垂体生长激素的调节激素对人B淋巴母细胞系IM-9中生长激素基因启动子活性的影响

目的：通过观察生长激素释放激素（GHRH）、山德定、生长激素（GH）和胰岛素样生长因子I（IGF－I）对IM－9细胞中GH基因启动子活性的影响，以了解这些激素对B淋巴细胞中GH表达的调节作用。方法：把人外周血淋巴细胞GH基因上游－484～ 2bp调控序列克隆到荧光素酶报告基因质粒中，然后把克隆得到的质粒转染到IM－9细胞内，通过测量荧光素酶泊活性观察垂体GH的各种调节激素对GH基因启动子活性的影响。结果：0．001nmol/L的人GHRH能够显著抑制荧光素酶在IM－9细胞中的表达（P＜0．002），但0．1nmol/L和10nmol/L的GHRH对荧光素酶的抑制作用反而减弱。人IGF－I（1、10和100nmol/L可增强荧光素酶的表达（均P＜0．05）。GH（1．0ng/ml）可显著增加荧光素酶的表达，但作用的强度不大。不同浓度的生长抑素类似物山德定对IM－9细胞中荧光素酶的表达均无显著影响。结论：GHRH、IGF－I和GH对IM－9细胞中GH启动子的活性有一定的调节作用,说明调节垂体GH分泌的GHRH、IGF－I和GH等能够调节G淋巴细胞内irGH的表达。     

激素对T淋巴细胞生长激素基因表达调控的影响

目的 :探讨各种激素对T淋巴细胞GH基因表达的影响。方法 :构建含人GH调控序列的荧光素酶报告基因质粒PGL2 GH luc,然后转染入T淋巴细胞系———JurkatE6 1细胞中 ,在培养液中分别加入各种激素。结果 :各浓度的GH、GHRH对Jurkat细胞中荧光素酶的表达有抑制作用 (P <0 0 1) ,而高剂量的TRH( 10、10 0nmol/L)和SS( 10 0nmol/L)对Jurkat细胞中荧光素酶的表达也有抑制作用 ( P<0 0 5 )。结论 :T淋巴细胞中GH基因的表达受下丘脑激素和GH的调节。     

激素对T淋巴细胞生长激素基因表达调控的影响1

目的探讨各种激素对T淋巴细胞GH基因表达的影响.方法构建含人GH调控序列的荧光素酶报告基因质粒PGL2-GH-luc,然后转染入T淋巴细胞系--JurkatE6-1细胞中,在培养液中分别加入各种激素.结果各浓度的GH、GHRH对Jurkat细胞中荧光素酶的表达有抑制作用(P＜0.01),而高剂量的TRH(10、100nmol/L)和SS(100nmol/L)对Jurkat细胞中荧光素酶的表达也有抑制作用(P＜0.05).结论T淋巴细胞中GH基因的表达受下丘脑激素和GH的调节.     

催乳素对小鼠脾脏CD11c+树突状细胞合成细胞因子的调控

本研究采用逆转录-多聚酶链反应(RT-PCR)方法,在mRNA水平上了解不同浓度的催乳素(PRL)对小鼠脾脏树突状细胞CD11c+(spleen CD11c-positive dendritic cells,SDC)合成细胞因子的影响。结果表明,低(0.01 nmol/L)、中浓度(0.1nmol/L)的PRL可以上调IL-6、IL-10、IL-12和TNF-α的水平而高浓度(1 nmol/L)则降低它们的表达(IL-12除外)。这提示PRL可能通过改变抗原提呈细胞SDC细胞因子的合成,进而参与调节机体的生理或病理性免疫反应。     

miR-124靶向镁转运蛋白1调控活化后人T细胞功能耗竭

目的研究miR-124靶向镁转运蛋白1(MagT1)可否调控活化后T细胞功能耗竭。方法 1)分离外周血单个核细胞,用IFN-γ、IL-2、抗-CD3抗体、抗-CD28抗体和IL-1α活化T细胞;流式细胞仪检测T细胞活化标记分子CD25和CD69所占的比例。2)构建miR-124/miR-124 sponge慢病毒载体;包装慢病毒后感染活化T细胞,用RT-qPCR检测感染后T细胞内miR-124和MagT1基因表达。3)生物信息学分析miR-124与MagT1的靶向位点,并用双荧光素酶报告基因系统鉴定。4)Western blot法检测慢病毒感染前后T细胞MagT1和PD-1蛋白水平。5)CCK8法检测慢病毒感染前后T细胞增殖能力;ELISA法检测细胞T分泌细胞因子TNF-α和TGF-β的能力。结果流式细胞仪检测表明T细胞活化成功。RT-qPCR检测表明慢病毒构建成功,miR-124/miR-124 sponge载体分别显著上调或下调miR-124的表达(P0.01)。双荧光素酶报告基因系统证实miR-124靶向MagT1 3′UTR并抑制其表达。Western blot表明miR-124过表达组MagT1蛋白水平低于对照组(P0.05),PD-1蛋白水平高于对照组(P0.05),抑制miR-124后,MagT1蛋白水平高于对照组(P0.05),PD-1蛋白水平低于对照组(P0.05)。miR-124过表达使T细胞增殖能力和分泌TNF-α能力显著降低,而抑制miR-124使T细胞增殖能力和分泌TGF-β能力显著升高(P0.01)。结论 miR-124可以负向调节靶基因MagT1的表达,并对活化后T细胞功能耗竭具有重要的调节作用。     

