重组人IL-18的基因克隆与表达的研究

目的：利用基因重组技术构建人IL-18(rhIL-18)的真核表达载体，并在大肠杆菌中表达。方法：利用RT-PCR技术从外周血细胞扩增得到IL-18的cDNA并测定其核酸序列。将扩增产物酶切后克隆到pORF5质粒的Nco Ⅰ／Nhe Ⅰ 酶切位点，转化大肠杆菌JM109，筛选阳性重组子，构建真核表达质粒pORF5-hIL-18。结果：真核表达质粒pORF5-hIL-18在大肠杆菌JM109表达，能够诱导PBMC合成GMCSF，具有协同rhIL-2增强NK细胞细胞毒作用的能力。结论：重组表达的rhIL-18具有生物活性，可用于进一步的功能研究。     

人BDNF重组逆转录病毒载体的构建

目的 构建PLEGFP-BDNF真核表达重组逆转录病毒，为进一步在细胞中表达及移植治疗作准备。方法 按照hBDNF基因全长编码序列设计合成引物，从人基因组DNA中扩增出744bp的hBDNF基因片段，插入到PMD18-T载体上，转化DH5α大肠杆菌菌株，获得PMD18T-BDNF克隆，对该克隆进行限制性酶切分析和DNA序列测定；从阳性克隆中获取hBDNF全长编码片段，与真核表达载体PLEGFP质粒连接，构建PLEGFP-BDNF真核表达重组质粒。重组质粒转化大肠杆菌JMl09，再经氨苄LB培养基筛选，酶切，PCR与测序鉴定。结果PLEGFP-BDNF重组逆转录病毒构建成功。结论hBDNF在大肠杆菌中的成功表达在转基因治疗CNS病变方面具有潜在应用价值。     

pcDNA3.1/DLC-1 重组质粒的构建及其在HT-29细胞中的表达

 目的 构建和表达DLC-1(deleted in liver cancer)基因重组质粒。 方法 用PCR法得到DLC-1基因片段,此基因片段带有Xba Ⅰ和BamH Ⅰ两个酶切位点,然后将此片段 和真核表达载体pcDNA3.1连接,转化大肠杆菌DH5a,利用脂质体介导将pcDNA3.1/DLC-1重组质 粒转染到结肠癌HT-29细胞中,再用RT-PCR法检测重组质粒的表达。 结果 重组质粒经Xba Ⅰ和BamH Ⅰ双酶切和测序与DLC-1基因序列一致;利用脂质体介导将 pcDNA3.1/DLC-1重组质粒导入HT-29细胞,获得了DLC-1基因的表达。 结论 成功构建pcDNA3.1/DLC-1重组质粒并在HT-29细胞内表达。     

人组织型基质金属蛋白酶抑制剂-1基因真核表达重组质粒的构建

目的构建人基质金属蛋白酶组织抑制剂-1(TIMP-1)真核表达重组质粒,并进行序列分析.方法利用人卵巢癌组织进行RP-PCR扩增TIMP-1基因cDNA,获目的片段(631bp)连接至pcDNA4.载体,转化大肠杆菌TOP10筛选阳性克隆共鉴定,并对克隆的全长片段进行DNA序列测定.结果经与GeneBark分布的序列进行分析比较证实,构建的真核表达重组质粒pcDNA4-TIMP-1含人TIMP-1全长cDNA编码序列,仅592位的碱基发生错义突变.结论成功构建了TIMP-1的真核表达质粒,为下一步研究TIMP-1在卵巢癌侵袭转移中的作用打下物质基础.     

