲线与培养板上无明显差异.再生丝素蛋白膜适合人嗅鞘细胞的贴附、增殖、生长,再生丝素蛋白膜与hOECs有较好的相容性,可以作为hOECs生长的候选组织工程支架用于修复神经损伤.     

McAb共激活T细胞介导肝癌细胞前后的膜分子与TCR Vβ基因的 …

Ｔ淋巴细胞活化是一个涉及多种膜表面分子和受体以及一系列相关多肽的复杂过程，为了使Ｔ细胞发挥更好的识别和杀伤癌细胞的功能，采用抗ＣＤ３、ＣＤ２８、ＣＤ８０（Ｂ７．１）、ＣＤ２、ＣＤ５８ＭｃＡｂ分别刺激健康人ＰＢＬｓ后作用肝癌细胞，对作用前后ＰＢＬｓ用ＦＡＣＳ进行表型分析，结果发现：作用后ＣＤ３和ＣＤ８分子表达比作用前明显增高，而ＣＤ４分子无显著变化，同时基因家族采用ＲＴ－ＰＣＲ－Ｓｏｕｔｈｅｒｎ印迹     

丝素蛋白材料对免疫细胞激发作用的研究进展

丝素蛋白是一种源于蚕丝的天然高分子材料,其无毒、无刺激、透氧渗水性好.该材料作为诱导型医用生物材料的应用研究已成为热点课题;与此同时,丝素蛋白材料作为＂异种＂抗原物质进入人体后触发免疫应答的研究也正深入开展.机体的免疫系统具有对＂异己＂物质进行识别和攻击的特性,通过激活免疫细胞,产生免疫效应物质包括效应细胞、抗体及细胞因子等,从而对进入机体的＂异己＂物质进行攻击与清除.就丝素蛋白材料对免疫细胞的激发作用研究作一综述.     

Activation of human alloreactive cytotoxic precursor T lymphocytes

The requirements for allogeneic T-cell activation have been studied in experiments with T and/or B cells as stimulator. Although target determinants (TDs, defined by CTL effectors in CML) are present on B and T cells used as target cells, this study indicates that TDs are functionally different when expressed on B and T cells used as stimulator cells, as only B cells can activate CTL precursors. Further, the study confirms that inducing TDs and strong lymphocyte-activating determinants (LADs, defined by proliferation in MLC) can be distinct structures found on two different stimulator B cells. The study suggests that binding of cytotoxic precursor T cells to TDs per se does not allow any detectable activation or start of proliferation and differentiation but requires another function of the stimulator cells in the non-T-cell compartment. The nature of this function is unknown, but it is the background for the first signal received by the TD-specific clones of CTL precursors, resulting in the expression of growth receptors for T-cell growth factor or interleukin 2 which is the second signal necessary for clonal expansion and differentiation.     

Activation of resting T lymphocytes by cross-linked anti-CD3 (T3)

In this study we have examined the role of small numbers of adherent cells in the stimulation of highly purified resting T lymphocytes by cross-linked monoclonal anti-CD3 antibodies (T3). T cells were obtained by 3 cycles of purification, using adherence to plastic surface, nylon wool column separation and rosetting with 2-aminoethyl isothiouronium bromide-treated sheep red blood cells. They were stimulated either with T3 antibodies or with solid-phase rabbit anti-mouse IgG-T3 complexes. In standard high density cultures (1 X 10(5)-2 X 10(5) cells), soluble T3, interleukin (IL) 1 and IL 2, used separately, did not induce proliferation of T cells. Soluble T3 together with IL 2 slightly activated T cells to proliferate. Rabbit anti-mouse IgG-T3 complexes attached to the plastic wells induced stimulation in some cultures. The response was markedly increased after addition of IL 2, but not IL 1. The irregular response of these cultures suggested the presence of another variable signal. When cell numbers were reduced (12 X 10(3) cells), neither cross-linking of the CD3-Ti complex nor addition of exogenous IL 1 or IL 2, alone or in combination, stimulated the T cells to increase DNA synthesis. A positive response, comparable to complete peripheral blood mononuclear cells, was restored with 0.5% supplemented adherent cells, provided that IL 2 was present.     

