造血干细胞(CD34+)表达谱数据分析

造血干细胞分化的潜能及自我更新的分子机制是造血干细胞研究中最重要的领域。通过对来自造血干细胞（CD34＾＋）的EST（Expressed　Sequence　Tags）和SAGE（Serial　Analysis　of　Gene　Expression）数据进行系统生物信息学分析,发现了造血干细胞除表达大量下游分化细胞的分子标志外,还表达一些非造血组织的特异基因。在分子调控方面,反义RNA可能是一种非常重要的调控手段。     

造血干细胞(CD34+)表达谱数据分析

造血干细胞分化的潜能及自我更新的分子机制是造血干细胞研究中最重要的领域.通过对来自造血干细胞(CD34+)的EST(Expressed Sequence Tags)和SAGE(Serial Analysis of GeneExpression)数据进行系统生物信息学分析,发现了造血干细胞除表达大量下游分化细胞的分子标志外,还表达一些非造血组织的特异基因.在分子调控方面,反义RNA可能是一种非常重要的调控手段.     

高血压病气虚血瘀证患者血清对CRL-1730分泌凋亡相关蛋白的影响

探讨高血压病气虚血瘀证患者血清诱导人脐静脉内皮细胞株CRL-1730凋亡的机制,为高血压病气虚血瘀证的实质研究提供实验依据。以高血压病气虚血瘀证患者血清作用于体外培养的人脐静脉内皮细胞株,用酶联免疫法(ELISA)检测细胞培养上清液凋亡相关蛋白Caspase-3、P53、Fas、Fas L、Bax、Bcl-2含量,并观察补阳还五汤含药血清的干预作用。与健康组相比,高血压病气虚血瘀证组、高血压病非气虚血瘀证组细胞上清液中的Caspase-3、P53、Fas、Fas L、Bax表达量升高,差异有统计学意义(P0.05或P0.01)。高血压病气虚血瘀证组、高血压病非气虚血瘀证组细胞上清液中的Bcl-2表达量降低,差异有统计学意义(P0.05或P0.01)。与高血压病非气虚血瘀证组相比,高血压病气虚血瘀证组Caspase-3、P53表达量升高,差异有统计学意义(P0.01)。另外,与高血压病气虚血瘀证组相比,补阳还五汤高、中、低剂量组细胞上清液中的Caspase-3、P53、Fas、Fas L、Bax表达量降低,差异有统计学意义(P0.05或P0.01),Bcl-2表达量升高,差异有统计学意义(P0.05)。高血压病气虚血瘀证(QDBS)患者血清诱导内皮细胞凋亡的机制可能与调节Caspase-3、P53、Fas、Fas L、Bax、Bcl-2的表达量有关,补阳还五汤(BYHWD)对内皮细胞凋亡有一定的保护作用。     

大熊猫白血病抑制因子RNAi干扰载体的构建及应用

旨在构建大熊猫白血病抑制因子(LIF)RNAi重组质粒,以期进一步研究LIF对大熊猫骨髓间充质干细胞(PDBM-MSCs)的调控作用.试验设计3组大熊猫LIF shRNA慢病毒pLKO.1 puro-LIF-down干扰载体,通过慢病毒的方式感染PDBM-MSCs,经q-PCR鉴定重组质粒pLKO.1 puro-LIF-down-1抑制效果最显著.pLKO.1 puro-LIF-down-1实验组和空质粒对照组比较发现,LIF mRNA表达水平抑制后能够显著下调细胞周期调控基因CDK2、CCNB2、CCND3的mRNA表达水平(P<0.05);显著上调凋亡基因P53、P16、P27、caspase3的mRNA表达水平(P<0.05);显著下调干性基因KLF4、SOX2的mRNA表达水平(P<0.05).以上结果表明,此次研究成功构建出大熊猫LIF RNAi干扰载体,LIF对PDBM-MSCs的生长和干性维持具有重要的调控作用,为大熊猫干细胞的体外培养及干性机制的研究奠定基础.     

