Study of recombinant human interleukin-12 for treatment of complications after radiotherapy for tumor patients

AIM To evaluate the treatment effects of recombinant human interleukin-12(rh IL-12) on radiotherapy complications, such as severe myelosuppression or pancytopenia, the decline or imbalance of immune function, etc.METHODS The patients received high-dose and short-course precise radiotherapy, such as Cyber knife and image-guided radiotherapy(IGRT), which can cause myelosuppression or pancytopenia and immune function decline within a short time. One-hundred subjects were enrolled in the study, and 50 were randomized to a treatment group which used rh IL-12 and 50 were randomized to a control group which used symptomatic and supportive therapy after radiotherapy. The 50 subjects in the treatment group were further divided into five subgroups and intervenedwith rh IL-12 at a dose of 50, 100, 150, 200 or 250 ng/kg respectively. The dose-effect relationship was observed. RESULTS Rh IL-12 significantly attenuated the decrease of peripheral blood cells in the treatment group, and immune function was improved after treatment. Due to the different radiation doses, there was a fluctuation within 12 h after treatment but mostly showing an increasing trend. As to the clinical manifestations, 2 patients in the 250 ng/kg subgroup showed low fever after administration, 1 patient in the 200 ng/kg subgroup and 2 patients in the 250 ng/kg subgroup showed mild impairment of liver function during the observation period.CONCLUSION Rh IL-12 has effective therapeutic and protective effects on complications following radiotherapy, such as the decline of blood cells, myelosuppression and the decline or imbalance of immune function, which indicated good prospects for development and application.

6-Gingerol, asarinin, and deoxyschizandrin improve bronchial epithelium functions in an interleukin-13einduced BEAS-2B cell model

Objective:To explore the effects of 6-gingerol,asarinin,and deoxyschizandrindthe main components of Zingiber officinale(Willd.)Rosc.(Gan Jiang),Asarum heterotropoides f.var.mandshuricum(Maxim.)(Xi Xin),and Schisandra chinensis(Turcz.)Baill.(Wu Wei Zi),respectivelydon an interleukin(IL)-13einduced BEAS-2B cell model in vitro.Methods:The BEAS-2B cell model was established using 25 ng/mL IL-13 combined with 1%fetal bovine serum(FBS)in vitro.Mitoquinone mesylate(Mito-Q)treatment was used as a positive control group,and different concentrations of 6-gingerol,asarinin,and deoxyschizandrin were used to treat the models.The level of reactive oxygen species(ROS)production was detected by flow cytometry.The expression levels of LC3B,Beclin1,adenosine 50-monophosphate(AMP)eactivated protein kinase(AMPK),phosphory-lated-AMPeactivated protein kinase(P-AMPK),dynamin-related protein 1(DRP1),and mitochondrial fusion protein 2(MFN2)were detected by Western blot.Mitochondrial membrane potential(MMP)assay kit with JC-1 was utilized to detect the level of MMP.Results:The BEAS-2B cells exposed to 25 ng/mL IL-13 with 1%FBS showed an increased ROS level and a decreased MMP.6-Gingerol,asarinin,and deoxyschizandrin were able to downregulate ROS level and upregulate the MMP in the BEAS-2B model.Asarinin and deoxyschizandrin reduced the expression of autophagy protein LC3B,while deoxyschizandrin significantly increased the expression of DRP1 in the BEAS-2B model.Conclusion:6-Gingerol,asarinin,and deoxyschizandrin can reduce ROS generation and increase MMP,but have different regulatory effects on the expression of autophagy protein and mitochondrial mitotic protein.The three components have both synergistic and complementary effects in classic medicine compatibility.This study may provide an innovative strategy to reduce the lung inflammation related to IL-13.

