环孢素A体外诱导HL—60细胞凋亡的研究

目的：观察环孢素Ａ是否有诱导白血病细胞凋亡的作用。方法：应用细胞形态学检查、二苯胺法ＤＮＡ片段化定量，ＤＮＡ凝胶电泳及流式细胞术等方法观察细胞凋亡。结果：ＣｓＡ５０ｍｇ／Ｌ作用ＨＬ－６０细胞４ｈ，ＤＮＡ片段率显著增高，达（２８．２±５．８）％，而对照组仅为（１２．５±１．７）％（Ｐ＜０．０１，ｎ＝１０）。光镜及电镜检查见细胞核固缩，碎裂，有凋亡小体形成。ＤＮＡ电泳显示典型的ＤＮＡｌａｄｄｅｒ。流式细胞术检测发现ＣｓＡ５０ｍｇ／Ｌ作用ＨＬ－６０细胞６ｈ凋亡细胞率为４９．７％，而空白组仅为９．１％。ＣｓＡ诱导ＨＬ－６０细胞ＤＮＡ片段化呈剂量和时间依赖性。结论：ＣｓＡ在体外能诱导ＨＬ－６０细胞凋亡。     

双氢青蒿素对卡氏肺孢子虫肺炎大鼠肺泡巨噬细胞凋亡的影响

目的：检测双氢青蒿素对卡氏肺孢子虫肺炎（PCP）大鼠肺泡巨噬细胞凋亡的影响。方法：以醋酸可的松皮下洲Wistar大鼠建立PCP动物模型，对60mg/kg双氢青蒿素治疗实验大鼠，杀鼠取肺，用胶原酶消化法分离肺泡巨噬细胞，可PI和TUNEL法检测其凋亡，同时设有正常大鼠对照组。结果：感染组和治疗组大鼠肺泡巨噬．细胞凋亡率显著高于正常对照组，治疗组大鼠肺泡巨噬细胞凋亡率明显低于感染组。结论：卡氏肺孢子虫感染引起大鼠肺泡巨噬细胞发生凋亡，经双氢青蒿素治疗后PCP大鼠肺泡巨噬细胞凋亡降低。     

RNAi介导Caspase-3基因及其抑制LPS诱导肾脏上皮细胞凋亡的研究

目的：探讨RNA干扰Caspase-3基因及抑制LPS诱导的肾脏上皮细胞凋亡的作用效果。方法：构建针对大鼠Caspase-3基因编码区的短发夹状RNA（shRNA）真核表达载体质粒pRNAT—U6．1-Caspase-3shRNA，用电穿孔法转染RK3E细胞，经G418筛选后，形成稳定的表达Caspase-3shRNA的细胞系。实验分为3组：①正常对照组：未转染的RK3E细胞；②阴性对照组：转染空载体pRNAT-U6．1的RK3E细胞系；③实验组：转染Caspase-3shRNA的RK3E细胞系。经脂多糖（LPS）孵育12小时后，流式细胞仪检测各组细胞凋亡率，RT-PCR检测mRNA的表达，以及各组Caspase-8蛋白的活性。结果：与正常对照组和阴性对照组相比，实验组的细胞凋亡率显著降低（P〈0．01），Caspase-3的mRNA水平和蛋白活性显著降低（P〈0．01）。结论：针对Caspase-3的特异性短发夹RNA可以明显引起靶基因的沉默，进而抑制LPS诱导的肾脏上皮细胞凋亡的发生。     