大肠杆菌嘌呤核苷磷酸化酶基因真核表达载体的构建及其在MKN-45细胞中的表达

目的克隆大肠杆菌嘌呤核苷磷酸化酶(EPNP)基因并构建真核表达载体pcDNA3.1-EPNP,将重组质粒转染MKN-45细胞获得高表达大肠杆菌PNP基因的MKN-45细胞克隆。方法根据Gen-bank中大肠杆菌PNP基因的核苷酸序列,设计并合成了一对引物,以大肠杆菌基因组DNA为模板,进行PCR扩增,并将扩增产物定向插入pMD-18T克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pcDNA3.1,利用脂质体将重组质粒转染MKN-45细胞。结果PCR扩增出716bp大小的片段,测序结果与已报道的EPNP基因序列一致;亚克隆经酶切鉴定正确;RT-PCR证明经G418筛选得到的转基因MKN-45细胞克隆中存在大量EPNPmRNA,前体药MePdR对转染EPNP的MKN-45细胞有较强的细胞毒作用。结论成功构建了大肠杆菌PNP基因的真核表达载体,获得了稳定表达大肠杆菌PNP基因的MKN-45细胞克隆,为PNP/MepdR自杀基因系统在胃癌基因治疗中的应用奠定了良好的基础。     

人白细胞介素-2真核表达质粒的构建及鉴定

目的构建高效表达人IL－2的真核表达质粒,为IL－2的肿瘤基因治疗提供重要的工具。方法运用PCR扩增出人IL－2基因,经BamHI及EcoRI双酶切后,回收片段与BamHI及EcoRI处理的表达载体pcDNA3在T4DNA连接酶的作用下连接,并用多种限制性内切酶对重组质位进行酶切鉴定。结果PCR扩增片段的长度约为480bp,重组质粒经酶切显示,构建的质粒中含480bp的IL－2插人片段,将此重组质粒命名为pcDNA3－IL－2。结论本研究结果为进一步研究表达IL－2质粒的肿瘤基因治疗及免疫调节作用打下了基础。     

白细胞介素-18转导人大肠癌细胞株的建立及其生物学活性的研究

目的 :建立转白细胞介素 18(IL 18)基因人大肠癌细胞株 ,并研究IL 18的表达情况。方法 :将携带人白细胞介素 18的质粒pcDNA3 1 hIL 18转染到人大肠癌细胞系SW 4 80中 ,通过药物GENETI CIN(G 4 18Sulfate)进行筛选 ,利用PCR、RT PCR及ELSIA法对IL 18的表达进行检测。结果 :IL 18基因成功的转导入SW 4 80细胞中 ,10 6个转染细胞在 2 4h内分泌IL 18的含量是 112 9± 17 6pg ;空载体转染的细胞和未转染的SW 4 80细胞不产生IL 18。结论 :人IL 18基因成功的整合到大肠癌细胞系SW 4 80细胞基因组中 ,且能持续大量表达     

PMG-36e-CD::upp重组质粒的构建及鉴定

目的:探讨利用分子生物学基因重组技术构建PMG-36 e-CD::upp自杀基因重组质粒的方法。方法:用小量制备质粒DNA的方法分别提取表达型质粒载体PMG-36 e和自杀基因pORF-CD::upp,以质粒pORF-CD::upp为模板PCR扩增CD::upp基因,表达型质粒载体PMG-36 e与自杀基因CD::upp分别用限制性内切酶SacⅠ与SalⅠ进行双酶切,T4DNA连接酶16℃恒温水浴过夜连接,转化入感受态JM109,筛选单克隆提取重组质粒PMG-36 e-CD::UPP进行1%琼脂糖凝胶电泳鉴定,双酶切鉴定,PCR鉴定,及测序鉴定。结果:重组质粒PMG-36 e-CD::upp经1%琼脂糖凝胶鉴定,分子量为5.5Kb,与其标准分子量大小一致;重组质粒PMG-36 e-CD::upp经双酶切鉴定及PCR初步鉴定构建成功;送上海生物工程技术服务有限公司测序认证,同源性达100%。结论:重组质粒PMG-36 e-CD::upp可成功构建,可用于后续实验的表达研究。     

T细胞淋巴瘤TCRγV1重排基因真核表达载体的构建

目的　构建含TCRγV1重排基因的真核表达质粒。方法　用PCR法扩增含XbaI与BglII酶切位点的TCRγV1基因序列 ,对VR10 12载体及TCRγV1基因PCR产物经双酶切 ,用连接酶将两者连接并转化到大肠杆菌DH5α ,对重组质粒经序列测定 ,称VR10 12 /TCRγ。结果　已构建的质粒VR10 12 /TCRγ经序列测定含有完整的TCRγV1基因片段。结论　VR10 12 /TCRγ表达载体的构建为基因治疗淋巴瘤奠定了基础。     