积雪草苷调控miR-342-5p/AQP3轴保护IL-1β诱导的软骨细胞损伤

目的 探讨积雪草昔(Ass)对白细胞介素1B(IL-1B)诱导的软骨细胞损伤的影响及作用机制。方法 不同 浓度的Ass作用IL-1B诱导软骨细胞24 h,流式细胞仪检测细胞凋亡,酶联免疫吸附法检测细胞培养上清液中白细 胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平,实时荧光定量PCR检测细胞中miR-342-5p和水通道蛋白3 (AQP3)mRNA表达水平,Western Blot法检测B淋巴细胞瘤-2 (Bcl-2)、B淋巴细胞瘤-2相关蛋白(Bax)和AQP3蛋 白表达水平。双荧光素酶报告基因实验验证miR-342-5p与AQP3靶向关系。转染miR-342-5p抑制剂或AQP3过表达载体至软骨细胞,上述相同方法检测抑制miR-342-5p表达或AQP3过表达对IL-1β诱导的软骨细胞凋亡及IL- 6和TNF-α表达的影响。结果 Ass可抑制IL-1β诱导的软骨细胞凋亡率、Bax蛋白、IL-6和TNF-α表达及miR- 342-5p表达(P <0. 05),促进Bcl-2蛋白、AQP3 mRNA和蛋白表达(P <0.05)。miR-342-5p在软骨细胞中负调控 AQP3表达。抑制miR-342-5p或AQP3过表达后,IL-1&诱导的软骨细胞凋亡率、Bax蛋白、IL-6和TNF-α表达降低 (P <0. 05) ,Bcl-2蛋白表达升高(P <0.05)。抑制AQP3表达逆转了抑制miR-342-5p对IL-1β诱导的软骨细胞凋 亡、Bax和Bcl-2蛋白及IL-6和TNF-α表达的影响。结论 Ass可抑制IL-1β诱导软骨细胞凋亡和炎症反应,其可 能通过调控miR-342-5p/AQP3保护软骨细胞损伤。     

miR-34a靶向MMP-13调控大鼠软骨细胞凋亡的机制

目的:探讨调控miR-34a的表达对大鼠软骨细胞凋亡的影响.方法:大鼠关节软骨原代细胞培养,慢病毒转染miR-34a、miR-34a inhibitor分别使大鼠关节软骨细胞miR-34a过表达和抑制表达,另设无关序列对照组,Annexin V/PI双染流式细胞术分析对比各组大鼠软骨细胞凋亡率,通过生物信息学方法预测miR-34a调控软骨细胞凋亡潜在基因靶点,采用实时荧光定量逆转录聚合酶链反应(real time-PCR)检测各组软骨细胞miR-34a的表达,检测软骨凋亡相关因子MMP-13、Col2a1的mRNA转录水平;免疫印迹法检测各组的MMP-13、Col2a1、caspase-3的蛋白表达水平.结果:miR-34a过表达后大鼠软骨细胞凋亡增多,抑制miR-34a表达后大鼠软骨细胞凋亡减少,同时miR-34a过表达后caspase-3的表达增高(P0.05),miR-34a过表达后Col2a1在mRNA和蛋白水平表达下降,而MMP-13的表达增加(P0.05).沉默miR-34a的表达后Col2a1在mRNA和蛋白水平表达增加,而MMP-13的表达下降.MMP-13是miR-34a调控大鼠软骨细胞凋亡的潜在靶点.结论:miR-34a的表达调控大鼠软骨细胞凋亡,MMP-13是miR-34a其机制中的潜在靶点.     

过表达IL-8受体对脐带源间充质干细胞生物活性的影响

目的探究腺病毒介导的IL-8受体过表达对脐带源间充质干细胞生物活性的影响。方法本研究利用含有IL-8RA和IL-8RB的c DNA和绿色荧光蛋白(GFP)基因的重组腺病毒载体(p Ad-IL-8RA-GFP、p Ad-IL-8RB-GFP和p Ad-Null-GFP)分别转染分离培养的P3-5代脐带源间充质干细胞,倒置荧光显微镜观察GFP的表达,流式细胞仪检测干细胞标签蛋白CD29、CD44、CD34、HLA-DR的表达,CCK-8实验检测转染后细胞的增殖能力,粘附实验及Transwell趋化实验观察过表达IL-8RA/B的脐带源间充质干细胞的迁移能力。结果脐带源间充质干细胞形态均一、生长状态良好;干细胞特异性相关蛋白CD29、CD44阳性表达,CD34、HLA-DR阴性表达;与对照组比较,IL-8RA/B过表达的脐带源间充质干细胞的增殖能力无明显改变,但是过表达IL-8RA/B的间充质干细胞粘附在损伤内皮上以及迁移到小室下方的细胞数目更多。结论以上结果表明IL-8RA/B的过表达不影响脐带源间充质干细胞的增殖能力,但是能够促进脐带源间充质干细胞向IL-8趋化和向损伤内皮细胞粘附。     

