人白细胞介素-15与18的协同刺激体外活化肿瘤浸润淋巴细胞的作用

目的 观察白细胞介素(IL)-15与IL-18协同刺激活化肿瘤浸润淋巴细胞(TIL)的作用.方法 用IL-15与IL-18共同刺激从结肠癌患者分离得到的肿瘤浸润淋巴细胞,通过测定其分泌转化生长因子(TGF)-β、IL-4、干扰素(IFN)-γ水平以及肿瘤浸润淋巴细胞的杀伤功能,观察IL-15与IL-18活化肿瘤浸润的淋巴细胞的功能.结果 IL-15与IL-18共同作用于肿瘤浸润淋巴细胞,能够抑制TGF-β、IL-4的分泌(P＜0.05),促进IFN-γ的分泌(P＜0.05),同时能提高肿瘤浸润淋巴细胞的杀伤活性.结论 IL-15与IL-18协同刺激能活化肿瘤浸润淋巴细胞.     

肿瘤浸润淋巴细胞对结肠癌细胞的杀伤作用及机制

目的探讨肿瘤浸润淋巴细胞在结肠癌细胞杀伤中的作用及机制。方法从6例结肠癌患者转移淋巴结中分离TIL细胞,采用^51Cr释放法测定其杀伤结肠癌SW480细胞的作用;采用流式细胞术测定结肠癌细胞的凋亡;采用固定化蛋白印迹法（Western blot）测定TIL细胞和结肠癌细胞Fas、FasL蛋白的表达。结果TIL细胞能有效杀伤结肠癌SW480细胞,其杀伤作用明显强于淋巴因子活化的杀伤细胞（100：1,P=0.004;50：1,P=0.002;10：1,P=0.006）和细胞因子诱导的杀伤细胞（100：1,P=0.001;50：1,P=0.002;10：1,P=0.008）,差异具有统计学意义（P〈0.01）;TIL细胞能促进结肠癌细胞的早期凋亡;TTL细胞高表达FasL分子,而SW480细胞高表达Fas分子,不表达FasL分子。结论TIL细胞能有效杀伤不表达FasL分子的结肠癌细胞,其机制可能通过Fas/FasL通路诱导结肠癌细胞的凋亡。     

肿瘤浸润淋巴细胞、重组白细胞介素2治疗对手术后原发性肝癌病人细胞免疫功能的影响

对５例经手术切除的原发性肝癌病人用肿瘤浸润淋巴细胞（ＴＩＬ）和重组白细胞介素２（ｒＩＬ－２）治疗。治疗前１周及治疗后１个月检测细胞免疫功能，治疗后外周血淋巴细胞的ＮＫ活性、白细胞介素２产生及活性均明显升高；植物血凝素（ＰＨＡ）皮试阴性转阳性。提示；ｒＩＬ－２和ＴＩＬ应用可明显提高原发性肝癌病人的细胞免疫功能，对杀灭残留癌细胞，抗肿瘤复发、转移，提高远期疗效有一定作用。可能成为肝癌术后抗肿瘤复发，转移的重要疗法。     

白细胞介素-15对放射线诱导T淋巴细胞凋亡的保护作用

目的观察重组人白细胞介素(rhIL)-15对放射线照射的T淋巴细胞损伤的保护作用。方法从30例正常人的骨髓分离淋巴细胞,随机分为实验组及对照组各15例。实验组加入100μg/L的IL15,两组分别放射线照射,流式细胞术和细胞核形态检测等方法观察照射前后T细胞亚群、bclXL、bax和bcl-2蛋白水平变化和淋巴细胞凋亡率。结果放射线照射后淋巴细胞出现大量凋亡;实验组的淋巴细胞凋亡率较对照组明显降低,T细胞亚群表达率较对照组明显升高,差异有统计学意义(P<0.01);实验组的细胞凋亡相关基因bcl2、bclXL的表达水平较对照组上调,bax表达则较对照组下调(P<0.01)。结论IL-15能抑制放射线引起的淋巴细胞凋亡,改善急性放射病引起的免疫损伤。     

