Functional expression of CD95/Fas Antigen and Bcl-2 on Cord blood Hematopoietic Progenitor Cells

The cell surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood (CB) were studied. The CD34 + cells freshly isolated from CB displayed low CD95 expression. The combinations of cytokines such as SCF+FL could up regulate the expression of CD95 in vitro culture and tumor necrosis factor α (TNF α) and interon γ (IFN γ) further increased the CD95 expression induced by positive cytokines. The functional status of CD95 mediated apoptosis were analyzed by incubation of CD34 +CB cells in the presence of anti CD95 monoclonal antibodies (McAbs). The effects of anti CD95 McAbs were measured by viable cell counting, flow cytometry, LTIC and CFU C assays. A decrease of viable cells, CFU C and LTIC numbers were observed in the presence of anti CD95 McAbs and TNF α or IFN γ. However, growth factor deprivation or the early acting cytokine such as SCF and FL cross linking to CD95 caused low apoptosis of CD34 + cells. The correlation of increased intracytoplasmic levels of bcl 2 and the presence of CD95 on fresh CB CD34 + cells suggested that bcl 2 might be involved in protecting against CD95 mediated apoptosis of CB CD34 + cells.

应用免疫磁珠法分离脐血CD_(34)^+造血细胞

目的：分离脐血CD细胞，以了解其造血增殖活性。方法：应用免疫磁珠法（Isolex50系统）分离脐血CD细胞，并对其实验方法进行了改进。结果：常规方法CD纯度较低，经过改进，脐血CD细胞由分离前的（1．39±0．76）％增加至（59．46±17．35）％，CD细胞富集倍数为（62．31±55．19）倍。分离后组分CFU－GM、BFU－E、CFU－Mix较分离前分别增加了（41．05±15．77）、（13．14±8.66）及（34．80±19．58）倍，而阴性组分所形成的集落显著减少。结论：免疫磁珠法可以有效地分离CD细胞，造血祖细胞集落大多来源于CD细胞。

造血细胞生长因子对脐血CD_(34)^+细胞短期体外扩增的作用

目的 探讨造血细胞生长因子 (HGF)的不同组合对脐血 CD34 + 细胞短期体外扩增的作用。 方法 用免疫磁珠法分离纯化脐血 CD34 +细胞 ,培养于无血清培养液中。加入不同的细胞因子 ,于第 4 ,7,10 ,14天检测 CD34 +细胞百分率和有核细胞 (NC)总数。 结果 和对照组相比 ,各组的 NC扩增倍数随着培养时间的延长呈不同程度的增加 ,干细胞在 FL、TPO、GM- CSF、SCF、IL - 6、G- CSF、EPO和 IL - 3等因子作用下 ,NC扩增倍数在培养第 14天达高峰 ,为32 8.96倍 ;而 CD34 +细胞扩增倍数在培养第 7天达最高峰 ,但在 FL、TPO、GM- CSF、SCF、IL- 6、G- CSF等因子作用下 ,CD34 + 细胞扩增倍数最大 ,7d后达 2 5 .2 3倍 ,而后随着培养时间的延长而逐渐下降。 结论 脐血 CD34 + 细胞在HGF作用下 ,扩增时间以 7～ 10

大肠癌患者外周血细胞CD_(44)s和CD_(44)V_5及CD_(44)V_6表达的研究

目的 :探讨大肠癌患者外周血细胞CD44s、CD44V5和CD44V6的表达与肿瘤转移和预后的关系。方法 :采用流式细胞术对 6 2例大肠癌患者外周血细胞CD44s、CD44V5和CD44V6的表达率进行检测 ,并分析他们之间的相关性。结果 :大肠癌患者外周血细胞的CD44V5+ 细胞和CD44V6+ 细胞检出率均显著高于正常对照组 ,P <0 0 1;且转移者显著高于未转移者 ,P <0 0 1;而CD44s+ 细胞检出率却显著低于正常对照组 ,P <0 0 5 ;且伴转移者显著低于未转移者 ,P <0 0 1;肿瘤患者外周血细胞CD44V5+ 和CD44V6+ 细胞表达率与CD44s+ 细胞检出率呈显著的负相关 ,r1=0 84 ,P1<0 0 5和r2 =- 0 .87,P2 <0 .0 1。结论 :CD44V5及CD44V6的表达与肿瘤转移密切相关 ,检测大肠癌患者外周血中的CD44s、CD44V及CD44V6的表达可为肿瘤的发生、发展及预后判断提供依据。

