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25μg/LrhIL-13处理过的实验组Dami细胞c-mplmRNA的表达比对照组高24.8%。经100μg/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28.5%(P＜0.05)。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-mpl的表达来实现的。     

STAT6介导白细胞介素—13诱导巨核细胞系HEL细胞血小板膜糖蛋白Ⅱb和血管性血友病因子的表达

目的:探索信号转导子和转录激活因子6(STAT6)介导白细胞介素-13(IL-13)对巨核细胞系-人红白血病HEL细胞(HEL)血小板膜糖蛋白Ⅱ b(GPⅡ b)和血管性血友病因子(vWF)表达的作用,部分阐明IL-13的信号转导通路.方法:RT-PCR检测GPⅡ b和vWF mRNA的表达,免疫印迹法检测磷酸化的STAT6、 GPⅡ b和vWF蛋白的表达,图像分析检测电泳条带吸光度.结果:IL-13(100μg/L)作用HEL细胞,使HEL细胞STAT6磷酸化,磷酸化抑制剂A77-1726(50μmol/L)阻断了IL-13诱导的HEL细胞STAT6磷酸化.IL-13(100μg/L)上调了HEL细胞GPⅡ b和vWF mRNA和蛋白的表达,A77-1726(50μmol/L)抑制了STAT6介导的IL-13诱导HEL细胞GPⅡ b和vWF mRNA和蛋白的表达.结论:IL-13上调了HEL细胞GPⅡ b和vWF的表达,STAT6介导了IL-13上调HEL细胞GPⅡ b和vWF的表达.     

白细胞介素13诱导Dami细胞分化并下调转录因子c-myc表达

目的：研究重组人白细胞介素13（recombinant human interleukine-13，rhIL-13）对巨核细胞白血病细胞株Dami细胞分化的影响，以及Dumi细胞白细胞介素13受体α1（Interleukine-13 receptor alpha 1，IL-13 Rα1）的表达，探讨IL-13诱导Dami细胞分化的机制。方法：Dumi细胞经无血清培养20小时后，以rhIL-13分别作用不同时间，提取总RNA，用RT—PCR检测Dumi细胞IL-13受体α1 mRNA、GPⅡb mRNA、c-myc mRNA表达水平。流式细胞仪检测在rhIL-13作用下，Dami细胞GPⅡb表达。免疫组化法检测在rhIL-13作用下，Dumi细胞c—myc的表达。图像分析仪对结果进行分析。结果：①Dami细胞表达IL-13 Rα1 mRNA，rhIL-13作用Dumi细胞4小时，IL-13 Rα1 mRNA表达降低，12小时IL-13 Rα1 mRNA表达恢复。②rhIL-13作用Dami细胞后，巨核细胞的分化标志物GPⅡb mRNA和蛋白质表达增加。③rhIL-13作用Dumi细胞，c—myc mRNA和蛋白质表达降低。结论：白细胞介素13下调c—myc表达。     

人脐血源基质细胞促进巨核细胞增殖的实验研究

目的： 探索体外共培养条件下人脐血源基质细胞（hUCBDSCs）促进巨核细胞系HEL增殖的作用。方法: 建立HEL与hUCBDSCs共培养体系,以HEL/人骨髓基质细胞（human bone marrow stromal cells,hBMSCs）和悬浮培养的HEL细胞为对照。通过光镜、扫描电镜观察共培养条件下HEL细胞与hUCBDSCs的位象关系;并通过CCK8、流式细胞仪等方法检测共培养条件下HEL细胞的生长曲线和增殖率。结果: 共培养条件下,HEL细胞可通过伪足黏附/嵌合于hUCBDSCs层,甚至移行到hUCBDSCs层下,包裹于hUCBDSCs层内。与hUCBDSCs共培养的HEL细胞的增殖速度和增殖率均明显高于同期培养的HEL细胞/hBMSCs共培养组和HEL细胞悬浮组。结论: 人脐血源基质细胞能够促进巨核细胞增殖,与对照hBMSCs组相比具有明显的优势,有望为造血功能损伤中血小板数量和功能的恢复提供新的治疗思路。     

