丁卵巢癌患者自体肿瘤细胞疫苗的临床应用

生物治疗已成为肿瘤继手术、化学药物治疗(化疗)、放射治疗之后的第4种重要的治疗方法。本研究对34例行肿瘤细胞减灭术后的卵巢癌患者，进行以新城鸡瘟病毒(NDV)修饰的自体肿瘤细胞疫苗(ATV，NDV．ATV)的特异性主动免疫治疗(免疫治疗)，检测患者的免疫功能，并探讨应用NDV-ATV的时间选择。     

泼尼松治疗原发性肾病综合征对患儿CD4+CD25+调节性T细胞的影响分析

目的　观察原发性肾病综合征（PNS）患儿泼尼松治疗前后CD4+CD25+ 调节性T细胞（CD4+CD25+ Tr）的变化,阐明肾上腺糖皮质激素治疗儿童PNS的免疫机制。方法　选择2004—2007年在深圳市儿童医院住院治疗的初发PNS患儿42例,其中激素敏感型32例,激素耐药型10例。同期25例健康体检儿童作为对照组。流式细胞术检测泼尼松治疗前后外周血CD3+CD4+CD8-、CD3+CD4-CD8+、CD4+CD25+Tr的比例,荧光定量聚合酶链反应（Real-time PCR）检测泼尼松治疗前后外周血单个核细胞（PBMC）叉头型基因P3（Foxp3）、细胞毒性T淋巴细胞相关抗原4（CTLA-4）和糖皮质激素诱导的肿瘤坏死因子受体 （GITR）基因mRNA的表达。结果 　与对照组比较,PNS患儿外周血CD3+CD4+CD8- T细胞、CD3+CD4-CD8+ T细胞、CD4+CD25+ Tr比例无明显改变（P > 0.05）。激素敏感型PNS患儿CD4+CD25+Tr比例在泼尼松治疗后明显增高,差异有统计学意义（P < 0.01）;激素耐药型PNS患儿CD4+CD25+Tr比例在泼尼松治疗前后无明显改变（P > 0.05）。激素敏感型PNS患儿在泼尼松治疗后PBMC细胞Foxp3、CTLA-4和GITR基因mRNA的表达明显增高;而激素耐药型PNS患儿泼尼松治疗前后Foxp3、CTLA-4基因表达无明显改变,仅GITR表达明显增高。 结论 泼尼松等肾上腺糖皮质激素类药物可通过上调激素敏感型PNS患儿CD4+CD25+调节性T细胞的表达发挥免疫治疗作用。     

主动免疫治疗复发性流产妊娠结局与调节性T细胞关系初探

目的:探讨主动免疫治疗原因不明复发性流产患者妊娠结局与调节性T细胞(CD4+CD25+Treg)的关系.方法:采用流式细胞仪测定主动免疫治疗前后复发性流产患者外周血CD4+CD25+Treg比例.结果:主动免疫治疗后CD4+CD25+Treg 的表达较治疗前增高(P＜0.05),且治疗后妊娠成功组与治疗后妊娠失败组比较,差异有统计学意义(P＜0.05).结论:主动免疫治疗可上调CD4+CD25+Treg的表达,可能与妊娠结局有一定相关性.     

子宫内膜异位症患者T辅助细胞亚群功能失衡的研究

目的　探讨T辅助细胞 (Th)失衡与子宫内膜异位症 (内异症 )的关系。方法　对 30例内异症患者 (内异症组 )及 15例健康生育年龄妇女 (对照组 )的外周血单个核细胞 (PBMC)加入植物血凝素进行体外培养诱生。采用酶联免疫吸附试验测定血浆及PBMC培养上清液中Th1类因子干扰素γ(IFN γ)、Th2类因子白细胞介素 4(IL 4)的水平。结果　 ( 1)在PBMC诱生培养上清液中 ,内异症组IL 4的诱生水平 [( 32 .8± 12 .5 )ng/L]比对照组 [( 2 4.3± 3.5 )ng/L]明显升高 (P <0 .0 1) ,而IFN γ的诱生水平内异症组 [( 10 95 .6± 375 .6 )ng/L]较对照组 [( 15 49.3± 36 1.9)ng/L]下降 ,内异症组的IFN γ/IL 4比值 ( 37.7± 17.5 )下降 ,与对照组 ( 71.1± 12 .1)比较 ,差异均有极显著性 (P <0 .0 1)。 ( 2 )在血浆中 ,除内异症组IL 4水平 [( 32 .0± 2 2 .6 )ng/L]高于与对照组 [( 2 3.3± 4.3)ng/L](P <0 .0 5 )外 ,IFN γ的诱生水平及IFN γ/IL 4比值与对照组比较 ,差异均无显著性 (P >0 .0 5 )。结论　内异症患者体内存在Th1/Th2失衡 ,使子宫内膜能够逃避免疫监视及杀伤而在异位种植 ,引起内异症的发生发展     

