非小细胞肺癌组织中CD44s、CD44v6、Fas的表达及意义

目的 探讨非小细胞肺癌组织中CD44s、CD44v6、Fas的表达及临床意义.方法 采用SP免疫组化法检测CD44s、CD44v6及Fas基因蛋白在58例非小细胞肺癌、18例癌旁正常组织中的表达变化,分析其表达与肿瘤TNM分期、分化程度及淋巴结转移关系.结果 Fas基因蛋白在非小细胞肺癌组织中的表达低于癌旁肺组织,CD44s和CD44v6在癌旁正常肺组织未见表达;CD44s在鳞癌和腺癌中的表达阳性率分别为83.3％及45.5％,CD44v6,在鳞癌和腺癌的表达阳性率分别为86.1％及40.9％,CD44s、CD44v6在鳞癌中的表达均高于腺癌（P均＜0.05）.Fas、CD44s和CD44v6的表达均与肿瘤TNM分期、分化程度及淋巴结转移相关.结论 非小细胞肺癌通过CD44分子的构型改变,降低Fas表达,逃逸Fas-FasL介导的细胞凋亡.非小细胞肺癌CD44s、CD44v6及Fas蛋白的检测可能为评价肿瘤预后的重要指标,也为非小细胞肺癌的治疗提供新的靶点.     

Smoothened shRNA载体的构建及其对乳腺癌细胞增殖的影响

目的构建并筛选Smoothened（SMO）基因的RNAi表达质粒,了解Hedgehog信号通路影响乳腺癌细胞增殖的作用机制。方法以脂质体Lipofectamine^TM2000介导转染乳腺癌MCF-7细胞。转染后48 h,采用实时定量RT-PCR技术检测转染细胞中SMO基因mRNA的转录水平,筛选有效的SMO RNAi质粒,Western blot检测SMO蛋白在乳腺癌MCF-7细胞中的表达,CCK-8法比较转染前后乳腺癌细胞增殖变化,实时定量RT-PCR检测SMO短发夹状RNA（shRNA）对cyclinD1 mRNA表达的影响。结果 4个SMO shRNA表达载体SMO shRNA-1、SMO shRNA-2、SMO shRNA-3和SMO shRNA-4经测序鉴定证明插入正确。转染48 h后,与MOCK组相比,SMO shRNA-1、SMOshRNA-3、SMO shRNA-4组SMO mRNA表达量均下降（P=0.015、0.002、0.000）,其中转染SMO shRNA-4的MCF-7细胞中SMO mRNA表达水平低于SMO shRNA-1、SMO shRNA-2、SMO shRNA-3组（P=0.007、0.000、0.046）。SMOshRNA-4干扰抑制效率为87%。Western blot可检测到SMO在MCF-7中的表达,干扰片段SMO shRNA-4的抑制效果较好。转染SMO shRNA-4的MCF-7细胞增殖受到明显抑制（P〈0.01）,转染SMO shRNA后cyclinD1 mRNA表达下降（P=0.000）。结论已成功构建并筛选出SMO基因的RNAi表达质粒,SMO shRNA对乳腺癌细胞增殖有明显的抑制作用,Hedgehog信号通路可通过活化cyclinD1诱导细胞恶性增殖。     

VEGF和CD44v6过表达与乳腺癌浸润转移的关系

目的检测血管内皮生长因子(VEGF)和转移相关黏附分子44v6(CD44v6)在乳腺癌中的表达情况,探讨两者与淋巴结转移的关系。方法采用免疫组化SP法检测92例乳腺浸润性癌中VEGF、CD44v6的表达情况。结果 VEGF在正常乳腺组织和乳腺癌中的阳性表达率分别为6.7%(3/45)和90.2%(83/92),且VEGF在淋巴结转移组中的阳性表达率(53/55)明显高于无淋巴结转移组(P<0.05)。CD44v6在正常乳腺组织及乳腺癌中的阳性表达率分别为8.9%(4/45)和85.9%(79/92),而且CD44v6在淋巴结转移组中的阳性表达率(51/55)明显高于无淋巴结转移组(P<0.05)。VEGF和CD44v6表达呈正相关关系(r=0.497,P<0.05)。结论 VEGF、CD44v6在乳腺癌组织中高表达,且均与淋巴结转移有关(P<0.05),两者可能在乳腺癌的远处转移中起协同作用。     