肿瘤浸润淋巴细胞及重组白细胞介素2治疗原发性肝细胞癌

目的探讨联合应用肿瘤浸润性淋巴细胞（ｔｕｍｏｒｉｎｆｉｔｒａｔｉｎｇｌｙｍｐｈｏｃｙｔｅｓ,ＴＩＬ）和重组白细胞介素２（ｒＩＬ２）治疗对原发性肝细胞癌（ｐｒｉｍａｒｙｈｅｐａｔｏｃｅｌｕｌａｒｃａｒｃｉｎｏｍａ,ＰＨＣＣ）术后患者细胞免疫功能的影响和其临床疗效。方法对１６例ＰＨＣＣ术后患者进行了ＴＩＬ和ｒＩＬ２治疗,１２例从肝动脉插管输入,４例从门静脉插管输入。结果ＴＩＬ治疗后１６例患者外周血白细胞介素２（ＩＬ２）,Ｔ细胞亚群及比率均有不同程度上升;肿瘤坏死因子（ＴＮＦ）,白细胞介素６（ＩＬ６）多有不同程度下降。随访６～２４个月,１４例肝癌根治性切除术患者,肿瘤无复发迹象,１例肝癌根治切除术患者ＴＮＦ上升了８ＩＵ,该患者治疗８个月后死于肝功能衰竭;１例肝癌姑息性切除,残癌无水酒精注射病人,ＴＩＬ治疗１８个月后肿瘤直径扩大２ｃｍ。结论ＴＩＬ治疗对提高ＰＨＣＣ术后患者抗肿瘤细胞免疫功能,防止肿瘤复发,抑制残瘤生长,延长患者生存期具有一定作用。     

人体肿瘤浸润淋巴细胞杀伤胃癌细胞的形态学观察

在光镜和电镜下观察人肿瘤浸润淋巴细胞（ＴＩＬ）杀伤ＭＫＮ４５传代人胃癌细胞的形态学变化。发现ＴＩＬ细胞与细胞共同孵育１小时后，前者向癌细胞靠近，并伸出伪足接触之３小时后，胃细胞结构模糊，胞浆内出现空泡，胞膜绒毛脱落。６小时后，癌细胞呈空泡状，膜裂解，细胞呈“凋零状”坏死，结果表明，ＴＩＬ细胞的主要作用方式是通过与癌细胞接触进行杀伤。     

肿瘤浸润淋巴细胞及重组白细胞介素2联合化疗在大肠癌？…

目的 研究进展期大肠癌根治术后应用肿瘤浸润淋巴细胞（ＴＩＬ）及重组白细胞介素－２（ｒＩＬ－２）联合化疗的临床治疗效果。方法 将４８例Ｄｕｋｅｓ Ｂ及ＤｕｋｅｓＣ期大肠癌患者于大肠癌根治术后采用前瞻性随机分组的方法分为２组：一组４４例,术后使用自身航ｒＩＬ－２联合－５氟脲嘧淀及丝裂霉素Ｃ治疗;另一组４０例,术后单纯采用相同方案的化疗。随访３年。结果 ＴＩＬ及ｒＩＬ－２联合化疗组患者３年内局部复发率为     

胃腺癌上皮细胞、癌旁上皮细胞及其浸润淋巴细胞白细胞介素-12和白细胞介素-13的表达

白细胞介素 (IL) 12是一种多功能免疫促进因子[1] ,主要由Th1细胞产生 ,先前对其抗肿瘤的基础和临床 ,尤其是应用于肿瘤的免疫疗法有大量研究 ;近年来 ,发现肿瘤细胞也可表达IL 12。IL 13由Th2细胞产生 ,是多功能免疫抑制因子[2 ] ,为探讨IL 12、IL 13与胃癌的发生发展关系 ,我们应用即用型SABC免疫组织化学法 (SP法 )观察胃癌上皮细胞及其浸润淋巴细胞 (TIL)白细胞介素 12和白细胞介素 13的表达 ,并探讨其意义。一、材料和方法1.材料 :10例胃腺癌组织及其对照癌旁组织 ,均用石蜡标本 ,所有标本术前未经化疗、放疗和免疫治疗。…     

