大鼠皮肤移植排异反应中T淋巴细胞亚群的观察

为了了解在皮肤移植组织排异反应中浸润的不同T淋巴细胞亚群 ,分析其与排异反应的关系。采用HE及免疫组化方法观察大鼠III度烫伤后 2 0例同种异体 自体皮肤混合移植和 15例大张同种异体皮肤移植组织在移植后 4或 5、 7、 14、 2 1和 2 8d时CD4+ 和CD8+ 淋巴细胞的比例。结果表明同种异体 自体皮肤混合移植组 7～ 14d时CD8+ 淋巴细胞明显高于大张同种异体皮肤移植组 (P <0 0 5 ) ,在移植后 4或 5dCD4+ 淋巴细胞明显高于CD8+ 淋巴细胞。大张同种异体皮肤移植组在移植后 7～ 14dCD4+ 淋巴细胞明显高于CD8+ 细胞 ,在植皮后 7～ 2 8d高于同种异体 自体皮肤混合移植组 ,2 1d时差异有统计学意义 (P <0 0 5 )。在 7～ 14d时CD4/CD8比值高于同种异体 自体皮肤混合移植组 (P <0 0 5 )。提示同种异体 自体皮肤混合移植排异反应以CD8+ 淋巴细胞为主 ,而在大张同种皮肤移植排异反应中CD4+ 淋巴细胞起主要作用。     

RDP1258对淋巴细胞功能及移植物存活的影响

目的 探讨RDP1258对T细胞功能及大鼠移植心脏存活时间的影响。方法 人工合成RDP1258，用MTT法分别进行淋巴细胞增殖试验、单向混合淋巴细胞反应(Mixed lymphocyte reaction，MLR)和淋巴细胞毒(Cytotoxic T lymphocyte，CTL)试验。建立大鼠心脏移植模型，观察不同组大鼠移植心脏存活时间。结果 RDP1258显著抑制ConA刺激的淋巴细胞增殖，抑制单向MLR反应体系中应答细胞对刺激细胞的反应能力和CTL的细胞毒功能。RDP1258组大鼠移植心脏存活时间长于对照组。结论 RDP1258体外能抑制由丝裂原或同种异体抗原引起的淋巴细胞增殖反应和CTL的杀伤功能，体内明显延长大鼠移植心脏的存活时间。     

调节性T细胞在大鼠小肠移植急性排斥反应中的作用

目的 分析调节性T细胞在大鼠急性排斥反应中的作用。方法 采用免疫组化SABC染色法，测定BN→LEW大鼠小肠移植急性排斥反应时，外周血及移植肠浸润淋巴细胞中调节性T细胞：CD4^ ,CD8^ ,CD25^ T淋巴细胞及相关细胞因子IL－4和IFN－γ的表达，并与同基因大鼠间小肠移植（BN→BN）作比较。结果 外周血淋巴细胞分析显示，大鼠小肠移植急性排斥反应时，以CD4^ ,CD25^ ,T细胞为主，CD8^ 淋巴细胞只占少部分；分泌IL－4的细胞在术后4，7，14d分别只占14．3％，16．2％和16．9％。移植肠基底浸润的淋巴细胞以CD4^ ,CD25^＋和分泌IFN－γ的淋巴细胞为主。结论 在大鼠同种小肠移植中，急性排斥反应与CD25^ ,CD4^ T细胞及Th1相关细胞因子IFN－γ的表达增加相关。而CD8^＋T淋巴细胞和Th2相关细胞因子IL－4可能具有保护作用。     

抗IL-2受体单抗选择性保留的T抑制细胞

抗IL-2R 单抗是一种免疫抑制剂,体内作用可通过清除灭活IL-2R 阳性细胞来实现,并同时保留了部分抑制细胞。这类T 抑制细胞,可抑制混合淋巴细胞反应,抑制同种免疫排斥反应;协同抗IL-2R 单抗清除灭活IL-2R 阳性细胞的免疫抑制作用,阻止初次排异反应的发生。这类T 抑制细胞可为CD_4或CD_8表型,它们不表达IL-2R 或不表达为抗IL-2R 单抗所识别的表位,从而被选择性地保留下来。     

