体外转染小鼠白细胞介素12基因抑制膀胱癌EJ细胞的实验研究

目的将含有小鼠白细胞介素12（mIL-12）全长基因的质粒转染到膀胱癌EJ细胞中,观察脾细胞对转染mIL-12膀胱癌细胞的杀伤作用。方法用脂质体转染法将含有mIL-12全长基因质粒转染到EJ细胞中,ELISA法检测IL-12表达水平。在脾细胞作用0～2 d时ELISA法连续检测上清干扰素γ（INF-γ）水平变化。转染后40 h加入制备好的小鼠脾细胞悬液约1×10^6作用24 h后,MTT法检测对膀胱癌细胞的抑制率。结果转染后40、60 h检测到IL-12的高表达（375±15） pg／ml和（400±50）pg／ml,较未转染组9～15 pg／ml明显增高;在EJ细胞表面可以检测到IL-12的表达,而对照组则无;加入脾细胞悬液1 d后,脾细胞加转染后的EJ细胞组的抑制率为58．7％（P〈 0．025）,而脾细胞加未转染EJ细胞组、单纯EJ细胞组以及转染的EJ细胞组差异无统计学意义（P〉 0．05）。脾细胞加入转染后EJ细胞上清24 h的。INF-γ为（155±21）pg／ml,与未转染组（65±8）pg／ml比较差异有统计学意义（P〈0．05）。结论体外转染mIL-12基因到膀胱癌EJ细胞中能够表达IL-12,在小鼠脾细胞存在的条件下,通过一系列的免疫调节作用,诱导T细胞、NK细胞等产生INF-γ等细胞因子从而杀伤膀胱癌细胞。     

重组腺病毒介导IL-12、IL-2联合基因治疗前列腺癌的实验研究

目的　探讨转移性前列腺癌的治疗方法 ,为前列腺癌免疫基因治疗提供实验依据。　方法　采用重组腺病毒介导IL 12、IL 2联合免疫基因疗法 ,对 6 6只C5 7BL/ 6小鼠前列腺癌模型进行致瘤性和抑瘤性观察。　结果　腺病毒AdmIL 12、AdhIL 2能有效表达目的基因。接种野生型 ,转AdLacZ及转AdmIL 12、AdhIL 2混合RM 1细胞的小鼠致瘤比例分别为 10 / 10、10 / 10及 2 / 10 ;成瘤时间分别为 (12 .3± 1.5 )d、(12 .8± 1.0 )d、(2 2 .5± 2 .1)d ;接种 30d后肿瘤结节直径分别为 (35 .0±2 .0 )mm、(34 .0± 2 .6 )mm、(10 .5± 3.5 )mm。与对照组相比 ,转AdmIL 12、AdhIL 2基因RM 1细胞小鼠致瘤率下降 ,成瘤时间延迟 (P <0 .0 1)、瘤结节小 (P <0 .0 1)。AdmIL 12、AdhIL 2瘤体注射还可抑制肿瘤生长 (P <0 .0 1) ,减少肺转移灶数目 (P <0 .0 1)。　结论　IL 12、IL 2联合基因治疗可诱发前列腺癌小鼠的肿瘤特异性免疫反应。     

IL-4,IL-12在宫颈病变中的表达及意义

目的:探讨IL-4,IL-12在宫颈病变发生发展中的作用以及对机体免疫功能的影响.方法:应用RT-PCR技术检测IL-4mRNA,IL-12p35以及IL-12p40 mRNA在正常宫颈组、CINⅠ、CINⅡ-Ⅲ和宫颈癌组中的表达.结果:在宫颈癌组中IL-4表达高于CIN和正常宫颈组,CI-NⅡ-Ⅲ组高于CINⅠ和正常宫颈组(P＜0.05),IL-12p35和IL-12p40mRNA表达均低于CIN和正常宫颈组,CIN各组均低于正常宫颈组(P＜0.05).结论体内IL-4,IL-12水平与肿瘤的发生密切相关,体内IL-12降低和(或)IL-4升高可促进宫颈癌的发生发展.     