氢化可的松对人外周血来源树突状细胞的诱导凋亡作用研究

旨在探讨氢化可的松对人外周血单核细胞来源树突状细胞 (DC )的凋亡诱导作用及其表型、功能的变化 ;采用细胞因子体外诱导人外周血来源DC ,加入不同浓度的氢化可的松进行培养 ;以细胞计数、PI染色测定细胞周期、间接荧光表型分析、ELISA法测定DC分泌的IL 12和3H TdR掺入测定DC对T细胞的激发作用 ;实验结果表明 ,外周血贴壁的单核细胞在细胞因子的诱导下可以分化为成熟的DC ,不同浓度 (2 μg/ml～ 5 0 μg/ml)的氢化可的松可下调DC表达的CD80和HLA DR分子 (分别下降 41 1%和 10 9% ) ,并使其分泌IL 12的能力明显下降 (由 38 1pg下降为 0 ) ,DC对自体T细胞的激发功能明显降低 ,而且一定浓度的氢化可的松还可以使DC发生凋亡 (凋亡率可达 5 6 6 % )。糖皮质激素不仅通过下调DC表达的协同刺激分子及其分泌的IL 12从而影响DC对T细胞的激发作用 ,而且可以直接诱导DC发生凋亡 ,从而下调免疫应答的发生。     

雌激素对SLE模型小鼠脾脏细胞凋亡的影响及其机制

目的研究雌激素对系统性红斑狼疮（SLE）模型小鼠的脾脏细胞凋亡及其相关调控机制。方法采用ConA活化的同系脾脏细胞诱导BALB／c小鼠SLE，同时肌注一定剂量的苯甲酸雌二醇。于第4周、第6周、第8周和第10周采用ELISA法检测血清雌二醇水平，TUNEL法检测脾脏细胞凋亡，免疫细胞化学法检测脾脏细胞Bcl-2和NFκB的表达。结果与对照组比较，肌注雌激素的SLE模型小鼠脾脏细胞凋亡率明显降低，Bcl-2和NFKB的表达均增加（P〈0．01）。结论雌激素抑制SLE模型小鼠脾脏细胞凋亡，可能是通过促进Bel-2和NFKB的表达所致。     

T细胞疫苗诱导异种特异性免疫耐受的作用探讨

目的：探讨T细胞疫苗诱导异种特异性免疫耐受的作用。方法：制备SD大鼠针对脉鼠的T细胞疫苗(TCV)，然后用T细胞疫苗免疫正常SD大鼠，连续3周，每周1次，同时设空白对照组。以被免疫SD大鼠的脾细胞作为反应细胞，以豚鼠的脾细胞作为刺激细胞(经丝裂霉素处理)，于接种前和每次接种后第5天进行单向混合淋巴细胞反应(MLR)，同时对外周血T细胞凋亡和CD4^ 、CD8^ T细胞亚群进行检测。结果：MLR及结果显示，在TCV组，SD大鼠的脾细胞反应程度比接种前显著减弱(P＜0．01)，接种后相同时点组间比较，TCV组显著低于对照组(P＜0．01)；接种T细胞疫苗后，外周血T细胞凋亡率比接种前显著增加(P＜0．01)，并随着接种次数的增加而升高；随着接种T细胞疫苗次数的增加，CD/CD8逐渐减小，与MLR及的cpm值的递减趋势一致。结论：T细胞疫苗成功地诱导出异种特异性免疫耐受。T细胞疫苗可能通过诱导抗原特异性的反应性T细胞凋亡去除独特型T细胞克隆，进而诱导出抗原特异性免疫耐受；CD4/CD8与免疫耐受的产生有关，CD4/CD8降低有利于特异性免疫耐受的形成。     

用原位缺口翻译法比较两种细胞死亡的DNA状态

用原位缺口翻译法比较了细胞凋亡和坏死所伴随的ＤＮＡ断裂类型。ＩＮＴ实验条件为：取大鼠小肠制备冰冻切片，乙醇／醋酸固定，加２０μｍｏｌ／Ｌｂｉｏｔｉｎ－１１－ｄＵＴＰ、０．２ＩＵ／ＬＤＮＡ聚合酶Ｉ，小于３７℃中孵育３ｈ。正常大鼠小肠绒毛顶端上细胞可迅速被染色，而位于肠绒毛基底部的Ｓ期上皮细胞则不着色，仅在用蛋白酶Ｋ处理后才着色。细胞凋亡体本取自皮下注射氢化可的松的大鼠胸腺；细胞坏死用ＣＣＬ４（１００     

