脐带源间充质干细胞对异源性脐带血T淋巴细胞激活与增殖的抑制作用

目的：〖HT5"SS〗观察人脐带源间充质干细胞（hUCMSC）对异源性脐带血T淋巴细胞激活与增殖的影响,探讨其在免疫调控中的作用。〖HT5W〗方法： 〖HT5"SS〗建立分离、扩增hUCMSC的方法,检测其MHC表型;实验分3组：(1)单纯脐带血组（阴性组）;（2)脐带血+丝裂原刺激组（对照组）;（3）脐带血+丝裂原刺激 +MSC共培养组（实验组）;流式细胞术检测各组脐带血T细胞及其CD4＋和CD8＋亚型细胞共培养8 h后早期激活标志CD69的表达, MTT法检测各组脐带血T细胞共培养5 d后增殖情况。 〖HT5W〗结果： 〖HT5"SS〗成功建立分离、扩增hUCMSC的方法,免疫表型分析显示hUCMSC不表达HLAⅡ抗原,低表达HLAI抗原;流式细胞术检测示在hUCMSC共培养情况下,经丝裂原刺激后,脐带血T淋巴细胞CD69表达与对照组\[(22. 6±5.2)%\]的阳性率相比较,下降至(7.8±3.5)%（P<0.01）,而且这种抑制对不同亚型T细胞（CD4+/CD8+）均起作用;MTT检测显示在hUCMSC共培养条件下,丝裂原刺激T细胞增殖受到抑制,抑制的程度与hUCMSC的剂量呈依赖性。 〖HT5W〗结论： 〖HT5"SS〗hUCMSC对异源性脐带血T淋巴细胞激活和增殖具有抑制作用,而且这种作用为非选择性的,呈剂量依赖性。     

PLA表位肽免疫微球诱导CTL对肝癌细胞的杀伤作用

目的〖HT5"SS〗：考察两种载肽聚乳酸（(polylatic acid, PLA)免疫微球M1（hAFP158~166）和M2（hAFP218~226）在体外诱导特异性CTL的能力及其对肝癌细胞的杀伤作用。〖HT5W〗方法： 〖HT5”SS〗制备分别荷载表位肽hAFP158~166、、hAFP218~226的PLA免疫微球M1和M2,体外刺激HLAA2+健康志愿者的外周血单个核细胞（PBMC）作为效应细胞,实验分为3组：对照组、hAFP表达组和hAFP阴性组。采用标准51Cr释放法检测CTL杀伤活性。〖HT5W〗结果：〖HT5"SS〗两种免疫微球在体外均能刺激人PBMC增殖,形成大量可见克隆;两者诱导的效应细胞对hAFP＋的荷肽T2细胞、HepG2和Alexander的杀伤率均达75％以上,均显著高于不表达hAFP的膀胱癌细胞BTT和未荷肽的T2细胞,差异非常显著（P<0.01）,而两者杀伤活性之间没有明显差异（P>0.05）。〖HT5W〗结论：〖HT5"SS〗两种载肽聚乳酸免疫微球均能在体外诱导产生特异性CTL,并对表达靶抗原的肝癌细胞有较强杀伤作用。     