Activation of influenza-specific memory cytotoxic T lymphocytes by Concanavalin A stimulation

Traditionally, the in vitro activation of virus-specific memory cytotoxic T lymphocytes (CTLs) has been achieved by stimulating the CTLs with antigen-presenting cells (APCs) infected with an appropriate virus or pulsed with virus-specific antigenic peptides. Here, we describe the utilization of the polyclonal activator Concanavalin A (ConA) for in vitro restimulation of memory CTLs from virus-primed mice. Using this simple method, the activation of splenocytes with ConA for 3 days (i) eliminates the need to stimulate with virus-pulsed APCs and (ii) generates CD8⁺ CTLs that exhibit virus specificity and MHC-restricted lytic activity similar to CTLs obtained by conventional viral restimulation. In vitro ConA stimulation of splenocytes from BALB/c mice primed with the A/Texas/77 or A/Japanese/57 strain of influenza virus and from C57L/J mice infected with the A/Texas strain, generated CTLs with specific lytic activity. Hence reactivation of memory CTLs by this method is a general phenomenon rather than a mouse or viral strain-specific one. The ConA stimulation method used here had a recall of long-term (1 year) memory CTLs that effectively lysed virally infected targets. Further ConA-stimulated effector lymphocytes from virally primed animals have been shown to recognize and subsequently lyse target cells pulsed with virus or virus-derived peptides. The ConA reactivation of specific anti-viral CTLs may facilitate (i) studying anti-viral CTL responses and (ii) identifying of viral epitopes when unknown or when appropriate viral stimulation is impossible.     

Activation of functionally distinct subsets of CD4+ T lymphocytes.

The studies summarized in this review have established that cloned lines of CD4+ T cells that produce distinct cytokines differ markedly in their responses to different forms of antigenic stimulation. Furthermore, we are beginning to develop experimental systems for better defining the signals that stimulate the differentiation of resting T cells into functionally distinct subsets. From these studies it is possible to construct the following hypothetical model for the differentiation of mature CD4+ T cells. Resting cells produce IL2 as the principal growth factor, IFN gamma, and little or no IL4. Antigenic stimulation in the presence of IL4 (which may be produced by non-T cells) leads to the preferential expansion of IL4-producing cells. These cells secrete their cytokines maximally when stimulated with antigens presented by B cells, which are also the principal targets of these cytokines. Continued expansion of IL4-producing T cells may require antigen exposures that also stimulate the production of IL1 by macrophages. In the absence of IL4 and IL1 (and in the presence of costimulators that are not yet defined) the T cells that are preferentially expanded belong to the IL2-producing subset. In addition, each subset may produce cytokines that stimulate the expansion of that subset and inhibit the other (Fiorentino et al., 1989). It is apparent that a number of assays and reagents need to be developed if these results are to be extended to physiologic immune responses. First, it is important to identify surface molecules that may serve as phenotypic markers for functionally distinct subsets of CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of human T lymphocytes by crosslinking of anti-CD3 monoclonal antibodies

The proliferation of human T lymphocytes induced by anti-CD3 monoclonal antibodies (mAb) is used as a model for antigen-induced activation via the T cell receptor-CD3 complex. Since both systems are accessory cell (AC)-dependent, an understanding of the role of AC in anti-CD3-induced proliferation may provide an understanding of physiological activation via the T cell receptor. Previous work has implicated receptor crosslinking as an important AC function. To determine its necessity in anti-CD3-induced lymphocyte proliferation, we prepared highly purified T lymphocytes and found that these cells did not respond to the anti-CD3 mAb UCHT1, either alone or with interleukin 1 (IL1), interleukin 2 (IL2), or tetradecanoyl phorbol acetate (TPA). However, the response, as measured by appearance of IL2 receptors and proliferation, was restored by crosslinking with immobilized goat anti-mouse antibodies (GAM) and did not require the addition of IL1, IL2, or TPA. Thus, crosslinking of CD3 receptors was a sufficient signal for proliferation of these cells. Cyclosporine A (CsA) inhibited the activation induced by immobilized UCHT1. Since macrophages are the principle targets of CsA-mediated suppression of mitogen-induced proliferation, but macrophages do not participate in the response to immobilized anti-CD3, this may indicate that CsA was inhibiting crosslinking or a signal generated by it.     