CD_4^+CD_(25)^(high)T细胞在小细胞肺癌患者外周血中的表达及意义

目的:探讨CD4+CD25h igh调节(抑制)性T细胞在小细胞肺癌患者外周血中的表达情况及其意义。方法:应用流式细胞技术检测40例初诊小细胞肺癌患者,18例CE及CAP方案交替化疗后完全缓解的小细胞肺癌患者外周血中的CD4+CD25h ighT细胞,计算它们占CD4+T细胞的比率。结果:初诊小细胞肺癌患者CD4+CD25h ighT细胞占CD4+T细胞的比率(6.69±2.32)%,高于健康对照组(4.13±1.25)%(P<0.01);经化疗后完全缓解的小细胞肺癌患者外周血CD4+CD25h ighT细胞占CD4+T细胞的比率与初诊时相比无明显变化。结论:小细胞肺癌患者外周血中CD4+CD25h igh调节T细胞占CD4+T细胞的比率增高,它们对小细胞肺癌患者具有免疫抑制作用。

脐血CD_(34)^+细胞体外扩增的实验研究

目的 探讨脐血造血细胞体外扩增及其移植的最佳时期。方法 用免疫磁珠法分离纯化人脐血CD+34细胞 ,从CD+34细胞不同体外培养系中取样检测细胞总数、CD+34细胞百分率和CFC(包括CFU -GM和BFU-E)。结果 A组 (加rhSCF、rhIL - 3、rhG -CSF、rhEpo及rhTPO)与B组 (加rhFL、rhIL - 3、rhG -CSF、rhEpo及rhTPO)对各阶段造血细胞扩增效果无显著差异 ,C组 (加rhSCF、rhFL、rhIL - 3、rhG -CSF、rhEpo及rhTPO)各项结果均显著优于A组 (P <0 0 1)和B组 (P <0 0 1) ,rhCSF与rhFL有明显的协调效应 ;培养 7d时开始增多 ,培养 14d进入高峰 ,培养 2 1d开始下降 ,培养 2 8d明显下降。结论 C组能明显扩增人脐血造血细胞 ,培养 7～ 14d造血细胞达高峰 ,为移植最佳时间。

Transplantation of microencapsulated umbilical-cord-blood-derived hepatic-like cells for treatment of hepatic failure

AIM: To investigate intraperitoneal transplantation of microencapsulated hepatic-like cells from human umbilical cord blood for treatment of hepatic failure in rats. METHODS: CD34+ cells in umbilical cord blood cells were isolated by magnetic cell sorting. In the in vitro experiment, sorted CD34+ cells were amplified and induced into hepatic-like cells by culturing with a combination of fibroblast growth factor 4 and hepatocyte growth factor. Cultures without growth factor addition served as controls. mRNA and protein levels for hepatic-like cells were analyzed by reverse transcription-polymerase chain reaction, immunohistochemistry and immunofluorescence. In the in vivo experiment, the hepatic-like cells were encapsulated and transplanted into the abdominal cavity of acute hepatic failure (AHF) rats at 48 h after D-galactosamine induction of acute hepatic failure. Transplantation with PBS and unencapsulated hepatic-like cells served as controls. The mortality rate, hepatic pathological changes and serum biochemical indexes were determined. The morphology and structure of microcapsules in the greater omentum were observed. RESULTS: Human albumin, alpha-fetoprotein and GATA-4 mRNA and albumin protein positive cells were found among cultured cells after 16 d. Albumin level in culture medium was significantly increased after culturing with growth factors in comparison with culturing without growth factor addition (P < 0.01). Compared with the unencapsulated group, the mortality rate of the encapsulated hepatic-like cell-transplanted group was significantly lower (P < 0.05). Serum biochemical parameters, alanine aminotransferase, aspartate aminotransferase and total bilirubin in the encapsulated group were significantly improvement compared with the PBS control group (P < 0.01). Pathological staining further supported these findings. At 1-2 wk post-transplantation, free microcapsules with a round clear structure and a smooth surface were observed in peritoneal lavage fluid, surviving cells inside microcapsules were found by trypan blue staining, but some fibrous tissue around microcapsules was also detected in the greater omentum of encapsulated group by hematoxylin and eosin staining. CONCLUSION: Transplantation of microencapsulated hepatic-like cells derived from umbilical cord blood cells could preliminarily alleviate the symptoms of AHF rats.