抗人P185 erbB2 scFv-Fc-IL-2调变肿瘤细胞表面分子和激活免疫效应细胞

将抗人P185erbB2scFvFcIL2融合蛋白（HFI）作用于人卵巢癌细胞株SKOV3细胞和人外周血单个核细胞（PBMC）,通过体外实验阐明HFI调变肿瘤细胞表面分子和激活免疫效应细胞的抗肿瘤机制,为HFI临床应用提供实验依据。〖HT5W〗方法： 〖HT5"SS〗MTT法检测细胞增殖、杀伤活性;流式细胞术观察细胞表面分子的表达水平;生物活性法检测细胞膜相关TNF杀伤活性;RTPCR检测细胞穿孔素表达水平。〖HT5W〗结果： 〖HT5"SS〗HFI处理后,未观察到对SKOV3细胞增殖活性的直接抑制作用;SKOV3细胞表面杀伤相关分子ICAM1、Fas表达率分别由24.85%、0.53%增高到85.36%、59.19%（P<0.01);人PBMC的增殖活性增强,CD3+CD8+T细胞和CD3CD16+CD56+NK细胞分别由24.37%、6.90%提高到38.80%、13.45%（P<0.01);CD25、LFA1、FasL表达水平分别由399%、86.52%、5.02%提高到12.96%、99.06% 16.19%（P<0.01);穿孔素基因、膜相关TNF均表达增强,LAK样、NK样杀伤活性在各效靶比时均明显增高（P<0.01)。〖HT5W〗结论： 〖HT5"SS〗HFI提高SKOV3细胞杀伤相关分子ICAM1、Fas表达水平,并且对人PBMC有明显的增殖活化作用,通过激活LFA1/ICAM1、Fas/FasL途径提高杀伤介质穿孔素和膜相关TNF的释放,增强LAK样、NK样杀伤活性。     

CpG-ODN持续刺激对小鼠骨髓树突状细胞成熟的影响

目的〖HT5"SS〗：研究CpGODN持续刺激对小鼠骨髓树突状细胞（DC）成熟的影响。〖HT5W〗方法〖HT5"SS〗： 小鼠骨髓细胞用GMCSF培养7 d,持续刺激组全程加入CpGODN,短期刺激组在培养的最后36 h加入CpGODN,对照组不加CpGODN。流式细胞仪检测细胞表型和细胞摄取抗原的能力,ELISA检测细胞产生的细胞因子,混合淋巴细胞培养检测细胞提呈抗原的能力。〖HT5W〗结果〖HT5"SS〗： 用CpGODN持续刺激的小鼠骨髓DC表达MHCⅡ、CD86和CD40等分子和分泌IL12 p70的能力并未增加,吞噬FITCOVA的能力显著升高,刺激同种异基因T细胞和刺激同种同基因T细胞增殖的能力显著低于CpGODN短期刺激组。〖HT5W〗结论〖HT5"SS〗：CpGODN持续刺激可抑制DC的发育成熟,可能是持续严重感染时免疫功能低下的原因。     

腺病毒介导的HBsAg基因修饰树突状细胞的体外生物学特性

目的： 〖HT5"SS〗探讨腺病毒介导乙型肝炎病毒表面抗原（AdVHBsAg）基因修饰树突状细胞（dendritic cell,DC）瘤苗体外生物学活性。〖HT5W〗方法〖HT5"SS〗：将腺病毒表达载体AdVHBsAg转染人单个核细胞来源的DC,构建AdVHBsAgDC肝癌瘤苗,采用Western blotting法鉴定转染基因表达,FACS检测表面分子和内吞功能,3HTdR法检测T细胞增殖反应的能力,MTT法检测CTL活性。〖HT5W〗结果〖HT5"SS〗：HBsAg基因转染后,Western blotting法检测结果示HBsAg基因表达于转染的DC,表明腺病毒介导的HBsAg基因转染的有效性。AdVHBsAgDC可高表达CD1a、CD11c、 CD83、CD86和HLADR,但内吞功能较DC组降低(P<0.05)。AdVHBsAg DC刺激自体T细胞增殖功能均明显高于DC对照组和AdVLacZDC组(P<0.05)。AdVHBsAg DC体外诱导CTL对HepG2215肿瘤细胞的杀伤作用具有特异性。〖HT5W〗结论〖HT5"SS〗：肝癌相关基因HBsAg可作为乙型肝炎病毒相关性肝癌的切入点,该研究为HBV相关肝癌DC体内免疫治疗提供了实验依据。     