紫杉醇联合卡铂对荷瘤鼠细胞免疫功能影响的研究

目的:揭示紫杉醇联合卡铂对荷瘤大鼠免疫系统的影响,探讨化疗后是否存在免疫功能逆转的"窗口期"。方法:取25只Fischer 344大鼠,随机分为5组,取4组于右腋下种植NuTu-19卵巢癌细胞株,待荷瘤体积生长至0.5cm3后,以牺牲大鼠的时间为第0天,取3组分别于-15天(化疗晚期)、-10天(化疗中期)、-6天(化疗早期)在荷瘤的大鼠腹腔内注射紫杉醇、卡铂。流式细胞技术检测化疗后各时期淋巴细胞数量及刺激活化后外周血Tc1淋巴细胞的功能亚群,H3-TdR掺入法检测化疗后淋巴细胞的增殖。结果:荷瘤鼠与正常大鼠相比,淋巴细胞数量无显著差异,荷瘤组淋巴细胞亚群CD3+T、CD4+T、CD8+T、NK细胞下降,但仅CD3+T细胞较正常组有差异(P<0.05)。荷瘤组肿瘤抑制性Treg细胞明显增高(P<0.05),而具有杀伤功能的Tc1细胞在荷瘤组明显下降(P<0.05),NK的杀伤功能在两组间无显著差异。移植瘤大鼠化疗后免疫功能变化:(1)在用药后不同阶段淋巴细胞数呈先下降后逐渐恢复至正常的变化规律,在化疗后6天降到最低点,15天恢复至化疗前水平;(2)化疗后6天淋巴细胞降到最低,其增殖力最强;(3)化疗后机体外周血中CD3+T、CD4+T、CD8+T细胞、肿瘤抑制性Treg细胞绝对数均先下降后恢复,化疗后6天降到最低,较未化疗组均有统计学意义,化疗后10天开始恢复,化疗后15天均恢复到化疗前水平;(4)化疗后CD8+T细胞中Tc1细胞比例逐渐增高,化疗后6天较未化疗组增高且有统计学意义,化疗后15天达到最高,但较化疗后6天无显著性差异,表明化疗促进具有杀伤功能Tc1的表达。结论:荷瘤鼠的机体呈负调节免疫状态,肿瘤的发生引起了免疫功能的逃逸,肿瘤的免疫治疗应该具有针对性;紫杉醇联合卡铂打破机体内原有的抗肿瘤免疫抑制状态,机体有可能通过免疫重建诱生了增强的抗肿瘤免疫应答,从而使抗肿瘤免疫抑制状态得到暂时逆转,表明机体经化疗后存在免疫"窗口期"。     

干预CD86协同刺激信号在诱导母胎界面Th2型免疫偏倚中的作用

目的 :探讨干预CD86协同刺激信号在诱导母胎界面局部形成Th2型免疫偏倚中的作用。方法 :将正常妊娠模型 (CBA×BALB/c)和自然流产模型 (CBA×DBA/ 2 )CBA孕鼠均分为两组 ,于孕第 4、6、8天 ,对照组腹腔注射大鼠IgG ,实验组腹腔注射大鼠抗小鼠CD86mAb ;孕第 9天 ,ELISA测定母胎界面组织培养上清中Th1型 (IFN γ、TNF α) /Th2型(IL 4、IL 10 )细胞因子表达水平 ,并计算IL 4 /IFN γ、IL 10 /IFN γ比值 ;孕第 12天比较两种模型各组的胚胎吸收率。结果 :正常妊娠模型中 ,干预CD86协同刺激信号对母胎界面原有的Th2型免疫偏离及妊娠预后均无显著影响。自然流产模型中 ,干预CD86协同刺激信号能够诱导母胎界面局部形成Th2型免疫偏倚并显著改善其妊娠预后。结论 :于孕早期 ,干预CD86协同刺激信号能够改善母胎界面局部细胞因子微环境 ,形成维持正常妊娠所需的Th2型免疫偏倚 ,诱导母胎免疫耐受     