DR5在TRAIL诱导Jurkat细胞凋亡中的作用

目的：研究DR5在介导TRAIL凋亡信号中的作用。方法：用含人DR5细胞外全长结构域重组DR5免疫BALB／C小鼠，制备抗DR5单克隆抗体；流式细胞仪检测Jurkat细胞表面DR5表达水平；采用TRAIL凋亡检测试剂盒，检测Jurkat细胞凋亡率及抗DR5单克隆抗体对TRAIL诱导细胞凋亡的阻断率。结果：DR5在Jurkat细胞表面的表达率为94．83％，TRAIL和抗TRAIL单克隆抗体能够诱导Jurkat细胞凋亡，呈现明显的剂量相关性，TRAIL浓度在50-100ng/ml时，杀伤率达90％以上。预先用抗DR5单克隆抗体与Jurkat细胞作用后，TRAIL对Jurkat细胞的杀伤功能几乎完全被mAb所阻断，其平均阻断率达90．49％。结论：DR5在TRAIL诱导Jurkat细胞凋亡中起着十分关键的作用。     

放线菌酮诱导大鼠脾淋巴细胞凋亡的初步探讨

近来研究认为，细胞凋亡（Ａｐｏｐｔｏｓｉｓ）与免疫细胞分化发育有着密切关系。但目前，凋亡与淋巴细胞的关系的研究大多采用体外实验方法，为了更有利于研究淋巴细胞凋亡的发生机制，我们建立了体内诱导大鼠脾脏淋巴细胞凋亡的方法。健康雌性ＳＤ大鼠禁食２４小时后，用乙醚麻醉后从腹腔或静脉注射５．０ｍｇ，ｋｇ－１的放线菌酮（Ｃｙｃｌｏ－ｈｅｘｉｍｉｄｅ）。４小时后处死动物，取出脾脏，常规固定、脱水和石蜡包埋。细胞凋亡判断依据光镜下典型的凋亡细胞形态变化并结合原位３′－末端标记方法。脾脏组织还进一步作免疫组化染色…     

IFN-γ对TRAIL诱导HBV相关性肝癌细胞凋亡的调节作用

目的：以转染了HBV全基因组、并稳定表达HBV病毒颗粒的HepG2.2.15细胞为细胞模型，探讨IFN-γ对新型凋亡分子TRAIL（TNF相关的诱导凋亡配体）诱导凋亡的影响，并初步探索其发生机制。方法：以流式细胞仪检测IFN-γ和TRAIL作用前，HepG2.2.15细胞的凋亡率，分别用流式细胞术和半定量RT－PCR的方法检测IFN-γ用0、6、12和24h后，细胞表面膜结合型TRAIL和TRAIL受体mRNA的表达。用HBsAg和HBeAg ELISA检测试剂盒测定IFN-γ作用0、6、12和24h后，HBV病毒颗粒的分泌表达状况。结果：TRAIL单独作用、IFN-γ单独作用、TRAIL和IFN-γ联合作用后，HepG2.2.15细胞的凋亡率分别为9．12％、5．84％和46．68％，表明IFN-γ可以显著上调TRAIL诱导的细胞凋亡。IFN-γ作用后，细胞表面膜型TRAIL的表达有显著上调，而部分与TRAIL诱导凋亡相关的TRAIL受体的表达也有不同程度的上调，并且IFN-γ可以抑制HepG2.2.15细胞HBV病毒颗粒的分泌。结论：转染HBV全基因组的HepG2.2.15细胞对TRAIL诱导的凋亡相对耐受,而IFN-γ却可以逆转这种耐受，使其变得对TRAIL诱导的凋亡敏感，其发生机制可能是通过IFN-γ上调细胞表面TRAIL及其受体的表达，以及抑制HBV病毒颗粒的分泌实现的。     

Hydrocortisone reverses the suppression of immunoglobulin synthesis by concanavalin A-activated spleen cell supernatants.