白细胞介素24对人树突状细胞活化和成熟的诱导作用

目的: 〖HT5"SS〗研究白细胞介素24（interleukin24,IL24）对人外周血单核细胞来源树突状细胞（dendritic cell, DC）表型及抗原提呈功能的影响。〖HT5W〗方法： 〖HT5"SS〗利用Western blot检测DC培养上清中IL24的分泌水平;利用半定量RTPCR方法分析不同条件下DC表达IL24受体及趋化因子受体的情况;通过流式细胞术分析检测IL24共培养后DC细胞表面CD80、CD86、MHCⅡ类分子的表达水平;采用体外混合淋巴细胞实验分析经IL24共培养对DC抗原提呈功能的影响。〖HT5W〗结果：〖HT5"SS〗 体外培养过程中,用LPS刺激DC可诱导其分泌IL24;同时,LPS还能上调DC表达IL24受体亚单位IL22R1。IL24作用于DC可上调DC细胞表面CD80、CD86及MHCⅡ类分子的表达,并增强DC对T细胞的抗原提呈功能。〖HT5W〗结论： 〖HT5"SS〗IL24这一新型的细胞因子在体外实验中能促进DC的表型成熟,增强DC的抗原提呈功能。     

紫杉醇与肿瘤疫苗联合应用对小鼠Lewis肺癌皮下移植瘤的治疗效果

目的： 〖HT5"SS〗评价化疗药物紫杉醇联合应用肿瘤疫苗对小鼠Lewis肺癌皮下移植瘤的治疗效果。〖HT5W〗方法： 〖HT5"SS〗以编码有GMCSF的腺病毒感染Lewis肺癌细胞3LL,制备肿瘤疫苗3LL/GMCSF。以2×104个3LL瘤细胞皮下注射C57BL/6（H2b）小鼠腹股沟部,制备小鼠皮下移植瘤。检测体内、外应用紫杉醇对Lewis肺癌细胞3LL的杀伤敏感性。在小鼠Lewis肺癌皮下移植瘤体内首先以紫杉醇化疗,随后以肿瘤疫苗3LL/GMCSF免疫;或反之,首先以肿瘤疫苗免疫,随后以紫杉醇化疗,观察肿瘤生长和小鼠生存状况,以及检测小鼠体内对肿瘤的特异性杀伤效率和免疫应答记忆反应。〖HT5W〗结果〖HT5"SS〗： 紫杉醇体外作用于3LL肿瘤细胞,24 h后在浓度为100 nmol/L时可使32.10 %的3LL肿瘤细胞发生死亡;但体内注射紫杉醇（5、10和25 mg/kg）不能使所有3LL荷瘤小鼠肿瘤消退。体内使用紫杉醇后应用3LL/GMCSF肿瘤疫苗,70%的荷瘤小鼠发生显著的肿瘤消退,与单纯化疗的小鼠相比生存时间明显延长（70.0 vs 27.5 d）; 化疗后应用肿瘤疫苗诱导了体内对3LL的特异性杀伤,第3天体内杀伤率达41.35%;同时,存活小鼠能抵抗2×104 3LL肿瘤细胞的再次攻击。接种3LL/GMCSF肿瘤疫苗后应用紫杉醇化疗却不能使肿瘤消退。〖HT5W〗结论： 〖HT5"SS〗化疗后应用肿瘤疫苗免疫可诱导出抗原特异性免疫效应,使荷瘤小鼠肿瘤消退并延长生存时间,为临床开展化疗后的肿瘤疫苗主动免疫提供了实验依据。     