海马生髓丸对S180荷瘤鼠、SKOV3卵巢癌模型鼠抑瘤机理的实验研究

以S180荷瘤鼠、SKOV3卵巢癌模型鼠为研究对象,进行海马生髓丸抗肿瘤的实验研究.通过对移破植瘤的抑瘤率、光、电镜技术及细胞凋亡调控基因P53、BcL-2等免疫组化的研究,表明海马生髓丸具有一定的抗肿瘤作用,可以抑制S180荷瘤鼠的肿瘤生长,明显增强SKOV3卵巢癌模型鼠DDP的杀伤肿瘤作用,对肿瘤细胞的凋亡具有明显促进作用.     

CD11b调控LPS刺激下巨噬细胞表达IL-12的分子机制

CD11b是白细胞整合素家族成员之一,在炎症的发生和发展中发挥重要作用.在内毒素血症小鼠模型,发现脂多糖(LPS)导致外周血白细胞介素12(IL-12)水平降低,而CD11b抑制剂能防止此现象发生.为阐明CD11b调控LPS刺激下IL-12产生的分子机制,本文利用RAW264.7细胞开展了进一步研究.酶联免疫吸附、实时定量PCR和Western Blot等检测结果表明,CD11b可抑制LPS刺激下RAW264.7细胞IL-12的表达;利用sh RNA敲低CD11b的表达则能提高LPS刺激下RAW264.7细胞IL-12p35、IL-12p40的转录水平和蛋白水平的表达;JNK和NF-κB信号通路与CD11b调控IL-12的产生关系密切,而p38、ERK信号通路影响较小.因此,研究结果提示,CD11b通过JNK和NF-κB信号通路抑制LPS刺激下巨噬细胞IL-12的产生,抑制CD11b的功能可能有助于削弱LPS引起的炎症反应.     

髓母细胞瘤中P53、bcl-2及Ki-67的表达及其意义

用免疫组化S P法检测 2 5例髓母细胞瘤中Ki 6 7、P53 及bcl 2基因的表达。结果表明 ,在 2 5例髓母细胞瘤中 ,bcl 2和P53 蛋白的阳性表达率分别为 72 % (18/ 2 5 )和 16 % (4/ 2 5 )。Ki 6 7阳性率平均为 (33.34± 4 .98) % ,其表达与组织学分型有关。说明在髓母细胞瘤中bcl 2蛋白与P53 蛋白可能参与调控细胞的凋亡     

藏猪白细胞介素-12基因表达对猪圆环病毒2 疫苗的免疫增强效应

为研究IL-12基因在猪体内表达对PCV2疫苗免疫应答的调控作用,本研究将藏猪IL-12基因克隆至VR1020载体,壳聚糖包裹重组质粒制备成壳聚糖纳米颗粒(VRIL-12-CNP)并与PCV2疫苗共同接种21日龄断奶仔猪.接种后第0d、7d、14d和28d采集猪前腔静脉血进行血细胞分析、PCV2抗体检测和相关免疫基因表达量检测,并记录体重.结果显示:接种VRIL-12-CNP和PCV2疫苗的实验组猪的CD3~+、CD4~+、CD8~+T细胞和PCV2抗体显著增长(P0.05),TLR2/7、IL-12/4/6/15、STAT1/3和Bcl-2基因的表达量显著高于对照组(P0.05);试验期间实验组体重净增长也明显高于对照组(P0.05).结果表明:VRIL12-CNP能增强PCV2疫苗的先天性和获得性体液、细胞免疫应答,是安全、有效的PCV2疫苗佐剂.     