Hedgehog通路抑制剂逆转人胰腺癌SW1990细胞获得性耐药的实验研究

目的 探讨人胰腺癌SW1990耐药细胞株中Hedgehog通路成员(Shh、SMO、Gli1、ABCB1、ABCG2)表达的变化及应用Hedgehog信号通路抑制剂对其耐药性的影响.方法 建立人胰腺癌SW1990耐吉西他滨细胞株(SW1990/GZ).采用Hedgehog信号通路抑制剂环巴明作用耐药细胞,应用实时PCR、蛋白质印迹法检测用药前后细胞Shh、SMO、Gli1、ABCB1、ABCG2 mRNA和蛋白的表达及CD44+ CD24+、CD133+细胞亚群比例.以100 μmol/L吉西他滨作用环巴明处理后的SW1990/GZ细胞,应用Annexin V加PI双染法检测细胞凋亡率.结果 SW1990/GZ细胞中CD44+ CD24+细胞比例从亲代的(29.45±1.99)％增加到(93.16±2.46)％、CD133+细胞比例从(59.37±1.69)％增加到(95.88±1.47)％(P＜0.01);ABCB1、ABCG2、Shh、Gli1 mRNA的表达分别增加(274.90±31.44)、(3.48±0.33)、(12.07±1.71)、(4.15±0.42)倍(P＜0.01),并检测出SMO mRNA表达.蛋白表达结果与mRNA表达一致.2μmol/L环巴明作用SW1990/GZ细胞1周后,CD44+CD24+比例下降到(36.68±2.44)％、CD133+细胞比例下降到(62.76±1.28)％(P＜0.05).2、5μmol/L环巴明处理SW1990/GZ细胞后再用100 μmol/L吉西他滨处理细胞,细胞的凋亡加死亡率分别为(53.68±5.24)％和(69.99±3.16)％,显著高于对照组的(4.55±0.87)％(P＜0.01).结论 人胰腺癌SW1990耐药细胞株中高富集肿瘤干细胞,高表达Hedgehog信号通路成员SMO及Gli1.抑制耐药株的Hedgehog通路,可以降低其肿瘤干细胞比例,下调SMO及Gli1表达,部分恢复对吉西他滨的敏感性.     

骨桥蛋白及 CD44 v6在肺腺癌侵袭转移中的作用

目的：探讨骨桥蛋白（ OPN）及其受体CD44v6在肺腺癌侵袭转移中的作用。方法应用免疫组化方法检测129例癌旁组织、肺原位腺癌（AIS）、伏壁样生长为主型腺癌（LPA）患者手术切除标本中OPN、CD44v6的表达情况。检测H358细胞（原位癌细胞系）和A549细胞中OPN、CD44v6蛋白及mRNA的表达,Transwell 试验观察OPN、CD44 v6对H358细胞、A549细胞侵袭力的影响。结果 OPN、CD44 v6在LPA组织中的表达阳性率显著高于AIS及癌旁组织,AIS高于癌旁组织,OPN与CD44v6的表达呈显著正相关（ P＜0．05）。 OPN、CD44v6及OPN mR-NA、CD44v6 mRNA在A549细胞中的表达水平高于H358细胞。 Transwell试验提示OPN对A549细胞的趋化作用高于H358细胞。使用OPN抗体阻断OPN对两种细胞的趋化作用,两种细胞穿过数均减少;阻断CD44 v6受体通道后,OPN对两种细胞的趋化作用明显减弱。结论 OPN-CD44 v6复合体对肺腺癌的侵袭能力具有重要作用,OPN有望成为评估肺腺癌进展及预测肿瘤转移潜能的指标。     

E—CD和CD44V6在膀胱癌组织中的表达及临床意义

应用免疫组化SABC法对81例膀胱移行细胞癌患者癌组织中细胞粘附分子上皮钙粘附素（E－CD）和CD44V6的表达进行检测。结果显示,E－CD的异常表达与膀胱癌临床分期、病理分级、淋巴结转移显著相关（P均＜0．05）。CD44V6的阳性表达与膀胱癌临床分期、病理分级有显著关系（P均＜0．05）,伴淋巴结转移组的CD44V6阳性表达率显著低于未转移组（P＜0．01）。认为E－CD和CD44V6均与膀胱癌浸润转移明显相关,两者结合分析能够更准确地反映膀胱癌的生物学行为。     