TLSFJM对小肠移植大鼠外周血及移植肠浸润T细胞的影响

目的：观察TLSFJM对大鼠小肠移植受体外周血及移植肠浸润T细胞的影响和对排斥反应的抑制效应。方法：应用免疫组化SABC法检测受体外周血及移植肠CD4^ 、CD8^ 、CD25^ 淋巴细胞和IL-4、IFN-γ的表达。结果：TLSFJM显著抑制外周血及移植肠浸润淋巴细胞CD4^ 、CD8^ 、CD25^ 和IFN-γ的表达，并提高IL-4的表达，显著延长受体大鼠的生存时间，但不能防止体质量下降，移植肠仍有排斥反应的病理组织学征象。结论：TLSFJM通过影响受体外周血及移植肠浸润T淋巴细胞对大鼠小肠移植发挥免疫抑制作用，但单独使用尚不足以作为预防和控制急性排斥反应的免疫抑制剂。     

小鼠FasL基因的克隆和在软骨细胞中表达

目的：获得小鼠FasL(mFasL)的cDNA克隆，通过逆转录病毒表达系统pLNCX2在软骨细胞中进行表达，并分析其功能。方法：应用RT—PCR和T-A克隆技术，从激活的小鼠脾脏淋巴细胞总RNA中，扩增mFasL cDNA并克隆到T载体中，再亚克隆到pLNCX2中。转染软骨细胞后，以流式细胞仪检测mFasL蛋白的表达，以单向混合淋巴细胞反应鉴定其活性。结果：成功地克隆了0．855kb的mFasL cDNA，并经测序确证。通过pLNCX2转染软骨细胞，转染率为60．64％。流式细胞仪检测到FasL蛋白的表达，并能显著抑制同种异体的单向混合淋巴细胞反应，刺激指数下调到未转染软骨细胞的11．71％。结论：克隆到的mFasL基因，并能通过pLNCX2有效表达。表达产物具有显著抑制同种异体单向混合淋巴细胞反应的活性。     

抗CD71人-鼠嵌合抗体对活化淋巴细胞的效应

目的　通过体外实验探讨抗CD71人 鼠嵌合抗体对活化淋巴细胞效应的影响 ,并与其鼠源性单克隆抗体进行比较。方法　以丝裂原诱导的人外周血单个核细胞 (PBMC)为靶细胞 ,测定嵌合抗体和鼠源性单抗对其增殖抑制率 ;在新鲜补体存在下 ,测定补体依赖性嵌合抗体介导的细胞毒效应(CDC)。以EBV转化的B细胞为刺激细胞 ,测定两种抗体对其诱导的同种异体PBMC的增殖抑制率 ;以同种异体的PBMC为刺激细胞 ,测定两种抗体在单向、双向混合淋巴细胞培养 (MLC)中的增殖抑制率。结果　嵌合抗体和鼠源性单抗均可明显抑制丝裂原诱导的PBMC的增殖反应 ,且二者抑制率差异无显著性(P >0 .0 5 ) ,其抑制作用随抗体浓度增加而增强 ,PBMC和丝裂原共同孵育 12h后加入抗体的增殖抑制效应最明显 ;在新鲜补体存在下 ,嵌合抗体对丝裂原诱导增殖的PBMC具有CDC作用 ,而鼠源性单抗CDC作用较弱 ;两种抗体对混合淋巴细胞培养反应有明显的抑制作用 ,且嵌合抗体组抑制率明显高于鼠源性单抗组 (P <0 .0 5 )。结论　抗CD71人 鼠嵌合抗体在体外实验中可抑制淋巴细胞的活化及其效应 ,其作用明显强于抗CD71鼠源性单克隆抗体。     

