以逆转录病毒为载体将人IL—2基因插入大鼠纤维肉瘤细胞

应用基因工程的技术将外源性基因引入肿瘤细胞成为在基因水平深入研究肿瘤分子生物学特性及探索可能的有效的防治途径的重要手段。白细胞介素—2(interleukin 2,IL—2)是T细胞分泌产生的一种多功能免疫活性调节因子,具有广泛的抗肿瘤活性。我们用逆转录病毒pLXSN载体成功地将人IL—2基因导入WKA大鼠WBT纤维肉瘤细胞系并得以持续稳定地高效表达。     

逆转录病毒介导IL—2基因导入肝癌细胞的研究

利用逆转录病毒载体系统ＰＡ３１７／ＬＮＣ－ＩＬ－２所产生的含有ＩＬ－２基因及新霉素抗性基因的重组病毒，经共培养转染ＳＭＭＣ－７７２１肝癌细胞株。ＩＬ－２基因受巨细胞病毒早期启动子调控，转染后的细胞置于含新霉素类毒性药物Ｇ４１８（４００ｕｇ／ｍｌ）培养基内培养、筛选３０天，结果果转染率约为１￣２％。对存活下来的转染有ＩＬ－２基因和新霉素抗性基因的细胞形态和生长情况进行观察，同时从水平及蛋白质水平对外     

33例头颈部恶性肿瘤患者局部过继免疫治疗的疗效观察

目的评价ＩＬ－２／ＬＡＫ细胞局部过继免疫疗法在头颈部恶性肿瘤治疗中的疗效。方法对３３例头颈部恶性肿瘤患者进行局部过继免疫治疗,采用ＩＬ－２每日１０～２０万单位局部注射,共１０天;于ＩＬ－２治疗的第４～８天同时于局部注射ＬＡＫ细胞１．０×１０８～５．０×１０８／ｄ。结果完全缓解１例,部分缓解６例,好转２０例,稳定６例,治疗总缓解率２１．２％,总有效率８１．８％。治疗后１,２,３年生存率分别为９６．３％、８３．３％、和７５．０％。组织病理学检查证实免疫治疗后肿瘤局部大量ＣＤ３、ＣＤ４阳性Ｔ淋巴细胞浸润。治疗过程中未见严重的毒副作用。结论局部应用ＬＡＫ细胞与ＩＬ－２治疗头颈部恶性肿瘤疗效明显,方法安全。     

重组人白细胞介素-2治疗晚期恶性肿瘤临床观察

目的　观察重组人白细胞介素 - 2治疗晚期恶性肿瘤疗效。方法　 2 1例经反复化疗或放疗失败的晚期恶性肿瘤患者 ,其中小细胞肺癌 2例 ,非小细胞肺癌 3例 ,胃腺癌 3例 ,食管鳞癌 3例 ,肝癌 1例 ,卵巢癌 3例 ,非霍奇金淋巴瘤 3例 ,恶性黑色素瘤 1例 ,每日静滴白细胞介素 - 2 10 0万 U ,连续 2 8天。结果　 CR 0例 ,PR 3例 ,MR 2例 ,NC 8例 ,PD 8例 ,总有效率 14 .3%。结论　单独应用白细胞介素 - 2治疗某些晚期肿瘤有一定疗效 ,可以改善部分病人的临床症状     

淋巴瘤化疗后行大剂量IL-2免疫治疗对长期生存的影响

目的探讨恶性淋巴瘤化疗缓解后行大剂量白介素-2（IL-2）免疫治疗的临床疗效。方法 2005年1月～2010年5月共40例淋巴瘤患者（治疗组）化疗缓解后进行大剂量IL-2免疫治疗,随机挑选的40例患者（对照组）,化疗结束缓解后不进行任何免疫治疗,检测两组患者外周血T淋巴细胞亚群,并对所有患者进行随访观察。结果治疗组外周血T淋巴细胞亚群水平明显提升,随访结束时统计复发率：治疗组17.5%,对照组32.5%;中位生存期：治疗组（34.5±2.0）（20～60）个月,对照组（23.2±2.0）（12～60）个月。结论恶性淋巴瘤化疗缓解后行大剂量IL-2治疗是有效的免疫治疗手段,能提高患者的免疫功能,减少复发率,延长生存期,具有潜在临床应用前景。     