Tat蛋白转导结构域介导HSV1-TK导入肝癌细胞及其效应

目的： 〖HT5"SS〗探讨HIV1编码的反式激活蛋白Tat与HSV1TK融合表达对肝癌细胞的杀伤效果。〖HT5W〗方法：〖HT5"SS〗 合成编码HIVTat47~57（Tat 11）的2条寡核苷酸单链,两端分别引入BamHⅠ和Hind Ⅲ 两个酶切位点,退火形成寡核苷酸双链,15%非变性聚丙烯酰胺凝胶电泳判断退火效果;以rpAs16Dr为模板,通过PCR扩增HSV1TK基因,定向克隆至原核表达载体pET32中。将含pET32cTat 11TK的BL21菌通过IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,免疫组化分析重组蛋白Tat 11TK的膜结合特性,蛋白印迹技术分析该融合蛋白的穿膜能力,检测和分析TK/GCV、Tat 11TK/GCV对肝癌细胞株HepG2的杀伤作用。 〖HT5W〗结果〖HT5"SS〗： 表达产物SDSPAGE在相对分子质量60 700左右显示条带,符合Tat 11TK与表达标签融合蛋白的理论值,并证明以包涵体的形式表达;通过Ni2+螯合柱亲和纯化获得的Tat 11TK重组融合蛋白,经免疫组化证实能结合到肝癌细胞表面,蛋白印迹结果说明Tat 11TK能有效穿过细胞膜进入HepG2细胞内,同时应用低浓度前药更昔洛韦（GCV,150 μmol/L）能有效抑制HepG2细胞生长。〖HT5W〗结论〖HT5"SS〗： Tat 11TK在原核系统获得高效表达,具有较强的穿膜能力和前药酶活性。     

肝动脉栓塞化疗联合CIK细胞疗法治疗原发性肝癌

目的:〖HT5"SS〗 评价肝动脉栓塞化疗联合细胞因子诱导的杀伤细胞(CIK)疗法治疗原发性肝癌的临床疗效。〖HT5W〗方法: 〖HT5"SS〗以生存期为观察终点指标,采用同期非随机对照方法,对2003年1月至2005年12月接受肝动脉栓塞化疗联合异体CIK细胞疗法的21例原发性肝癌患者(治疗组)与单纯肝动脉栓塞化疗46例患者(对照组)比较,观察两组的生存期差异。〖HT5W〗结果:〖HT5"SS〗 治疗组与对照组中位生存期分别为22个月(95％CI, 7~37)、10个月(95％CI, 8~12)。两组的半年、1年、2年生存率分别为8571%、5835%、48.62%和69.05%、32.74%、3.97%,治疗组生存期明显长于对照组（P＜0.05)。〖HT5W〗结论: 〖HT5"SS〗肝动脉栓塞化疗联合CIK细胞疗法较单纯肝动脉栓塞化疗有可能提高原发性肝癌患者的远期生存率。     

几种免疫辅助治疗对晚期复治肺癌疗效的对比

目的:〖HT5"SS〗 探讨几种以免疫为主的非手术治疗方法对晚期复治肺癌的疗效及其临床意义。〖HT5W〗方法: 〖HT5"SS〗102例晚期复治肺癌患者按主要治疗方法分单纯免疫治疗(A)、介入化疗(B)、雾化吸入免疫治疗(C)及热化疗(D)4组,观察4组的肿瘤缓解有效率、患者中位生存期及生存率。〖HT5W〗结果: 〖HT5"SS〗A、B、C、D 4组的肿瘤缓解有效率分别为：18.8%、45.4%、23.1%、20.0%;中位生存期分别为：6.09、7.35、5.46、10.24个月,B组及D组较高; ２年以上生存率A组及D组较高,但4组间无统计差异。 〖HT5W〗结论：〖HT5"SS〗单纯性全身免疫疗法肿瘤缓解率较低,微创介入化疗有较高的肿瘤缓解率,但并非一定可延长生存率。局部吸入性免疫疗法及热化疗有积极意义。     

Functional analysis of mononuclear cells infiltrating into tumors: lysis of autologous human tumor cells by cultured infiltrating lymphocytes