白细胞介素13抑制肾小球系膜细胞增殖及白细胞介素6表达

目的:探讨白细胞介素13(IL-13)对体外培养的大鼠系膜细胞的增殖及其产生白细胞介素6(IL-6)的影响。方法:用四甲基偶氮唑(MTT)法测定系膜细胞增殖,用逆转录聚合酶链反应(RT-PCR)及酶联免疫吸附法(ELISA)测定系膜细胞IL-6mRNA表达及其蛋白水平。结果:IL-13在1、10、100μg/L浓度范围呈剂量依赖性地抑制系膜细胞的增殖;5%FCSRPMI1640培养条件下系膜细胞IL-6mRNA表达及IL-6分泌水平较低,脂多糖(LPS)可刺激系膜细胞IL-6mRNA的表达及提高IL-6分泌水平,而IL-13可抑制LPS诱导的系膜细胞IL-6分泌及其mRNA表达。结论:IL-13抑制体外培养的系膜细胞增殖及LPS诱导的系膜细胞IL-6的产生,IL-13可能对于肾小球肾炎的系膜细胞炎症反应具有拮抗作用。     

RACK1高表达促进人结肠癌细胞的生长和增殖

目的：研究RACK1高表达对人结肠癌细胞增殖能力的影响。方法：采用脂质体转染术分别建立 RACK1表达下调的SW620细胞系、RACK1表达上调的SW480细胞系以及对照细胞系;采用CCK-8细胞增殖测定、软琼脂集落形成实验、EdU掺入实验以及流式细胞术检测RACK1表达改变对 SW620和SW480细胞增殖的影响;采用Western blot分析RACK1表达改变对G1/S期限制点调控蛋白Cyclin D1和p27蛋白表达的影响。结果：下调RACK1的表达抑制SW620细胞的生长、软琼脂集落形成能力,降低EdU标记的S期细胞数目,阻滞细胞周期于G1/S期;而上调RACK1的表达增强 SW480细胞的生长、软琼脂集落形成能力,增加EdU标记的S期细胞数目,促进细胞周期G1/S期进程。结论：RACK1高表达促进人结肠癌细胞的生长和增殖。     

LAIR-1对人急性髓系白血病细胞HEL增殖、凋亡和迁移的影响

研究人白细胞相关免疫球蛋白样受体1(leukocyte-associated immunoglobulin-like receptor 1,LAIR-1)对人红白血病细胞HEL增殖、凋亡和迁移的影响,探讨LAIR-1在急性髓系白血病(acute myeloid leukemia,AML)中的作用。采用LAIR-1慢病毒表达载体(LV-LAIR-1)转染HEL细胞,建立稳定高表达LAIR-1的HEL细胞株,分别采用流式细胞术、激光扫描共聚焦显微技术、western blotting和RT-PCR鉴定LAIR-1分子的表达效率。采用CCK-8试剂盒、FITC Annexin V凋亡试剂盒和Transwell迁移实验分别检测LAIR-1对HEL细胞增殖、凋亡和迁移能力的影响。实验结果显示,经LVLAIR-1转染的HEL细胞高表达LAIR-1分子,阳性率达到85%以上;功能实验结果显示,与对照组细胞相比,LAIR-1能够显著抑制HEL细胞的增殖(P<0.001),并且促进其凋亡(P<0.01),但是对细胞迁移能力无明显影响(P>0.05)。上述研究结果显示,LAIR-1分子的表达能够影响人AML细胞的生物学功能。     

Wnt/β-catenin信号通路在低氧促进骨肉瘤细胞体外增殖和侵袭中的作用

目的探讨缺氧条件下,Wnt/β-catenin信号通路在骨肉瘤细胞增殖和侵袭中的作用和机制。方法分别在常氧(21%O_2)和缺氧(1%O_2)环境下培养骨肉瘤SaOS-2细胞系24 h,采用CCK-8法检测细胞在缺氧环境下的增殖能力变化,Transwell侵袭实验检测细胞侵袭能力,Real time-PCR和Western blot法检测细胞中Wnt/β-catenin mRNA和蛋白的表达水平,进一步使用β-catenin特异性siRNA阻断其表达并检测。结果缺氧可明显促进骨肉瘤SaOS-2细胞的增殖以及体外侵袭能力的增强,同时上调Wnt/β-catenin mRNA和蛋白表达水平。靶向沉默Wnt/β-catenin之后,缺氧失去了对骨肉瘤SaOS-2细胞增殖和侵袭的诱导作用。结论缺氧条件下,骨肉瘤SaOS-2细胞系中的Wnt/β-catenin信号通路显著激活,从而促进细胞的异常增殖与侵袭能力增强。     