补肾痹通方联合骨髓间充质干细胞对损伤软骨细胞的保护机制及SOX9、MMP-13表达的影响

目的:探讨补肾痹通方(BSBT)联合骨髓间充质干细胞(BMSCs)对白介素(IL)-1β诱导的损伤软骨细胞的保护机制及性别决定区Y框蛋白9(SOX9)、基质金属蛋白酶13(MMP-13)表达的影响。方法:使用10 ng/ml的IL-1β建立损伤软骨细胞模型,实验分为对照组、模型组(IL-1β)、中药组(IL-1β+BSBT)、干细胞组(IL-1β+BMSCs)和联合组(IL-1β+BSBT+BMSCs);CCK-8法检测各组软骨细胞增殖情况;RT-qPCR法和Western blot法分别检测软骨细胞内SOX9、MMP-13、Ⅱ型胶原α1重组蛋白(COL2A1)、IL-10的基因和蛋白表达;ELISA检测各组细胞培养上清中肿瘤坏死因子-α(TNF-α)、IL-6、胰岛素样生长因子1(IGF-1)、碱性成纤维细胞生长因子(bFGF)的水平。结果:与模型组比较,各组软骨细胞活性显著增强(P<0.01),软骨细胞中的SOX9、COL2A1、IL-10的基因和蛋白水平显著升高(P<0.01),MMP-13的基因和蛋白水平明显降低(P<0.01),软骨细胞培养上清中TNF-α、IL-6的含量显著降低(P<0.01),IGF-1、bFGF的水平升高(P<0.01),其中联合组变化最明显(P<0.05)。结论:BSBT联合BMSCs可有效保护损伤软骨细胞,其机制可能是通过上调SOX9,下调MMP-13,抑制炎症,改善软骨细胞微环境,促进关节软骨的修复与再生。

Neuronal changes in the retinal ganglion cell layer following recombinant human interleukin-2 intravitreal injection in a rat model of chronically elevated intraocular pressure

Intraperitoneal injection of recombinant human interleukin-2（rhIL-2）inhibits neuronal apoptosis in the chronic ocular hypertension retinal ganglion cell layer.Intravitreous injection was performed on retinal ganglion cells in a Wistar rat model of chronically elevated intraocular pressure to observe the effects of LY294002 and AG490 on retinal ganglion cell survival,macrophage activation,and PI3K/Akt and JAK/STAT activation.The number of retinal ganglion cells in the rhIL-2 treatment group was much greater than in the normal control and phosphate-buffered saline groups.Western blot analysis revealed low Akt and STAT3 protein expression in the retina after 3-hour intravitreous injections of rhIL-2.However,protein expression was increased at 12 hours,but decreased again at 24 hours,with very low expression at 96 hours.LY294002 and AG490,which are inhibitors of the PI3K/Akt and JAK/STAT3 signal pathways,prevented upregulation of Akt and STAT3 protein expression in the retina,respectively.Intravitreous injection of rhIL-2 exhibited neuroprotective effects by decreasing retinal ganglion cell layer damage in a rat model of chronic glaucoma.These results suggest that intravitreal injection of rhIL-2 could induce the PI3K/Akt and JAK/STAT3 signaling pathways to protect retinal ganglion cells in chronically elevated intraocular pressure models.

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective： Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin （IL） -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells （HMCs）. Methods： The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay （RPA）. Activity of nuclear factor-kappa B （NF-κB） and activa- tor protein-1 （AP-1） was examined by electrophoretic mobility shift assay （EMSA）. NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase （JNK） activity were assayed by immunoblot. Results： Recombinant IL-13 inhibited tumor necrosis factor-α （TNF-u）, IL-1α, IL-1β, monocyte chemoattractant protein-1 （MCP-1）, IL-8, and transforming growth factor-β1 （TGF-β1） mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides （LPS） dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion： IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.

Interleukin-13 promotes cellular senescence through inducing mitochondrial dysfunction in IgG4-related sialadenitis

Immunoglobulin G4-related sialadenitis(IgG4-RS)is an immune-mediated fibro-inflammatory disease and the pathogenesis is still not fully understood.The aim of this study was to explore the role and mechanism of interleukin-13(IL-13)in the cellular senescence during the progress of IgG4-RS.We found that the expression of IL-13 and IL-13 receptorα1(IL-13Rα1)as well as the number of senescent cells were significantly higher in the submandibular glands(SMGs)of IgG4-RS patients.IL-13 directly induced senescence as shown by the elevated activity of senescence-associatedβ-galactosidase(SA-β-gal),the decreased cell proliferation,and the upregulation of senescence markers(p53 and p16)and senescence-associated secretory phenotype(SASP)factors(IL-1βand IL-6)in SMG-C6 cells.Mechanistically,IL-13 increased the level of phosphorylated signal transducer and activator of transcription 6(p-STAT6)and mitochondrial-reactive oxygen species(mt ROS),while decreased the mitochondrial membrane potential,ATP level,and the expression and activity of superoxide dismutase 2(SOD2).Notably,the IL-13-induced cellular senescence and mitochondrial dysfunction could be inhibited by pretreatment with either STAT6 inhibitor AS1517499 or mitochondria-targeted ROS scavenger Mito TEMPO.Moreover,IL-13 increased the interaction between p-STAT6 and c AMP-response element binding protein(CREB)-binding protein(CBP)and decreased the transcriptional activity of CREB on SOD2.Taken together,our findings revealed a critical role of IL-13 in the induction of salivary gland epithelial cell senescence through the elevated mitochondrial oxidative stress in a STAT6–CREB–SOD2-dependent pathway in IgG4-RS.