Lymphocytes separated from surgically resected tumor tissue, uninvolved lung tissue, and peripheral blood of lung cancer patients were investigated for cytotoxic potential and analyzed for their phenotypes at the time of surgery and after having been propagated for 4 to 5 wk in the presence of interleukin-2. Most of the tumor lymphocyte infiltrates examined were shown to have a shift in favor of T8 subsets from those found in peripheral blood. No natural killer activity and low cytotoxicity against the autologous tumor were found to characterize the tumor-derived lymphocyte population. Propagation of lymphocytes from the different tissues of the cancer patient in the presence of interleukin-2 preparation induced widespread lytic activity against K562 cells, autologous and allogeneic tumors, but not autologous normal lung or lymphoblasts. However, cytotoxic activity against autologous tumor cells exerted by cultured tumor-infiltrating lymphocytes was found to be significantly higher than the activity of cultured lymphocytes isolated from peripheral blood or uninvolved lung tissue of the same patient. The elevated lytic activity of cells derived from the tumor tissue indicates the accumulation at the tumor site of precursors of natural killer-like cells and specifically stimulated antitumor effectors. Our results suggest the coexistence of two types of anti-autotumor cytotoxic lymphocytes at the tumor site: natural killer-like and specific cytotoxic T-cells.     

FOLFOX方案化疗对结直肠癌患者免疫细胞数的影响

背景与目的：化疗在杀伤肿瘤的同时,也损害了机体的正常免疫。很多文献曾报道晚期肠癌的化疗,常使细胞免疫功能抑制加重。本研究探讨FOLFOX方案即奥沙利铂联合亚叶酸钙及氟尿嘧啶联合化疗对结直肠癌患者免疫细胞数的影响及与健康人的差异。方法：80例结直肠癌患者行FOLFOX方案化疗（奥沙利铂85mg/m2,静脉滴注,第1天;亚叶酸钙200mg/m2,静脉滴注,第1天;氟尿嘧啶400mg/m2,第1天,2400mg/m2,持续静脉滴注46h）,2周1次,2次为1个疗程,采用流式细胞仪测定化疗前及化疗后2周、4周外周血T淋巴细胞亚群和NK细胞的活性,比较化疗前后的变化,同时根据临床分期进行亚组分析比较,以健康人作对照分析。结果：本组结直肠癌患者第1天、第2周及第4周外周血CD3＋、CD4＋、CD8＋、CD4＋/CD8＋及NK细胞活性比较均无显著下降（P〉0.05）,但患者外周血CD3＋、CD4＋、NK细胞数量及CD4＋/CD8＋比值与健康人相比下降,而CD8＋细胞比例显著升高,差异有显著性（P〈0.05）,提示免疫功能较健康人下降,而且外周血T淋巴细胞亚群和NK细胞数量的改变与结直肠癌临床病理分期有关,分期越晚,CD3＋、CD4＋及NK细胞数量CD4＋/CD8＋细胞比值越低,CD8＋细胞比例越高;Ⅰ、Ⅱ期结直肠癌患者与Ⅲ、Ⅳ期患者之间差异有显著性（P〈0.05）。结论：FOLFOX方案治疗结直肠癌疗效肯定,可改善患者生存质量,而且对机体免疫力影响小。检测淋巴细胞亚群对判断患者的免疫功能、预测肿瘤患者的预后以及指导临床是否需要应用免疫增强剂均有重要意义。     

化疗对晚期肺癌患者T淋巴细胞亚群和红细胞免疫功能的影响

目的：分析42例晚期肺癌者化疗前后T淋巴细胞亚群和红细胞免疫功能的变化,并探讨其与病情的关系。方法：对42例晚期肺癌化疗前后的血标本采用流式细胞术检测T淋巴细胞亚群和采用交体粘附法检测红细胞免疫功能,并与体检健康者作比较,结果：本组晚期肺癌患者治疗前后T淋巴细胞亚群和红细胞免疫功能均低于对照组（P＜0．05,P＜0．01）。治疗后,化疗有效率总T细胞（CD3^ ）、辅助／诱导T淋巴（CD4^ ）、CD4^ 与CD8^ 比值均显著升高（P＜0．01）,细胞毒／抑制性T淋巴细胞（CD8^ ）降低（P＜0．05）;化疗无效者CD3^ 、CD4^ 、CD4^ ／CD8^ 显著降低（P＜0．05,P＜0．01）,而CD8^ 升高（P＜0．01）,直向肿瘤红细胞花环（direct tumor erythrocyte rosette,DTER）,红细胞C3b受体花环（red blood cell C3b receptor rosette,RBC-C3bRR)和红细胞免疫复合物花环（red blood cell immunity complex rosette,RBC-ICR),化疗前后变化不明显（P＞0．05）。结论：晚期肺癌患者免疫功能低下,有铲化疗能提高患者的T淋巴细胞亚群免疫功能,临床上对免疫功能的观察对肺癌患者的治疗和预后有一定的监测作用。     

