逆转录病毒介导的小鼠IL-23基因在小鼠结肠癌细胞中的表达及其抗肿瘤活性

目的：获得表达小鼠IL-23(mIL-23)基因的小鼠结肠癌细胞株。方法：应用逆转录病毒载体，将mIL-23基因导入小鼠结肠癌细胞株Colon26，经G418筛选后获得表达mIL-23的阳性细胞克隆(Colon26／IL-23)。用PCR和RT—PCR检测目的基因的表达，用ELISA法检测mIL-23的产生及mIL-23诱导的小鼠脾细胞IFN-γ的产生，用MTT比色法检测Colon26／IL-23细胞和Colon26细胞的体外增殖，将Colon26／IL-23细胞接种于BALB／c小鼠的右侧背部皮下，观察其致瘤性结果：建立了可表达mIL-23基因的小鼠结肠癌细胞株。分泌至培养上清中的IL-23，可诱导小鼠脾细胞产生IFN-γ在体外Colon26／IL-23细胞的生长与Colon26细胞无明显不同，但其在体内的致瘤性下降，具有抗瘤作用。结论：Colon6／IL-23细胞可分泌IL-23并证明其具有抗瘤活性。     

真核表达的重组猪单链IL-12生物学活性的研究

目的:真核表达重组猪单链IL-12(pscIL-12)并鉴定表达的pscIL-12的生物学活性。方法:采用梭华-SofastTM将pcDNA3.1(+)-pscIL-12转入CHO-K1细胞,ELISA法测定细胞培养上清中pscIL-12的表达水平;MTT法检测细胞培养上清对NK细胞杀伤作用的影响;ELISA法测定细胞培养上清刺激人PBMC分泌IFN-γ的水平;流式细胞术(FCM)检测细胞培养上清对人淋巴母细胞增殖水平的作用。结果:融合基因pscIL-12在CHO-K1细胞中表达产物pscIL-12融合蛋白含量较高;生物学活性鉴定表明:pscIL-12融合蛋白可增强NK细胞活性、提高IFN-γ的产生和促进人淋巴母细胞的增殖。结论:转染pcDNA3.1(+)-pscIL-12质粒的CHO-K1细胞成功表达了具有良好生物学活性的pscIL-12。     

转染IL-27基因的食管癌细胞在裸鼠体内的抗肿瘤作用

目的将小鼠IL-27基因转染人食管癌细胞株,研究其在裸鼠体内的抗肿瘤活性.方法以逆转录病毒为载体,将IL-27基因转染人食管癌细胞株Eca109,获得表达IL-27的阳性细胞克隆(Eca109/IL-27),用ELISA法检测细胞IL-27的分泌.将Eca109/IL-27移植裸鼠腹腔,观察瘤细胞的成瘤性和裸鼠的生存期.用RT-PCR检测IL-27基因在裸鼠体内瘤组织中的表达,用流式细胞仪检测CD204表达、细胞周期和细胞凋亡变化.结果成功建立了表达IL-27基因的人食管癌细胞株Eca109/IL-27,该细胞培养上清中IL-27含量为(137.73±7.00)pg/ml,而Eca109/LXSN和Eca109细胞培养上清中未检出IL-27(P＜0.01),但Eca109/IL-27与Eca109/LXSN和Eca109细胞周期和细胞凋亡率无明显差异(P＞0.05).将Eca109/IL-27、Eca109/LXSN 和Eca109细胞分别接种裸鼠腹腔15天后,各组裸鼠均于腹腔形成多数瘤结节,Eca109/IL-27组明显少于Eca109/LXSN 和Eca109细胞组(P＜0.05);Eca109/IL-27组裸鼠的生存期(35天时均无死亡)明显长于Eca109/LXSN 和Eca109细胞组[分别为(23.33±1.52)、(24.00±1.37)天](P＜0.01);Eca109/IL-27组肿瘤组织中有p28和EBI3亚基基因表达,而Eca109/LXSN 和Eca109细胞组瘤组织中未见表达(P＜0.01);其CD204表达水平、细胞凋亡率也明显高于接种Eca109/LXSN与Eca109细胞组(P＜0.05);G0/G1和G2/M期细胞增多,S期细胞明显减少,与接种Eca109/LXSN和Eca109细胞组比较均有显著差异(P＜0.05).结论IL-27可于体内促进肿瘤细胞凋亡,阻滞细胞周期发展,提高CD204的表达,延长生存期,发挥抗肿瘤免疫活性.     