Supernatants of concanavalin A (Con A)-activated human spleen cells have been previously shown to inhibit polyclonal immunoglobulin (Ig) biosynthesis by pokeweed mitogen (PWM)-stimulated human spleen and peripheral blood mononuclear cells. In the present study, hydrocortisone was added at the beginning of in vitro culture to determine whether it might influence the immunoregulation of polyclonal IgG, IgA and IgM biosynthesis by PWM-stimulated human spleen and peripheral blood mononuclear cells. Hydrocortisone (10(-5) m) mildly increased (15 +/- 9%; mean +/- s.e.m.) polyclonal Ig biosynthesis when added to PWM-stimulated human mononuclear cells. The addition of supernatants from Con A-activated human spleen cells to PWM-stimulated human spleen and peripheral blood mononuclear cells significantly (P less than 0 . 001) suppressed (94 +/- 2%) polyclonal Ig biosynthesis. In contrast, when hydrocortisone (10(-5) m) was added together with Con A supernatants to PWM-stimulated cells, there was no significant suppression (6 +/- 13%) of polyclonal Ig synthesis. Thus, one mechanism by which hydrocortisone can influence Ig biosynthesis is by blocking the suppressive effect of a soluble suppressor factor secreted by Con A-activated human spleen cells.     

Suppression of xenogeneic local graft versus host reaction by rat fetal spleen cells

Xenogeneic local graft versus host reaction was induced by the injection of 5 X 10(7) adult rat spleen cells under the capsule of cyclophosphamide-treated adult mice. A significant decrease in the reaction, measured by the kidney enlargement index, was obtained by adding to the reacting cells 5-20% of fetal rat spleen cells. Fetal rat thymocytes of fetal mouse splenocytes were not found to be inhibitory. The suppressive activity of rat spleen cells waned completely on day 3 of the postnatal life.     

Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, phenylbutazone and oxyphenbutazone in equine serum by high-performance liquid chromatography

Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite the occurrence of significant degradation of OPB and PB during the analysis) and precision (intra-assay and inter-assay C.V.'s of about 3-6 and 9-15%, respectively) of the method appeared appropriate for anti-doping control of racehorses.     

Effect of hydrocortisone on the reactivity of thymus and spleen cells of mice to in vitro stimulation

Treatment of mice with hydrocortisone reduced the number of thymus-cells to 6 per cent and the number of spleen-cells to 32 per cent of matched controls. Per spleen, an absolute decrease of cells carrying immunoglobulin receptors on the surface as determined by immunofluorescence was found and a smaller decrease in cells with theta-antigen. Equal numbers of thymus and spleen cells from hydrocortisone-treated mice and matched controls were cultured and stimulated with phytohaemagglutinin (PHA), pokeweed mitogen (PWM), allogeneic cells, and, following immunization, with keyhole limpet haemocyanin (KLH). Stimulation was assessed by incorporation of [³H]thymidine into the acid precipitable fraction of the cultured cells. With thymus cells, hydrocortisone treatment increased the reaction to PHA and allogeneic cells. Thymus cells from untreated animals already gave a good response to PWM, further increased by treatment with hydrocortisone. With spleen cells, hydrocortisone treatment reduced the reaction to KLH, PWM, allogeneic cells and PHA in decreasing order. The results are discussed with reference to cells affected by hydrocortisone treatment and to the mechanism of in vitro lymphocyte stimulation.     

Analysis of rat hemopoietic cells using monoclonal antibodies

Rat hemopoietic cells were analyzed with immunohistochemical technique, binding inhibition assay and flow cytometer using a monoclonal antibody (UB-12) to rat fetal liver hemopoietic cells. UB-12 positive cells were recognized in only red pulp but not in white pulp of spleen. The number and fluorescence intensity of UB-12 positive cells in spleen appeared to reach to peak at 6 weeks old occupying about 60 to 70% of total cells in red pulp. On the other hand, OX-7 (anti-Thy-1) positive and W3/13 (anti-leuko-sialoglycoprotein) positive cells were found in both red and white pulp, but not in marginal zone of spleen. UB-12 antigen was found on the surface of the cells only in the early stages of hemopoiesis: relatively large nuclei of UB-12 positive cells were rich in heterochromatin. There were a large number of free-ribosomes and some mitochondria in cytoplasm, and a centriole was observed in cytoplasm at some sections of UB-12 positive cells. From the EPICS analysis of adult rat bone marrow cells using UB-12, OX-7 and W3/13 monoclonal antibodies, the percent of UB-12, OX-7 and W3/13 positive cells was 82%, i.e., 18% was negative from these monoclonal antibodies. UB-12 single positive, OX-7 single positive and W3/13 single positive cells were 7%, 7% and 47%, respectively. The percent of triple positive cells with these antibodies was about 2%.     