初诊肺癌患者多次化疗外周血免疫相关指标动态变化的研究

  目的  探索免疫治疗与化疗相结合的有利切入点,为临床免疫治疗介入提供试验依据。  方法  收集2015年11月至2016年12月新乡医学院第一附属医院初诊的23例肺癌患者,全部患者均完成连续5个周期的化疗,采用流式细胞术方法检测化疗过程中患者外周血免疫相关指标如CD4+T细胞、CD8+T细胞、CD19+B细胞和CD16+、CD56+、NK细胞比率,T细胞表面共信号分子PD-1、PD-L1、CD137、CTLA-4、CCR-4、LAG-3以及细胞因子表达情况。分析多周期过程中,上述指标动态变化趋势。  结果  在肺癌患者多个周期的化疗过程中,机体CD8+T淋巴细胞、CD19+B淋巴细胞和CD16+、CD56+、NK细胞水平下调,CD4+T淋巴细胞数量增加,差异具有统计学意义（P＜0.05）;T细胞表面共抑制分子PD-1、CTLA-4和CCR-4随治疗进行表达下调,差异具有统计学意义（P＜0.05）。  结论  肺癌患者在多个周期的化疗中,实施淋巴细胞亚群数量及T细胞表面共抑制分子监测具有重要意义,化疗中后期针对免疫检查点高表达患者,联合应用PD-1抗体、PD-L1抗体、CTLA-4抗体或CCR-4抗体可能具有更优的治疗效果。      

Antitumor Activity of Lentivirus-mediated Interleukin -12 Gene Modified Dendritic Cells in Human Lung Cancer in Vitro

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associatedantigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whethertransduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigenspecificcytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocytederivedDCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated targetof the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCswas measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry.DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag).Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulatedby LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methylthiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfullyconstructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstratedgood stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumoreffects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have anenhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.     

Effects of Interleukin-12 on the Induction of Cytotoxic T Lymphocytes from the Regional Lymph Node Lymphocytes of Patients with Lung Adenocarcinoma

Lung cancer-specific cytotoxic T lymphocytes (CTL) were induced by repeated stimulations of regional lymph node lymphocytes (RLNL) in lung cancer patients with either autologous or HLA-A-locus-matched tumor cells. To investigate the effect of interleukin-12 (IL-12), IL-12 was added during the stimulation of RLNL from HLA A24 / adenocarcinoma patients with either autologous tumor cells or HLA A24-positive adenocarcinoma cells (PC-9) in combination with, or instead of interleukin-2 (IL-2), and then the cytotoxic activity, cytokine production and populations of the lymphocyte subsets were examined. The addition of IL-12, or the substitution of IL-2 by IL-12 was found to enhance the cytotoxic activity and the cytokine production (IFN-γ, GM-CSF) of the CTL as compared with IL-2 alone. The cytotoxic activity and cytokine production were both partially inhibited by anti-MHC-class I monoclonal antibody. The CTL thus induced by IL-12 had a higher proportion of CD3⁺/CD56⁺ cells than the CTL induced with IL-2 alone. The positively selected CD8⁺/CD56^– lymphocytes showed PC-9-specific cytotoxic activity, because the population did not show any cytotoxicity to K562 or A549 (HLA-A26/A30). However, the CD3⁺/CD56⁺ lymphocytes were cytotoxic to both PC-9 and K562. In conclusion, IL-12 is considered to be a useful cytokine for both the induction of lung-cancer specific CTL and the augmentation of non-MHC-restricted cytotoxicity against tumor cells, and may be applicable for adoptive immunotherapy using CTL.     