干扰3T3-L1脂肪细胞生长激素受体对生长激素诱导的细胞炎症因子表达和分泌的影响

目的:探讨干扰3T3-L1脂肪细胞生长激素受体(GHR)对生长激素(GH)诱导的脂肪细胞核因子κB(NF-κB)激活及炎症细胞因子mRNA表达和分泌的影响。方法:采用RNA干扰技术抑制3T3-L1脂肪细胞中GHR的表达;Western blot检测GHR的蛋白表达;双萤光素酶报告基因系统分析GHR对GH激活的3T3-L1脂肪细胞NF-κB转录活性的影响;real-time RT-PCR和ELISA技术检测GHR对GH诱导的3T3-L1脂肪细胞炎症因子mRNA表达和分泌的影响。结果:干扰3T3-L1脂肪细胞GHR的表达能够显著抑制生长激素诱导的细胞NF-κB的激活,并减少TNF-α、IL-1β和IL-6等炎症细胞因子的mRNA表达和分泌。结论:干扰3T3-L1脂肪细胞GHR可抑制GH诱导的炎症细胞因子表达和分泌。     

心肌细胞内人脑利钠肽启动子的IL-1β调节涉及Ras-、Rac和P38激酶依赖通路

脑利钠肽(BNP)基因和细胞因子白介素-1β(IL-1β)均在梗死心肌中诱导产生,故局部产生的IL-1β可调节BNP基因。本实验验证1IL-β是否调节人BNP启动子;2在近端启动子的cis成分是否对IL-1β有反应;3分裂素激活的蛋白激活酶(MAPK)信号通路[P42/44,c-jun(JNK)和P38激酶]是否参与。hBNP启动子连接在荧光素酶报告基因或在近端启动子构建GATA和M-CAI突变,本实验将它们转移到新生大鼠心室肌细胞并使用IL-1β处理24h。结果显示:IL-1β剌激的hBNP荧光酶活性经转录抑制物放线菌D预处理而被清除。P38激酶抑制物SB205380(SB)和P38激…     

生长激素基因

生长激素基因与人绒毛膜生长催乳素基因密切相关,由5个同源性程度很高的基因共同组成基因簇,位于人第17号染色体长臂的q22—24带上。其中只有生长激素结构基因(GH—N)能在垂体中表达,表达产物90%为有活性的22kDa hGH,10%为20kDa hGH。GH—N基因的启动子是5′端上游的前289bp,它含有GHF-1因子。反式作用因子、甲状腺素和糖皮质激素的结合位点,它们控制着GH—N基因表达的组织特异性和产量。GH—N基因的缺失导致了单纯性生长激素缺乏症I_A型的发生,这类患者可用Southern印迹法和PCR方法进行检测。     

HSG通过Wnt/β-catenin通路抑制COPD大鼠气道成纤维细胞增殖

目的研究增殖抑制基因(HSG)在大鼠慢性阻塞性肺疾病(COPD)气道重建中与Wnt/β-catenin信号传导通路的关系及其调控气道重建的机制。方法将大鼠随机分为对照组及COPD模型组,模型组采用气道内注射脂多糖加烟熏方法建立。HE染色鉴定COPD组织,测定气道管壁总面积/气道基底膜周长(WAt/Pbm)评价气道重建情况。组织块贴壁法培养大鼠气道成纤维细胞,real time-PCR检测成纤维细胞HSG和β-catenin mRNA表达。Western blot联合ELISA法检测HSG、β-catenin、GSK-3β、TGF-β1和MMP-9蛋白表达。分析HSG、β-catenin基因表达与TGF-β1和MMP-9细胞因子分泌的相关性。结果模型组大鼠小气道重建显著。COPD大鼠气道成纤维细胞中HSG mRNA和蛋白表达均低于对照组。β-catenin mRNA和蛋白表达均高于对照组,P-GSK-3β、TGF-β1和MMP-9蛋白表达也高于对照组。HSG mRNA的表达与β-catenin mRNA表达及与TGF-β1和MMP-9细胞因子分泌呈负相关。结论 HSG可能通过调控Wnt/β-catenin信号通路,抑制大鼠气道成纤维细胞的增殖从而参与气道重建。     