PPAR-γ在LDL诱导的大鼠系膜细胞增殖和基质硬化中的作用

目的探讨PPAR-γ在LDL诱导的系膜细胞增殖和基质硬化中的作用。方法以体外培养的大鼠系膜细胞为研究对象,应用不同浓度的LDL刺激系膜细胞。应用MTT法检测细胞增殖;应用RT-PCR的方法检测LDL对MMP-2、TIMP-2、PPAR-γmRNA表达的影响;应用WesternBlot的方法检测LDL对MMP-2、TIMP-2、PPAR-γ蛋白表达的影响。结果以浓度为3.125~100μg/ml的LDL刺激系膜细胞,可促进系膜细胞的增殖,在LDL3.125~50μg/ml的浓度范围内,系膜细胞的增殖和LDL的浓度呈正相关(r=0.865,P<0.05);以浓度为12.5~100μg/ml的LDL刺激系膜细胞,可下调MMP-2/TIMP-2mRNA和蛋白的表达(P<0.01);以浓度为6.25~100μg/ml的LDL刺激系膜细胞,可下调PPAR-γmRNA和蛋白的表达(P<0.01)。结论在LDL诱导下,PPAR-γ的下调促进了系膜细胞的增殖和系膜基质的增生。     

重组人白细胞介素-13刺激Dami细胞增殖

目的和方法：研究重组人白细胞介素－１３（rhＩＬ－１３）对Ｄami细胞的作用，在血浆 凝决培养基中分别加入rhＩＬ－１３５０ng/mＬ、１００ng/mＬ、１５０ng/mＬ，计数Ｄami细胞ＤＮＡ中的５－溴－２－脱氧尿嘧啶（５－ＢrdＵ）。结果：血浆凝块培养基中加入rhＩＬ－１３能ami使细胞扩增，５０ng/mＬ、１００ng/mＬ、１５０ng/mＬ rhＩＬ－１３ 实验组Ｄami细胞集落数分别是对照组的１．４２倍、１．８５倍、２．３１倍。５－Ｂrd     

NF-κB活化在IL-1β诱导的小鼠肾小球系膜细胞表达IL-6中的作用

目的:研究NF-κB在rhIL-1β刺激引起的体外培养的鼠肾小球系膜细胞表达IL-6中的作用。方法:NF-κΒ活性检测采用电泳迁移率改变法(EMSA),IL-6mRNA表达采用逆转录/聚合酶链反应(RT/PR)检测,培养上清IL-6蛋白含量采用ELISA检测。结果:rhIL-1β刺激肾小球系膜细胞时,在上调IL-6蛋白和基因表达的同时亦激活NF-κB,而且这种上调作用可被NF-κB特异性抑制剂PDTC所阻抑。结论:IL-1β诱导鼠肾小球系膜细胞表达IL-6是通过NF-κB调控,NF-κB可能参与肾小球肾炎的免疫炎症反应。     

NF-κB活化在IL-1β诱导的小鼠肾小球系膜细胞表达IL-6中的作用

目的：研究NF-κB在rhIL-1β刺激引起的体外培养的鼠肾小球系膜细胞表达IL-6中的作用。方法： NF-κΒ活性检测采用电泳迁移率改变法(EMSA),IL-6 mRNA表达采用逆转录/聚合酶链反应(RT/PR)检测,培养上清IL-6蛋白含量采用ELISA检测。结果：rhIL-1β刺激肾小球系膜细胞时,在上调IL-6蛋白和基因表达的同时亦激活NF-κB,而且这种上调作用可被NF-κB特异性抑制剂PDTC所阻抑。结论： IL-1β诱导鼠肾小球系膜细胞表达IL-6是通过NF-κB调控,NF-κB可能参与肾小球肾炎的免疫炎症反应。     

Detection of mRNA in megakaryocytes using in situ hybridization]