分泌型核心蛋白聚糖真核表达载体的构建及在HepG2细胞中的表达

目的构建分泌型核心蛋白聚糖(decorin)真核表达载体,并在肝癌细胞HepG2中表达,为研究核心蛋白聚糖的抗肿瘤作用奠定基础。方法利用特异性引物,应用PCR技术扩增核心蛋白聚糖全长基因cDNA片段,与pcDNA3.1载体进行连接,并转化到大肠杆菌JM109中扩增以获得重组载体,应用双酶切、PCR以及测序鉴定此重组载体;脂质体介导重组载体转染HepG2,经G418筛选建立稳定转染细胞株,分别采用RT-PCR、免疫组化法和westernblot检测其表达。结果获得了约1080bp大小的特异性DNA片段;PCR产物与真核表达载体进行连接,经过双酶切、PCR以及测序鉴定证实分泌型核心蛋白聚糖cDNA片段正确插入真核表达载体pcDNA3.1中。RT-PCR方法可见转染组mRNA表达明显增多,免疫组化和westernblot方法可见转染组细胞decorin蛋白表达明显增高。结论成功构建了真核表达载体pcDNA3.1-decorin,建立了稳定转染decorin的HepG2细胞株。     

重组人促血小板生成素与重组人白细胞介素-11治疗肿瘤患者血小板减少症的疗效比较

目的比较重组人促血小板生成素（rhTPO）与重组人白细胞介素-11（rhIL-11）治疗肿瘤患者血小板减少症的临床疗效。方法将肿瘤患者合并血小板减少症62例随机分为TPO组和IL-11组,分别为32例和30例,分别给予rhTPO 15 000 U/d、rhIL-11 3 mg/d皮下注射5-10天,同时监测血常规。结果TPO组、IL-11组血小板开始恢复时间分别为（7.19±0.88）天和（8.52±2.65）天,TPO组低于IL-11组（P〈0.05）。TPO组、IL-11组血小板减少持续时间分别为（11.89±4.46）天和（12.57±6.67）天,两组间差异无统计学意义（P〉0.05）。TPO组、IL-11组治疗开始前血小板值分别为（35.27±6.31）×109/L、（34.73±5.71）×109/L,用药第14天血小板值分别为（103.33±16.31）×109/L、（115.33±20.43）×109/L,两组间差异无统计学意义（P〉0.05）。结论rhTPO和rhIL-11治疗肿瘤患者血小板减少症疗效相似,但rhTPO提升血小板时间早于rhIL-11。     

重组人白介素2梯度胶的肽图分析

白细胞介素17(IL-17)是一新近发现的由活化T淋巴细胞产生的细胞因子,初步研究表明它可能参与T淋巴细胞与造血系统的相互作用,由于IL-17的研究工作刚刚起步,对其确切功能并不完全清楚.为了进一步了解IL-17的生物学功能及可能机制,我们从活化的人外周血单个核细胞中克隆了人IL-17基因的编码序列,将其克隆至本室构建的克隆、表达、测序一体化载体 pLCM182中,经序列测定结果与报道基因序列一致后在大肠杆菌中进行表达,结果发现经诱导后其表达量可达30%以上.IL-17的表达产物在体外能刺激原代培养的人成纤维细胞分泌IL-6和GM-CSF,证明所表达的IL-17具有生物学活性.     

人白细胞介素17(IL-17)的克隆及其在大肠杆菌中的表达

树突状细胞(DC)是一类能特异性启动T淋巴细胞介导的免疫反应的抗原提呈细胞,与其它抗原提呈细胞不同的是,树突状细胞能激活初始型T淋巴细胞(naive T cells).近年来,人们试图通过刺激血液中的前体细胞以提高DC的量.最近,有学者通过GM-CSF、IL-4及MCM的培养单个核细胞中获得了DC.本研究对此法加以改良并从恒河猴血中获得了形态、表型及功能均与人成熟DC非常类似的DC.去除了T细胞的单个核细胞在含有1%人血清及GM-CSF和IL-4的培养基中培养,在第7天,50%的培养基换成单核细胞的条件培养基MCM.此培养基是获得成熟的具有免疫刺激作用的DC所必需的,这样可从2Oml血中获得0.5-1.0×10~6DC.对这些DC与在同样培养基中培养产生的巨噬细胞的比较表明,这些DC刺激同种异体T细胞的能力较巨噬细胞明显增强,而且这些DC的形态和表型很典型且能表达高水平的p55及与细胞内CD68.对比之下,贴壁的巨噬细胞表达非常低水平的p55及高水平的弥漫性CD68,用此方法制备的恒河猴     

A dose-escalation study of recombinant human interleukin-18 using two different schedules of administration in patients with cancer.