Expression of Lewisx sugar structure in the liver metastasis of mouse colon carcinoma (colon 26) cells

Immunohistochemical aspects of the process of experimentally induced metastasis were examined by light and electron microscopy employing a series of labeled carbohydrate-specific monoclonal antibodies as probes. Liver metastasis was induced by injecting mouse colon carcinoma cell (colon 26) into the spleen of Balb/c mice. Labeled anti-Lewis^x (Le^x) antibody stained the metastasized colon 26 cells strongly compared with the heterogeneous and faint staining in non-metastasized tumor foci in the spleen or in the subcutaneous space. Other antibodies having specificities for Lewis-related antigens other than Le^x, e.g. those against Le^y, Le^a, Le^b, sialyl Le^x and sialyl Le^a, did not show any differences in binding between metastasized cells and non-metastasized tumor foci. Immunoelectron microscopy revealed the expression of Le^x-carbohydrate in the plasma membranes as well as in the intercellular spaces of metastasized colon 26 cells in the liver. Based on these results, it is likely that sugar chains containing the Le^x-carbohydrate structure are involved in the interactions between colon 26 cells and hepatic cells during the process of liver metastasis.     

Effects of immunization with tumor cells double transfected with interleukin-2 (IL-2) and interleukin-12 (IL-12) genes on artificial metastasis of colon26 cells in BALB/c mice

In order to induce tumor specific cytotoxicity, the poorly immunogenic murine colon cancer cell colon26 was transfected with murine IL-2 cDNA and/or IL-12 cDNA and their anti-tumor effects were investigated. Double transfectants produced murine IL-2 and murine IL-12, the same as single transfectants. Intraperitoneal administration of double transfectants inhibited pulmonary metastasis of colon26 inoculated intravenously to a stronger degree than that of single transfectants. Splenocytes from mice administered double transfectants intraperitonealy showed higher cytolytic activity against colon26 than those from mice administered single transfectants, and also showed cytolytic activity against murine B16-BL6 melanoma. In the NK cell-depleted mice, double transfectants inhibited pulmonary metastasis from the control markedly, but could not do completely, the same as in the NK cell-reserved mice. The difference of the metastatic colonies between NK cell-depleted mice and the control was much greater than that between NK cell-depleted mice and NK cell-reserved mice. These results suggested that cytotoxic T lymphocytes might participate in this anti-tumor effect.     

Differential activity of IL-12 and IL-23 in mucosal and systemic innate immune pathology

The CD40-CD154 pathway is important in the pathogenesis of inflammatory bowel disease. Here we show that injection of an agonistic CD40 mAb to T and B cell-deficient mice was sufficient to induce a pathogenic systemic and intestinal innate inflammatory response that was functionally dependent on tumor necrosis factor-alpha and interferon-gamma as well as interleukin-12 p40 and interleukin-23 p40 secretion. CD40-induced colitis, but not wasting disease or serum proinflammatory cytokine production, depended on interleukin-23 p19 secretion, whereas interleukin-12 p35 secretion controlled wasting disease and serum cytokine production but not mucosal immunopathology. Intestinal inflammation was associated with IL-23 (p19) mRNA-producing intestinal dendritic cells and IL-17A mRNA within the intestine. Our experiments identified IL-23 as an effector cytokine within the innate intestinal immune system. The differential role of IL-23 in local but not systemic inflammation suggests that it may make a more specific target for the treatment of IBD.     