丙泊酚对肺癌患者外周血IL-12、IFN-γ及IL-4的影响

目的探讨丙泊酚对肺癌患者外周血细胞因子白细胞介素(IL)-12、γ-干扰素(IFN-γ)、IL-4的影响。方法30例行非小细胞型肺癌肺叶切除术患者随机分为丙泊酚(IV)组和异氟醚(IH)组,每组15例。分别于麻醉诱导前(T0)、麻醉诱导后10min(T1)、切皮后1h(T2)、停药即刻(T3)、术后1h(T4)及术后24h(T5)采集肘静脉血,测定血清IL-12、IFN-γ、IL-4及皮质醇(Cor)浓度。结果与T0时比较,IV组T5时IL-12、IFN-γ/IL-4及T4、T5时IFN-γ明显增高(P<0.05或P<0.01);且T5时IL-12、IFN-γ/IL-4及T4、T5时IFN-γ均高于IH组(P<0.05)。两组IL-4均有增高趋势,但组内、组间差异无显著意义。与T4时相比,T5时IV组IFN-γ、IFN-γ/IL-4增高明显(P<0.05)。结论丙泊酚可以促进外周血IL-12、IFN-γ的分泌,升高IFN-γ/IL-4比值,诱导围术期Ⅰ型辅助性淋巴细胞(Th1)反应,有利于抗肿瘤、抗感染免疫。     

白细胞介素12基因治疗结肠癌的实验研究

目的观察携白细胞介素12(mIL-12)基因逆转录病毒载体的包装细胞(PA317-mIL-12)瘤内注射后,对肿瘤生长的抑制及对荷瘤动物生存的影响。方法在非免疫缺陷的结肠癌动物模型上分别施以化疗、放射免疫(放免)导向治疗、PA317-mIL-12基因治疗以及放免导向与IL-12基因联合治疗,比较各治疗组的肿瘤抑制效果和荷瘤动物的生存情况。结果经筛选后的携有mIL-12逆转录病毒载体包装细胞系PA317-mIL-12的上清,mIL-12浓度48h达27ng/106细胞。治疗3周后,IL-12基因治疗组(GT组)和IL-12基因与RIT联合治疗组(GT加RIT组)无论瘤体积还是瘤重量均明显低于对照组,生存率明显高于对照组(P<0.01)。结论PA317-mIL-12瘤内注射可有效抑制肿瘤生长,延长荷瘤动物的生存时间。基因治疗疗效优于化疗、放免导向治疗。放免导向与IL-12基因联合治疗疗效更佳。     

白细胞介素12对肝癌基因治疗的实验研究

目的 研究携带白细胞介素１２（ＩＬ－１２）基因逆转录病毒载体对体内肝癌生长抑制作用,探索基因治疗肝癌新途径。方法 构建携带ＩＬ－１２的逆转录病毒载体,转染逆转录病毒包装细胞系ＰＡ３１７,应用该包装细胞对实验性肝癌大鼠进行治疗,观察抗肿瘤作用,免疫功能变化。     

血清IL-6、IL-10、IL-12在消化道肿瘤中的表达及其意义

目的探讨消化道肿瘤患者血清IL-6、IL-10、IL-12的变化及其意义.方法应用酶联免疫法测定了胃癌、结肠癌及健康者血清IL-6、IL-10、IL-12含量,比较其相互间的关系.结果胃癌和结肠癌组IL-10、IL-12含量低于对照组;IL-6含量则高于对照组,且随着肿瘤临床病理分期的进展而不断升高;胃癌血清IL-6、IL-10、IL-12含量与结肠癌接近.结论消化道肿瘤患者血清细胞因子IL-6、IL-10、IL-12的变化可能与肿瘤的生长及机体抗肿瘤免疫功能的受损有关.     

小鼠心脏移植模型及IL-12抗体抗排斥反应作用初步观察

大鼠异位心脏移植模型是目前器官移植实验研究中最常用的模型。由于移植免疫研究对供受体间种系纯化程度要求越来越高，而纯种小鼠易于得到，因而异位小鼠心脏移植模型有其优点。笔者于１９９５～１９９６年在德国基尔大学器官移植中心研究室工作期间，探索了小鼠心脏移植...     

白细胞介素12基因转染抑制人胰腺癌细胞血管生成及肿瘤的生长

目的　探讨白细胞介素 (IL) 12基因转染人胰腺癌细胞对肿瘤的抑制作用及机制。方法　用逆转录病毒MFG作为载体 ,通过逆转录PCR和磷酸钙沉淀法将鼠IL 12基因转染导入人胰腺癌细胞PK 1,得到PK1/IL 12细胞。单纯用MFG感染PK1所得细胞命名为PK1/MFG。用ELISA方法检测IL 12产生量 ,用NK细胞抗体阻断NK细胞作用 ,比较活体及离体条件下两种细胞的增长曲线。活体显微镜下观察肿瘤细胞的血管生成及IL 12的抑制效应。结果　PK1/IL 12细胞株的IL 12产生量为 1 12 μg·ml-1·48h-1。离体条件下两种细胞的生长曲线相似 ,但在活体条件下PK1/IL 12细胞的生长被显著抑制 (P <0 0 1) ,其血管生成被完全阻断 ,正常毛细血管亦被破坏。结论　IL 12基因转染可以抑制人胰腺癌细胞的生长。在免疫缺陷条件下 ,IL 12的抗血管生成作用足以抑制肿瘤细胞的血管生成及肿瘤的生长。IL 12的血管生成抑制效应缺乏特异性。     