CD44s mRNA在胃癌组织中的表达及其临床意义

目的探讨CD44s mRNA在胃癌组织中的表达及其临床意义。方法采用核酸原位杂交技术检测66例胃癌（胃癌组）、25例浅表性胃炎（胃炎组）和25例胃上皮不典型增生（增生组）中CD44s mRNA的水平。结果胃炎组无阳性表达;增生组中阳性率为20％;在胃癌组中阳性率为62．12％,胃癌组低分化组织中阳性率明显高于高分化组织（P〈0．05）,有淋巴结转移组阳性率明显高于无淋巴结转移组（P〈0．05）。结论CD44s mRNA表达与胃癌组织分化程度和转移有相关性;CD44s mRNA基因水平可作为判断胃癌病情和预后的指标。     

原发性乳腺癌患者外周血循环肿瘤细胞及肿瘤干细胞的检测及临床意义

目的探究原发性乳腺癌患者外周血中循环肿瘤细胞(CTCs)及肿瘤干细胞(TSCs)与乳腺癌临床、病理特征的关系。方法采集44例初治原发性乳腺癌患者和15例正常女性志愿者外周血,制备外周血单个核细胞悬液,以CK19单克隆抗体标记,免疫磁性激活细胞分选获得的CTCs,将提取的CK19+细胞标记单克隆抗体CD44、CD24进行免疫荧光抗体双染色,免疫荧光显微镜检测表达CD44+/CD24-/low的TSCs含量。结果 15例正常女性志愿者PBMC中未检测到CK19+细胞。44例原发性乳腺癌患者中33例检测到CK19+细胞,按临床分期和淋巴结转移分组:每组患者均能检测到CK19+细胞,差异具有明显统计学意义。33例CK19阳性样本中21例(63.63%)检测到CD44+/CD24-/low细胞,按临床分期、病理学指标及分子亚型分组均无明显统计学差异。结论原发性乳腺癌患者外周血中可检测到CTCs及TSCs,CTCs的阳性率与临床分期、淋巴结转移密切相关。临床分期越晚,TSCs阳性率越高。     

内镜活检组织E-钙粘蛋白和CD44V6水平与肺癌生物学特征的相关性研究

目的 探讨E-钙粘蛋白和CD44V6在肺癌组织中的表达及其与肺癌侵袭、转移及预后的关系。方法 经纤维支气管镜肺活检或经皮肺穿刺获取肺组织，用免疫组化法检测组织中E-钙粘蛋白和CD44V6的表达，并与术后肺癌组织、癌旁组织及正常肺组织的表达作比较。分析其与肺癌分期、类型及淋巴结转移之间的关系。结果 CD44V6在肺癌组织中的阳性表达率高，显著高于癌旁组织，在非小细胞肺癌中表达显著高于小细胞肺癌，与PTNM分期呈显著正相关，与淋巴结转移呈正相关。E-钙粘蛋白在肿瘤细胞中的表达低于正常细胞，有的甚至缺失。结论 肺癌组织中的CD44V6高表达与肺癌的发生发展、淋巴结转移及预后相关。而E-钙粘蛋白在肺癌组织中的表达降低，并与分化不良呈显著相关性。     

非小细胞肺癌外周血CD44和CD54表达的定量研究

目的 探讨非小细胞肺癌患者外周血淋巴细胞中CD44和CD54表达与临床病理的关系。方法 应用流式细胞术对50例肺癌患者外周血淋巴细胞中CD44和CD54表达与临床病理的关系。方法 应用流式细胞术对50例肺癌患者外周血淋巴细胞中CD44和CD54表达进行荧光免疫检测,并与正常对照组（30名）及肺部良性病变组（25例）进行对比研究。结果 50例肺癌患者外周血淋巴细胞中CD44和CD54表达明显高于正常对照组及良性病变组（P＜0．01）。良性病变组和正常对照组之间CD44和CD54的表达比较,差异无显著性（P＞0．05）。肺癌伴淋巴结转移CD44和CD54高于不伴淋巴结转移者（P＜0．01）;Ⅲ期、Ⅳ期和Ⅰ期、Ⅱ期之间CD44表达比较,差异有显著性（P＜0．01）;Ⅳ期和Ⅰ期、Ⅱ期、Ⅲ期之间CD54表达比较,差异有显著性（P＜0．01）。CD44和CD54表达与肺癌组织学分级有明显相关性（P＜0．05或0．01）;与鳞癌和腺癌没有相关性。结论 应用流式细胞仪检测CD44和CD54的表达水平可作为肺癌转移和预后的指标。     