Immunohistochemical expression of metallothionein in resected hepatic primary tumors and colorectal carcinoma metastases

Metallothionein is a protein with affinity for metals and is present in several tumors. We examined its immunohistochemical expression in 37 resected primary liver tumors and 117 colorectal metastases. The reaction was intense in the two fibrolamellar hepatocellular carcinomas and in many of the hepatocytes of the pseudotumor case of focal nodular hyperplasia. The reaction was low or moderate in 5 of 17 ordinary hepatocellular carcinomas and in 4 of 14 cholangiocellular carcinomas. There was no reaction in one case each of spindle cell hepatocellular carcinoma, oncocytic adenoma and hemangioendothelial sarcoma. In the metastases, the reaction was low or moderate in 14 cases and negative in 103. Surrounding hepatocytes and stromal cells were more or less positive in all cases.     

In vitro levels of interleukin 10 (IL-10) and IL-12 in response to a recombinant 32-kilodalton antigen of Mycobacterium bovis BCG after treatment for tuberculosis

Cell-mediated immunity plays a major role in conferring protection against tuberculosis (TB) on an individual. It is not known whether the immune status correlates with the bacterial load or whether the immunity improves after treatment. Also, it may be important to monitor treatment by being able to discriminate between active disease and successfully treated TB. The main aim of this study was to investigate the usefulness of a recombinant 32-kDa antigen (r32-kDa Ag) of Mycobacterium bovis BCG (Ag85A-BCG) as a diagnostic marker in patients being treated for TB. Specifically, the in vitro T-cell assays and the release of interleukin-12 (IL-12) (Th1-type cytokine) and IL-10 (Th2-type cytokine) in response to the r32-kDa Ag of BCG were assayed in patients with either pulmonary (sputum positive/negative, n = 74) or extrapulmonary TB (n = 49) and healthy controls. The proliferative responses of stimulated cells at 0, 2 to 4, and 6 months of treatment increased and were highly significant (P < 0.000) compared to the responses in controls. The increase in IL-12 and decrease in IL-10 release suggest that there is cytokine expression modification during different stages of TB, and treatment seems to have an influence on the levels of these cytokines, suggesting an augmentation in the protective responses. The in vitro response to the M. bovis BCG r32-kDa Ag may be useful in monitoring treatment of TB.     

Antitumor effect of FP3 in combination with cetuximab on patient-derived tumor tissue xenograft models of primary colon carcinoma and related lymphatic and hepatic metastases

FP3 is an engineered protein which contains the extracellular domain 2 of vascular endothelial growth factor (VEGF) receptor 1 (Flt-1) and the extracellular domain 3 and 4 of VEGF receptor 2 (Flk-1, KDR) fused to the Fc portion of human immunoglobulin G1. Previous studies have demonstrated its antiangiogenic effects in vitro and in vivo, and its antitumor activity in vivo. Cetuximab is a monoclonal antibody against epidermal growth factor (EGF) receptor. Combined inhibition of VEGF and EGF signaling may act additively or synergistically. In this study, patient-derived tumor tissue (PDTT) xenograft models of primary colon carcinoma and lymphatic and hepatic metastases were established for assessment of the antitumor activity of FP3 in combination with cetuximab. Xenografts were treated with FP3 and cetuximab, alone or in combination. After tumor growth was confirmed, volume and microvessel density in tumors were evaluated. Levels of VEGF, EGFR and PCNA in the tumor were examined by immunohistochemical staining, and levels of related cell signaling pathway proteins were examined by western blotting. FP3 in combination with cetuximab showed significant antitumor activity in three xenograft models (primary colon carcinoma, lymphatic metastasis and hepatic metastasis). The microvessel density in tumor tissues treated with FP3 in combination with cetuximab was lower compared to that of the control. Antitumor activity of FP3 in combination with cetuximab was significantly higher than that of each agent alone in two xenograft models (colon carcinoma lymphatic metastasis and hepatic metastasis). This study indicated that addition of FP3 to cetuximab significantly improved tumor growth inhibition in the PDTT xenograft models of colon carcinoma lymphatic and hepatic metastases. Combination anti-VEGF (FP3) and anti-EGFR (cetuximab) therapies may represent a novel therapeutic strategy for the management of metastatic colon carcinoma.     

Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-{gamma} in vivo

2 Is a heterodlmerlc cytoklne that has been shown to enhancenatural killer (NK) and cytotoxic T lymphocyte (CTL) responses,and to induce IFN- production in vitro. In this study, we haveexamined the effects in vivo of administering purified murinerlL-12 to normal mice. Dally injections of riL-12 I.p. (1 ngto 10 µg/day) caused dose-dependent enhancement of NKcell lytic activity in the spleens and livers of treated mice.Histologlc examination of the livers of IL-12-treated mice revealedfocal mononuclear cell infiltrates, and flow cytometry studiesindicated that the livers of IL-12-treated mice contained increasednumbers of NK cells, CD8⁺ T cells, and monocytes. Liver andsplenic lymphold cells from IL-12-treated mice, unlike liverand splenic lymphoid cells from control mice, spontaneouslysecreted IFN- in vitro, suggesting that they had been inducedby IL-12 to produce IFN- in vivo. Consistent with this, IFN-could be detected in the serum of IL-12-treated mice. In micewhich had been immunized by footpad injection of allogenelcsplenocytes, dally administration of rlL-12 I.p. was shown toenhance the specific CTL response in the draining lymph nodes.Thus, these studies demonstrate that IL-12 can enhance NK andCTL activity and induce IFN- production in vivo, as well asin vitro, and suggest possible mechanisms by which IL-12 mayexert therapeutic effects in the treatment of some tu more andinfectious diseases.     

pDC316-hIL-12-IRES-CKb双顺反子重组腺病毒载体的构建及其在肝细胞中的表达

目的: 构建含肌酸激酶(CK)基因和人白细胞介素12(hIL- 12 )基因的双顺反子重组腺病毒载体pDC316-hIL-12-IRES-CKb,并包装成重组腺病毒,检测其在肝细胞中蛋白表达和肝脏中磷酸肌酸的水平。方法: 将PCR 扩增的产物CK片段和hIL- 12 片段顺次插入双顺反子腺病毒载体(IES)中,经同源重组、包装和扩增获得重组腺病毒子。再用该病毒感染体外培养的兔肝细胞,经Western blotting检测到 CK 蛋白的表达和ELISA检测培养液中hIL-12水平。结果: 体外实验在感染肝细胞后检测到CK和hIL-12蛋白的表达;同时体内实验应用核磁共振波谱(MRS)的方法能检测到肝脏特异性CK产物磷酸肌酸的水平。结论: 携带CK和hIL- 12 基因的双顺反子重组腺病毒能使CK 基因异源性地表达在肝脏并能用非侵袭性的MRS技术检测其表达。此载体的构建为进一步研究CK基因作为一个影像报告基因动态监测肝脏治疗基因hIL-12的表达奠定基础。     

