抗Fas锤头状核酶阻抑小鼠急性粒-单核白血病细胞免疫逃逸的研究

为了研究抗Fas锤头状核酶对T细胞Fas表达及其凋亡的影响和探讨增强供者淋巴细胞输注时移植物抗白血病（GVL）效应的新策略，构建可有效切割Fas mRNA的锤头状核酶真核质粒，用电穿孔法将其导入小鼠CTL细胞株CTLL-2之后，借助RT—PCR和Western blot检测其Fas的表达，同时检测转染前后其胱冬酶-3（Caspase-3）活性和凋亡（Annexin V—FITC法）的改变，并用MTT法检测空白对照组、空载体转染组及pU6-RZ596转染组CTLL-2细胞的增殖情况和体外杀伤小鼠急性粒-单核白血病细胞（WEHI-3）的活性。结果表明：构建的U6嵌合型锤头状核酶RZ596在细胞内能有效切割Fas，明显降低小鼠活化CTLL-2的Fas水平，与高表达Fas配体的WEHI-3孵育后，其存活率和体外杀伤WEHI-3活性明显高于对照组。结论：抗Fas核酶能显著降低小鼠活化CTLL-2的Fas表达，使其免于WEHI-3的膜Fas配体经Fas途径所致的凋亡，并提高CTL对小鼠急性粒-单核白血病细胞的杀伤力，从而阻抑小鼠急性粒-单核白血病细胞的免疫逃逸。     

腺病毒介导的hVEGF165基因治疗对骨髓移植后骨髓造血干细胞恢复的影响及其意义

目的：探讨重组腺病毒Ad—EGFP／hVEGF165介导的血管内皮细胞生长因子在骨髓移植后小鼠体内的表达效率及对骨髓造血干细胞恢复的影响。方法：利用细菌内同源重组法快速构建复制缺陷型腺病毒Ad—EGFP／hVEGF165，经尾静脉注射给同基因骨髓移植BALB／c小鼠；在移植后不同时间，通过荧光显微镜、RT—PCR、免疫组化染色和ELISA方法检测外源基因在小鼠体内的表达；取移植后30d小鼠骨髓单个核细胞并计数，随后进行脾集落形成单位试验(CFU—S)以评价VEGF基因转移组小鼠骨髓造血干细胞数量恢复情况。结果：重组腺病毒AdEGFP／hVEGF165介导的VEGF基因转移广泛分布在骨髓移植小鼠心、肺、肝、脾、肾、小肠和骨髓组织；小鼠血浆中VEGF浓度高峰出现在移植后2d；接受重组腺病毒AdEGFP／hVEGF165基因干预治疗组小鼠骨髓单个核细胞计数及其形成的脾集落数量明显高于其他对照组。结论：腺病毒成功介导了hVEGF165基因在骨髓移植小鼠体内的稳定表达，明显促进骨髓移植小鼠骨髓造血干细胞数量的恢复．是骨髓单个核细胞数量增加的根本原因。     

右旋糖酐酶对牙菌斑作用的研究

牙菌斑是龋齿和牙周病重要的致病因素之一。因此为了预防和控制龋齿和牙周病,抑制牙菌斑的发生和发展是至关重要的。利用右旋糖酐酶抑制牙菌斑,近年来受到各国口腔医学研究的重视。国处已有这方面的一些报道。本研究利用中国科学院微生物研究所提供的右旋     

抑制mll-af9融合基因表达对急性单核细胞白血病细胞THP-1增殖的影响

本研究探讨siRNA(small interfering RNA)对人急性单核细胞白血病细胞系THP-1的mll-af9融合基因的影响.选择THP-1细胞特有的m11-af9融合基因为靶基因,设计并合成siRNA片段.以人急性单核细胞白血病细胞系THP-1为靶细胞,应用脂质体转染方法,将siRNA导入细胞,通过流式细胞仪检测siRNA转染效率,用RTPCR法比较转染前后mll-af9 mRNA表达水平的变化,以MTT法检测细胞增生抑制率,流式细胞术检测细胞周期的改变和细胞凋亡水平.结果表明siRNA转染效率为(69.1±1.8)%,转染siRNA后THP-1细胞生长受到明显抑制,m11-af9融合基因表达量大幅度减少,细胞周期发生了明显的变化,主要表现为G0/G1期细胞增多,S期细胞减少,并使细胞周期阻滞于G0/G1期,细胞凋亡水平增高,而对照组siRNA转染后则无上述改变.结论利用siRNA靶向干扰mll-af9融合基因的表达可以有效抑制人急性单核细胞白血病细胞THP-1的增殖.     