Determination of Serum Interleukin-13 and Nerve Growth Factor in Patients with Systemic Lupus Erythematosus and Clinical Significance

The changes in the levels of serum interleukin-13 (IL-13) and nerve growth factor (NGF) in patients with systemic lupus erythematosus (SLE) and their clinical significance were investigated. Sandwich ELISA was used to determine the levels of serum IL-13 and NGF in 35 SLE patients and 15 normal controls. The results showed that the levels of serum IL-13 (92.69±9.87 pg/ml) and NGF (339.69±25.60 pg/ml) in active SLE patients were significantly higher than those in inactive SLE patients (IL-13, 54.22±9.31 pg/ml; NGF, 300.89±33.51 pg/ml)(P<0.01). The inactive patients also had significantly increased serum levels of IL-13 and NGF as compared with normal controls (IL-13, 35.20±12.70 pg/ml; NGF, 111.40±32.54 pg/ml; P<0.05 and P<0.01, respectively). Spearman correlation analysis revealed that the serum IL-13 levels were correlated with disease activity index of SLE (SLEDAI), ESR and serum levels of C_3 (r= 0.813, 0.504, -0.605, respectively). The serum NGF levels were also correlated with above markers (r=0.442, 0.338, -0.463, respectively). The serum levels of IL-13 and NGF had a positive correlation (r=0.506, P<0.01). It was suggested that IL-13 and NGF might be involved in the pathogenesis of SLE and closely correlated with disease activity.

Interleukin-13 and age-related macular degeneration

AIM：To identify the effects of interleukin（IL）-13 on retinal pigment epithelial（RPE） cells and the IL-13 level in aqueous humor of age-related macular degeneration（AMD） patients.METHODS：IL-13 levels in aqueous humor specimens from AMD patients were detected with enzyme-linked immunosorbent assay（ELISA）.ARPE-19 cells were treated with 10 ng/m L IL-13 for 12,24,and 48 h.The cell proliferaton was evaluated by the MTS method.The m RNA and protein levels of α-SMA and ZO-1 were evaluated with quantitative real-time polymerase chain reaction（q RT-PCR） and Western blot respectively.The expression of tumor necrosis factor-α（TNF-α）,transforming growth factor-β（TGF-β） and vascular endothelial growth factor（VEGF） were assessed by ELISA.RESU LTS：IL-13 levels in the aqueous humor of patients with AMD were significantly higher than those in the control（167.33±17.64 vs 27.12±5.65 pg/m L;P〈0.01）.In vit ro,IL-13 of high concentrations（10,15,and 20 ng/m L） inhibited ARPE-19 cell proliferation.α-SMA m RNA in ARPE-19 cell were increased（1.017±0.112 vs 1.476±0.168;P〈0.001） and ZO-1 decreased（1.051±0.136 vs 0.702±0.069;P〈0.001） after treated with 10 ng/m L IL-13 for 48 h.The protein expression of α-SMA and ZO-1 also showed the same tendency（α-SMA：P=0.038;ZO-1：P=0.008）.IL-13 significantly reduced the level of TNF-α（44.70±1.67 vs 31.79±3.53 pg/m L;P=0.005） at 48 h,but the le vel of TGF-β2 was significantly increased from 34.44±2.92 to 57.61±6.31 pg/m L at 24h（P=0.004） and from 61.26±1.11 to 86.91±3.59 pg/m L at 48h（P〈0.001）.While expressions of VEGF didn＇t change after IL-13 treatment.CONCLUSION：IL-13 in vitr o inhibit ARPE-19 cell proliferation and expression in the aqueous may be associated with AMD.