化疗对肺癌患者外周血中CD4~＋ CD25~＋调节性T细胞数目的影响及意义

目的研究化疗药物对肺癌患者外周血中Treg（CD4＋ CD25＋调节性T细胞）的影响及意义。方法采集60例肺癌术后患者化疗前1天及化疗后第10天外周静脉血,应用流式细胞技术检测外周血中Treg细胞以及CD3＋、CD4＋、CD8＋T、NK细胞占T淋巴细胞百分比,CD4＋T/CD8＋T比值。结果化疗前NSCLC患者外周血CD4＋ CD25＋调节T细胞比率明显高于健康对照组（P〈0.05）;且Ⅳ期患者调节T细胞比率明显高于Ⅲ期患者（P〈0.05）。化疗后NSCLC患者外周血CD4＋ CD25＋调节T细胞较化疗前显著降低（P〈0.05）。化疗前后不同病理分型患者外周血中CD4＋CD25＋细胞变化差异无统计学意义。化疗后CD8＋T细胞占T淋巴细胞比例（28.129±10.900）%较化疗前（24.876±6.631）%升高（P〈0.05）。化疗后CD4＋/CD8＋（1.506±0.691）较化疗前（1.680±0.704）降低（P〈0.05）。结论肿瘤负荷可显著促进肺癌患者外周血Treg细胞分化,化疗后肺癌患者Treg细胞比例下降。     

Reinfusion of autologous lymphocytes with granulocyte-macrophage colony-stimulating factor induces rapid recovery of CD4+ and CD8+ T cells after high-dose chemotherapy for metastatic breast cancer.

PURPOSE: Repeated high-dose chemotherapy (HDCT) followed by peripheral-blood progenitor cell (PBPC) transplantation can induce a complete remission in patients with metastatic breast cancer sensitive to standard chemotherapy (CT), but the majority of patients relapse within 1 to 2 years. The immune system is seriously compromised after HDCT, which precludes the development of effective immunotherapy. We investigated whether autologous lymphocytes, reinfused after HDCT, could induce a rapid recovery of T cells. PATIENTS AND METHODS: Three patients were monitored for immune recovery without reinfusion of lymphocytes. In the next 11 patients, stem cells were harvested after CT + granulocyte colony-stimulating factor (G-CSF) and lymphocytes were harvested after CT + granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2. These patients received stem cells and G-CSF after the first HDCT; stem cells, G-CSF, and lymphocytes after the second; and stem cells, GM-CSF, and lymphocytes after the third HDCT. RESULTS: Patients not receiving lymphocyte reinfusion had a very slow recovery of lymphocytes. In particular, CD4 counts remained low (< 200/microL for 9 months). Lymphocyte reinfusion had a significant effect on the recovery of lymphocytes, T cells, and CD8+ T cells (normalized on day 25). Recovery of CD4+ T cells was significantly accelerated by lymphocyte reinfusion and GM-CSF, leading to counts of 500/microL at 25 days. CONCLUSION: Lymphocyte reinfusion with G-CSF had a significant effect on the recovery of CD8+ T cells, whereas rapid recovery of CD4+ T cells required lymphocyte reinfusion and GM-CSF, which possibly acts as a survival factor through activation of antigen presenting cells. Whether the rapid recovery of CD4+ and CD8+ T cells prevents or delays relapse of the disease should be further investigated.     