异体角膜刺激引起的人外周血T细胞及其亚群CD25分子表达

目的：观察异体角膜体外对人外周血T细胞及其亚群CD25分子表达的影响。方法： 异体角膜与外周血淋巴细胞体外共同培养后，进行单克隆抗体标记和流式细胞分析。结果： 对照组T细胞CD25表达为25.2％；受角膜或沸波醇酯（PDB）激活后T细胞CD25表达分别为56.8％和80.9％；受角膜和沸波醇酯共同激活后T细胞CD25表达为70.2％；受异体角膜激活后，CD4和CD8 T淋巴细胞CD25表达分别为67.3％和52.3％。结论： 异体角膜组织体外能够刺激人外周T细胞CD25表达和T细胞活化，CD4 T细胞比CD8 T细胞活化更明显。     

霉酚酸及其衍生物对人T淋巴细胞及混合淋巴细胞免疫应答的影响

目的探讨霉酚酸及霉酚酸衍生物对人T淋巴细胞和混合淋巴细胞免疫应答的影响。方法从正常人外周血中分离单个核淋巴细胞(PBMC),在抗CD3刺激下和混合淋巴细胞反应体系中,加入或不加霉酚酸及其衍生物共培养,应用ELISA和流式细胞术检测CD4+和CD8+T细胞细胞因子的产生、细胞活化和增殖情况。结果经抗CD3刺激后,PBMC产生的细胞因子明显增加,加入霉酚酸及其衍生物后,IFN-γ及TNF-α明显降低,IL-2产生略有增加。在混和淋巴细胞反应体系中,3种细胞因子产生皆被抑制。抗CD3刺激和混和淋巴细胞反应后,T细胞表面活化分子CD25及CD69表达增加,加入霉酚酸及其衍生物共培养24、48 h后,T细胞CD25及CD69的表达并未被抑制。霉酚酸及其衍生物可抑制抗CD3刺激下和混和淋巴细胞反应后T细胞的增殖。结论霉酚酸及其衍生物对T淋巴细胞和混合淋巴细胞体系中因子的产生和细胞增殖都有抑制作用,为其在临床自身免疫疾病及移植排斥中的广泛应用提供了理论依据。     

天花粉蛋白诱发CD8阳性细胞参与的人体免疫抑制

已证明0.05～5μg/ml 剂量范围内的天花粉蛋白对同种异体抗原、可溶性抗原、致分裂原诱发的人体淋巴细胞体外增殖的强烈抑制作用,与细胞裂解无关。本文提供的下列证据进一步表明,天花粉蛋白诱发的免疫低反应性属于免疫抑制:①经该蛋白脉冲处理过的 PBMC 照射后加到不含天花粉的培养组合中可抑制新鲜PBMC的增殖;②天花粉蛋白脉冲处理过的PBMC培养上清液显示抑制功能;③去除CD8阳性细胞抑制缓解;回加 CD8阳性细胞抑制再现,提示有CD8抑制性T细胞参与反应。     

山茱萸总甙抑制免疫的体内效应及其对移植心脏存活的延长

腹腔注射山茱萸总甙(COG)50mg/kg·d×5和100mg/kg·d×5,小鼠淋巴细胞产生白细胞介素2(IL—2),分别由2.28±0.48U/ml下降为1.65±0.29U/ml和0.90±0.20U/ml。小鼠的MLR也因预先注射COG而显著下降。小鼠移植心脏存活时间,在COG治疗组由9.2±1.2天延长到17.4±6.7天,而且COG可在环孢霉素A(CsA)10mg/kg·d×4基础上,由15.0±1.6天延长到19.0±3.2天。     

间充质干细胞对混合淋巴细胞反应体系中T淋巴细胞的免疫调节作用

目的:探讨间充质干细胞(MSC)在体外对T淋巴细胞免疫调节作用的特点。方法:通过Ficoll梯度密度离心法分离出正常人骨髓单个核细胞,体外培养扩增MSC,获取第3代细胞。将其按照不同的比例加入双向混合淋巴细胞培养(MLC)体系中,在第3、5天,采用MTT比色法检测各组MLC中的T淋巴细胞的增殖情况,再用流式细胞术分析其与MSC共孵育前后细胞表面标记的变化情况。结果:加入了MSC的MLC体系中,MSC对T淋巴细胞的增殖抑制具有剂量依赖性,且随着时间延长,抑制程度增强;其中,CD4 T细胞亚群受抑不如CD8 T细胞亚群显著;另外,T淋巴细胞表面CD25的表达虽然较对照组有所下降,但是CD4、CD25共表达的细胞却较对照组有明显的上升趋势。T淋巴细胞表面活化抗原HLA-DR的表达较对照组有轻度减低。结论:MSC在体外能够明显抑制T淋巴细胞的增殖,其主要是针对CD8 T细胞(CTL)。另外,它还能下调活化T淋巴细胞上一些较特异的表面标记CD25和HLA-DR的表达。     