乌苯美司对肺鳞癌化疗患者免疫功能的影响

目的 评价乌苯美司对肺鳞化疗患者免疫功能的影响。方法 将56例未手术肺鳞癌患者按3：2的比例随机分入试验组（34例）及对照组（22例）,患者均接受CAP方案化疗,每21天为1周期,共3个周期;试验组加服乌苯美司30mg,每日1次,共6个月。分析化疗疗效,观察治疗前、治疗后3、6个月淋巴细胞绝对计数（LC）、OKT4／T8、NK细胞活性、血清IL－2、SIL－2R水平的变化情况。结果 在可评价的52例（试验组32例,对照组20例）患者中,两组间近期化疗疗效无统计学差异（P＞0．05）。治疗后3、6个月,试验组OKT4／T8比值、NK细胞活性及IL－2水平均较治疗前明显提高（P＜0．05）,但前二项指标与对照组比较无统计学差异（P＞0．05）,而后一项则有明显统计学差异（P＜0．05）;治疗后3、6个月,试验组患者血清SIL－2R水平均较治疗前降低（P＜0．05）,但组间差异比较无统计学意义,而对照组除治疗后6月SIL－2R水平较治疗前明显降低外（P＜0．05）,其余各项指标与治疗前比较均无显著性差异。结论 乌苯美司对肺鳞癌化疗患者OKT4／T8比值、NK细胞活性、血清IL－2和SIL－2R有一定程度的改善作用,但其临床应用价值仍需进一步观察确定。     

重组人白细胞介素-2联合化疗治疗多发性骨髓瘤

 目的　研究重组人白细胞介素-2（rIL-2）联合化疗治疗多发性骨髓瘤（MM）的临床疗效及患者免疫功能变化。方法　分单纯化疗组和化疗+rIL-2组。化疗+rIL-2组于常规化疗（MP或VAD方案为主的方案）同时联合应用rIL-2,用法为100万U,皮下注射,5次／周,连用4周为1个周期,连续应用4个周期后检测免疫指标（T细胞亚群、NK细胞、CD+3 CD+69细胞百分率）变化,采用Bladé标准评价疗效,按照NCI CTCAE（第3版）判断不良反应。结果　化疗+rIL-2组治疗后CD3、CD4和CD56（NK）阳性百分率及CD4／CD8比值均明显高于化疗前和单纯化疗组化疗后水平,总有效率（CR+nCR+PR）、缓解率（CR+nCR）分别为66.7 %和25.0 %,而单纯化疗组为50.0 %和8.3 %。rIL-2不良反应轻微。结论　rIL-2能够改善MM化疗患者的免疫功能,提高有效率,降低感染率,值得继续应用。     

人白细胞介素2重组逆转录病毒载体的构建与表达

通过RT-PCR克隆到含有全部编码序列的人IL-2 cDNA,并经核苷酸测序加以证实。将其克隆至逆转录病毒载体pLXSN,构建成人IL-2的重组逆转录病毒表达载体pLXSN-hIL2,体外经CRIP细胞包装后病毒滴度达7.6×10~5CFU/ml。NIH3T3小鼠成纤维细胞感染hIL-2重组逆转录病毒后,分泌IL-2水平达118.2U/ml;PCR从其基因组DNA中扩增到NeoR基因片段,提示重组逆转录病毒载体己整合至宿主细胞的基因组DNA中。本研究为开展人IL-2基因治疗奠定了基础。     

IL—2和（或）IL—3基因治疗对大剂量化疗后小鼠免疫功能恢复的影响

目的探讨ＩＬ－２、ＩＬ－３对化疗后小鼠免疫功能恢复的影响。方法正常小鼠腹腔注射大剂量环磷酰胺２４小时后,直接腹腔注射ＩＬ－２重组腺病毒（Ａｄ－ＩＬ－２）和（或）ＩＬ－３重组腺病毒（Ａｄ－ＩＬ－３）,观察小鼠腹腔巨噬细胞数量、杀伤活性、Ｉａ抗原表达、脾细胞增殖及ＮＫ细胞杀伤活性。结果腹腔注射Ａｄ－ＩＬ－３组小鼠,腹腔巨噬细胞数量明显增加,杀伤活性及Ｉａ抗原表达显著增强。腹腔注射Ａｄ－ＩＬ－２组小鼠,脾细胞增殖能力显著升高,ＮＫ细胞杀伤活性增强,但对小鼠腹腔巨噬细胞无激活作用。联合应用Ａｄ－ＩＬ－２及Ａｄ－ＩＬ－３组小鼠,腹腔巨噬细胞、脾细胞、ＮＫ细胞均被激活。结论腹腔直接注射Ａｄ－ＩＬ－２和Ａｄ－ＩＬ－３可促进化疗后小鼠免疫功能的恢复。     