肿瘤浸润性淋巴细胞在白细胞介素-2、-4协同下局部过继免疫治疗膀胱癌的实验研究

目的　观察膀胱癌肿瘤浸润性淋巴细胞 (TIL)联合不同细胞因子瘤灶内过继免疫抗癌的效应及对机体全身抗肿瘤免疫机制的影响。方法　建立BTT73 9动物模型 ,分离、培养TIL。采用正交设计实验方法 ,将TIL、白细胞介素 (IL) 2、 4及三因素交互组合悬液分别直接注射至瘤体内 ,定期测量肿瘤体积 ,免疫治疗 2周后检测NK细胞活性、T淋巴细胞转刺激指数 ,观察组织学及超微结构变化。结果　比较对照组 ,治疗 2周时各TIL相关组均不同程度抑制了膀胱肿瘤体积的增长 ,且NK细胞活性及T淋巴细胞转化增殖能力得以提高 (P <0 .0 5 )。TIL/IL 2疗法明显抑制了瘤体的增长 ,免疫治疗 1周后即表现出协同增强效应 (P <0 .0 5 ) ,而NK细胞活性及T淋巴细胞转刺激指数也显著提高 (P <0 .0 5 )。TIL/IL 2 /IL 4组获得了较强的抗癌功效 ,但与TIL/IL 2组差异无显著性 (P >0 .0 5 )。超微结构变化显示出TIL强烈的溶癌现象。结论　TIL在细胞因子特别是IL 2协同下瘤灶内注射的局部免疫疗法 ,具有较强的抗膀胱癌效应 ,并显著提高了机体全身抗瘤免疫功能。     

The effects of interleukin-6 on tumor-infiltrating lymphocytes derived from human renal cell cancer

Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of T cells with potent antitumor activity against a wide variety of tumors. TIL from renal cell cancer (RCC) typically exhibit diminished growth and antitumor activity after four weeks in vitro. We have therefore investigated effects of varying doses of interleukin-6 (IL-6) (0, 25, 100 units/ml.) on in vitro expansion, proliferation, cytotoxicity, and expression of cell surface phenotypes of long term renal TIL cultures from three RCC patients. Among the various conditions tested, three of three TIL cultures displayed a mild increase in cell expansion when grown in IL-2 with the addition of 100 units/ml. of IL-6. Two of three TIL cultures grown in IL-2 and 100 U/ml. of IL-6 demonstrated enhanced proliferation as determined by 3H-thymidine uptake. TIL could not be isolated or maintained in vitro when grown in the presence of IL-6 alone without IL-2. IL-6 was also found to enhance the long term non-specific cytotoxicity against an allogeneic nonrenal tumor target. No consistent effect on autologous tumor-specific cytotoxicity was demonstrated. We conclude that IL-6, when used in combination with IL-2, may modestly enhance the long-term growth of RCC-derived TIL.     

Regulatory effects of interleukin-4 on tumor-infiltrating lymphocytes derived from human renal cell carcinoma.

We have studied the effects of interleukin-4 (IL-4) on the expansion, proliferation, phenotype, and antitumor activity of tumor-infiltrating lymphocytes (TIL) derived from human renal cell carcinoma. Cultures were obtained from three primary renal tumors and one group of tumor-invaded, regional lymph nodes. IL-4 induced a significant increase in lymphocyte expansion and proliferation, but the response was dependent on the concurrent dose of IL-2 in culture. Increased growth activity was only observed in those cultures receiving low doses (20 U/ml) of IL-2 (average increase of fold expansion of 6.5, P < 0.01) with no changes in growth activity in the high dose (1000 U/ml) cultures. The combination of low dose IL-2 and IL-4 (200 U/ml) promoted lymphocyte growth significantly better than high dose IL-2 alone, the current standard growth regimen for in vitro expansion of TIL. TIL grown in the presence of IL-4 significantly reduced the level of non-specific, non-major histocompatibility complex-restricted antitumor activity (P < 0.01 for allogeneic renal, nonrenal, and NK-sensitive K562 cells), while exhibiting no effect on the level of autologous killing. This is in contrast to previous findings of significant enhancement of autologous antitumor activity using IL-4 on tumor-specific, melanoma-derived TIL. We also evaluated the effects of irradiated autologous tumor stimulation (TIL:tumor ratio, 10:1) on TIL cultures. Addition of autologous tumor cells increased expansion and proliferation of all cultures regardless of concurrent lymphokines present in the culture media (average increase fold expansion of 2.21, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