不同转移潜能肝癌细胞株中CD标志物的表达差异及意义

目的探讨不同转移潜能的肝癌细胞株中几种CD标记物的表达差异及其意义,为进一步利用CD分子标志物分离多潜能肝癌干细胞奠定基础。方法常规培养三种不同转移潜能的肝癌细胞株HepG2、MHCC-97H、SK-HEP-1,胰酶消化获得单细胞悬液后,经流式抗体染色,通过流式细胞仪分析三种肝癌细胞株中CD133、CD90、CD44、CD34、CD24的表达差异及意义。结果 CD133+细胞在HepG2细胞株仅占0.1%,而在MHCC-97H、SK-HEP-1细胞中表达也较低;CD90+、CD44+、CD90+CD44+表达水平随细胞转移潜能的增加呈递增趋势(P<0.05)。CD44+CD24+在HepG2、MHCC-97H、SK-HEP-1细胞中的阳性表达率分别为0.05%、10.3%和0.3%(P<0.05);三者中均无CD34+和CD133+CD90+细胞表达。结论 CD90和CD44与肝癌细胞的转移潜能有一定的相关性,可能成为潜在的肝癌肿瘤干细胞标记物。     

自体造血干细胞移植治疗系统性红斑狼疮随访

目的 探讨自体造血干细胞移植治疗系统性红斑狼疮(SLE)的远期疗效.方法 分析48例接受自体造血干细胞移植的SLE患者于移植后的随访情况,自诊断时间计算,病程均超过10年.其中24例为CD34+细胞纯化移植,24例非CD34瑚胞纯化移植.采用驴榆验进行统计分析.结果 死亡5例,移植相关死亡4例,其中3例为CD34+移植.自诊断病程超过10年的生存率为90%(43/48).可评价的43例中,复发7例,6例为非CD34+细胞纯化移植,1例为CD34+细胞纯化移植.CD34+细胞纯化移植复发率为5%(1/21),非CD34+细胞纯化移植复发率为27%(6/22),2组问复发率差异有统计学意义(x2=3.995,P=0.046).随访中,8例上大学,26例恢复正常工作与生活,其中4例生育子女.结论 自体造血干细胞移植联合抗人胸腺球蛋白(ATG)治疗SLE的远期疗效确切,CD34+植复发率低于非CD34+移植,尤其是SLE患者移植后生活质量好于传统治疗.

Abstract：

Objective To investigate the long-term efficacy of autologous stem cell transplantation for systemic lupus erythematosus (SLE). Methods Long-term follow up of 48 SLE patients with autologous stem cell transplantation were studied. All patients were followed up for 10 years. Among the patients, 24 cases were treated with purified CD34+ cells transplantation and 24 cases were treated with non-CD34+ cell transplantation. Comparison between groups was performed by x2 test. Results Among 5 dead patients, 4 died of transplantation related complications including 3 cases treated with CD34+ transplantion. The survival rate of those patients with more than 10 years duration of lupus was 90%(43/48). Among 43 patients, 7 had disease flare, 6 were treated with non-CD34+ cell transplantation. Eight patients went to college, 26 returned to normal life and 4 of them had children. Conclusion The long-term effect of SLE treated with autologous hematopoietic stem cell transplantation and anti-thymocyte globulin (ATG) is good. The recurrence of CD34+ transplant patients is lower than those treated with non-CD34+ transplantation. The quality of life in SLE patients treated with transplantation is better than those treated with conventional therapy.     