胡桃楸树皮抗肿瘤有效成分对H22荷瘤小鼠T细胞亚群的影响

 为测定胡桃楸树皮提取物(HT)抗肿瘤有效成分对H₂₂荷瘤小鼠T细胞亚群的影响,将昆明小鼠随机分为HT高剂量组、HT低剂量组、阳性药环磷酰胺组、模型组和正常组,采用接种法复制H₂₂肿瘤移植模型。利用荧光标记抗体-流式细胞术检测HT对建立的H₂₂荷瘤小鼠模型外周血中CD4⁺/CD8⁺细胞亚群比值变化; ELISA法检测CD4⁺细胞所分泌细胞因子IL-4和IFN-γ的含量,以测定Th1/Th2细胞亚群比例。结果显示,与阳性药注射用环磷酰胺(CTX)相比,HT高、低剂量组能显著降低机体CD8⁺亚群比例(P<0.01),HT低剂量组可明显提高CD4⁺/CD8⁺比值(P<0.05)。HT高、低剂量组的IL-4含量显著低于阳性药CTX组(P<0.001),明显低于模型组(P<0.05)。HT高剂量组的IFN-γ/IL-4水平明显高于阳性药CTX组(P<0.05)。研究表明,胡桃楸树皮提取物具有明显的抑瘤作用,能保护胸腺和脾脏,能减轻荷瘤机体的T细胞免疫功能的损伤。     

Formation of haematopoietic microenvironment and haematopoietic stem cells from single human bone marrow stem cells.

Haematopoietic stem cells are a population of cells capable both of self renewal and of differentiation into a variety of haematopoietic lineages. Enrichment techniques of human haematopoietic stem cells have used the expression of CD34, present on bone marrow progenitor cells. But most CD34+ bone marrow cells are committed to their lineage, and more recent efforts have focused on the precise characterization of the pluripotent subset of CD34+ cells. Here we report the characterization of two distinct subsets of pluripotent stem cells from human fetal bone marrow, a CD34+, HLA-DR+, CD38- subset that can differentiate into all haematopoietic lineages, and a distinct more primitive subset, that is CD34+, HLA-DR-, CD38-, that can differentiate into haematopoietic precursors and stromal cells capable of supporting the differentiation of these precursors. These data represent, to our knowledge, the first identification of a single cell capable of reconstituting the haematopoietic cells and their associated bone marrow microenvironment.     

谷氨酰胺对大强度跑台训练大鼠免疫功能影响的实验研究

目的:探讨补充谷氨酰胺对大强度跑台训练大鼠免疫功能的影响。方法:适应性训练后,30只6周龄健康雄性SD大鼠,随机分为安静组(C,6只),训练组(T,12只)和服药训练组(TG,12只)。进行6周大强度跑台训练,跑台坡度5°,前2周每周训练6d,每次训练25min,速度依次为20 m/min和24 m/min,后4周每天训练,速度和时间不变,分别为32 m/min和35min。训练期间每日给药前称体重,TG组大鼠按O.3g/kg.d剂量灌胃给药,C、T组喂以相同体重比例的自来水。最后一次训练12h后进行大鼠脾脏T淋巴细胞亚群CD4+、CD8+和脾细胞IL-2表达测定。结果:T组与C组比较,CD8+含量显著升高,CD4+/CD8+比值及IL-2表达显著下降;TG组与C组比较,CD8+含量显著下降,CD4+/CD8+及IL-2表达升高显著。结论:本该实验的大强度跑台训练造成大鼠免疫功能显著下降,而谷氨酰胺补充可明显改善大强度跑台训练造造成的大鼠免疫抑制,增强大鼠免疫功能。     

Induction of embryonic stem cells to hematopoietic cells in vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright's staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34+ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34-CD38+. Wright's staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