肠道病毒脑炎患儿脑脊液中IL 4/IFN γ失衡与NSE异常表达的相关性探讨

目的通过检测肠道病毒脑炎患儿脑脊液中细胞因子IL 4、IFN γ和神经元特异性烯醇化酶(NSE)的表达质量浓度，并进行相关分析，探讨肠道病毒脑炎的免疫损害机制。 方法2005 03—2006 03应用ELISE方法检测青岛大学医学院附属医院和滨州医学院附属医院40例肠道病毒脑炎患儿和30例对照组脑脊液中细胞因子IL 4、IFN γ及NSE的质量浓度,并对检测结果进行了统计学处理。 结果肠道病毒脑炎患儿急性期脑脊液中IFN γ和NSE质量浓度高于对照组,IL 4浓度低于对照组,差异有显著性(P<0.01)。相关关系分析结果表明，IFN γ和IL 4及IL 4与NSE均呈负相关(r=-0.615，r=-0.656；P<0.01)，而IFN γ与NSE呈正相关(r=0.765，P<0.01)，IFN γ/IL 4比值与NSE相关性最好(r=0.799，P<0.01)。 结论细胞因子IL 4和IFN γ参与肠道病毒脑炎的免疫损害过程，IL 4和IFN γ之间失衡与肠道病毒脑炎时神经元的损害密切相关。     

孕酮在人正常早期妊娠及自然流产中对外周血单个核细胞(PBMC)产生INF-γ、IL-4的作用

目的　探讨孕酮 (P)在人正常早期妊娠及自然流产中 ,对外周血单个核细胞 (PBMC)产生IFN -γ、IL - 4的作用及意义。方法　 2 1例早期不明原因自然流产患者和 2 6例正常早孕者的外周血单个核细胞(PBMC) ,经植物血凝素 (PHA)在有或无孕酮诱导培养后 ,用双抗体夹心酶联免疫吸附试验 (ELISA)测定培养上清液中的γ干扰素 (IFN -γ)和白介素 4 (IL - 4 )的水平。结果　 1 PBMC经PHA诱导 (无孕酮刺激 )培养后 ,流产组IL - 4含量显著低于正常早孕组 (P <0 0 1 ) ;IFN -γ含量两组间差异无显著性。 2 流产组PBMC经PHA和P刺激培养后的IFN -γ含量显著低于无孕激素刺激组 (P <0 0 1 ) ,而IL - 4含量两组间差异无显著性 ;正常早孕组PBMC经PHA和P刺激培养后及的IFN -γ含量显著低于无孕激素刺激组 (P <0 0 1 ) ,而IL - 4含量两组间差异无显著性。结论　正常早期妊娠免疫以Th2反应为主 ;孕酮对人妊娠早期PBMC产生Th1型细胞因子IFN -γ有抑制作用     

主动免疫治疗对不明原因习惯性流产患者辅助T细胞因子水平的影响

目的　探讨主动免疫治疗对不明原因习惯性流产 (UHA)患者辅助T细胞 (Th) 1 /Th2型细胞因子水平的影响。方法　采用酶联免疫吸附法 ,检测 30例半年内接受过淋巴细胞主动免疫治疗的UHA患者 (治疗组 ) ,及 2 5例未经治疗的UHA患者 (未治疗组 ) ,外周血单个核细胞 (PBMC)经滋养细胞抗原刺激产生的Th1型细胞因子白细胞介素 (IL) 2、γ干扰素 (IFN γ)及Th2型细胞因子产生IL 4、IL 1 0水平。并选取 1 5例正常非妊娠妇女作为对照 (对照组 )。结果　 (1 )在最佳诱导时间内 ,治疗组IL 2、IFN γ的水平分别为 (1 0 8± 37)ng/L、(1 1 0± 52 )ng/L ,明显低于未治疗组的 (2 2 3± 85)ng/L、(32 6±92 )ng/L(P值均 <0 .0 5) ;IL 4、IL 1 0水平分别为 (50± 1 1 )ng/L、(1 4 0± 37)ng/L ,明显高于未治疗组的(2 3± 1 1 )ng/L、(52± 2 8)ng/L(P值均 <0 .0 5)。未治疗组IL 2、IFN γ水平明显高于对照组的 (92± 32 )ng/L、(1 0 2± 35)ng/L(P值均 <0 .0 5) ;IL 4、IL 1 0水平低于对照组的 (62± 2 1 )ng/L、(1 50± 42 )ng/L(P值均 <0 .0 5)。治疗组与对照组各细胞因子水平比较 ,差异均无显著性 (P值均 >0 .0 5)。 (2 )治疗组30例患者治疗后半年内妊娠 2 6例 ,其中 8例自然流产 ,IL 2、IFN γ水平明显高于 1 8例妊娠     