The usefulness of in situ hybridization for detecting mRNA encoding various kinds of proteins in megakaryocytic cell lines (K562, HEL and CMK) was examined. Using in situ hybridization, glycoprotein (GP) IIIa mRNA was found to be present in K562, HEL and CMK cells. Both GPIb and GPIIb mRNA were also present in HEL and CMK cells, while only HEL cells expressed platelet factor 4 mRNA. These findings were identical to those obtained with Northern blotting. It should be emphasized that in situ hybridization was useful to clearly define each cell expressing platelet specific protein mRNA. The expression of interleukin-6 (IL-6) and IL-6 receptor mRNA in mononuclear cells prepared from bone marrow aspirates was examined. In situ hybridization study demonstrated the presence of IL-6 receptor mRNA in the recognized megakaryocytes only, while IL-6 mRNA was found to be present in the megakaryocytes and a few mononuclear cells. These findings suggest that differentiation and proliferation of normal megakaryocytes might be controlled by an IL-6 autocrine loop, and detection of IL-6 receptor mRNA might be useful to identify megakaryocytes.     

Effects of interleukin-12 and interleukin-15 on measles-specific T-cell responses in vaccinated infants

Understanding the infant host response to measles vaccination is important because of their increased mortality from measles and the need to provide effective protection during the first year of life. Measles-specific T and B-cell responses are lower in infants after measles vaccination than in adults. To define potential mechanisms, we investigated age-related differences in measles-specific T-cell proliferation, CD40-L expression, and IFN-gamma production after measles immunization, and the effects of rhIL-12 and rhIL-15 on these responses. Measles-specific T-cell proliferation and mean IFN-gamma release from infant PBMCs were significantly lower when compared with responses of vaccinated children and adults. Infant responses increased to ranges observed in children and adults when both rhIL-12 and rhIL-15 were added to PBMC cultures. Furthermore, a significant rise in T-cell proliferation and IFN-gamma release was observed when infant PBMCs were stimulated with measles antigen in the presence of rhIL-12 and rhIL-15 compared to measles antigen alone. CD40-L expression by infant and adult T cells stimulated with measles antigen was comparable, but fewer infant CD40-L(+) T cells expressed IFN-gamma. These observations suggest that lower measles-specific T-cell immune responses elicited by measles vaccine in infants may be due to diminished levels of key cytokines.     

IL-13对LPS诱导人系膜细胞分泌IL-12的影响

目的: 探讨白细胞介素13（IL-13）对肾小球系膜细胞分泌白细胞介素12（IL-12）的影响作用。方法: 用酶联免疫吸附（ELISA）法和逆转录聚合酶链反应（RT-PCR）法检测系膜细胞IL-12蛋白和L-12p40 mRNA表达。结果: LPS诱导系膜细胞的IL-12p40 mRNA表达及其蛋白分泌（P<0.01）。IL-13在1-100 μg/L浓度范围内呈剂量依赖性抑制LPS诱导的系膜细胞IL-12分泌及其mRNA表达（P<0.05或P<0.01）。结论: IL-13可能通过抑制LPS诱导系膜细胞分泌IL-12，而调整了体内Th1/Th2细胞因子平衡。     

rhIL-2与阿霉素长循环热敏脂质体靶向治疗肿瘤的协同作用

目的:观察重组人白细胞介素-2(rhIL-2)与阿霉素长循环热敏脂质体(ALTSL)联合靶向治疗荷H22瘤小鼠的协同作用,并探讨其抑瘤作用的机制。方法:以荷H22瘤小鼠的瘤重为指标,评价药物的抑瘤活性。以荷H22瘤小鼠的存活天数计算生命延长率。以乳酸脱氢酶释放法测定NK细胞的杀伤活性。以MTT比色法检测淋巴细胞的转化率。以流式细胞术检测肿瘤细胞的凋亡及p53、Fas、FasL和caspase-3的表达。用RT PCR法测定IL-2mRNA及IL-12mRNA的表达。镜下观察荷H22瘤小鼠肿瘤、心、肝及肾脏组织的病理变化。结果:rhIL2 ALTSL的抑瘤率显著高于单用ALTSL,分别为73.5%和67.0%;ALTSL和rhIL2 ALTSL均可显著延长荷瘤小鼠的存活时间(P<0.05～P<0.01)与对照组和游离阿霉素(FADM)组相比较,ALTSL组NK细胞的杀伤活性显著增加,rhIL2 ALTSL组NK细胞的杀伤活性更高。与FADM组比较,rhIL-2 ALTSL组淋巴细胞的转化率明显提高(P<0.01)。应用ALTSL组和rhIL-2 ALTSL组均可增强脾细胞中IL-2mRNA和IL-12mRNA的表达,但后者的增强作用高于前者。病理切片检查显示,热敏脂质体配合肿瘤局部加热,使肿瘤细胞凝固坏死,联合应用rhIL-2肿瘤组织溶解,细胞破碎,可见大量的淋巴细胞浸润。结论:ALTSL能提高ADM的抑瘤效果,降低ADM心肺的毒性。rhIL-2 ALTSL可诱导肿瘤     