表达白细胞介素18的条件增殖腺病毒在肾癌Ketr-3细胞中的生物活性及抗肿瘤作用

目的 观察表达白细胞介素18(IL-18)基因的条件增殖腺病毒在肾癌Ketr-3细胞中的生物活性及其对Ketr-3细胞的杀伤作用.方法 通过荧光显微镜观察表达绿色荧光蛋白的条件增殖腺病毒(ZD55-EGFP)在肾癌Ketr-3细胞中的感染和增殖情况.分别将表达IL-18的条件增殖腺病毒(ZD55-IL-18)及表达IL-18的普通腺病毒(Ad-IL-18)感染人肾癌Ketr-3细胞系,通过Western blot法检测病毒E1A和IL-18蛋白的表达;免疫细胞化学染色检测IL-18抗原表达;TUNEL法检测Ketr-3细胞的凋亡情况;噻唑蓝(MTT)比色法检测Ketr-3细胞存活情况.结果 ZD55-EGFP能有效感染肾癌Ketr-3细胞并在其中大量增殖.Westem blot检测结果 发现ZD55-IL-18能在肿瘤细胞内表达E1 A并有效介导IL-18表达,在病毒感染Ketr-3细胞48 h后,ZD55-IL-18、Ad-IL-18处理组的IL-18蛋白表达量分别为255.6±3.1、118.7±2.90免疫组织化学检测显示ZD55-IL-18、Ad-IL-18处理组的IL-18抗原阳性率分别为(82.4±3.2)%和(23.4±1.9)%.TNUEL检测结果 显示ZD55-IL-18、Ad-IL-18处理组的细胞凋亡率分别为(52.2±3.5)%和(25.5±1.9)%.病毒感染4 d后,MTT检测结果 显示ZD55-IL-18、Ad-IL-18处理组细胞存活率分别为(32.6±2.3)%和(73.3±2.5)%,表明ZD55-IL-18对Ketr-3细胞有显著的杀伤作用.结论 ZD55-IL-18能在Kerr-3细胞中高效特异性表达IL-18基因并显示出良好的抗肿瘤作用.     

Antitumor effect of simultaneous transfer of interleukin-12 and interleukin-18 genes and its mechanism in a mouse bladder cancer model

BACKGROUND: The objectives of this study were to evaluate the antitumor effects of the simultaneous introduction of interleukin 12 (IL-12) and IL-18 genes into a mouse bladder cancer cell line (MBT2). We intended to compare these with those of either gene alone and to investigate the mechanism of the effects induced by the transfer of IL-12 and/or IL-18 genes in this model system. METHODS: We transfected the IL-12 and/or IL-18 genes into MBT2 cells by the liposome-mediated gene transfer method. We confirmed the secretion of IL-12 and/or IL-18 by enzyme-linked immunosorbent assay. Parental (MBT2/P), IL-12-transfected (MBT2/IL-12), IL-18-transfected (MBT2/IL-18) or both IL-12- and IL-18-transfected (MBT2/Both) cells were subcutaneously or intravenously injected into syngeneic C3H mice. To analyze the mechanism of tumor rejection, these clones were subcutaneously injected into naive nude mice and those depleted with natural killer (NK) cells by antibody. RESULTS: MBT2/IL-12, MBT2/IL-18 and MBT2/Both were completely rejected when they were injected subcutaneously or intravenously into syngeneic mice. However, MBT2/IL-12, but not MBT2/IL-18, could grow in nude mice. Moreover, the antitumor effect of MBT2/IL-18 was partially abrogated when injected into nude mice of which NK cells were depleted by antibody treatment. MBT2/Both was completely rejected in both nude mice with and without NK cells. CONCLUSION: The results of the present study indicate that T cells and NK cells seem to play important roles in the antitumor effects by the secretion of IL-12 and IL-18, respectively, and MBT2/Both possesses both mechanisms.     