GLS和IL-12偶联重组耻垢分枝杆菌免疫效果的观察

目的：研究携带编码人天然颗粒溶素（GLS）和小鼠IL-12基因质粒（pZM03）偶联重组耻垢分枝杆菌（ATCC607）经鼻黏膜免疫小鼠后,小鼠体内的免疫状况.方法：BALB/c小鼠36只,随机分为生理盐水、pZM03、ATCC607、卡介苗（BCG）、重组ATCC607（即携带pZM03的ATCC607）、灭活重组ATCC607组;采用滴鼻法免疫小鼠,BCG组0、14天各1次,其它组0、4、14天各1次,第0天免疫后4周处死小鼠,用ELISA检测血清中IFN-γ、IL-12、IgG2a、lL-4的分泌水平和淋巴细胞PPD诱导的IFN-γ分泌水平以及肺泡灌洗液特异性SIgA的水平,用MTS法检测免疫小鼠脾淋巴细胞增殖情况.结果：重组ATCC607血清中IFN-γ、IL-12水平与BCG组无明显差异但明显高于其他组（P＜0.05）,IgG2a水平重组ATCC607组高于BCG组和其他组（P＜0.05）,各组间IL-4水平差异无统计学意义.重组ATCC607、BCG、灭活重组ATCC607及ATCC607组用PPD均可诱导小鼠脾淋巴细胞增殖,各组间差异无显著意义,但与生理盐水和pZM03组间差异有显著意义（P＜0.05）.重组ATCC607组PPD诱导淋巴细胞IFN-γ明显高于其它组（P＜0.05）,与BCG组比较无显著差异.重组ATCC607等含菌组经鼻黏膜免疫可产生黏膜特异性SIgA.结论：重组ATCC607经鼻黏膜免疫小鼠后,机体特异性免疫特别是细胞免疫和黏膜免疫增强,为重组ATCC607治疗结核病的研究奠定了基础.     

Age-associated decline in IL-2 and IL-12 induction of LAK cell activity of human PBMC samples

Previously we reported that young and elderly natural killer (NK) cell activity against the standard NK sensitive K562 cell line can be augmented to the same degree by IL-2 and IFN-. We have extended these studies to include IL-12. Similar to IL-2 and IFN-, IL-12 can enhance NK cytotoxicity to the same degree in both young and elderly samples over a wide range of doses and incubation times when K562 cells are used as targets. However, in contrast to our findings with the NK system, we have observed that induction of lymphokine activated killer (LAK) cell activity, as defined by the ability of peripheral blood mononuclear cells (PBMC) samples to lyse the normally NK resistant Daudi cell line, was significantly decreased in the elderly samples compared to young samples. Comparable age-associated differences were observed in LAK activity after induction with IL-2, IL-12, and IFN- at varying doses and incubation times. We hypothesize an age-associated deficiency either in the mechanism of LAK induction or in target cell recognition.     

Induction of the activity of the histamine-forming enzyme,histidine decarboxylase,in mice by IL-18 and by IL-18 plus IL-12

OBJECTIVE AND DESIGN: IL-18 shares functional properties with IL-12, which induces an elevation of histidine decarboxylase (HDC) activity in mouse tissues. Therefore, we examined the effects of IL-18 and IL-18+IL-12 on HDC activity. METHODS: IL-18, IL-12 or IL-18+IL-12 was intraperitoneally injected into BALB/c and C3H/HeN mice and their HDC activities were measured. RESULTS: IL-18, at effective antitumour doses, induced HDC activity in lung, liver, spleen and bone. The IL-18-induced HDC elevation was more rapid than that induced by IL-12. As with IL-12, repeated injections of IL-18 produced more HDC activity than a single injection. Repeated injections of IL-18+IL-12 induced serious illness and produced marked HDC activity. However, an inhibitor of HDC did not prevent the illness. CONCLUSIONS: IL-18 may be involved in the stimulation of histamine synthesis during the course of immune responses, though an elevation of HDC activity is not itself the direct cause of the serious illness induced by IL-18+IL-12.     

白介素-12及白介素-23对胃癌组织中CD4^＋记忆T细胞的影响

目的:探讨胃癌组织中IL-12、IL-23对CD4+记忆T细胞(CD4+Tm)的影响.方法:ELISA检测TNM不同期胃癌组织匀浆中IL-12、IL-23含量;不连续密度梯度离心法分离胃癌组织中的肿瘤浸润淋巴细胞(TIL),流式细胞仪检测TIL中CD4+ Tm及其亚群TEM和TCM在不同期胃癌组织中的分布状况.结果:TNM-Ⅰ、Ⅱ期胃癌组织中IL-12含量差别不明显,但均显著高于TNM-Ⅲ、TNM-Ⅳ期;TNM-Ⅰ期胃癌组织中IL-23的含量高于TNM-Ⅱ、TNM-Ⅲ、TNM-Ⅳ期.胃癌TIL中CD4+ Tm及TEM比例随TNM分期增加逐渐降低,而TCM比例逐渐升高.结论:胃癌组织中IL-12、IL-23含量的变化与胃癌TNM分期有关,且影响胃癌TIL中CD4+记忆T细胞及其亚群TCM和TEM的分布.     