人脐带间充质干细胞分泌白细胞介素6促进骨肉瘤细胞增殖和迁移

 目的: 探讨人脐带间充质干细胞(hUC-MSCs)对骨肉瘤Saos-2细胞增殖和迁移的作用及分子机制。方法: 组织块贴壁法分离培养hUC-MSCs,流式细胞术鉴定细胞表面标记物;CCK-8法和细胞计数法检测hUC-MSCs条件培养基(CM)、重组人白细胞介素6(rhIL-6)及IL-6中和抗体对Saos-2细胞增殖的作用;ELISA检测hUC-MSCs分泌IL-6的量;RT-PCR检测增殖相关基因增殖细胞核抗原(PCNA)、cyclin D1和survivin的转录水平;Transwell实验检测hUC-MSCs和Saos-2细胞迁移能力的变化。结果: hUC-MSCs可向Saos-2细胞迁移;hUC-MSCs-CM含有高浓度的IL-6,可达(1 835.5±134.1)ng/L;hUC-MSCs-CM和rhIL-6均能促进Saos-2细胞增殖和迁移,IL-6中和抗体能明显削弱hUC-MSCs-CM的促Saos-2细胞增殖和迁移作用;RT-PCR显示hUC-MSCs-CM和rhIL-6均能上调Saos-2细胞增殖相关基因PCNA、cyclin D1和survivin的表达,而IL-6中和抗体则削弱了这一作用。结论: 脐带间充质干细胞能向骨肉瘤Saos-2细胞迁移,并通过分泌IL-6促进其增殖和迁移。     

Differences in proliferation of the hematopoietic cell line TF-1 and cytokine production by peripheral blood leukocytes induced by 2 naturally occurring forms of human IL-3

BACKGROUND: A naturally occurring polymorphism in the coding region of the human IL3 gene leads to a change in amino acid residue 8 from proline to serine. OBJECTIVE: We sought to determine whether the 2 different forms of IL-3 varied in function. These different forms are available as recombinant proteins (recombinant human IL-3/proline 8 [rhIL-3/P8] and recombinant human IL-3/serine 8 [rhIL-3/S8]). METHODS: The erythroleukemic cell line TF-1 was incubated with varying concentrations of rhIL-3/P8 or rhIL-3/S8 to determine the capacity of each type of IL-3 to induce proliferation. Human leukocytes were primed with rhIL-3/P8 or rhIL-3/S8 for up to 24 hours and then stimulated with anti-IgE and assessed for leukotrienes (LTs), IL-4, and TNF-alpha. RESULTS: Proliferation of TF-1 cells was induced by both forms of IL-3 at 48 and 72 hours but to a greater degree by rhIL-3/P8. In contrast, the mean fold increase over control values of LT and IL-4 production was higher after priming the cells with rhIL-3/S8 versus rhIL-3/P8. Additionally, TNF-alpha production was greater (and reached significance only) for rhIL-3/S8. This activity was independent of IgE and thus directly stimulated by IL-3. Studies with basophil-enriched and basophil-depleted cell preparations revealed that LT production was evident only from the former and TNF-alpha only from the latter. CONCLUSION: We conclude that the 2 naturally occurring forms of human IL-3 have similar spectra of activities on cells with IL-3 receptors, but the 2 forms have reversed relative efficacies for promoting proliferation (rhIL-3/P8 > rhIL-3/S8) compared with priming or inducing mediator secretion (rhIL-3/S8 > rhIL-3/P8).