J2对同种异体穿透性角膜移植模型大鼠CD4+和CD8+T细胞免疫功能的抑制

BACKGROUND: J2 takes functional domain (MHC CD4-D1/) of complex conjugate of CD4 molecule and MHC class II molecule as a target, and is a small molecule compound obtained by computer screening from a chemical data containing hundreds of thousands of organic compounds. In the previous study, J2 was used in mouse models of skin transplantation and keratoplasty by oral and intraperitoneal injection. Results verified that J2 could prolong the survival time of grafts, and suppress occurrence of rejection. To better play the role of a drug targeting and to reduce systemic toxicity, J2 will be further utilized in local treatment of keratoplasty rejection. OBJECTIVE: To investigate the inhibitory effect of new immunosuppressive agent J2 on CD4+ and CD8+ T cell immune functions in rat models receiving allogenic penetrating keratoplasty. METHODS: Allogeneic penetrating keratoplasty model was established using the adult female Wistar rats as donors and Sprague-Dawley rats as recipients. Group A: normal Sprague-Dawley rats were injected with 0.05 mL placebo subconjunctivally. Surgery rats were randomly divided into three groups. Group B: allograft rats were injected with 0.05 mL placebo subconjunctivally after autologous keratoplasty. Group C: allograft rats were injected with 0.05 mL placebo subconjunctivally. Group D: allograft rats were injected with 1% J2-nanosuspension 0.05 mL subconjunctivally. The distribution of T cell subsets in peripheral blood was detected using flow cytometry at 3 days, 1, 2 and 3 weeks after transplantation and compared among groups.  RESULTS AND CONCLUSION: There was no significant difference in total CD3+ T cells, CD4+ T cells, CD8+ T cells and CD4+/CD8+ in peripheral blood lymphocytes in group B at various time points. At 3 days and 1 week after surgery in group C, no significant difference in total CD3+ T cells, CD4+ T cells and CD8+ T cells was detected. At 1 and 2 weeks, the number of total CD3+ T cells, CD4+ T cells and CD8+ T cells increased, showing significant differences (P < 0.05). In group D, no significant hyperplasy was found in CD4+ T cells and CD8+ T cells at 1 and 2 weeks. The horizontal comparison of the same time point: the total CD3+ T lymphocytes of group D was significantly less than group C at 3 days, 1 and 2 weeks after operation (P < 0.05), whereas there was no significant difference at 3 weeks between the group D and group C. The number of CD4+ T lymphocytes in group D was less than in group C at 3 days and 1 week, but with no significant difference. The ratio of CD4+/CD8+ had no significant difference in group D compared with group C at 3 days, 1 and 3 weeks. J2 inhibits T lymphocyte proliferation and then inhibits T cell-mediated corneal allograft rejection.       

山茱萸总甙滴眼液防治角膜移植免疫排斥反应的实验研究

目的:观察山茱萸总甙(COG)滴眼液局部应用对角膜移植免疫排斥反应的影响。方法:建立封闭群大鼠角膜移植模型,用裂隙灯显微镜记录及比较各组角膜排斥反应发生时间;并进行病理学和扫描电镜检查。结果:病理学检查B、C组15d时的角膜植片淋巴细胞浸润及新生血管明显少于A组。免疫组化显示:ICAM-1的表达在15d时B、C组明显少于A组。结论:COG滴眼液能有效地防治角膜移植免疫排斥反应。     

Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA-stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA-stimulated lymphocytes dose-dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor-beta (TGF-beta). Stromal-cell-derived factor-1 (SDF-1) is not expressed on the cell surface. A recent report suggested that T-cell suppression by MSC is mediated by HGF and TGF-beta. MSC suppression was not restored by the addition of neutralizing antibodies against SDF-1, OPG, HGF or TGF-beta, alone or in combination. Addition of guanosine to PHA-stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA-activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.     