Responses of lymphocytes of hydrocortisone-stimulated and adrenalectomized mice to h alloantigens

The ability of spleen, bone marrow, and thymus cells of intact and adrenalectomized CBA mice, and of CBA mice receiving single or repeated doses of hydrocortisone, to induce a lymph node graft versus host reaction (GVHR) in (CBA×C57BL)F₁ hybrids was determined. The ability of the spleen and bone marrow cells to induce GVHR was increased two days after administration of 2.5 mg hydrocortisone, whereas the ability of the thymus cells was unchanged. Seven days and, in particular, 15 days after injection of hydrocortisone, the spleen cells were less active. Activity of the thymocytes in GVHR was increased two days after repeated daily injections of the hormone in a dose of 0.25 mg for 18 days, whereas activity of the spleen and bone marrow cells was unchanged.Laboratory of Pathophysiology, Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 7, pp. 58–60, July, 1979.     

Induction of apoptosis in cord blood lymphocytes by HHV-6

Apoptosis induced by human herpesvirus 6 (HHV-6) in cord blood lymphocytes was investigated. Cord blood mononuclear cells (CBMC) prestimulated with phytohemagglutinin (PHA) were infected with HHV-6 and cultured with interleukin 2 (IL-2) for 5 days. Apoptosis was investigated by cell cycle analysis, terminal deoxytransferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, and staining with monoclonal antibody APO2.7 reacting with 7A6 antigen. The percentage of the hypodiploid fraction by cell cycle analysis and the percentage of apoptosis determined by TUNEL assay were significantly higher in HHV-6-infected CBMC compared with uninfected CBMC. 7A6 antigen, induced on the mitochondria membrane in apoptotic cells, were mainly expressed in CD4+ cells. 7A6 antigen was also detected in HHV-6-infected cells determined by monoclonal antibody OHV-3 reacting with HHV-6 glycoprotein. These data indicated that HHV-6 induced apoptosis in HHV-6-infected cells after stimulation with IL-2 for 5 days. The addition of anti-Fas antibody, anti-Fas ligand antibody, and anti-TNF-alpha antibody did not affect the induction of apoptosis by HHV-6, indicating that the Fas-Fas ligand pathway and TNF pathway did not contribute to the apoptosis induced by HHV-6.     

死亡受体5在内毒素诱导人肝细胞凋亡中的作用

探讨内毒素(LPS)在体外直接作用于LO2细胞引起凋亡及可能机制。将LO2细胞常规培养7 h贴壁后,分为对照组、内毒素处理组、抗人DR5单抗mDRA-6处理组及联合组(内毒素+mDRA-6)。然后在倒置显微镜下观察LO2细胞形态变化;MTT法检测内毒素对LO2细胞生长的影响;流式细胞术检测LO2细胞表面DR5的表达率;Annexin-V和PI双染法检测细胞凋亡率。结果:经内毒素处理的LO2细胞呈现明显的凋亡形态特征,出现核固缩、染色质边缘化、凋亡小体形成。流式细胞术分析表明:对照组LO2细胞表面DR5的表达率为4.8%,用内毒素(10 mg/L)处理12 h后,DR5表达率上调为37.76%。LO2细胞12 h凋亡率:内毒素处理组、抗人DR5单抗mDRA-6处理组、内毒素联合mDRA-6组细胞凋亡率分别为37.78%(早期凋亡21.65%,晚期凋亡16.13%)、29.58%(早期凋亡18.21%,晚期凋亡11.37%)和65.81%(早期凋亡50.61%,晚期凋亡15.20%)(P<0.05)。细胞凋亡率与内毒素的剂量及细胞表面DR5的表达呈相关性。结论:LPS对LO2细胞的杀伤作用主要通过诱导凋亡,细胞表面DR5受体的表达上调可促进这种凋亡作用。