PURPOSE: Interleukin-18 (IL-18) is an immunostimulatory cytokine with antitumor activity in preclinical models. A phase I study of recombinant human IL-18 (rhIL-18) was done to determine the toxicity, pharmacokinetics, and biological activities of rhIL-18 administered at different doses in two different schedules to patients with advanced cancer. EXPERIMENTAL DESIGN: Cohorts of three to four patients were given escalating doses of rhIL-18 as a 2-h i.v. infusion either on 5 consecutive days repeated every 28 days (group A) or once a week (group B) for up to 6 months. Toxicities were graded using standard criteria. Blood samples were obtained for safety, pharmacokinetic, and pharmacodynamic measurements. RESULTS: Nineteen patients (10 melanoma and 9 renal cell cancer) were given rhIL-18 in doses of 100, 500, or 1,000 microg/kg (group A) or 100, 1,000, or 2,000 microg/kg (group B). Common side effects included chills, fever, headache, fatigue, and nausea. Common laboratory abnormalities included transient, asymptomatic grade 1 to 3 lymphopenia, grade 1 to 4 hyperglycemia, grade 1 to 2 anemia, neutropenia, hypoalbuminemia, liver enzyme elevations, and serum creatinine elevations. No dose-limiting toxicities were observed. Biological effects of rhIL-18 included transient lymphopenia and increased expression of activation antigens on lymphocytes. Increases in serum concentrations of IFN-gamma, granulocyte macrophage colony-stimulating factor, and IL-18-binding protein were observed following dosing. CONCLUSIONS: rhIL-18 can be given in biologically active doses by either weekly infusions or daily infusions for 5 days repeated every 28 days to patients with advanced cancer. Toxicity was generally mild to moderate, and a maximum tolerated dose of rhIL-18 by either schedule was not determined.     

Immunotherapeutic Approach for Better Management of Cancer - Role of IL-18

Interleukin-18 (IL-18) is an immune-stimulatory cytokine with antitumor activity in preclinical models. Itplays pivotal roles in linking inflammatory immune responses and tumor progression and is a useful candidatein gene therapy of lymphoma or lymphoid leukemia. A phase I study of recombinant human IL-18 (rhIL-18) inpatients with advanced cancer concluded that rhIL-18 can be safely given in biologically active doses to patientswith advanced cancer. Some viruses can induce the secretion of IL-18 for immune evasion. The individualcytokine activity might be potentiated or inhibited by combinations of cytokines. Here we focus on combinationaleffects of cytokines with IL-18 in cancer progression. IL-18 is an important non- invasive marker suspected ofcontributing to metastasis. Serum IL-18 may a useful biological marker as independent prognostic factor ofsurvival. In this review we cover roles of IL-18 in immune evasion, metastasis and angiogenesis, applicationsfor chemotherapy and prognostic or diagnostic significance.     

血管基膜衍生多功能肽基因克隆、表达及空间构象分析

目的：构建血管基膜衍生多功能肽克隆和原核表达载体，并对血管基膜衍生多功能肽氨基酸序列进行空间结构分析预测。方法：用人源性IgG3上游铰链区连接肽连接的Tumstatin的2个功能区片段，即血管基膜衍生多功能肽(VBMDMP)，利用合成的3条长引物片段，进行PCR扩增。克隆到pUC19载体，酶切和测序鉴定。亚克隆构建原核表达载体pGEX4T1VBMDMP,IPTG诱导表达，聚丙烯酰胺凝胶电泳鉴定表达产物。用Glutathione Sepharose 4B层析柱进行了亲和层析鉴定。将VBMDMP序列输入计算机，利用Antheprot软件分析。结果：血管基膜衍生多功能肽（VBMDMP）基因经限制性内切酶酶切和测序鉴定，其大小和核苷酸序列正确，与设计完全一致。获得了原核表达融合蛋白GSTVBMDMP。Antheprot分析显示，人IgG3上游铰链区连接后的Tumstatin的2个功能区域能自由伸展。结论:成功构建了血管基膜衍生多功能肽克隆和原核表达载体，并对其进行了空间结构分析     

Phase I trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain IFN-gamma induction is associated with clinical response.