鸡白痢沙门菌C79-13ΔcrpΔasd平衡致死系统的构建及其生物学特性的研究

目的:为了研制更加安全的鸡白痢沙门菌弱毒株,并将其开发为疫苗活载体。方法:本研究构建了鸡白痢沙门菌C79-13ΔcrpΔasd缺失株。双基因缺失株的构建是以含有重组自杀性质粒pREΔasd的大肠杆菌χ7213为供体菌,C79-13Δcrp为受体菌进行接合转移,两步法筛选crp/asd基因双缺失突变株。结果:PCR及测序结果表明C79-13ΔcrpΔasd构建成功。进一步研究表明,该缺失株的生长需要外源的二氨基庚二酸(DAP),且能稳定遗传缺失的asd基因,与亲本株C79-13相比,其血清型未发生变化,但其生长速度明显减慢,毒力明显降低。结论:C79-13ΔcrpΔasd双缺失株可接受asd+质粒作为宿主平衡致死系统高效稳定地表达外源基因,并为研制安全有效的鸡白痢沙门菌疫苗弱毒株以及进一步将其开发为适于黏膜免疫的口服活载体疫苗奠定了基础。

血清白介素-13、白介素-4水平及吸入过敏原在新疆维吾尔族哮喘儿童相关研究

目的探讨新疆维吾尔族哮喘儿童白介素-13(IL-13)、白介素-4(IL-4)水平及吸入过敏原检测的意义。方法采取双抗体夹心酶联免疫吸附法(ELISA)对新疆维吾尔族哮喘儿童及维吾尔族健康儿童进行血清IL-13、IL-4水平的检测,酶免法定性检测吸入过敏原,并进行比较。结果 (1)哮喘组IL-13水平[(8.12±4.78)ng/L]较对照组[(5.69±3.62)ng/L]高,差异有统计学意义(P<0.05),哮喘组IL-4水平[(10.64±27.90)ng/L]较对照组[(36.06±82.68)ng/L]低,但差异无统计学意义(P>0.05);(2)哮喘组IL-13、IL-4呈负相关(r=-1.116,P=0.514),对照组IL-13、IL-4呈正相关(r=0.121,P=0.575),但差异均无统学意义(P>0.05);(3)哮喘组儿童过敏原阳性率[76.5%(26例)]较对照组[50%(12例)]高,差异有统计学意义(P<0.05)。结论 IL-13、吸入过敏原在新疆维吾尔族哮喘儿童的发病过程中起着重要的作用。

白介素13基因多态性与哮喘发病易感性的关系

目的探讨白介素13(IL-13)基因内含子区+1923C/T多态性与哮喘发病易感性的关系。方法采用聚合酶链反应—限制性片段长度多态性技术对150例哮喘患者(哮喘组)和150例健康对照者(对照组)IL-13基因内含子区+1923C/T单核苷酸多态性进行检测,比较其基因型和等位基因分布频率。结果对照组IL-13基因内含子区+1923C/T基因型CC、CT和TT的分布频率分别为41.33%(62/150)、44.00%(66/150)和14.67%(22/150),在哮喘组分别为21.33%(32/150)、41.33%(62/150)和37.34%(56/150),两组各基因型分布频率比较差异有统计学意义(χ2=24.52,P<0.01)。CT、TT基因型者患哮喘的危险性高于CC基因型者(χ2=27.38,P<0.01)。结论 IL-13基因内含子区+1923C/T多态性是影响哮喘发病的重要候选基因,T等位基因与哮喘易感性相关。

MMP-13基因真核表达载体的构建及其功能研究

目的为了研究MMP-13蛋白在瘢痕形成的作用,我们构建MMP-13基因真核重组表达质粒,检测并鉴定了MMP-13基因在转染了重组质粒的皮肤角质形成细胞HaCat中的表达。方法通过RT-PCR方法将MMP-13基因克隆到真核表达载体pcDNA3.1(+)中,构建其真核表达重组质粒pcDNA3.1(+)/MMP-13,基因测序鉴定。重组质粒以阳离子脂质体2000介导转染HaCat细胞,RT-PCR检测外源基因的表达,间接免疫荧光和Western blot检测外源蛋白的表达。以明胶酶谱法和MTT法分别检测MMP-13的活性以及其对HaCat细胞增殖的影响。结果通过RT-PCR检测到MMP-13基因在重组质粒转染后的HaCat细胞中大量表达,以间接免疫荧光反应和Western blot可检测到MMP-13蛋白在在重组质粒转染后的HaCat细胞中呈阳性,明胶酶谱法和MTT法检测显示真核表达质粒pcDNA3.1(+)/MMP-13表达的MMP-13蛋白具有活性,并且可以抑制HaCat细胞的增殖。结论构建的重组质粒pcDNA3.1(+)/MMP-13能在Hacat细胞中表达活性蛋白MMP-13,并可以抑制HaCat细胞的增殖,这为研究MMP-13在瘢痕形成中的作用奠定了基础,从而为瘢痕治疗提供有效靶标。