基因修饰树突状细胞诱导前列腺癌患者外周血T细胞亚群重排的研究

目的探讨以腺相关病毒（AAV）为载体,前列腺特异性抗原（PSA）基因转染树突状细胞（DC）诱导前列腺癌患者外周血T细胞亚群变化特点及临床意义。方法抽取30例前列腺癌患者外周血,采用密度梯度离心法分离外周血单个核细胞,以rAAV/PSA感染DC前体细胞,采用系列细胞因子诱导DC前体细胞成熟。第6天收集成熟DC并与T细胞按比例混合培养,诱导细胞毒性T淋巴细胞（CTL）。分别于DC与T细胞混合培养前后应用流式细胞术分析外周血T细胞亚群及调节性T细胞（CD4^＋CD25^＋FoxP3^＋Treg）的表达水平。结果 PSA基因转染DC刺激T淋巴细胞爆发增殖,与培养前比较,混合培养6d后CD8^＋、CD8^＋CD69^＋、CD8^＋CD28^＋T细胞的比例和CD8^＋/CD4^＋比值均明显增高,差异有统计学意义（P〈0.01）;而CD8＋CD28-T细胞和Treg细胞的比例均显著降低,差异有统计学意义（P〈0.01）。CD4^＋T细胞比例较前略有升高,但差异无统计学意义（P〉0.05）。结论 PSA基因转染DC能够有效地激活CD8＋抗原特异性CTL,下调免疫抑制性T细胞,提高患者的细胞免疫功能,为前列腺癌的免疫治疗提供新的有效策略。     

Novel mode of action of the calcium antagonist mibefradil (Ro 40-5967): potent immunosuppression by inhibition of T-cell infiltration through allogeneic endothelium.

Cyclosporin A reduces the mitotic activity of allosensitized lymphocytes, but fails to limit emigration of these cells into the donor organ. However, the modulation of both lymphocyte proliferation and infiltration are desirable characteristics of immunosuppressive therapy. The calcium-channel blocker, verapamil, has recently been shown to effectively prevent the transmigration of CD4+ and CD8+ T cells through allogeneic endothelium. Mibefradil (Ro 40-5967) represents a new generation of calcium antagonists with high potency and long-term activity. To evaluate the immunosuppressive potential of this drug, the influence of mibefradil on lymphocyte adhesion to, horizontal locomotion along, and penetration through allogeneic endothelium (HUVEC) was performed. When lymphocytes were prestimulated for 24 hr with mibefradil, adhesion and penetration were dose-dependently reduced. The adhesion ID50 values were 3.4 microM (CD4+ T cells) versus 9.2 microM (CD8+ T cells) and 2.1 microM (CD4+ T cells) versus 3.9 microM (CD8+ T cells) with regard to penetration. Mibefradil also effectively blocked horizontal locomotion. Specific down-regulation of T-cell binding to the P-selection receptor (ID50: CD4+ T cells, 0.8 microM: CD8+ T cells, 1.2 microM) and to the intracellular adhesion molecule-1 (ICAM-1) receptor (ID50: CD4+ T cells, 1.9 microM; CD8+ T cells, 1.5 microM) by mibefradil seems to be responsible for the decreased adhesion and penetration rates. Reduction of intracellular F-actin in T lymphocytes could diminish cell locomotion. In conclusion, the potent suppressive properties of mibefradil support its use as a co-medication in cyclosporin A-based immunosuppressive therapy.     