Antibodies to CD44 enhance adhesion of normal CD34+ cells and acute myeloblastic but not lymphoblastic leukaemia cells to bone marrow stroma

The role of CD44 in the adhesion of haemopoietic cells to bone marrow stromal layers has not been clearly defined in humans, although its importance in the murine system has been well documented. We have demonstrated that the CD44 antibody, NIH44-1, enhances the adhesion of haemopoietic cells to bone marrow stroma. Normal human CD34⁺ haemopoietic progenitors and blasts from patients with acute myeloblastic, but not lymphoblastic, leukaemia responded to NIH44-1. All CD44 antibodies tested which bound the same epitope as NIH44-1 also augmented haemopoietic cell adhesion to bone marrow adherent layers; however, antibodies which bound to other CD44 epitopes showed mixed responses. Augmented adhesion was independent of cell metabolism, suggesting that antibody binding resulted in direct activation of the CD44 molecule. However, hyaluronic acid was not the ligand for induced adhesion, nor could we show a role for other CD44 ligands including fibronectin, laminin, collagen or chondroitin sulphate proteoglycan. Similarly, none of the 22 CD44 antibodies tested inhibited the stimulatory effect of the NIH44-1. Expression of CD44 was not sufficient to determine NIH44-1 responsiveness since cell lines and leukaemic cells which failed to respond to NIH44-1 expressed high levels of CD44. Neither CD44 isoforms nor glycosylation patterns could be identified as predictive of response. CD44 antibodies enhanced binding of normal and leukaemic haemopoietic progenitors to bone marrow fibroblasts via an unidentified stromal ligand.     

Differential antigen expression and aberrant signaling via PI3/AKT,MAP/ERK,JAK/STAT,and Wnt/β catenin pathways in Lin−/CD38−/CD34+ cells in acute myeloid leukemia

Acute myeloid leukemia is often called as stem cell disease that presents with treatment failure and poor disease outcome. Leukemic stem cells in acute myeloid leukemia (AML) are enriched in Lineage‐/CD38?/CD34+ compartment of CD34?positive AML. Many markers important for stem cell biology have been reported for their association with leukemic stem cell population, but what remains clinically most important is a rapid identification of prognostic information. In this study, we evaluated four signal transduction pathways and thirteen markers on Lin?/CD38?/CD34+ population in AML. Expressions were compared in different AML subtypes, survival, and treatment outcome groups. We observed that markers important in homing, cell quiescence, and signal propagation such as CD44, CD96, CD90, WT‐1, CD123 and CD25 were most significantly differentially expressed on Lin‐/CD38?/CD34+ population in AML from their normal counterparts (P < 0.05, Mann–Whitney). Constitutive activation of phospho ERK, AKT, and STAT5 in these cells was associated with poor outcome. Also, an increased frequency of putative leukemic stem cell population shows negative impact on treatment outcome and overall survival, suggesting that initial evaluation of AML samples for pLSC frequency and constitutively activated signaling pathway can provide prognostic and therapeutic information at the time of diagnosis.     

Pathological role of CD44 on NKT cells in carbon tetrachloride-mediated liver injury

Aim: CD44 has a variety of functions in immune regulation and signal transduction. Although CD44 is involved in the induction of several inflammatory diseases, it remains unknown whether CD44-targeting therapies are useful for liver diseases. Here, we examined whether CD44 blockade is effective in a chemical-induced liver injury model. Methods: We injected CD44 knock out (KO) or wild type mice with carbon tetrachloride (CCl(4)) and examined the difference of liver injury by immunological or histological analysis. Results: Although CD44KO mice exhibited suppressed liver injury at 6 h after CCl(4) injection with decreased inflammatory cell numbers and cytokine production, these mice showed severe liver injury at 24 h. We found that NKT cells played an important role in liver injury with increased infiltration of theliver after migration, which was independent of the CD44 pathway. In CD44NKT double-KO mice, liver injury was suppressed with reduced cytokine production and macrophage infiltration compared with CD44KO mice. Furthermore, MIP-2 derived from NKT cells or tumor necrosis factor alpha from macrophages contributed to exacerbation of the liver injury, since neutralization of MIP-2 provided significant protection against liver injury in CD44KO mice. Finally, we found that CD44KO mice exhibited excessive liver fibrosis compared with wild-type mice after repeated CCl(4) injections. Conclusion: We found that CD44 has unique characteristics for inflammatory liver diseases associated with NKT cell infiltration and activation. Furthermore, CD44-targeting therapies may need to be viewed with caution for liver diseases due to the actions of the liver immune system.     