抗CD3单抗促进肿瘤细胞凋亡的研究

目的 研究抗CD 3单抗活化T细胞后促进肿瘤细胞发生凋亡的作用。方法 应用流式细胞术并结合AnnexinⅤ FITC/PI双染色法检测细胞凋亡情况并对凋亡因子Fas抗原 (CD 95 )和Apo 2 .7进行定量分析。结果 加入抗CD 3单抗后可明显诱导肿瘤细胞的凋亡产生 (AnnexinⅤ FITC/PI染色 ) (P <0 .0 5 ) ,凋亡因子Fas抗原 (CD 95 )表达也明显增高 (P <0 .0 5 ) ,而Apo 2 .7仅略增高 (P >0 .0 5 )。结论 加入抗CD 3单抗活化的T细胞可明显促进肿瘤细胞发生凋亡 ;介导凋亡发生主要通过Fas抗原 (CD 95 )

条件培养液对脐血CD_(34)^+细胞扩增效应的实验研究

目的探讨并分析不同条件培养液对脐血CD+ 34 细胞的体外扩增效应及影响因素。方法利用 4种条件培养液 (胎儿肌肉、胎盘组织、外周血、脐血单个核细胞 )对脐血CD+ 34 细胞进行 2周的体外扩增 ,并和三细胞因子组合 :干细胞因子 (stemcellfactor ,SCF)、血小板生成素 (thrombopoietin ,TPO)、flt 3配基 (flt 3ligand ,FL) ,对脐血CD+ 3 4 细胞扩增结果进行了比较 ;利用ELISA方法检测 4种条件培养液中细胞因子 [白细胞介素 1(interleukin 1,IL 1)、白细胞介素 6 (interleukin 6 ,IL 6 )、FL、TPO]的浓度。结果脐血单个核细胞条件培养液及细胞因子组合对脐血CD+ 3 4 细胞均有明显的扩增效应 ,脐血组优于细胞因子组合 ;4种条件培养液中 ,脐血组细胞因子FL的含量明显高于其余条件培养液组。结论脐血条件培养液可作为体外培养扩增脐血CD+ 34 细胞有效而廉价的扩增剂 ;细胞因子FL可能是造成不同条件培养液对脐血CD+ 34

Serum concentration of sFas and sFasL in healthy HBsAg carriers,chronic viral hepatitis B and C patients

AIM:To estimate the amount of apoptosis among healthy HBsAg carriers,patients with chronic HBV infection treated wibh lamivudine and patients with chronic HCV infection treated with interferon alpha and ribavirin.Activity of apoptosis was evaluated by serum sFas/sFasL concentration measurement. Moreover dependence between apoptosis and HBV-DNA or HCV-RNA levels was studied. METHODS:Eighty-six persons were included into study:34 healthy HBsAg carders,33 patients with chronic HBV infecl^on and 19 patients with chronic HCV infection.Serum levels of sFas/sFasL were measured by ELISA assay.HBV-DNA and HCV-RNA were measured by RT-PCR assay.Levels of sFas/sFasL were determined before and 2 and 12 wk after therapy in patients with chronic hepatitis B and C infection. HBV-DNA or HCV-RNA was detected before treatment and 6 mo after treatment. RESULTS:Twenty-four (71%) healthy HBsAg carders showed HBV-DNA over 10~5/mL,which was comparable to the patients with chronic hepatitis B.independently from HBV-DNA levels, the concentration of sFas among healthy HBsAg carders was comparable to healthy persons.Among patients with chronic hepatitis B and C,the concentration of sFas was significantly higher in comparison to healthy HBsAg carriers and healthy persons.In chronic hepatitis B patients the concentration of sFas was decreased during lamivudine treatment.Among chronic hepatitis C patients the concentration of sFas was increased during IFN alpha and ribavirin treatment,sFasL was not detected in control group.Furbhermore sFasL occurred more frequently in chronic hepatitis C patients in comparison to chronic hepatitis B patients. CONCLUSION:There are no correlations between apoptosis and HBV-DNA levels.However ther is an association between apoptosis and activity of inflammation in patients with chronic HBV infection.Apoptosis can be increased in patients with chronic hepatitis C by effective treatment which may be a result of apoptosis stimulation by IFN-α.