子痫前期患者外周血协同刺激分子CD28/CTLA-4的表达研究

目的:研究子痫前期患者外周血协同刺激分子CD28、CTLA-4、CD28/CTLA-4在CD3+CD4 +T细胞和CD3+ CD8+T细胞上的表达,探讨CD28/CTLA-4在子痫前期发病中的作用.方法:采集22例正常妊娠妇女(正常妊娠组),45例子痫前期患者(子痫前期组)外周血,用流式细胞技术分别检测外周血T淋巴细胞CD3+、CD4+、CD8+的表达和CD28、CTLA-4、CD28/CTLA-4在CD3+ CD4+T细胞和CD3+ CD8+T细胞上的表达.结果:子痫前期组外周血T淋巴细胞中CD3+及CD8+的表达低于正常妊娠组(P＜0.05),而CD4+及CD4 +/CD8+的表达高于正常妊娠组(P＜0.05).与正常妊娠组相比,子痫前期组CD28在CD3+ CD4+T细胞和CD3+ CD8+T细胞上的表达低于正常妊娠组,但差异无统计学意义(P＞0.05);CTLA-4在CD3+ CD4+T细胞和CD3+ CD8+T细胞上的表达高于正常妊娠组,且重度子痫前期组高于轻度子痫前期组,差异均有统计学意义(P＜0.05);CD28/CTLA-4比率在CD3+ CD4+T细胞和CD3+ CD8+T细胞上的表达低于正常妊娠组,且重度子痫前期组低于轻度子痫前期组,差异均有统计学意义(P＜0.05).结论:子痫前期患者外周血T细胞亚群明显偏移,且协同刺激分子CD28/CTLA-4表达异常,高表达的CTLA-4导致T淋巴细胞亚群失衡,这可能是促使子痫前期发生的重要原因之一.     

Induction of ovarian tumor-specific CD8+ cytotoxic T lymphocytes by acid-eluted peptide-pulsed autologous dendritic cells

OBJECTIVE: To evaluate the potential of dendritic cells pulsed with acid-eluted peptides derived from autologous ovarian cancer cells for eliciting a tumor-specific cytotoxic T cell response in women with advanced ovarian cancer. METHODS: CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous ovarian tumor peptide-pulsed dendritic cells were tested for their ability to induce an HLA class I-restricted cytotoxic T lymphocyte response against autologous tumor cells. To correlate cytotoxic activity by cytotoxic T lymphocytes with T cell phenotype, we used two-color flow cytometric analysis of surface markers and intracellular cytokine expression (interferon-gamma versus interleukin-4). RESULTS: CD8+ cytotoxic T lymphocyte responses against autologous ovarian tumor cells were elicited in three consecutive women who had advanced ovarian cancer. Although cytotoxic T lymphocyte populations from all women expressed strong cytolytic activity against autologous tumor cells, they did not lyse autologous lymphoblasts or Epstein-Barr virus-transformed cell lines, and they showed negligible cytotoxicity against the natural killer-sensitive cell line K-562. Cytotoxicity against the autologous tumor cells was significantly inhibited by anti-HLA class I (W6/32) and anti-HLA-A2 (BB7-2) monoclonal antibodies. CD8+ cytotoxic T lymphocytes expressed variable levels of CD56 and preferentially expressed interferon-gamma rather than interleukin-4. CONCLUSIONS: Peptide-pulsed dendritic cells induced specific CD8+ cytotoxic T lymphocytes that killed autologous tumor cells from women with advanced ovarian cancer. This finding might contribute to the development of active or adoptive immunotherapy for residual or resistant ovarian cancer after standard surgery and cytotoxic treatment.     