门静脉注射白细胞介素-12治疗肝转移肿瘤的实验研究

目的　探讨通过门静脉系统局部应用白细胞介素 12 (IL 12 )对于肝转移肿瘤的治疗作用。方法　通过门静脉注射 2× 10 5个MCA 2 0 5肿瘤细胞建立小鼠肝转移肿瘤模型 ,同时脾脏被移植到皮下 ,作为反复多次向门静脉系统注射的途径。第 3～ 7天 ,0 1μgIL 12通过腹腔或脾脏注射 ,同时对照组中通过脾脏注射等体积的平衡盐水。第 2 1天检查肝转移肿瘤的情况。结果　在肝转移模型中 ,IL 12腹腔注射组和IL 12脾脏注射组的肝脏重量 (1 33± 0 0 8)g和 (1 2 9± 0 0 7)g明显小于对照组 (1 92± 0 17)g ,P <0 0 5 ,IL 12腹腔注射组和IL 12脾脏注射组的肝脏转移结节数目 (1 5 3± 0 5 8,0 6 0± 0 89)明显少于对照组 (18 2 5± 5 71,P <0 0 5 )。在IL 12脾脏注射组中 (n =6 ) ,3只小鼠的肝脏肿瘤完全消失。结论　通过门静脉系统局部应用IL 12是治疗肝转移肿瘤的有效方法。     

Antitumor effect to IL-12 administration into the portal vein on murine liver metastasis

Background/Purpose: Interleukin (IL)-12 has been shown to possess potent antitumor activity, and an antitumor effect of systemic IL-12 administration has been reported in liver metastasis models. Methods: In this study, we examined the usefulness of local IL-12 administration into the portal system in the treatment of liver metastasis. First, we confirmed the antitumor effect of recombinant IL-12 (rIL-12) on MC-38 tumors in an intracutaneous model. In the murine liver metastasis model, 1 × 10⁵ of MC-38 cells were injected into the portal vein on day 0, and the spleen was transpositioned subcutaneously for administration of rIL-12 continually into the portal system. From days 3 to 7, 0.1 μg rIL-12 was administered intraperitoneally or intrasplenicly, while Hanks' Balanced Salf Solution (HBSS) was injected intrasplenicly in the control group. Results: The liver weight in the rIL-12 intraperitoneal treatment group (1.88 ± 0.37 g) and that in the rIL-12 intrasplenic treatment group (1.43 ± 0.21 g) were significantly less than that in the HBSS group (2.86 ± 0.74 g; P < 0.05). The numbers of metastatic nodules in the rIL-12 intraperitoneal treatment group (22.3 ± 17.1) and in the rIL-12 intrasplenic treatment group (12.4 ± 13.8) were significantly less than that in the HBSS group (137.1 ± 44.9; P < 0.05). Complete regression of the tumor was observed in one of six mice in the rIL-12 intrasplenic treatment group. This antitumor effect of rIL-12 on MC-38 liver metastasis was not observed in interferon (IFN)-γ knockout mice. Intraportal administration of IL-12-transduced fibroblasts, which were syngeneic to C57BL/6 mice, had an antitumor effect in the MC-38 liver metastasis model. Conclusions: These results suggested that the local administration of IL-12 into the portal system would be a useful strategy for the treatment of liver metastasis. Received: April 4, 2002 / Accepted: July 1, 2002 Acknowledgment. The authors are grateful to Dr. H. Okamura (Hyogo Medical College, Japan), for kindly providing recombinant mIL-12, and Ms. Reiko Tsubouchi for her excellent technical assistance. Offprint requests to: T. Kitagawa     

Treatment of patients with lung abscess by local administration of papain

Under study were results of treatment of 109 patients with lung abscess aged from 17 to 76 years. All the patients were treated by active antiinflammatory therapy, bronchosanitation measures. In addition, local treatment by transthoracal punctures and drainage of the abscess cavity was used. Patients of the main group (52 patients) were given transthoracal injections of 0.5% solution of papain. The inclusion of papain in the complex therapy of patients with a lung abscess allowed to improve results of the treatment of this severe disease. The method may be recommended for wide use in pulmonology.     