三氧化二砷对rIL-13诱导肺成纤维细胞增殖和炎性介质表达的影响

目的探讨三氧化二砷(As2O3)的抗肺纤维化机制。方法将体外培养的肺成纤维NIH3T3细胞株随机分为对照组及观察组,对照组仅予DMEM培养基,观察组予80 ng/ml重组白细胞介素(rIL)-13及不同浓度As2O3处理24、48 h。MTT法检测细胞增殖;原位杂交方法检测巨噬细胞炎症蛋白(MCP)-1αmRNA表达;放射免疫法检测细胞培养上清液IL-6、IL-8水平。结果 As2O3处理后细胞增殖明显受抑,且呈时间和剂量依赖性(P<0.01);rIL-13处理后细胞MCP-1αmRNA和IL-6、IL-8表达增强,联用As2O3后此作用明显受抑(P<0.01)。结论 As2O3可能通过抑制rIL-13诱导的肺成纤维细胞增殖及炎性介质表达而发挥抗肺纤维化作用。

重组白细胞介素13对成纤维细胞增殖的影响

目的:观察重组白细胞介素13(rIL-13)对NIH3T3细胞增殖作用的影响,探讨肺纤维化的发病机制。方法:NIH3T3细胞分为rIL-13组和不含rIL-13的对照组r,IL-13组加入不同浓度的rIL-13,作用24及48h。透射电镜观察3T3细胞的超微结构,Hoechst试剂盒观察细胞DNA形态变化,噻唑蓝比色试验(MTT法)测定细胞增殖率。结果r:IL-13呈剂量依赖方式刺激3T3细胞细胞生长。经rIL-13 80 ng/ml孵育的细胞DNA合成增加,细胞核增大,细胞质中可见较多核糖体及线粒体。rIL-13组细胞增殖率增加,与对照组比较有显著性差异(P<0.05)。结论r:IL-13可能通过促进成纤维细胞增殖,在肺纤维化的发病机制中发挥重要作用。

IL-13和IL-33重组蛋白的原核表达和纯化

目的:利用PET101/D-TOPO蛋白表达系统,表达人IL-13和IL-33重组蛋白。方法:以健康人外周血单个核细胞(PBMC)的mRNA为模板,采用RT-PCR扩增IL-13和IL-33开放阅读框,将扩增所得目的基因连接到质粒p ET101/DTOPO,构建出重组质粒并转化大肠杆菌BL21,诱导其表达IL-13和IL-33重组蛋白,纯化后进行鉴定。结果:RT-PCR扩增所得IL-13 c DNA为490 bp,IL-33 cDNA大小为800 bp,将其分别连接到质粒PET101/D-TOPO,构建出IL-13和IL-33重组质粒,分别转化大肠杆菌BL21,诱导其表达。SDS-PAGE电泳分析,发现目的条带大小分别为29 kDa和35 kDa左右,Western blotting结果证实重组表达正确。结论:成功构建出IL-13和IL-33重组质粒,成功表达并纯化出IL-13和IL-33重组蛋白。

白细胞介素-13上调巨核细胞系-Dami细胞原癌基因c-mpl的研究

目的 :研究重组人白细胞介素 - 13(rhIL - 13)对巨核细胞系 -Dami细胞原癌基因c -mpl表达的影响。方法 :用RT -PCR法检测c-mplmRNA的表达。用配体结合实验检测膜蛋白c-mpl的表达。结果 :经 2 5 μg/LrhIL - 13处理过的实验组Dami细胞c -mplmRNA的表达比对照组高 2 4 8%。经 10 0 μg/LrhIL - 13处理过的实验组Dami细胞膜蛋白c -mpl的表达比对照组高 2 8 5 % (P <0 0 5 )。结论 :rhIL - 13增强了Dami细胞原癌基因c -mpl表达 ;膜蛋白c-mpl与促血小板生成素 (TPO)的结合功能正常 ;rhIL - 13促进Dami细胞增殖的部分机制可能是通过上调原癌基因c