大肠癌旁移行粘膜的增殖活性及其分布

目的：为进一步认识大肠癌“移行粘膜”（ＴＭ）的性质及为手术切除范围提供病理学依据。方法：通过连续观察２５例大肠癌癌旁１０ｃｍ范围内粘膜上皮细胞增殖活性的变化与正常结肠粘膜进行比较，发现ＴＭ在癌旁两边６ｃｍ范围内和近切端多春唾液酸粘蛋白反应、ＡｇＮＯＲｓ核均数、ＰＣＮＡ 率、Ｐ５３蛋白阳性等增殖活性均介于癌和正常结肠粘膜之间。结论：ＴＭ是腺体粘蛋白合成异常、增殖活性较高的一种癌前病变。手术切除肠段地     

Survivin在结肠癌中的不同表达定位与预后的关系

目的探讨survivin在结肠癌中的不同表达定位与临床病理特征及预后的关系。方法应用免疫组化检测80例结肠癌中survivin的表达,并分析其不同表达定位与临床病理特征及预后的关系。结果 65(81.25%)例结肠癌表达survivin,其中胞核表达35例,胞质表达43例。survivin胞核表达与结肠癌淋巴结转移及组织学分级呈负相关(P0.05),而survivin胞质表达在Ⅰ、Ⅱ期结肠癌中明显高于Ⅲ、Ⅳ期(P0.05)。Kaplan-Meier生存分析显示survivin胞核阳性的结肠癌病人预后明显好于其胞核阴性病人预后,差异有统计学意义(P0.05),而survivin胞质阳性与阴性结肠癌病人之间预后差异无统计学意义(P0.05)。结论 survivin在结肠癌中的不同表达定位提示不同的预后价值,survivin在结肠癌细胞中核表达可作为肿瘤预后良好的一个参考指标。     

雄激素调节卵巢癌细胞IL-6基因启动子活性的研究

目的探讨雄激素诱导卵巢癌细胞IL-6基因启动子活化的分子机制。方法选择SKOV-3细胞作为研究模型，应用ELISA、RT-PCR及荧光素酶检测技术观察5廿二氢睾酮（5a-dihvdrotest—osterone，DHT）对IL-6表达水平及IL-6基因启动子活性的影响，并对DHT诱导IL-6基因启动子活化的机制进行研究。结果（1）DHT可促进SKOV-3细胞IL-6分泌及相应mRNA的表达，该作用可被雄激素受体（AR）阻断剂氟他胺（flutamide，nu）完全阻断，提示DHT诱导上述作用是AR途径介导的。（2）IL-6本身能以剂量依赖性的方式增强SKOV-3细胞IL-6基因启动子的转录活性，DHT也具有与IL-6相似的作用。DHT的这种作用可被Flu完全阻断，也可被抗IL-6中和抗体部分阻断。（3）转录因子NF-IL-6、NF-xB及其亚单位pSO、p65均可诱导SKOV-3细胞IL-6基因启动子的活性。DHT不改变NF-IL-6介导的IL-6基因启动子的转录活化，而增强NF-xB（pSO＋p65）及其亚单位pSO、p65介导的IL-6基因启动子的转录活化。结论雄激素增强的IL-6基因启动子转录活性是AR途径介导的，同时有部分是通过IL-6的作用而实现。雄激素很可能通过AR与转录因子NF-xB或其亚单位pSO和p65之间蛋白-蛋白的相互作用反式激活IL-6基因启动子的活性。