Natural killer and lymphokine-activated killer activities in stomach cancer patients with special emphasis on the effect of 5-fluorouracil, adriamycin and mitomycin-C chemotherapy

The cytotoxicities of peripheral blood lymphocytes (PBL) and lymphokine-activated killer (LAK) cells were studied to evaluate the effect of chemotherapy on cellular immunity, in 18 patients with unresectable stomach cancer before and after chemotherapy with 5-fluorouracil, adriamycin and mitomycin-C (FAM), and in 21 healthy volunteers. LAK cells were generated in vitro by culturing PBL with 100 U recombinant human interleukin-2 (rH-IL-2)/ml for 72 h. K562 (human myelogenous leukemia), MKN-45 (human stomach adenocarcinoma) and PC-14 (human pulmonary adenocarcinoma) were used as target cells. The cytotoxicity of PBL to K562 and MKN-45 was suppressed in patients with stomach cancer before chemotherapy, compared with that in healthy volunteers (P less than 0.05). The cytotoxicity of LAK cells was significantly higher to all three cell lines tested than that of PBL in both the healthy volunteers and stomach cancer patients (P less than 0.01); however, a lower level of LAK activity was generated in patients with cancer compared to that in the healthy volunteers. FAM therapy did not suppress the cytotoxicities of PBL and LAK cells. The surface markers of PBL and LAK cells were measured, demonstrating that there was no significant change in the percentage of lymphocytes with CD3+, CD4+, CD8+, CD16+ or CD19+ after chemotherapy. The ratios of CD4+ to CD8+ cells in PBL and LAK cells were also not significantly changed after chemotherapy. In the present study, we have demonstrated that the PBL of stomach cancer were defective in generating LAK activity compared to those of controls, but the LAK activity generated from PBL receiving chemotherapy was similar to that from PBL without chemotherapy in stomach cancer patients.     

晚期消化道肿瘤患者化疗前后T细胞亚群的变化

背景与目的:肿瘤患者免疫功能低下,对其进行化疗将进一步抑制其机体的免疫系统。因此,合理的免疫调节将是对肿瘤患者重要的辅助治疗手段。本研究旨在检测消化道肿瘤患者化疗前后外周血T细胞亚群及相关细胞因子IL-2的变化规律,研究化疗及化疗后不同反应患者机体免疫功能状态的改变,并探讨对晚期消化道肿瘤患者进行适时、合理免疫治疗的临床意义。方法:采用流式细胞仪检测(flow cytometry,FCM)分析2005年9月至2006年4月苏州大学附属第一医院收治的104例消化道肿瘤患者化疗前后外周血中CD3 、CD4 、CD8 、CD4 CD28 、CD8 CD28 和CD4 CD25 T细胞百分数;采用酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测其血浆相关细胞因子IL-2的表达水平。结果:消化道肿瘤患者组CD4 、CD4 CD28 、CD8 CD28 和CD4 CD25 T细胞百分数及CD4/CD8比值分别为(36.52±3.85)%、(32.87±4.98)%、(6.87±3.11)%、(9.68±3.42)%、0.98±0.17;对照组各指标分别为:(45.23±9.20)%、(40.12±5.85)%、(15.8±4.50)%、(5.67±2.90)%、1.43±0.12。晚期消化道肿瘤化疗有效组。化疗前CD4 CD28 和CD8 CD28 T细胞百分数及CD4/CD8比值分别为:(22.93±3.98)%、(7.08±1.23)%、0.90±0.22,化疗3周后,各指标分别为(28.25±4.03)%、(12.10±3.45)%、1.24±0.22;化疗无效组,化疗前CD4 CD28 、CD8 CD28 和CD4 CD25 T细胞百分数分别为(24.08±4.02)%、(6.35±1.23)%、(8.20±2.34)%,化疗3周后,各指标分别为(16.45±3.27)%、(3.20±0.82)%、(20.34±3.69)%。结论:晚期消化道肿瘤患者化疗1～2周后加重了免疫抑制的程度,化疗有效组患者化疗3周后全身免疫状况改善;而化疗无效组未出现相应的免疫功能的改善,甚至免疫功能进一步恶化。