白细胞介素-18基因多态性与其表达量关系的研究

目的:探讨IL-18基因启动子-607C/A、-137G/C位点多态性是否影响其表达量。方法:选择80例体检健康个体,确定IL-18基因启动子区-137G/C、-607C/A位点基因型,分离上述研究对象外周血单个核细胞(PeripheralBloodMonocytes,PBMC),统一浓度培养24h后,收集培养细胞和上清液。采用ELISA检测上清液中IL-18的含量,并用RT-PCR检测培养细胞中IL-18mRNA的水平。结果:IL-18基因启动子-607位点CC、CA和AA基因型个体PBMC分泌IL-18的浓度分别为(11.54±6.48)ng/ml、(10.92±5.16)ng/ml和(11.79±3.18)ng/ml,表达IL-18mRNA水平分别为0.878±0.633、0.877±0.521和0.881±0.400;IL-18基因启动子-137位点GG、GC或CC基因型个体PBMC分泌IL-18的浓度分别为(11.27±5.42)ng/ml和(11.31±4.62)ng/ml,表达IL-18mRNA水平分别为0.835±0.485和0.984±0.613。两个位点各基因型间个体PBMC分泌和表达IL-18水平比较,差异无显著性(P>0.05)。结论:IL-18基因外显子1上游启动子-607C/A、-137G/C位点基因多态性对IL-18分泌和表达量没有明显影响。     

细胞因子对HLA-B27启动子的调节作用及其在强直性脊柱炎发病机制中的意义

目的： 构建含HLA-B27启动子的HeLa稳定转染细胞株，观察7种细胞因子对HLA-B27启动子及其上游NF-κB及ISRE顺式作用元件活性的调节作用，探讨强直性脊柱炎（AS）等B27相关疾病的发生机制。方法:转染HeLa细胞，抗生素筛选单克隆构建含HLA-B27启动子的稳定转染细胞株。构建HeLa-B27稳定细胞株和HeLa-NF-κB稳定细胞株，在瞬时转染pISRE-luc的HeLa细胞中加入白细胞介素1α（IL-1α）、 白细胞介素1β（IL-1β）、 肿瘤坏死因子α（TNF-α）、干扰素α（IFN-α）、干扰素β（IFN-β）、 干扰素γ（IFN-γ）和转化生长因子β（TGF-β），观察7种细胞因子对B27启动子及其上游NF-κB和ISRE作用元件的调节作用。另外在HeLa-B27 稳定细胞株培养液中同时加入3种细胞因子单克隆抗体和相应细胞因子，观察其对启动子活性的调节作用。结果:TNF-α、IFN-α、IFN-β 和 IFN-γ 均能明显增强HeLa细胞B27启动子活性。细胞培养96 h 后，IFN-β为最强的启动子诱导剂（5.4倍，P<0.05）；细胞培养8 h 内，TNF-α、IL1-α 和 IL1-β,可诱导NF-κB的活性增加30倍左右（P<0.05），IFN-α 和IFN-β 可诱导ISRE的活性增加12倍左右（P<0.05），抗TNF-α 抗体对于I类IFN 增加的B27 启动子活性没有明显的抑制作用。结论：TNF-α和IFN 可通过结合于B27 启动子中各种转录因子结合元件调控HLA-B27启动子的转录活性，IFN-β 可能在强直性脊柱炎等B27 相关的脊柱关节病的发病机制中起着重要作用。     

Somatotropes Maintain Their Immature Cells Through Insulin-like Growth Factor I (IGF-I)

A pituitary tumor is considered to be composed of a heterogeneous population of hormone-producing endocrine cells, folliculo-stellate (FS) cells, and potential hormone-inactive progenitor cells to maintain a microenvironment such as that in angiogenesis for tumor development cooperatively. However, the system that maintains such a heterogeneous cell population has not been clarified yet. In the present study, we examined the mechanism for maintaining a heterogeneous cell population using two rat cell lines, MtT/S and MtT/E cells, which are known growth hormone (GH)-producing cells, and their progenitor cells, respectively. We found that conditioned medium of MtT/S cells could stimulate the growth of MtT/E cells. In addition, GH and insulin-like growth factor I (IGF-I) stimulated the growth of MtT/E cells. The messenger RNAs (mRNAs) of receptors for IGF-I and GH were expressed in the MtT/E cells. Moreover, IGF-I receptor inhibitor AG1024 could abolish the growth stimulatory activity in the conditioned medium of MtT/S cells. Therefore, we concluded that somatotropes (MtT/S) maintain their progenitor cells (MtT/E) through the GH–IGF-I signaling and IGF-I directly, which might be involved in the maintenance of progenitors of GH-producing cells and might contribute to pituitary tumor development.     