The aim of this study was to examine the tolerability, antitumor activity, and biological effects of a new schedule of i.v. recombinant human interleukin 12 (rhIL-12). Twenty-eight patients were enrolled in a Phase I trial in which rhIL-12 was administered twice weekly as an i.v. bolus for 6 weeks. Stable or responding patients were eligible to receive additional 6-week cycles until there was no evidence of disease or until tumor progression. Patient cohorts were treated with escalating doses of rhIL-12 (30-700 ng/kg). The maximum tolerated dose (MTD) was 500 ng/kg, with dose-limiting toxicities consisting of elevated hepatic transaminases and cytopenias. At the MTD (n = 14), there was one partial response occurring after 6 cycles of rhIL-12 in a patient with renal cell cancer. Two additional renal cell cancer patients treated at the MTD had prolonged disease stabilization, with one of these exhibiting tumor regression after 8 cycles of rhIL-12. IFN-gamma, IL-15, and IL-18 were induced in patients treated with rhIL-12. Whereas IFN-gamma and IL-15 induction were attenuated midway through the first cycle in patients with disease progression, those patients with tumor regression or prolonged disease stabilization were able to maintain IFN-gamma, IL-15, and IL-18 induction. The down-modulation of IFN-gamma induction during rhIL-12 treatment did not relate to IL-10 production or alterations in rhIL-12 bioavailability but was associated with an acquired defect in lymphocyte IFN-gamma production in response to IL-12, IL-2, or IL-15. This defect could be partially overcome in vitro through combined stimulation with IL-12 plus IL-2. These findings show that the chronic administration of twice-weekly i.v. rhIL-12 is well-tolerated, stimulates the production of IL-12 costimulatory cytokines and IFN-gamma, and can induce delayed tumor regression. Strategies aimed at maintaining IFN-gamma induction, such as the addition of IL-2, may further augment the response rate to this schedule of rhIL-12.     

Interleukin-1 receptor antagonist inhibits growth modulation of human tumor-cell lines by interleukin-1 in-vitro

In this study we report that recombinant human (rh) interleukin-1alpha (IL-1alpha) has direct and dose-dependent growth-modulating effects on human tumor cell lines in vitro as measured by a human tumor cloning assay (HTCA). Colony formation of the melanoma cell line A 375 was inhibited by rhIL-1alpha, whereas colony formation of the glioblastoma cell line HTB 14 was enhanced by this cytokine. Both growth-modulating effects were dose-dependent, however with some saturation. Subsequently, we have tested the activity of recombinant human IL-1 receptor antagonist (rhIL-1ra) on the tumor growth modulation by rhIL-1alpha. Tumor cells were incubated with increasing concentrations of rhIL-1ra and then added to the cultures containing rHIL-1alpha. Concentrations of rhIL-1ra were chosen to achieve a range between 0.01 and 100 ng/ml which includes a 1000-fold molar excess over IL-1alpha. The receptor antagonist was able to block both the inhibition and the stimulation of clonal growth of the respective tumor cell line by rhIL-1alpha. Furthermore, there was a direct dose dependent relationship revealing higher IL-1 antagonism of rhIL-1ra at higher concentrations with maximum efficacy at 1000-molar excess concentrations over IL-1alpha. In addition, rhIL-1ra alone did not reveal major modulation of the growth of A 375, but significantly decreased colony formation of HTB 14. We conclude that rhIL-1ra can counteract modulation of clonal growth of human tumor cells by IL-1alpha in vitro. Since our report provides first evidence that the stimulation of clonal tumor cell growth by IL-1alpha can be blocked by rhIL-1ra, this member of the IL-1 cytokine network should be further studied as a possible candidate for experimental cancer treatment.