转染增殖细胞核抗原肽表位融合白细胞介素-18基因的树突状细胞对T细胞增殖的影响

目的 观察转染增殖细胞核抗原(PCNA)肽表位、白细胞介素(IL)-18融合基因的树突状细胞(DCs)对T淋巴细胞增殖的诱导作用.方法 实验分为DCs组、空载体组、PCNA肽表位基因转染组、PCNA肽表位融合IL-18基因转染组共4组,分别将空质粒(pIPES-EGFP)、PCNA肽表位质粒[pIPES-EGFP-PCNA(6)]和PCNA肽表位融合IL-18基因质粒[pIRES-EGFP-PCNA(6)/IL-18]通过脂质体转染DCs.每组按照DCs∶T淋巴细胞1∶1、1∶10、1∶20、1∶40、1∶80的比例分为4个浓度梯度,将DCs与T淋巴细胞共培养.采用CCK-8法检测T细胞增殖.结果 各组DCs均能刺激T淋巴细胞增殖,DCs∶T为1∶10时,转染pIPES-EGFP-PCNA(6)/IL-18的DCs刺激指数为2.62,优于DCs组(1.70)、空载体组(1.78)和pIPES-EGFP-PCNA(6)组(1.93).结论 转染pIRES-EGFP-PCNA(6)/IL-18的DCs刺激T淋巴细胞增殖的作用最强,DCs:T为1∶10时最佳.     

转染增殖细胞核抗原肽表位融合白细胞介素-18基因的树突状细胞对T细胞增殖的影响

目的 观察转染增殖细胞核抗原(PCNA)肽表位、白细胞介素(IL)-18融合基因的树突状细胞(DCs)对T淋巴细胞增殖的诱导作用.方法 实验分为DCs组、空载体组、PCNA肽表位基因转染组、PCNA肽表位融合IL-18基因转染组共4组,分别将空质粒(pIPES-EGFP)、PCNA肽表位质粒[pIPES-EGFP-PCNA(6)]和PCNA肽表位融合IL-18基因质粒[pIRES-EGFP-PCNA(6)/IL-18]通过脂质体转染DCs.每组按照DCs∶T淋巴细胞1∶1、1∶10、1∶20、1∶40、1∶80的比例分为4个浓度梯度,将DCs与T淋巴细胞共培养.采用CCK-8法检测T细胞增殖.结果 各组DCs均能刺激T淋巴细胞增殖,DCs∶T为1∶10时,转染pIPES-EGFP-PCNA(6)/IL-18的DCs刺激指数为2.62,优于DCs组(1.70)、空载体组(1.78)和pIPES-EGFP-PCNA(6)组(1.93).结论 转染pIRES-EGFP-PCNA(6)/IL-18的DCs刺激T淋巴细胞增殖的作用最强,DCs:T为1∶10时最佳.     

Interleukin-18 and interleukin-12 synergistically inhibit osteoclastic bone-resorbing activity

The effect of interleukin (IL)-18 on osteoclastic bone-resorbing activity was investigated in vitro. Osteoclast-enriched cells, about 70% of which were tartrate-resistant acid phosphatase (TRAP)-positive, were cultured on dentine slices, and then the total volume of resorption pits on each dentine slice was measured as bone-resorbing activity. When the effects of IL-18 alone at 1, 10, 100, and 1000 ng/mL were examined, bone-resorbing activity was significantly reduced only at 1000 ng/mL, by about 50%. However, IL-18 plus IL-12 (10 ng/mL each) reduced bone-resorbing activity by about 70%, whereas IL-12 alone had no significant effect. When the concentration of interferon (IFN)-γ in the medium was measured, IL-18 or IL-12 was found to increase it slightly, and the combination of these two cytokines synergistically increased it. The inhibitory effect of the combination of the two cytokines was completely abolished by the addition of an anti-IFN-γ neutralizing antibody to the medium, but IFN-γ by itself did not inhibit osteoclastic bone resorption. IL-18 alone or in combination with IL-12 did not affect the number of TRAP-positive cells in culture of osteoclast-enriched cells. Osteoclasts prepared from osteoclast-enriched cells expressed mRNAs of IL-18 receptor, MyD88, and cathepsin K. Furthermore, IL-18 receptor protein was detected on the cell surface of osteoclasts. The present results indicate that the combination of IL-18 and IL-12 synergistically inhibits osteoclastic bone-resorbing activity, suggesting that IFN-γ participates in the mechanism underlying this inhibition.     