Analysis of prolactin and growth hormone production in the MtT/F4 transplantable pituitary tumor by the reverse hemolytic plaque assay.

The reverse hemolytic plaque assay (RHPA) was used to detect hormone secretion from normal pituitary cells and from the transplantable MtT/F4 pituitary tumor cells. Aliquots of the same cell suspensions were analyzed by immunocytochemistry (ICC). Normal pituitaries had more growth hormone (GH)-producing cells than tumors when analyzed by both the RHPA and ICC. However, the MtT/F4 tumor had significantly more prolactin (PRL)-secreting cells. Mammosomatotropic (MS) cells, which produced both PRL and GH, were identified in both normal and tumorous pituitaries with the RHPA and ICC. A combined procedure of RHPA followed by ICC staining on the same slide also revealed MS cells in both normal and tumorous pituitary cells, although the percentage of MS with this technique was less than with the other two methods. These results show that MS cells from a significant population of cells in the MtT/F4 tumor and that the RHPA and ICC can be used to study the regulation of this cell type.     

Pituitary Folliculo-Stellate-Like Cells Stimulate Somatotroic Pituitary Tumor Growth in Nude Mice

A new cell line (TtT/GF) established from a murine pituitary thyrotropic tumor having characteristics similar to those of pituitary folliculo-stellate cell (FS cell) was implanted into nude mice together with cells from a rat pituitary somatotrophic tumor cell line (MtT/S) to determine whether the former enhances pituitary tumor growth. For as long as 2-3 mo after implantation, MtT/S cells implanted either alone or together with fibroblasts formed either no tumors or only very small tumors in the nude mice. In contrast all of the nude mice that had received MtT/S cells implanted together with TtT/GF cells developed large tumors. Furthermore, the mice bearing the MtT/S and TtT/GF implants showed a significantly higher body weight and serum growth hormone level than those bearing only MtT/S cells or a combination of MtT/S cells and fibroblasts. The TtT/GF cell line itself had no tumorigenicity during the experimental period. Therefore, the TtT/GF cell line as a model of FS cells enhanced pituitary endocrine cell tumor formation. Additionally, immunocytochemistry showed that TtT/GF cells positive for glial fibrillary acidic protein (GFAP) or S-100 protein were present in the parenchymatous tissue elements or connective tissue surrounding the tumor nests. In the parenchymatous tissue, the TtT/GF cells exhibited a stellate appearance and surrounded neighboring tumor cells with their long cell processes. These results suggest that TtT/GF cells can serve as a model for pituitary FS cells, and are capable of stimulating pituitary tumor growth either by modifying the microenvironment or producing growth factors.     

携人IL-12基因腺病毒的构建及其对Hep3B细胞增殖及TGF-β表达的影响

目的:构建携带人IL-12的重组腺病毒,观察其对Hep3B增殖及TGF-β表达的影响。方法:PCR扩增IL-12基因,构建穿梭质粒pAdTrack-IL-12;穿梭质粒经Pme I线性化后转化AdEasier Cell,E.coli BJ5183内同源重组;PacⅠ酶切重组腺病毒质粒,转染AD293,包装病毒;病毒pAd-IL-12感染肝癌细胞Hep3B,以RT-PCR、Western blot检测白介素12的表达;MTT法检测肝癌细胞的增殖情况;RT-PCR检测转化生长因子-β(TGF-β)的表达。结果:成功构建携IL-12基因的重组质粒,并成功包装可高效感染Hep3B的腺病毒pAd-IL-12;RT-PCR以及Western blot结果表明,病毒pAd-IL-12感染Hep3B后可高表达白介素12,并且与对照组相比Hep3B增殖能力显著降低(P<0.05),TGF-β表达量也降低(P<0.05)。结论:可高表达IL-12的腺病毒包装成功,感染肝癌细胞Hep3B后抑制其增殖,同时也下调TGF-β的表达,为进一步研究IL-12的肿瘤治疗作用奠定了基础。     

人生长激素基因在小鼠体内的表达及其对血清中细胞因子浓度的影响

目的 :探讨体内高水平的人生长激素 (GH)对机体免疫功能的影响。方法 :构建人GH基因的真核表达质粒pcDNA3 hGH ,直接注射于小鼠的股四头肌中。注射后分别在第 5、15、2 5天分离血清 ,通过ELISA方法检测血清中GH的水平 ,同时检测血清中IL 2、IFN γ和TNF α水平的变化。结果 :所构建的质粒目的基因插入正确 ,转染细胞能分泌高水平的GH。接受质粒的小鼠血清中人GH的浓度与对照组相比较明显提高 ,但IL 2、IFN γ和TNF α的浓度没有显著变化。结论 :体内较高水平的GH对IL 2、IFN γ和TNF α的分泌没有显著影响