The administration of IL-12/GM-CSF and Ig-4-1BB ligand markedly decreases murine floor of mouth squamous cell cancer

OBJECTIVE: To assess immune-based gene therapy in a murine floor of mouth (FOM) squamous cell carcinoma (SCC) model. STUDY DESIGN: In vitro and in vivo testing of immune therapy for SCC. METHODS: Multiple SCC lines were infected by using advRSV-interleukin-12 (IL-12) and advCMV-interleukin-12/granulocyte macrophage colony-stimulating factor (IL-12/GM-CSF) and monitored for production of IL-12 and GM-CSF. Intratumoral injections of viral vectors were administered with systemic Ig-4-1BB ligand in an orthotopic murine FOM SCC model and followed for tumor size and survival. RESULTS: In vitro, all cell lines produced substantial levels of IL-12 and GM-CSF. In vivo, tumors treated with advCMV-IL-12/GM-CSF and Ig-4-1BBL showed a striking reduction in tumor volume (vs control P < 0.0001) and improved median survival (38 days vs 19 days for control, P < 0.0001). CONCLUSION: Combination immune-based therapies effectively improve survival in mice bearing FOM SCC over single-modality therapy.     

Effect of intravesical administration of tumor necrosis serum and human recombinant tumor necrosis factor on a murine bladder tumor

The effect of intravesical administration of tumor necrosis factor (TNF) on the implantability and growth of bladder cancer was studied using the murine tumor model, MBT-2. Crude TNF was prepared from serum (TNS) of BCG infected rats after injection of endotoxin or the recombinant product of human TNF-alpha was used. Treatment was begun 24 hr. or seven days after tumor instillation and repeated three times. Tumor implantability and tumor weight were determined on day 21. Low dose TNF (200 U), given in the form of TNS, failed to suppress the growth of established tumors (seven-day tumor). It did, however, significantly reduce tumor implantability. The growth of seven-day tumors was significantly suppressed by administration of higher dose of TNF (3,700 U) given in recombinant form.     

Uremic pericardial effusion. Treatment by catheter drainage and local nonabsorbable steroid administration.

Pericardial drainage via percutaneous catheter placement and local nonabsorbable steroid instillation was employed as definitive therapy for uremic patients who had intractable pericardial effusions. Twelve patients are reported. Prior daily dialysis, and in one case systemic steroids, were not curative. 11 of 12 cases suffered severe tamponade requiring pericardiocentesis. One patient had an organized pericardial effusion, making pericardiocentesis impossible. He required pericardiectomy with prolonged hospitalization (2 weeks) due to postoperative complications. There were no complications in the 11 patients where catheter drainage and local steroid instillation were employed. No patient had recurrence of his pericardial effusion (followed from 2 weeks to 32 months). Instillation of a relatively nonabsorbable steroid through an indwelling pericardial catheter provides immediate and lasting relief without either the inconvenience or postoperative complications and prolonged hospitalization associated with the surgical procedure of pericardial fenestration. This report offers initial evidence that the percutaneous approach may be a safe and effective alternative to pericardial fenestration in most uremic patients with pericardial effusion.     

Treatment of murine hepatic metastases with vaccinia colon oncolysates and IL-2

To evaluate the feasibility and utility of vaccinia colon oncolysates (VCO) and low-dose interleukin-2 (IL-2) immunotherapy for advanced colon cancer, we have developed a murine model and tested the efficacy of combined treatment regimens. We employed intrasplenic injection of cultured colon adenocarcinomas (C-C36) in syngeneic Balb/c mice to produce experimental hepatic metastases. In the first set of experiments, animals were challenged with 5 X 10(5) tumor cells and sacrificed 14 days following tumor challenge. In the second set of experiments, animals were challenged with 2 X 10(5) tumor cells and followed for survival over the ensuing 90 days. In the first set of experiments, animals were treated prophylactically with VCO (40 micrograms, sc, 14 and 7 days prior to challenge) and/or therapeutically with IL-2 (25,000 u, Hoffmann-LaRoche rIL-2, ip BID, on Days 1-3 following challenge). In the second set of experiments, animals were treated with either the identical regimens or therapeutically with VCO (same dose, sc, 2 and 10 days following challenge) and/or IL-2 (same dose, ip BID, on Days 9-11 following challenge). Tumor burden data from sacrificed animals was in agreement with survival data and showed significant tumor burden reduction in the combined treatment group as assessed by liver weight and tumor nodule enumeration. Survival data demonstrated highly significant survival advantage for animals treated with the two biological response modifiers: VCO (P less than 0.0021), and IL-2 (P less than 0.0017). The data presented suggest a synergistic effect for these two agents.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-12p40 and IL-18 in crescentic glomerulonephritis: IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 promotes local inflammation and leukocyte recruitment