白细胞介素-18与一氧化氮在非小细胞肺癌的生长与转移中的意义

目的探讨白细胞介素(IL)-18和一氧化氮(NO)在非小细胞肺癌(NSCLC)的生长与转移方面的影响作用。方法采用酶联免疫吸附法(ELISA)及Griess反应分别检测82例NSCLC肿瘤组与20例正常对照组血清IL-18和亚硝酸盐+硝酸盐浓度。结果肿瘤组血清IL-18浓度为(334.2±31.0)ng/L,对照组为(151.3±22.0)ng/L,肿瘤组亚硝酸盐+硝酸盐浓度为(237.1±21.0)μmol/L,对照组为(44.2±15.0)μmol/L,肿瘤组明显高于对照组。肿瘤组外周血血清IL-18和亚硝酸盐+硝酸盐浓度与患者的性别、年龄、病理类型无关。IL-18血清浓度与肿瘤的TNM分期、淋巴结转移及远处转移有关但与淋巴结转移的数量和部位及远处转移的脏器类别差异无统计学意义。而亚硝酸盐+硝酸盐浓度则与之无关。IL-18与亚硝酸盐+硝酸盐浓度之间无关联。结论研究提示血清中IL-18和亚硝酸盐+硝酸盐浓度在NSCLC患者对疾病的评估具有临床检测意义。     

表达白细胞介素-18的结肠癌瘤苗抗肿瘤作用及其机制

目的 观察表达人白细胞介素(hIL)-18的结肠癌移植瘤新生血管生成及肿瘤生长.方法 0.1 ml单克隆化的人结肠癌sw480细胞、携有绿色荧光蛋白表达质粒的pEGFP-sw480细胞、携有hIL-18基因表达质粒的hIL-18.pEGFP-sw480细胞(1×108个/ml)分别注射裸鼠,每组5只.ELASA法检测裸鼠血清IL-18含量;记录成瘤时间及瘤体大小;免疫组织化学法检测移植瘤组织VEGF、MMP-9及微血管密度(CD34表达);TUNEL法检测移植瘤组织凋亡.结果 hIL-18-pEGFP-sw480组成瘤时间为(26.2±1.8)d,而pEGFP-sw480、sw480组分别为(14.8±2.8)d和(14.6±1.9)d.注射42 d后,hIL-18-pEGFP-sw48组裸鼠血中IL-18含量为(63.58±13.75)ng/L.hIL-18-pEGFP-sw480、pEGFP-sw480、sw480组肿瘤平均体积(mm3)分别为:336.39±28.50、1029.08±114.84、957.96±91.55.ML-18-pEGFP-sw480组肿瘤明显小于sw480组和pEGFP-sw480组(P<0.01).微血管密度(CD34)分别为(9.08±4.01)、(32.48±8.84)、(33.32±3.39),MMP-9灰度值分别为143.2±6.8、114.6±5.4、110.2±5.9;VEGF灰度值分别为140.8±9.2、108.5±8.7、107.3±4.9,hIL-18.pEGFP-sw480组血管生成及促血管生成因子表达明显减少;hIL-18-pEGFP-sw480组凋亡细胞阳性率(30.32±7.23)%明显高于pEGFP-sw480组(6.32±1.18)%和sw480组(5.80±2.28)%(P<0.01).结论 IL-18可明显减少裸鼠结肠癌sw480细胞移植瘤的血管生成,促进肿瘤细胞凋亡,抑制肿瘤生长.