树突细胞疫苗诱发抗肿瘤免疫特异性的体内研究

目的:探讨卵巢肿瘤提取物负载的树突细胞(DC)体内诱发抗肿瘤免疫效应的特异性。方法:应用rrGM-CSF(终浓度40ng/ml)、rrIL-4(终浓度40ng/ml)及rrTNF-α(终浓度40ng/ml)体外培养Fischer344大鼠骨髓来源的单个核细胞,于培养第12天通过流式细胞仪检测细胞标志CD86、OX62抗原,用电镜观察细胞形态,证实获得树突细胞。分别利用NuTu-19卵巢肿瘤细胞和CBRH-7919大鼠肝癌细胞肿瘤提取物制备肿瘤抗原致敏Fischer344大鼠骨髓来源的树突细胞,得到两种肿瘤DC疫苗,即NuTu-DC和CBRH-DC。体内试验:Fischer344大鼠随机分为3组(1)对照组;(2)NuTu-DC治疗组;(3)CBRH-DC治疗组。于大鼠皮下接种NuTu-19细胞,5天后出瘤,开始于肿瘤接种对侧接种DC疫苗,每隔1周给予疫苗1次,共治疗2次。于第0天(未荷瘤时),第5天(出瘤后),第26天(治疗后1周),第40天(治疗后3周)天眼球取血分析各组动物外周血CD3/4/8表达水平;于荷瘤30,32,34,36,38,40天测量肿瘤体积,观察大鼠皮下肿瘤生长情况。于荷瘤40天处死各组实验动物,取肿瘤组织称重,评价治疗效果。结果:大鼠骨髓来源的骨髓单个核细胞(bone marrow mononuclearcells,BMMNC)在相关细胞因子的作用下可以培养出成熟的树突细胞,负载肿瘤提取物后,高表达表面抗原OX-62,CD-86。流式细胞仪检测外周血CD3/4/8水平显示:各组大鼠CD4/CD8比值升高,与未荷瘤时(第0天)相比,差异有统计学意义(P<0.05);CD3+CD4+T细胞表达百分率及CD4/CD8在治疗后3周时(第40天)NuTu-DC治疗组明显高于另两组(P<0.05),而对照组与CBRH-DC比较无差异。通过测量肿瘤生长曲线可见NuTu-DC治疗组各大鼠肿瘤体积较其它两组小,荷瘤第40天时,NuTu-DC治疗组为106.57±56.65mm3,对照组为299.78±118.17mm3,CBRH-DC治疗组为299.12±85.61mm3,与对照组和CBRH-DC组比较差异有统计学意义(P<0.01);而且NuTu-DC治疗组肿瘤平均瘤重为0.197±0.039g,与对照组和CBRH-DC治疗组有显著差异(P<0.01),其抑瘤率为64.4%;而CBRH-DC治疗组大鼠平均肿瘤体积、生长速度及瘤重与对照组无明显差异(P>0.05),其抑瘤率仅为0.22%。结论:应用大鼠卵巢癌细胞肿瘤提取物负载的树突细胞体内治疗卵巢癌荷瘤大鼠,能有效抑制肿瘤生长,且所诱发的抗肿瘤免疫具有组织特异性。     

卵巢上皮性癌肿瘤浸润淋巴细胞与腹水中肿瘤相关淋巴细胞生物？…

比较卵巢上皮性癌肿瘤浸润淋巴细胞及腹水中肿瘤相关淋巴细胞的生物学特性。方法收集１２例上皮性卵巢癌组织及腹水 ,并其为实体瘤,血性腹水和非血性腹水３组。将３组标本分别在体外进行ＴＩＬ及ＴＡＬ分离,并在自体肿瘤细以及重组白细胞介素２存在的条件下共同培养。其间     

YB1和CD44在上皮性卵巢癌中的表达及其与铂类耐药的关系

目的:检测上皮性卵巢癌中Y-盒结合蛋白1(YB-1)和黏附分子CD44的表达,分析其与卵巢癌化疗耐药的关系。方法:采用免疫组化SP法分别检测YB-1及CD44蛋白在铂类耐药或敏感的上皮性卵巢癌及正常卵巢组织中的表达情况,实时荧光定量PCR(qRT-PCR)、Western blot法分别检测上皮性卵巢癌顺铂敏感与耐药细胞中YB1及CD44 mRNA与蛋白水平,分析两者表达的相关性。结果:YB-1和CD44在上皮性卵巢癌组织中阳性率分别为56.3%和60.9%,耐药组织中表达均明显高于敏感组织,且两者表达呈正相关(r=0.456);顺铂耐药上皮性卵巢癌细胞株中YB-1和CD44 mRNA及蛋白水平明显高于敏感株(P均0.05)。结论:YB-1和CD44与上皮性卵巢癌铂类耐药相关,独立或联合检测可能预测化疗的敏感性。     