雷公藤对Ly－22．2~＋细胞（抑制性T淋巴细胞）的激活作用

本文用单克隆抗体观察了雷公藤对小鼠脾脏的Ｔｈｙ─１￣＋细胞、Ｌ３、Ｔ＿４￣＋细胞、Ｌｙｔ─２．３￣＋细胞和Ｌｙ─２２．２￣＋细胞（抑制性Ｔ淋巴细胞，Ｔｓ）的影响，发现Ｌｖｔ─２．３￣＋细胞增多的同时（Ｐ＜０．０１），Ｌｙ─２２．２￣＋细胞明显增加，Ｌ３Ｔ＿４￣＋细胞／Ｌｙｔ─２．３＋细胞（即Ｔｈ／Ｔｓ）明显下降（Ｐ＜０，０５），而Ｔｈｙ─１￣＋和Ｌ３Ｔ＿４￣＋细胞比例不变，表明雷公藤在小鼠体内直接激活脾脏Ｔｓ，而发挥其免疫抑制作用。     

Activation of T lymphocytes and autologous mixed lymphocyte reaction in allergic patients

In allergic patients the authors previously observed high proportions of circulating T lymphocytes bearing Ia antigens, assumed to be "activated" T cells. In the present investigation they employed other T cell activation markers (4F2, insulin receptor, MLR4) which differ in the kinetics of appearance upon the surface of stimulated T cells. They report high proportions of Ia and 4F2-positive T cells, normal levels of MLR4-positive T lymphocytes and no insulin binding on T cells. However, T cells of allergic subjects are able to express insulin receptors in PHA-induced culture, such as normal subjects do. The authors conclude that these data, supported by similar observations in autoimmune diseases, indicates differences between in vivo and in vitro features of expression of T cell activation markers. In addition the autologous mixed lymphocyte reaction (AMLR) in atopic patients was studied. The results indicate that AMLR responsiveness is defective in allergic patients.     

Activation of human T lymphocytes I. Requirements for mitogen-induced proliferation of antigen-specific T lymphocyte clones

In a model system for accessory cell (AC)-dependent mitogen-induced T cell proliferation the response of several human antigen-specific, HLA-restricted helper T lymphocyte clones (HTLC) to mitogens was studied. It was found that the HTLC themselves did not or only weakly respond to various mitogens or to oxidation by galactose oxidase, but that the response could be strongly increased if certain tumor cell lines were added to the assay as AC. Pretreatment with lectins or oxidation of either HTLC or AC was effective in stimulating the proliferation of the T cells in this system. Reduction of the aldehydes formed during oxidation completely abolished the stimulatory activity of oxidized B lymphoblastoid cell line. This shows that crosslinking of T cell and AC is required to induce proliferation. When several established cell lines were tested for their capacity to function as AC in this system, profound differences in AC activity were detected. The inability of cells with poor AC activity to stimulate the HTLC was not due to trivial reasons, such as requirements for different mitogen concentrations, a decreased binding of mitogens or suppressive effects. Furthermore, AC activity was not dependent on the presence of Ia antigens on the AC. These findings are discussed with regard to the mechanism of mitogen-induced T cell activation.     

Activation of regulatory T cells instigates functional down-regulation of cytotoxic T lymphocytes in human breast cancer

Regulatory T (Treg) cells are a subpopulation of T cells with the ability to control the responses of both CD4+ and CD8+ T cells. A case–control study was conducted in order to determine the functional attributes of Treg cells within the breast cancer milieu. Triple-color flow cytometry was utilized to study the phenotype expression of CD4+CD25+ Treg cells and CD8+ T cells in autologous tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) derived from 33 patients with stage I–III breast cancer. The prevalence of CD4+CD25+ T cells was significantly higher in TILs than in PBLs. The expressions of FOXP3 and GITR in CD4+CD25+ Treg cells were lower in PBLs than in TILs. Functional studies showed that both granzyme B and perforin were barely expressed in peripheral Treg cells but were highly expressed in Treg cells in the tumor microenvironment. On the contrary, down-regulation of both granzyme B and perforin expressed in the CD8+ cytotoxic T lymphocytes was significantly lower in TILs than in PBLs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules were synchronously up-regulated in CD8+ cytotoxic T cells. The in vitro kinetic study showed that adequate activation of TILs derived from breast cancer tissue could restore the appropriate antitumor immune response.