通过Fas-FasL途径体外清除小鼠同种异体反应性T细胞

目的　通过Fas Fas配体 (FasL)途径清除小鼠同种异体反应性T细胞 (ARTC) ,为减轻异基因骨髓移植 (allo BMT)后的移植物抗宿主病探索新的手段。方法　用磁性细胞分离系统分离BALB c小鼠 (H 2 d)Sca 1+早期造血细胞 (HC) ,然后借助逆转录病毒基因转移技术对其转染外源小鼠FasL(mFasL)cDNA基因 ;扩增 1周后与异基因BAC小鼠 (H 2 d ×b)脾细胞进行单向混合淋巴细胞培养(OWMLC) 6d ,观察处理后的BAC小鼠脾细胞对Na251 CrO4标记的BALB c小鼠脾细胞的杀伤作用。结果　成功分离出BALB c小鼠Sca 1+HC ,纯度为 (89.0± 6 .1) %。BAC小鼠脾细胞与转染外源mFasL的BALB cHC以 1∶5比例共培养 6d后 ,对BALB c源脾细胞杀伤率在不同效、靶比例时均呈显著降低 (P<0 .0 1)。结论　体外反应中 ,转染外源mFasLcDNA并高表达的早期HC可清除针对自身MHC抗原的ARTC。     

Bay 11-7082增强Fas介导的HL-60细胞凋亡作用

本研究观察NF-κB抑制剂Bay 11-7082对HL-60细胞Fas/FasL系统的作用及对Fas介导的HL-60细胞凋亡的影响。用RT-PCR和流式细胞术检测Bay 11-7082干预后HL-60细胞Fas、FasL和XIAP的mRNA和蛋白表达水平的变化;用ELISA方法检测Bay 11-7082干预前后sFasL水平的变化;流式细胞术检测Bay 11-7082干预前后CH-11(活性Fas抗体)诱导HL-60细胞凋亡的变化。结果表明:用Bay 11-7082干预后,HL-60细胞FasL和XIAP的mRNA和蛋白水平较对照组明显下降,差异具有显著性(p<0.05),Fas的mRNA、蛋白水平的变化和sFasL水平的变化无显著的差异(p>0.05);Bay 11-7082干预后HL-60细胞对CH-11诱导细胞凋亡敏感性显著增强。结论:Bay 11-7082可能通过下调FasL和XIAP水平增强HL-60细胞Fas介导的凋亡。     

FasL基因转移诱导同种异体反应性T细胞凋亡的实验研究

目的　研究FasL基因转染鼠骨髓单个核细胞 (BMMNC)后 ,诱导同种异体反应性T淋巴细胞凋亡的效率。方法　采用脂质体法将mFasL cDNA转入Balb/C小鼠造血细胞 ,Western Blot检测其是否表达 ,然后将转染mFasL cDNA的BMMNC与BAC鼠 (Balb/C×C5 7BL/6 ,H 2d×b ,F1)脾单核细胞 (SMNC)混合培养 ,通过混合淋巴细胞反应 (MLR)及流式细胞仪 (FCM)检测SMNC的凋亡效率。结果　基因转移后Western Blot分析Balb/C鼠BMNC显示明显抗FasL抗体识别的条带 (约 4 0 0 0 0 ) ,未转基因的BMMNC无阳性条带发现 ;转基因组进行MLR其每分钟脉冲数 (CPM)值为 5 6 2 8± 186 ,明显低于对照组 (96 74± 78) (t=4 4 86 ,P <0 0 0 1) ;FCM检测转基因组诱导SMNC凋亡率为 (14 8± 0 4 ) % ,明显高于对照组 [(1 8± 0 4 ) % ](t=3 0 1,P <0 0 1)。结论　采用脂质体法能将mFasL cDNA转入鼠BMMNC ,并能有效表达 ,转基因后的BMMNC有诱导同种异基因反应性T淋巴细胞凋亡的作用     

Lower Phosphorylation of p38 MAPK Blocks the Oxidative Stress-induced Senescence in Myeloid Leukemic CD34~+CD38~- Cells

Leukemia seems to depend on a small population of "leukemia stem cells (LSCs)" for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34+CD38- cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34+CD38- cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluo-rescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34+CD38- cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34+CD38- cells as compared with HSCs and H2O2-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34+CD38- cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.     

Selective Depletion of the Allo-Antigen Specific T Cells by Fas/FasL Pathway by Cytokine IFN-γ and IL-2

Severegraft versus hostdisease (GVHD)isthemajorcauseoffailureofallogeneichematopoieticstemcelltransplantation (allo HSCT) .Thedonormatu rateTlymphocytesareactivatedwhentheyrecognizeallo histocompatibilityantigenofrecipients ,anddi rectlyswitchongraft versus hostreactionandcausetargettissueinjury[1] .DepletionofTcellsingraftorinhibitionoftheirfunctioncoulddeceleratetheoccur renceanddevelopmentofGVHD[2 ] .However ,Tcellsareabletoresistinfection ,killtumorcellsandsecretecytokinessuchasIL 3…     

Cloning and Expression of the cDNA of a Murine Soluble Fas

Fas/Apo- 1 ( CD95 ) ,a cell surface moleculeexpressed in a variety of cells,can induce cellapop-tosis and plays some important role.The Fas re-ceptor has two isoforms:membrane form and solu-ble form.The membrane form,mainly expressedin activated lymphocytes,hepatocytes,endothelialcells and virus infected cells etc.,plays an impor-tant role in the regulation of the homeostasis of theimmune system and cells.Butnormally the solubleform Fas is excreted into the plasma and broughtinto full play i…