Experimental crescentic glomerulonephritis (GN) is characterized by T helper 1 (Th1) directed nephritogenic immune responses and cell-mediated glomerular injury. IL-12p40, the common cytokine chain for both IL-12 and IL-23, is important in the generation and potentially the maintenance of Th1 responses, whereas IL-18 is a co-factor for Th1 responses that may have systemic and local proinflammatory effects. For testing the hypothesis that both endogenous IL-12p40 and endogenous IL-18 play pathogenetic roles in crescentic GN, accelerated anti-glomerular basement membrane GN was induced in mice genetically deficient in IL-12p40 (IL-12p40-/-), IL-18 (IL-18-/-), or both IL-12p40 and IL-18 (IL-12p40-/-IL-18-/-). Compared with wild-type C57BL/6 mice, IL-12p40-/- mice failed to make a nephritogenic Th1 response and developed markedly reduced crescent formation and renal leukocytic infiltration, despite renal production of chemoattractants and adhesion molecules. IL-18-/- mice developed an intact antigen-specific systemic Th1 response, a similar degree of crescent formation, but fewer glomeruli affected by other severe histologic changes and fewer leukocytes in glomeruli and interstitium. IL-18 was expressed within diseased kidneys. Local production of TNF, IL-1beta, IFN-gamma, CCL3 (MIP-1alpha), and CCL4 (MIP-1beta) was reduced in IL-18-/- mice, demonstrating a local proinflammatory role for IL-18. Combined deletion of IL-12p40 and IL-18 did not result in synergistic effects. Consistent with the hypothesis that inflammation leads to fibrosis, all three groups of deficient mice expressed lower levels of intrarenal TGF-beta1 and/or alpha1(I) procollagen mRNA. These studies demonstrate that in severe experimental crescentic GN, IL-12p40 is the key Th1-defining cytokine chain, whereas IL-18 has local proinflammatory roles.     

Induction of cytotoxic T lymphocyte development from murine thymocytes by IL-1 and IL-6

Cytotoxic T lymphocytes (CTL) are believed to play an important role in the regression of advanced malignancies in response to adoptive immunotherapy with interleukin-2 (IL-2) and lymphokine-activated killer cells or tumor-infiltrating lymphocytes. Because the current limitations to the use of adoptive immunotherapy are the IL-2 dose-dependent toxicities and the difficulty in expanding the effector cell population, recent investigations have focused on the development of newer methods for generating CTL in vitro. IL-1 and IL-6 have been shown to synergistically promote thymocyte proliferation; however, their effect on CTL development has not been studied. We investigated the ability of these two cytokines to induce CTL development from immature thymocytes. Thymocytes from 5-week-old BALB/c mice were cultured for 72 hours in the presence of Con A and recombinant IL-1, IL-6, or IL-1 plus IL-6. Cytotoxicity against 51Cr-labeled P815 target cells was then measured in the presence of submitogenic doses of PHA. Neither IL-1 nor IL-6 induced a significant number of CTL from immature thymocytes. However, these two cytokines synergistically induced maximal CTL development. The monoclonal antibody to IL-4 completely abrogated CTL development induced by IL-1 and IL-6, but antibody to the IL-2 receptor had no effect. The data suggest that IL-1 and IL-6 can provide an additional method for in vitro CTL generation in adoptive immunotherapy of advanced tumors.     

皮下磨削术治疗腋臭

目的:探讨皮下磨削方法治疗腋臭的临床效果。方法:15例患者,共30侧。每一侧皮下及真皮层各注射60~100ml肿胀麻醉液,局部麻醉。30侧全部取腋窝皱褶切口长2~3cm,在浅筋膜层的浅层用皮刀刺入,剥离皮瓣,剥离范围到达腋毛区外1cm,皮瓣下需带有皮下脂肪。反转皮瓣,剪除皮瓣下脂肪层,将高速电动磨削机的金属磨削头伸入到皮瓣下,挑起皮瓣并绷紧,按顺序均匀磨削成全厚皮瓣。结果:15例30侧全部愈合,无皮瓣坏死。随访6~12月无复发,瘢痕不明显,几乎无腋毛生长,无臭味。结论:皮下磨削术治疗腋臭,手术时间短,治疗效果彻底,切口小,几乎无瘢痕,美容效果佳。