Expression of homing-associated cell adhesion molecule (H-CAM/CD44) on human CD34+ hematopoietic progenitor cells

We investigated the expression of CD44 molecule on CD34+ hematopoietic progenitor cells. Significantly lower expression of CD44 was observed on bone marrow (BM) CD34+ cells compared with circulating CD34+ cells in cord blood and peripheral blood. Using fluorescence-activated cell sorting, human CD34+ BM cells were fractionated into CD44+ and CD44- populations. Immunofluorescence analysis revealed that the majority of CD34+CD44- cells expressed B-lymphocyte-associated CD10 and CD19 antigens, whereas only a part of CD34+CD44+ cells were positive for CD19. Myeloid and erythroid progenitor cells were found predominantly in CD34+ CD44+ cell fractions. In short-term suspension cultures, cell proliferation and G1-->S transition in the cell cycle were enhanced in CD34+CD44+ cells. In contrast, a large part of CD34+CD44- cells underwent apoptotic cell death. Although co-culture with BM stromal cells could partially prevent CD34+CD44- cells from undergoing apoptosis, significant increase of apoptotic cells was consistently observed. Furthermore, CD34+CD44- cells plated on BM stromal cells could differentiate into CD34-CD44-CD10-CD19+ cells. These findings suggest that CD34+CD44- cells expressing CD19 would represent unique B-lymphocyte-committed precursors in BM, which might undergo apoptotic cell death in the early steps of B-cell differentiation.     

骨髓增生异常综合征患者骨髓CD34+细胞亚群及细胞周期分析

目的 探讨骨髓增生异常综合征(MDS)患者骨髓CD34+细胞亚群及细胞周期分布异常的临床意义.方法 采用流式细胞术检测50例(17例低危、33例高危)MDS患者、8例MDS转化的急性髓系自血病(MDS-AML)及25例对照组原代骨髓CD34+CD38+、CD34+CD38-细胞亚群及G0/G1期、S期和G2/M期细胞比例.结果 高危和MDS-AML组骨髓CD34+细胞比例[(2.29±2.17)%、(18.69±17.47)%]明显高于对照组[(0.36±0.49)%,P＜0.05].低危、高危及MDS-AML组CD34+CD38+细胞相对比例[(86.09±7.79)%、(81.68±11.82)%、(82.88±2.60)%]显著低于对照组[(92.21±3.85)%,P＜0.05],而CD34+CD38-细胞比例[(13.91±7.79)%、(18.32±11.82)%、(17.13±2.60)%]显著高于对照组[(7.79±3.85)%,P＜0.05].MDS组CD34+CD38-细胞比例与国际预后积分系统(IPSS)(r=0.493,P=0.001)、WHO分型预后积分系统(WPSS)积分(r=0.586,P=0.000)均呈正相关.低危、高危及MDS-AML组骨髓单个核细胞(BMMNC)中G0/G1期细胞比例[(94.52±4.32)%,(96.07±3.88)%,(94.65±4.55)%]明显高于对照组[(88.94±7.30)%,P＜0.01],而3组S期[(4.63±3.34)%,(3.45±3.80)%,(5.12±4.55)%]和G2/M期[(0.84±1.52)%,(0.48±0.74)%,(0.22±0.34)%]细胞比例明显低于对照组[(9.06±6.50)%,(2.00±2.93)%,P值均＜0.05],3组S+G2/M期细胞比例明显高于对照组(P＜0.01).CD34+CD38-细胞比例≤12.0%的MDS患者治疗有效率高于CD34+CD38-细胞比例＞12.0%的患者,但差异无统计学意义.结论 MDS患者原代骨髓CD34+细胞亚群分化异常,BMMNC存在G1期阻滞现象,提示CD34+细胞亚群和细胞周期测定可能有助于MDS患者的辅助诊断以及疗效和预后判断.     