In vitro induction of tumor-specific human lymphocyte antigen class I-restricted CD8 cytotoxic T lymphocytes by ovarian tumor antigen-pulsed autologous dendritic cells from patients with advanced ovarian cancer

OBJECTIVE: The purpose of this study was to evaluate the potential of dendritic cells pulsed with whole-tumor extracts derived from autologous ovarian cancer cells in eliciting a tumor-specific cytotoxic T-cell response in vitro from patients with advanced ovarian cancer. STUDY DESIGN: CD8(+) T lymphocytes stimulated in vitro with autologous ovarian tumor lysate-pulsed dendritic cells were tested for their ability to induce a human leukocyte antigen class I-restricted cytotoxic T-lymphocyte response able to specifically kill autologous tumor cells in standard 6-hour chromium 51 cytotoxicity assays. In addition, to correlate cytotoxic activity by cytotoxic T-lymphocytes with a particular lymphoid subset, 2-color flow cytometric analysis of intracellular cytokine expression (interferon gamma and interleukin 4) at the single-cell level was performed. RESULTS: Cytotoxic T lymphocytes specific for autologous ovarian tumor cells were elicited from 3 patients with advanced ovarian cancer. Although cytotoxic T-lymphocyte populations expressed strong cytolytic activity against autologous tumor cells, they did not lyse concanavalin A-stimulated autologous lymphocytes or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines and showed negligible cytotoxicity against the natural killer cell-sensitive cell line K-562. Cytotoxic effect against the autologous tumor cells was inhibited by an anti-human leukocyte antigen class I monoclonal antibody (W6/32). It is interesting that CD8(+) cytotoxic T lymphocytes expressed variable levels of CD56, a marker that may be associated with high cytotoxic activity. Finally, most of the tumor-specific CD8(+) T cells exhibited a T(H)1 cytokine bias, and a high percentage of interferon gamma expressors among cytotoxic T lymphocytes was correlated with higher cytotoxic activity. CONCLUSION: These data show that tumor lysate-pulsed dendritic cells can consistently induce in vitro specific CD8(+) cytotoxic T lymphocytes able to kill autologous tumor cells from patients with advanced stage ovarian cancer. This novel approach may have important implications for the treatment of residual or resistant disease with active or adoptive immunotherapy after standard surgical and cytotoxic treatment.     

Tumor Lymphocytes in Patients with Advanced Ovarian Cancer: Changes duringin VitroCulture and Implications for Immunotherapy

Tumor specimens and ascites of patients with advanced ovarian cancer were utilized to obtain both primary ovarian carcinoma cell cultures and lymphocytes: tumor-infiltrating lymphocytes (TILs) from solid tumor tissue and tumor-associated lymphocytes (TALs) from peritoneal fluid. Tumor lymphocytes were grown in coculture with autologous tumor cells and recombinant human IL-2 (rhIL-2) for up to 4 weeks and at weekly intervals these were examined with respect to phenotype and cytotoxicity. The phenotype was studied using flow cytometry for a variety of human immunocompetent cell surface markers (CD3, CD4 CD8, CD16, CD56, TCRαβ, TCRγδ). Cytotoxicity was investigated using 4-hr⁵¹Cr-release assays with the primary ovarian carcinoma cell cultures and the K562 cell line as target cells. The tumor lymphocytes did not demonstrate any obvious trend in phenotype changes during culture, although for different cultures a large range was noted for the various lymphocyte populations studied. Cytotoxicity against both autologous and allogeneic targets declined with culture length for the majority (6/7) of the lymphocyte cell lines tested (greatest at 1 week and least at 3 weeks). These initial results indicate that anin vitronon-MHC-restricted cytotoxic function of peritoneal lymphocytes can be effectively activated with IL-2 and autologous tumor cells. However, if activated lymphocytes are to be employed as a form of immunotherapy, they should be given within the first week of culture for maximum cytotoxic effect.     