环氧化酶-2、Fas、FasL表达与胃癌细胞凋亡的关系

目的 研究环氧化酶 - 2 (Cox- 2 )、Fas与 Fas L在胃癌中的表达及与细胞凋亡之间的关系。 方法 采用 SP免疫组织化学法检测胃癌及癌旁组织各 6 0例标本 Cox- 2、Fas与 Fas L的表达 ,用脱氧核糖核酸末端转移酶介导的 d UTP缺口末端标记法 (TUNEL )检测胃癌及癌旁组织的凋亡率。 结果 胃癌组织的 Cox- 2及 Fas L的表达明显高于癌旁组织 (P<0 .0 5 ) ,而 Fas的表达低于癌旁组织 (P<0 .0 5 )。胃癌组织的凋亡率明显低于癌旁组织 (P<0 .0 5 )。 结论 Cox- 2、Fas及 Fas

Fas-Fasl系统及凋亡与再生障碍性贫血关系的研究

目的 :研究Fas Fasl系统及凋亡与再生障碍性贫血 (Aplasticanemia ,AA)发生、发展的关系。方法 :分别用TUNEL方法和免疫组织化学标记方法检测凋亡细胞和Fas、Fasl表达细胞 ,并计算其百分率 ;用ELISA方法检测血清中的sFas含量。结果 :AA患者骨髓单核细胞 (BMMNC)中的凋亡细胞百分率为 2 9.6 3%± 17.71% ,Fas表达细胞百分率为 2 4 .81%± 9.4 5 % ,Fasl表达细胞百分率为 2 1.83%± 7.91% ,血清sFas含量为 5 .72± 0 .34μg/L ,与正常对照组 (分别为 2 .2 7%± 0 .74 %、1.93%± 0 .32 %、4 .18%± 1.0 2 %及 7.5 5± 0 .79μg/L)比较差异均有显著性意义 (P <0 .0 5 ) ;AA患者凋亡细胞百分率与Fas+ 细胞或Fasl+ 细胞百分率之间无直线相关关系 (P >0 .0 5 )。结论 :Fas Fasl系统介导的凋亡以及机体对其调控的失常可能参与了AA患者骨髓造血衰竭的病理生理过程 ,但体内可能还存在另外的机制 。

乙型肝炎病毒X基因在Hep G2肝癌细胞的转染及对Fas/FasL表达的影响

目的 探讨乙型肝炎病毒 (HBV)X基因在HepG2肝癌细胞中的转染及对Fas/FasL表达的影响。 方法 用分子克隆方法构建HBVX真核表达载体 pcDNA3 X ,用脂质体转染方法瞬时转染HepG2细胞 ,以转染空载体 pcDNA3的HepG2细胞及未转染质粒的HepG2为对照 ,抽提RNA ,进行RT PCR ,检测HBVX基因的表达。以 β actin为内参 ,用半定量RT PCR法检测三组细胞凋亡相关基因Fas/FasLmRNA相对表达量的变化。 结果 重组真核表达载体经RT PCR及DNA测序均检测出HBVX特异性片段 ,pcDNA3 X转染HepG2细胞后 ,RT PCR扩增出HBVX片段 ,空质粒组及正常对照组均未扩增出相应片段。转染HBx的细胞Fas与FasLmRNA相对表达量较转染空质粒组和未转染质粒组明显增高 ,差别有统计学意义 (P <0 0 5 )。 结论 成功构建HBVX真核表达载体 pcDNA3 X ,转染入HepG2肝癌细胞 ,并在该细胞中表达。HBx在肝癌细胞中的表达能上调Fas和FasL基因的表达。

Relationship between Fas/ FasL expression and apoptosis of colon adenocarcinoma cell lines

INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'

Transretinoic acid inhibits rats gastric epithelial dysplasia induced by N-methyi-N-nitro-N-nitrosoguanidine:influences on cell apoptosis and expression of its regulatory genes

INTRODUCTIONGastric epithelial dysplasia (GED) hypothetically is a straight-forward concept: dysplastic epithelium replacing the normal gastric epithelium of the stomach [1].In the stomach ,like any other segment of the gut ,it is defined as an unequivocal non-invasive epithelial change[2,3].The observation of gastric dysplasia as a cancerous lesion was recognized over a century ago ,but it is only after the advent of gastroscopy that its clinical significance has been stressed[4-7].