MiR-590-3p在胰腺癌干细胞中的表达

目的 应用无血清培养基分离胰腺癌干细胞,检测其miR-590-3p的表达。方法 运用无血清培养基克隆培养ASPC-1、PANC1细胞,检测其单克隆形成、分化及细胞周期、半数抑制浓度(IC50)和表面标记物CD24+、CD44+表达。实时定量PCR法检测细胞miR-590-3p的表达。结果 经无血清培养基培养,(0.94±0.53)％的ASPC-1细胞和(0.57±0.12)％的PANC1细胞能存活,呈克隆球样悬浮生长,并可以在体外连续传代。加入血清后细胞球又重新贴壁生长。ASPC-1细胞球G0/G1期比例和CD24+、C44+、CD24+ CD44+的细胞比例及IC50分别为(75.3±5.4)％、0.96％～2.01％、27.52％～34.47％、0.35％ ～0.44％和(224.37±5.71) μg/ml,均显著高于亲本细胞的(43.7±3.8)％、0.38％～0.42％、17.65％ ～ 18.25％、0.05％～ 0.08％、(11.43±2.10) μg/ml(P值均＜0.05)。PANC1细胞球G0/G1期比例和CD24+、CD44+、CD24 +CD44+的细胞比例及IC50分别为(80.1±4.7)％、5.31％ ～9.84％、72.05％ ～93.06％、4.91％ ～5.21％、(296.58±4.27) μg/ml,均显著高于亲本细胞的(46.1±5.3)％、4.09％～4.97％、47.71％～55.66％、1.48％ ～2.63％、(26.17±3.81) μg/ml(P值均＜0.05)。ASPC-1、PANC1细胞球miR-590-3p表达分别是亲本细胞的4.67和4.52倍。结论 应用无血清培养基可以从ASPC-1、PANC1细胞系中分离出具有干细胞特性的胰腺癌细胞球,其miR-590-3p表达上调,该基因可能是胰腺癌干细胞特性维持的关键基因。     

系统性红斑狼疮患者CD4+ CD8+T细胞caspase-3及TNF相关凋亡诱导配体受体的表达

目的探求系统性红斑狼疮（SLE）患者CD4^＋,CD8^＋T淋巴细胞caspase-3及肿瘤坏死闪子相关凋亡诱导配体（TRAIL）受体的表达。方法采用免疫磁珠的方法对20例系统性红斑狼疮患者及10名正常人外周血CD4^＋、CD8^T淋巴细胞进行分离，采用反转录聚合酶链反应（RT—PCR）对CD4^＋、CD8^＋T淋巴细胞caspase-3及TRAIL受体的表达进仃分析。结果TRAIL—R3及TRAIL—R4在两符之间的表达差异无统计学意义，但SLE患者CD8^＋T细胞caspase-3（P=0．003）及TRAIL-R2（P=0．024）的表达增加显著高于正常对照。结论TRAIL—R2介导的通路可能在TRAIL诱导SLE患者T淋巴细胞凋亡中发挥重要的作用。     

Distinct patterns of apoptosis in association with modulation of CD44 induced by thrombopoietin and granulocyte-colony stimulating factor during ex vivo expansion of human cord blood CD34+ cells

The insufficient number of haemopoietic stem cells (HSCs) in cord blood (CB) is the major potential limitation to widespread use of CB for marrow replacement. Cytokine-mediated ex vivo expansion has been proposed as a means of increasing the number of CB HSCs for transplantation. However, the biology of CB HSCs during cytokine-mediated ex vivo expansion, such as apoptosis or expression of adhesion molecules, has not yet been elucidated. We have investigated the patterns of apoptosis and CD44 expression on human CB CD34+ cells during ex vivo expansion. CD34+ cells isolated from human CB were cultured in a stroma-free liquid culture system with thrombopoietin (TPO), flt3-ligand (FL), stem cell factor (SCF), and/or granulocyte-colony stimulating factor (G-CSF). During the culture, for up to 5 weeks, apoptosis was measured by staining with 7-amino-actinomycin D (7-AAD) along with concurrent immunophenotyping of CD34 and CD44 with three-colour flow cytometry. In the cultures with TPO, an apoptotic fraction with down-regulated CD44 appeared from the fourth day up to the second week. G-CSF also induced apoptosis but in a different manner; the apoptotic fraction without down-regulation of CD44 appeared unremittingly for up to 5 weeks. FL did not induce apoptosis or down-regulation of CD44. These findings show that apoptosis is indeed involved in the regulation of CB CD34+ cells in ex vivo expansion and the patterns of apoptosis are dependent on the type of cytokines used. The distinct patterns of apoptosis suggest different mechanisms of TPO and G-CSF in inducing apoptosis, which still remains to be elucidated.