羧甲基茯苓多糖对IL-2促诱生效应的实验与临床研究

采用0.5％～1.0％低血清培养法较好地模拟了小剂量rhTNF-α(50U/ml)对人早幼粒白血病细胞株HL-60的增殖抑制效应,但未发现促分化作用,对细胞株c-myc基因的表达亦无明显影响;而TNF(50μ/ml)具有诱导HL-60细胞分化和抑制细胞增殖的作用,且显著抑制其c-myc癌基因的表达水平。结果表明c-myc基因表达减低主要与TNF诱导HL-60白血病细胞分化相关,并非细胞增殖受抑的结果,对癌基因表达水平的调控可能是小剂量TNF发挥分化诱导作用的重要机制。     

Clinical efficacy of adoptive immunotherapy by IL-4 activated tumor-infiltrating lymphocytes in patients with advanced cancer

Background Adoptive immunotherapy using tumor-infiltrating lymphocytes (TILs) has been used to treat malignant melanoma and renal cell carcinoma, although the clinical efficacy of this method has not yet been proved. To improve clinical efficacy, it is important to induce sufficient amounts of tumor-infiltrating lymphocytes to fight the tumor. In this study, we investigated the clinical efficacy of adoptive immunotherapy using interleukin (IL)-2 activated tumor-infiltrating lymphocytes combined with IL-4. Methods Tumor-infiltrating lymphocytes from patients with non-malignant melanoma and non-renal cell carcinoma were cultured with IL-2 monotherapy (n=10) and combination therapy with IL-4+IL-2 (n=20). Comparing the 2 groups, we investigated the clinical benefits of adoptive immunotherapy, considering safety, clinical efficacy, survival periods, and quality of life. Results By using the IL-4+IL-2 combination, we successfully transferred 6.5 times more activated tumor-infiltrating lymphocytes, as compared to cell counts using IL-2 monotherapy. The response rate achieved was 58.8% (2 complete and 8 partial responses) without any side effects in the IL-4+IL-2 group. Furthermore, the IL-4+IL-2 group had longer survival periods and improved quality of life, compared to the IL-2 monotherapy group. Conclusion The IL-4+IL-2 combination improved the clinical efficacy of adoptive immunotherapy, and improved quality of life in patients with non-malignant melanoma and non-renal cell carcinoma.     

A Clinical Study on Juheli (Recombinant Human Interleukin - 11) in the Second Prevention of Chemotherapy Induced Thrombocytopenia

Objective: to investigate the effect and side effects of recombinant human interleukin - 11 (rhIL - 11, in Chinese Juheli, produced by Qi Lu Biotechnology CO., LTD) in the second prevention of chemotherapy induced thrombocytopenia (CIT). Methods: Cancer patients with CIT were recruited and were treated with rhIL - 11 (treatment phase, TP), and in the following cycle, all these patients administered with rhIL - 11 24 hours immediately after chemotherapy (preventive treatment phase, PTP). Duration and severity of thrombocytopenia between two phases were compared. Results: for patients in TP or PTP, nadir values of platelet were (29.28±20.08)×109/L and (45.24±19.66)×109/L, duration of thrombocytopenia in TP and PTP was (11.52±4.33) and (8.20±+2.77)days, recovery time was (19.40±3.89)and (13.44±3.02)days, duration of rhIL - 11 administration was 10.68±2.46)and (6.28±1.77)days, number of patients needing platelet infusion was 16and4 respectively, all differences were statistically significant (p value were 0.007, 0.002, 0.000, 0.000, 0.034 respectively). For TP and PTP, number of patients with hemorrhage was 8 and 4, duration of bleeding was (5.00±0.82) and (4.50 ± 0.71) days respectively, with no statistically significant difference. Adverse reactions mainly included fever, edema, arrhythmia, joint pain, fatigue, skin rash, headache, dizziness, etc., all were not statistically significant between TP and PTP. Conclusion: rhIL - 11 could be well tolerated and is effective that could reduce the duration, severity of CIT, platelet transfusion, and incidence of bleeding, as well as shorten the recovery time, duration of rhIL - 11 administration. Thus, rhIL - 11 could be commended in the second prevention of CIT for patients with cancer.     