三氧化二砷抑制重组白细胞介素13促成纤维细胞胶原的表达

目的观察三氧化二砷(As2O3)对重组白细胞介素13(rIL-13)促成纤维细胞的增殖及分泌胶原是否有抑制作用。方法体外培养3T3成纤维细胞,细胞分组为DMEM对照组、rIL-13(80 ng/mL)组及rIL-13加不同浓度As2O3(2μmol/L和4μmol/L)组。24 h和48 h后,MTT法观察细胞生长抑制情况,用细胞抑制率表示;各组药物作用细胞48 h后,Western blot法检测I型胶原(CoI I)的表达。结果 As2O3可显著抑制3T3成纤维细胞增殖,且呈剂量-效应关系(P<0.01)。各组均可检测到Ⅰ型胶原表达。其中,rIL-13(80 ng/mL)组Ⅰ型胶原表达显著高于其他各组细胞;rIL-13加不同浓度As2O3(2μmol/L和4μmol/L)组成纤维细胞分泌I型胶原的量显著低于rIL-13组(P<0.01),但4μmol/LAs2O3+rIL-13组与2μmol/L As2O3+rIL-13组相比,细胞分泌I型胶原的水平差异无统计学意义(P>0.05)。结论 As2O3对白细胞介素13促3T3成纤维细胞增殖和分泌I型胶原有抑制作用。

瘤内注射重组人p53腺病毒对鼻咽癌组织中基质金属蛋白酶-13表达的影响及意义

目的探讨瘤内注射重组人p53腺病毒注射液(r Ad-p53)对鼻咽癌原发灶组织基质金属蛋白酶(MMP)-13蛋白表达的影响及临床意义。方法将61例确诊中晚期鼻咽癌的患者分为两组。治疗组32例给予r Ad-p53鼻咽部瘤内注射联合同步放化疗;对照组29例给予同步放化疗。收集r Ad-p53瘤内注射前后患者鼻咽部瘤体活组织标本,采用免疫组化检测标本中MMP-13的表达。结果治疗组患者治疗后MMP-13的阳性表达率下降,且存在淋巴结转移的患者MMP-13阳性表达率下降更明显,而对照组患者治疗后MMP-13阳性表达率升高;治疗后治疗组MMP-13的阳性表达率低于对照组(P<0.05)。治疗组和对照组总体生存率比较无明显差异(P>0.05)。结论 r Ad-p53明显影响鼻咽癌原发灶MMP-13蛋白表达,在淋巴结转移患者中更明显,但对总体生存率的影响尚不明确。

重组人白细胞介素-13对巨核细胞系-Dami细胞原癌基因c-mpl表达的影响

目的 :研究重组人白细胞介素 13 (rhIL 13 )对巨核细胞系 -Dami细胞原癌基因c mpl表达的影响。方法 :实验组细胞培养系统中分别加入 2 5ng/ml,5 0ng/ml,10 0ng/mlrhIL 13 ,对照组不加任何细胞因子。用RT PCR法检测c mplmRNA的表达。结果 :经 2 5ng/ml、5 0ng/ml、10 0ng/mlrhIL 13处理过的实验组Dami细胞较之对照组其c mplmRNA的表达分别提高 2 4 .8%、2 7.9%和 3 1.5 %。结论 :rhIL 13增强了Dami细胞原癌基因c mpl表达 ;rhIL 13促进Dami细胞增殖的部分机制可能是通过上调c mpl的表达来实现的。

重组人白细胞介素-13在大肠杆菌中的表达纯化及鉴定

采用大肠杆菌(Escherichia coli)作为宿主,通过300L发酵罐进行中试发酵表达重组人白细胞介素-13(rhIL-13)融合蛋白;经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导,离心发酵液收集菌体,菌体经溶解、变性、稀释复性及亲和层析捕获,获得融合蛋白;融合蛋白经肠激酶酶切后,再经亲和层析分离和分子筛精细纯化等步骤,获得高纯度rhIL-13原液.每升发酵液可获得高纯度的rhIL-13原液约30mg.通过本纯化工艺获得的rhIL-13原液经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法检测,纯度大于98.75%;经高效液相色谱分子排阻(HPLC-SEC)法检测,纯度为100%;质谱(MS)法测定rhIL-13分子质量为12 348u,与理论值基本一致;采用噻唑蓝(MTT)法进行比活性检测,比活大于8.0×106 IU/mg;采用HPLC-SEC法和MS法对rhIL-13二硫键数目和位置进行分析,结果显示二硫键数目和位置与理论一致.此操作方法可获得高纯度、高活性的rhIL-13原液,并且操作简单,易于放大,可实现工业化规模生产.