葡萄球菌肠毒素激活肿瘤浸润淋巴细胞及外周血淋巴细胞体外抗人卵巢癌的对比研究

目的:探讨葡萄球菌肠毒素A(SEA)对卵巢癌肿瘤浸润淋巴细胞(TIL)及其外周血淋巴细胞(PBL)抗瘤活性的诱导作用。方法:取10例卵巢癌伴腹水患者实体瘤、腹水及外周血标本,分离TIL和PBL。在SEA及IL-2作用下培养,定时计数,了解其增殖情况;流式细胞仪检测其CD3、CD4、CD8表达;噻唑蓝(MTT)比色法测定其对K562及自体肿瘤细胞的细胞毒活性;酶联免疫吸附试验(ELISA)测定培养上清液中TNF-a和IFN-γ浓度。结果:10例中8例成功分离实体瘤TIL、腹水TIL及PBL。(1)SEA刺激的实体瘤TIL、腹水TIL及PBL增殖速率明显较IL-2诱导组快(P<0.05),但增殖高峰后出现下降趋势,IL-2组未出现此现象;(2)CD3+CD4+及CD3+CD8+T表达率均明显上升,其中SEA诱导组比IL-2组增加比例明显(P<0.05),以SEA作用的CD3+CD8+T比例增加最快;(3)TIL对自体肿瘤细胞的杀伤活性明显高于对K562细胞的杀伤活性(P<0.05),PBL对自体肿瘤细胞的杀伤活性则明显低于对K562细胞的杀伤活性(P<0.05),SEA激活组比IL-2组杀伤率高(P<0.05);(4)各效应细胞分泌的TNF-a、IFN-γ分别在培养的第2天和第4天达到高峰,高峰后迅速下降,SEA诱导组在前10天明显高于IL-2诱导组(P<0.05)。结论:SEA可高效、迅速诱导卵巢癌TIL的抗瘤活性。     

T lymphocytes and cytokine production in ascitic fluid of ovarian malignancies.

The activation status of T lymphocytes and the presence of various cytokines in ascitic fluid were examined to test peritoneal immunity in women with ovarian malignancies. Peripheral blood and peritoneal fluid were collected from 12 patients with primary ovarian cancer with ascites and 27 normal control subjects during laparoscopic examination. Lymphocyte subpopulations and the expression of activation markers on T lymphocytes were analyzed by dual-color flow cytometry. The concentrations of various cytokines and soluble interleukin (IL)-2 receptor-alpha were measured. CD8 T lymphocytes were the main component of peritoneal lymphocytes. CD69 and HLA-DR, but not CD25, were highly expressed on peritoneal T lymphocytes compared to those in peripheral blood. In ascitic fluid of ovarian malignancies, CD4 T lymphocyte concentrations were further decreased, resulting in a decreased CD4/CD8 ratio. Decreased expression of CD69 and CD25 was also noted on T lymphocytes from ascites compared with T lymphocytes in normal peritoneal fluid. IL-1b, tumor necrosis factor-alpha, IL-6, and soluble IL-2 receptor-alpha concentrations were increased significantly in the ascitic fluid of women with ovarian cancer. The decrease in activation markers on T lymphocytes is suggestive of an immunosuppressive state, despite the presence of abundant stimulatory cytokines. The immunosuppression may be multifactorial, attributed, in part, to the increased concentrations of soluble IL-2 receptor-alpha and other inhibitors.     

S100A4与卵巢癌细胞顺铂耐药相关性的研究

目的:探讨钙结合蛋白S100A4与卵巢癌细胞顺铂耐药的关系。方法:MTT法测定顺铂对卵巢癌顺铂敏感细胞株A2780和耐药株CP70的半效抑制浓度(IC50);分别应用免疫细胞化学、Western blot和实时荧光定量PCR检测A2780和CP70细胞株中S100A4的差异表达,观察顺铂处理CP70细胞后S100A4在蛋白水平及基因水平的动态变化。结果:顺铂对A2780和CP70的IC50分别为20.88μmol/ml和57.10μmol/ml。S100A4蛋白在二者中均以胞浆表达为主;CP70中S100A4 mRNA水平和蛋白水平的表达均显著高于A2780;经顺铂处理CP70后,S100A4的表达呈剂量-时间依赖性。结论:S100A4的高表达与卵巢癌顺铂化疗耐药有关。