正常人和AITD患者对碘致甲状腺细胞凋亡的不同敏感性

目的:探讨碘对体外培养的Grave s病(GD)、桥本氏甲状腺炎(HT)、正常人甲状腺滤泡上皮细胞(TEC)凋亡及相关蛋白表达的影响并明确正常人和自身免疫性甲状腺疾病(AITD)患者甲状腺细胞对碘的敏感性.方法:分离培养正常、GD和HT甲状腺细胞,给予不同浓度碘化钠(NaI)干预,流式细胞仪检测细胞凋亡率,免疫组化检测各组Fas/Fasl表达的水平;比较各组细胞对碘致凋亡和凋亡蛋白表达的敏感性.结果:NaI可以使正常、GD、HT患者甲状腺细胞凋亡率增加(P<0.05),并使相关的凋亡蛋白及基因Fas/Fasl表达增加,但HT和GD甲状腺细胞凋亡和Fas/Fasl表达率较正常甲状腺细胞增加更明显,以HT患者甲状腺细胞加高碘培养后凋亡更多(P<0.05).结论:与正常人相比,AITD患者对高碘导致的甲状腺细胞凋亡更敏感.     

白癜风患者维医证候学特征及其临床流行病学调查与免疫功能变化的相关性

 通过观察新疆白癜风患者的维医证候特征及其临床流行病学特征,探讨白癜风患者各体液证型与免疫功能变化的关系。采用流式细胞技术、酶联免疫吸附试验(ELISA法)检测全血中CD3⁺、CD4⁺、CD8⁺、CD25⁺、白介素-2(IL-2)、白介素-4(IL-4)、白介素-6(IL-6)、白介素-8(IL-8)、细胞间附分子(ICAM-1)、γ干扰素(IFN-γ)、肿瘤坏死因子(TNF-а)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和干细胞因子(SCF)等指标,并进行分析。结果显示,183例白癜风患者中女性106例(57.92%),男性77例(42.08%),3~15岁之间的患者比例较高(36.61%),白癜风患者在不同异常体液中的构成比不同,黏液质型白癜风组107例(占58.47%)和非黏液质型白癜风组76例(占41.53%),其构成比大小顺序为:异常黏液质型白癜风>异常黑胆质型白癜风>异常胆液质型白癜风>异常血液质型白癜风。与正常对照组相比,CD8⁺、IL-2、IL-6、IL-8、IFN-γ、TNF-а、ICAM-1在异常黏液质型白癜风组和非异常黏液质型白癜风组均升高(P<0.05);CD3⁺、CD25⁺、IL-4在异常黏液质型白癜风组和非异常黏液质型白癜风组均降低 (P<0.05)。与非异常黏液质型白癜风组相比,CD8⁺、IL-6、IL-8在异常黏液质型白癜风患者升高明显(P<0.05);CD3⁺、CD25⁺、IL-4在异常黏液质型白癜风患者降低明显(P<0.05),CD4⁺、CD4⁺/D8⁺,在各组间比较无差异(P>0.05)。由此推断,异常黏液质型白癜风是白癜风维医主要证型,且白癜风患者存在自身免疫调节的异常,异常黏液质型白癜风患者的免疫功能紊乱程度较非异常黏液质型白癜风患者更为明显。     

Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis

Antigen receptor stimulation of mature alpha beta T lymphocytes can lead either to proliferation or death. Programmed cell death, termed apoptosis, leads to the clonal deletion of both thymocytes and mature T cells that establishes tolerance. How a mature T cell selects between proliferation and death is not understood. Here I show that interleukin-2 (IL-2) is a critical determinant of the choice between these two fates. Both CD4+ and CD8+ T cells previously exposed to IL-2 undergo apoptosis after antigen-receptor stimulation. Antibody blockade of IL-2 but not IL-4 reverses the marked reduction of lymph node V beta 8+ T cells caused in mice by the bacterial superantigen Staphylococcus aureus enterotoxin B. IL-2 may thus participate in a feedback regulatory mechanism by predisposing mature T lymphocytes to apoptosis.     

miR-143-3p通过调节TGIF2的表达在甲状腺癌中的作用研究

目的 探究过表达miR-143-3p对甲状腺癌(thyroid cancer,TC)细胞的影响及其作用机制.方法 Real-time PCR分析正常甲状腺上皮细胞和不同TC细胞中miR-143-3p的表达.Targetscan网站和双荧光素酶报告系统验证miR-143-3p和TGIF23′UTR的靶向关系;Real-t...