山东地区5844例脐血HLA I类等位基因多态性分析

目的:研究山东地区脐带血HLA I类(HLA-A,-B)等位基因的分布特征,探讨中国其他地区及世界不同种族利用山东脐血供者进行造血干细胞移植的可能性。方法:应用PCR-SSO方法对山东地区5 844例无血缘关系的汉族健康新生儿脐带血进行了HLA-A,-B等位基因的分布调查。结果:HLA-A等位基因,检出20种;HLA-B等位基因,检出46种。通过与不同地域汉族人群的比较显示:在不同种族的人群中有其各自独特的人群分布特征,在不同地域的同一民族人群中亦有其独特的地理分布特征。结论:山东脐血HLA I类基因具有多态性,能够代表山东汉族人群HLA的分布特征,北方人在山东脐血库中最容易寻找到HLA I类等位基因相合的异基因脐血供者,而南方人找到的机率较北方人稍低。

Effects of“Moxibustion Serum”on Proliferation and Phenotypes of Tumor Infiltrating Lymphocytes

Tumor infiltrating lymphocytes (TIL) were cultured with “moxibustion serum”(MS), and the results were examined by flow cytometry. The results indicated that MS could enhance the proliferation of TIL,accelerate it to reach the exponential growth phase, and assist recombinant interleukin 2 (rIL-2) to enhance successively the percentage of CD3^+ positive cells, maintain the number of CD4^+ positive T cells, promote greatly the percentage of CD8^+ positive T cells among TILs, and reverse the CD4^+/CD8^+ ratio. Such cooperative effects rely on relative specificity of acupoints. It is suggested that MS is beneficial to the growth of TIL both in the aspects of proliferation and phenotypes.

A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34^+ cells to improve combination chemotherapy tolerance

OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. RESULTS: Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. CONCLUSION: The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.

Fas/FasL系统与卵巢肿瘤的免疫逃逸的研究

目的 :检测Fas、FasL在上皮性卵巢癌及其肿瘤浸润淋巴细胞 (TIL)中的表达 ,探讨Fas系统在卵巢癌免疫逃逸中的作用。方法 :用流式细胞术检测了 31份上皮性卵巢癌、2 0份卵巢良性肿瘤、10份正常卵巢组织以及 31份卵巢癌TIL、12份卵巢良性肿瘤TIL的Fas及FasL的表达。结果 :卵巢癌组织的Fas表达明显低于卵巢良性肿瘤及正常卵巢组织 ,P <0 0 1;而其FasL表达明显高于后者 ,P <0 0 1。卵巢良性肿瘤及正常卵巢组织的Fas、FasL表达差异无显著意义 ,P >0 0 5。卵巢癌组织中含有丰富的TIL ,其Fas及FasL的表达明显高于卵巢良性肿瘤TIL ,差异有显著意义 ,P <0 0 5。卵巢癌组织Fas的表达与临床分期、组织学分级及淋巴结转移无关 ,P >0 0 5。FasL的表达与临床分期无关 ,但随组织学分级的升高而增加 ,P <0 0 5 ,有淋巴结转移者FasL的表达明显高于无淋巴结转移者 ,P <0 0 5。卵巢癌TIL中Fas及FasL的表达与各临床病理参数无关 ,P >0 0 5。结论 :上皮性卵巢癌组织中存在Fas表达的下调和FasL表达的增加 ,FasL高表达者预后不良。肿瘤细胞可能通过FasL的过度表达 ,逃避免疫监视 ,诱导Fas敏感的TIL凋亡 ,发生浸润和转移。

紫外线照射氧合疗法对淋巴细胞表面标志及其功能的影响

目的 :探讨紫外线照射氧合 (UBIO)疗法对淋巴细胞功能影响的机制。 方法 :①对紫外线照射及充氧前后血液中淋巴细胞的表面标志用流式细胞仪进行分析 ;②用淋巴细胞转化试验评估紫外线处理前后血液中淋巴细胞转化功能的变化情况。 结果 :①血液经紫外线照射及充氧后CD8+ 细胞数明显上升 (P <0 .0 5 ) ,HLA DR+ 细胞数显著降低 (P <0 .0 1 ) ,CD4 + /CD8+ 比值降低 (P <0 .0 5 ) ;②血液中的淋巴细胞转化能力降低 (P <0 .0 5 )。 结论 :U