化疗联合细胞因子诱导的杀伤细胞治疗中晚期胃癌临床研究

目的评估化疗联合细胞因子诱导的杀伤细胞（cytokine induced killers，CIK）治疗中晚期胃癌的临床疗效。方法参照美国斯坦福大学骨髓移植中心建立的CIK培养方法诱导扩增CIK联合化疗，培养14d后分次回输给病人，观察病人治疗前后瘤体变化、生活质量、卡氏评分等，同时记录生存期。结果21例胃癌单纯化疗后，完全缓解（CR）2例，部分缓解（PR）4例，好转（MR）8例，稳定（SD）3例，无改变（PD）4例，总有效率为28．6％。21例化疗联合CIK治疗后，完全缓解（CR）4例，部分缓解（PR）9例，好转（MR）6例，稳定（SD）1例，无改变（PD）1例，总有效率为52．38％。两组之间具有明显的差异（x^2=6．43，P〈0．05），胃癌单纯化疗后1a生存期为90．5％，2a生存期为81．0％，3a生存期为71．4％。胃癌化疗联合CIK治疗后1a生存期为95：2％，2a生存期为85．7％，3a生存期为81．0％。两组3a生存期没有明显差异（P〉0．05）。用Karnofsky评分及体重的改变来评估生存质量的改善。胃癌单纯化疗后Karnofsky评分平均提高10分，其中提高5例，稳定8例，下降8例，提高率为23．8％。化疗联合CIK治疗后Karnofsky评分平均提高10-20分，其中提高10例，稳定5例，下降6例，提高率为47．6％。两组生存质量变化没有明显差异（P〉0．05）。结论化疗联合CIK治疗对为那些手术、放疗、单纯化疗已无适应证的病人提供了一种可以继续延长生存期，提高生活质量的新的途径。     

过继免疫治疗联合放化疗治疗非小细胞肺癌的Meta分析

目的： 探讨过继免疫治疗联合放化疗与单纯放化疗相比对非小细胞肺癌（non-small cell lung cancer,NSCLC）患者疗效的差异。 方法: 通过检索PubMed、Medline、EMBASE、Cochrane 数据库、中国期刊全文数据库和维普中文数据库,收集1995年1月至2012年9月发表的符合要求的随机化对照试验（randomized controlled trial,RCT）或非随机同期对照试验（non-randomized concurrent controlled trail,NRCCT）,应用Revman5.0软件进行数据分析。 结果： 共纳入10项研究,共计1 326名患者。Meta分析结果显示：与单纯放化疗相比,过继免疫治疗联合放化疗能够提高患者2年无进展生存（progression-free survival,PFS）（OR=2.20,95%CI：1.44~3.36,P=0.0003）和2年总生存（OR=2.69,95%CI：1.92~3.78,P<0.00001）。接受过继免疫治疗后,早期和进展期NSCLC患者都能得到较大的获益\[（OR=3.24（1.65~6.35）;OR=2.86（1.37~5.98）\]。过继免疫治疗引起的不良反应多呈自限性,主要有发热、寒战、恶心、乏力等,未观察到严重毒性反应。 结论： 过继免疫治疗联合放化疗能延缓NSCLC复发,提高患者生存期,且早期患者接受免疫治疗获益更显著。     

Establishment of interleukin 2 dependent cytotoxic T lymphocyte cell line specific for autologous brain tumor and its intracranial administration for therapy of the tumor

Autologous brain tumor specific CTLs were induced from the patient's PBL by a mixed lymphocyte-tumor culture, and were maintained for more than 2 months in a medium containing exogenous ILr2. The autologous T cell line containing specific CTL was administered into the tumor-bed for the treatment of malignant glioma. In 2 cases out of 5, tumors regressed more than 50% in diameter. One of these patients is still alive now with full of his social activities, and it is 104 weeks after the initiation of the immunotherapy. Autologous T cell lines were safely administered in all cases without any complications nor toxicities.     

过继免疫疗法(AIT)与羟基喜树碱(HCPT)序贯应用治疗膀胱癌的实验研究

目的了解过继免疫疗法(AIT)与羟基喜树碱(HCPT)序贯应用对膀胱肿瘤的作用.方法体外实验测定TIL及TIL与HCPT联合应用对不同靶细胞的杀伤活性,体内实验了解不同治疗方法对小鼠膀胱肿瘤的治疗效果.结果TIL对同种异体膀胱肿瘤细胞有强大的杀伤活性,对正常组织细胞无毒性,TIL与HCPT联合应用对肿瘤细胞的杀伤毒性低于单独应用时的杀伤活性.体内实验说明同时应用TIL及HCPT无明显疗效,而合理联合应用即序贯疗法有强大的抗癌作用.结论通过本实验说明过继免疫疗法与HCPT序贯应用才能起到抗癌作用,其可能机理是序贯疗法充分发挥HCPT化疗作用又不影响TIL的杀伤活性,并且有促进TIL聚积于肿瘤周围的效果.     