Role of bone marrow stromal cells in the generation of human CD8+ regulatory T cells

Fibroblast-like stromal cells exert a strong inhibitory effect on lymphocyte proliferation, both directly by interacting with responding lymphocytes and indirectly by inducing the generation of regulatory T cells. Indeed, upon triggering via the CD3/TCR complex, highly effective CD8(+)regulatory cells (CD8(+)Reg(c)) are generated from cocultures of peripheral blood CD8(+)T cells and bone-marrow-derived stromal cells. When cell-to-cell interactions occur, CD8(+)Reg(c) strongly inhibit lymphocyte proliferation at a ratio of 1:1 to 1:100 between CD8(+)Reg(c) and responding lymphocytes. Phenotypic analysis indicated that CD8(+)Reg(c) are CD25(+)CD28(+) and express low levels of mRNA for Foxp3 but they do not bear CTLA4 and glucocorticoid-induced tumor necrosis factor receptor antigens. Soluble mediators such as interleukin-10, transforming growth factor-beta, and prostaglandin E(2) are not involved in the generation of CD8(+)Reg(c) from CD8(+) precursors or in the immunosuppressive mechanism mediated by CD8(+)Reg(c) on lymphocyte proliferation. Cyclosporin A (CSA) slightly downregulated generation of CD8(+)Reg(c) indicating that only a small fraction of precursors of CD8(+)Reg(c) are sensitive to this immune-suppressive drug. Along this line, treatment of effector CD8(+)Reg(c)with CSA does not affect their immunosuppressive effect, indicating that the molecular mechanism of CD8(+)Reg(c)-mediated regulation is independent of the function of CSA biochemical target molecules.     

急性心肌梗塞患者外周血T淋巴细胞表达CD25及其对刺激剂反应能力的变化

目的：研究急性心肌梗塞患者外周血T淋巴细胞表达CD25及其对刺激剂反应能力的变化。方法：流式细胞仪技术。结果：急性心肌梗塞患者外周血淋巴细胞CD25 、CD4 和CD4 ／CD8 比值明显低于对照组,CD8 高于对照组;CD3 CD25 、CD4 CD25 和CD4 CD25 /CD8 CD25 显著降低。在培养前后,CD3 CD25 表达均显著降低;在PDB刺激情况下,CD3 CD25 、CD8 CD25 表达均显著降低。结论：急性心肌梗塞患者存在细胞免疫功能紊乱,并存在对刺激剂PDB的反应能力降低。     

三种赤灵芝粉对免疫抑制模型小鼠免疫功能的调节作用

目的:为了探讨灵芝调节机体免疫功能的作用机制,并寻找出一种能有效改善机体免疫功能的灵芝制剂,本文研究了三种赤灵芝粉对免疫抑制小鼠免疫功能的调节作用。方法:实验动物随机分为5组(Ⅰ～Ⅴ组),Ⅱ～Ⅴ组采用腹腔注射环磷酰胺(CTX)70 mg.kg-1.bw-1,隔日注射,共4次,以制备动物模型,Ⅰ组注射等体积生理盐水。Ⅲ～Ⅴ组于注射CTX次日及末次注射CTX后11日内分别灌胃给予赤灵芝破壁孢子粉(D1)、赤灵芝孢子粉(D2)和赤灵芝精粉(D3)(1g.kg-1.bw-1),定期监测小鼠体重;给药后无菌取脾脏称重,计算脾指数;采用小鼠腹腔巨噬细胞吞噬鸡红细胞试验检测巨噬细胞吞噬功能;采用NK细胞介导的细胞毒试验检测NK细胞功能;采用淋巴细胞转化试验检测T、B淋巴细胞功能;采用流式细胞术检测CD4+T及CD8+T细胞比例。结果:与免疫抑制组相比,小鼠给予灵芝粉后,三组小鼠体重均升高(P<0.05);D3组脾指数降低;D1和D2组小鼠的腹腔巨噬细胞吞噬指数较免疫抑制组升高(P<0.05);三组小鼠NK细胞杀伤活性无明显变化;脾脏T、B淋巴细胞增殖能力无明显变化;D3组小鼠脾脏CD4+和CD8+T细胞比例明显升高,特别是CD4+T细胞升高最为显著且较正常对照高(P<0.05)。结论:三种灵芝对免疫抑制模型小鼠的免疫功能具有调节作用,尤其是赤灵芝精粉具有显著提高CD4+T细胞数量的作用,为其临床应用提供了实验依据。