Levels of soluble interleukin-2 receptors are predictive of response in patients treated with interleukin-2 and lymphokine-activated killer cells

Soluble interleukin-2 receptor (sIL-2R) α (CD25) levels were serially determined in the sera of 20 patients who had undergone adoptive immunotherapy with high-dose IL-2 and lymphokine-activated killer (LAK) cells for various types of metastatic solid tumors or Hodgkin’s disease. The treatment course consisted of 5 days of high-dose IL-2 priming followed by the collection of peripheral blood leukocytes by leukapheresis, andin vitro activation of mononuclear cells with IL-2, and the subsequent infusion of such prepared LAK-cells together with IL-2. sIL-2R levels increased in all patients following IL-2 administration, and the ratio of baseline sIL-2R levels to those measured after 5 days of IL-2 was signifıcantly correlated with pre-IL-2 levels (p=0.016) in that higher pre-IL-2 levels resulted in a larger increase upon IL-2 administration. In terms of treatment outcome, the variables analysed included sIL-2R levels, total IL-2 doses administered, the expression of membrane-bound CD25 onin vitro cultured cells (pre- and post-IL-2 exposure), the total number of LAK-cells infused andin vitro cytotoxic activity of LAK-cells against the natural killer cell-resistant cell line Daudi. In a multivariate analysis, low baseline sIL-2R levels (p=0.095) and highin vitro cytotoxic activity of LAK-cells against Daudi cells (p=0.082) were jointly associated with response. Our data suggest that serum sIL-2R levels provide a fast and non-invasive parameter for predicting the response in patients treated with IL-2 and LAK-cells.     

A Multicenter Randomized Clinical Trial Evaluating Interleukin-2 Activated Hematopoietic Stem Cell Transplantation and Post-transplant IL-2 for High Risk Breast Cancer Patients

Summary Purpose. This Phase III randomized multicenter trial compared progression-free (PFS) and overall survival (OS) for autologous peripheral blood stem cell (aPBSC) transplantation with or without immunotherapy in high-risk breast cancer patients. Methods. Eligible patients had American Joint Committee on Cancer (AJCC) 5th Edition Stage II/IIIA with ≥ 4 axillary nodes, Stage IIIB, or chemotherapy-sensitive or stable Stage IV disease. Following treatment with cyclophosphamide, thiotepa and carboplatin (STAMP V), patients were randomized to aPBSC transplant with or without immunotherapy. Patients on immunotherapy received cells that were incubated in interleukin-2 (IL-2) for 24 h followed by parenteral IL-2 for 5 days then 2 days of rest for 4 weeks. Results. Fifty-nine patients were treated (35 Stage II/IIIA; 13 Stage IIIB; 11 Stage IV), 30 patients were randomized to immunotherapy and 29 patients to no immunotherapy. Neutrophils engrafted a median of 10 days post-transplant in both groups. The median times to platelet engraftment were 9 and 10 days after transplant in the no-immunotherapy and immunotherapy groups, respectively (p = 0.03). There was no statistical evidence (p = 0.61) of a difference in progression-free and surviving (PFS) at 3 years for patients receiving immunotherapy (53%) compared with no immunotherapy (48%). There was some evidence of superiority in overall survival (OS) at 3 years for patients receiving immunotherapy (83%) compared with no immunotherapy (69%), but the difference between survival curves was not statistically significant (p = 0.08). Also, there was some evidence that patients developing acute graft versus host disease (aGVHD) had superior PFS (p = 0.02) but not OS (p = 0.19) than patients not developing aGVHD. Toxicities were transient and similar between groups, with no treatment-related deaths. Conclusions. This phase III study of high-risk breast cancer patients randomized to immunotherapy or no immunotherapy demonstrated that a well-tolerated immunotherapy regimen added to aPBSC transplant did not improve PFS, but there was some improvement in OS, but not by an amount that was statistically significant (p = 0.08).     

Effect of Krestin (PSK) on the induction of IL-2 activated killer cells.

The effects of Krestin (PSK) on the generation of lymphokine-activated killer (LAK) cells were examined in tumor-bearing mice. BALB/c mice were inoculated subcutaneously with methylcholanthrene-induced fibrosarcoma (Meth A) cells, and PSK was administered intraperitoneally every other day. The reduced LAK activity in tumor-bearing mice was restored by the administration of PSK. Since involvement of the humoral immunosuppressive factor in the impairment of LAK activity has been suggested, the effect of PSK on the impaired LAK activity in the presence of an immunosuppressive factor isolated from the ascites of X5563 (plasmacytoma)-inoculated mice was examined. The activity reduced by the immunosuppressive factor in an in vitro induction of LAK was restored by incubation with PSK. The antimetastatic effect of IL-2 was also augmented by its combined use with PSK. The data provide a rational basis for using PSK in combination with recombinant IL-2 in cancer immunotherapy.