外周血CD4^(+)PD-1^(+)Tcells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性分析

目的 探讨外周血CD4^(+)程序性细胞死亡受体-1(PD-1)^(+)T cells及CD4^(+)T淋巴细胞三磷酸腺苷(ATP)含量与复发性卵巢癌疗效的相关性。方法 选取30例复发性卵巢癌患者为复发组,30例未复发卵巢癌患者为非复发组;另选取30名同期体检健康者作为对照组。评估外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性。结果 复发组和非复发组的CD4^(+)PD-1^(+)T cells较对照组明显升高(P<0.05)。复发组和非复发组的CD4^(+)T淋巴细胞ATP含量较对照组明显降低(P<0.05)。复发组治疗后CD4^(+)PD-1^(+)Tcells显著低于治疗前(P<0.05),治疗后CD4^(+)T淋巴细胞ATP含量显著高于治疗前(P<0.05)。CD4^(+)PD-1^(+)T cells与复发性卵巢癌疗效成负相关(r=-0.393,P=0.039),CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效成正相关(r=0.449,P=0.031)。结论 复发性卵巢癌患者外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与疗效密切相关。

双特异性CAR-T细胞对EGFRvⅢ^(+)/CD133^(+)胶质瘤干细胞的靶向杀伤

目的:制备双特异性CAR-T(bs CAR-T)细胞,观察其对表达表皮生长因子Ⅲ型突变阳性(EGFRvⅢ^(+),简称vⅢ^(+))和CD133^(+)胶质瘤干细胞的靶向杀伤作用。方法:基于前期研制的vⅢ/CD133双特异性微抗体和二代CAR构建的双特异性CAR(bs CAR),制备慢病毒载体转染人外周血T细胞,FCM和WB法检测bs CAR转染效率和表达水平。bs CAR-T细胞和vⅢ^(+)/CD133^(+)U87胶质瘤干细胞共培养,乳酸脱氢酶(LDH)释放实验、IFN-γ分泌实验检测其特异性杀伤作用和对IFN-γ分泌的促进作用。制备裸鼠vⅢ^(+)/CD133^(+)U87干细胞移植瘤模型检测bs CAR-T细胞对移植瘤生长的抑制作用。结果:vⅢsc Fv和CD133sc Fv通过重叠PCR无缝连接入二代CAR表达框(S-vⅢscFv/CD133scFv-Hinge-TM-CD137-CD3ζ)中,然后克隆入p CDH-MSCV-MCS-EF1-copGFP载体的Eco RⅠ和Bam HⅠ位点(pbs CAR)。3种质粒(p VSV-G、p CMV-d R8.9和pbs CAR)共转染HEK293T细胞制备慢病毒载体,转染外周血T细胞,FCM检测bs CAR表达率为71.1%,WB法结果显示bs CAR表达正确。bs CAR-T细胞和vⅢ^(+)/CD133^(+)U87干细胞共培养检测结果显示,bs CAR-T细胞对胶质瘤干细胞具有特异性杀伤作用,与效靶比呈正比;IFN-γ分泌量为(2350.6±92)pg·m L^(-1),明显高于对照组(P<0.01)。裸鼠移植瘤动物模型显示,bs CAR-T细胞在体内具有明显的移植瘤抑制作用(P<0.01)。结论:bs CAR-T细胞能够特异性靶向杀伤vⅢ^(+)/CD133^(+)胶质瘤干细胞,实验结果为促进实体瘤的细胞免疫治疗提供了实验依据。

Peripheral CD4^(+)CD8^(+) double positive T cells:A potential marker to evaluate renal impairment susceptibility during systemic lupus erythematosus

Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^(+)CD8^(+)double positive T(DPT) lymphocytes and LN. The study included patients with SLE without renal impairment(SLE-NRI), LN, nephritic syndrome(NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group(t=4.012, P<0.001), NS group(t=3.240,P=0.001), and nephritis group(t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times(95% confidence interval, 2.115–12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.

CD133^(+)结肠癌细胞区域变异位点在结肠癌免疫逃逸中的作用研究

目的分析探讨CD133^(+)结肠癌细胞3-UTR区域rs2240688变异位点在结肠癌免疫逃逸中的作用。方法检测CD133^(+)结肠癌细胞3-UTR区域rs2240688位点单核苷酸多态性,并分别检测不同基因型CD133^(+)结肠癌细胞侵袭迁移能力以及NK-92细胞对不同基因型CD133^(+)结肠癌细胞的细胞毒力。结果基因型为G/G的CD133^(+)结肠癌细胞侵袭迁移能力最强,而基因型为T/T的CD133^(+)结肠癌细胞侵袭迁移能力最弱(P均<0.05);NK-92细胞对基因型为G/G的CD133^(+)结肠癌细胞的细胞毒力最弱,而对基因型为T/T的CD133^(+)结肠癌细胞的细胞毒力最强(P均<0.05)。结论CD133^(+)结肠癌细胞3-UTR区域单核苷酸多态性位点基因多态性能够参与机体的免疫逃逸,对CD133^(+)肿瘤干细胞进行深入研究,有望成为结直肠癌临床治疗的新靶点。

Autologous CD34^+ and CD133^+ stem cells transplantation in patients with end stage liver disease

AIM:To assess the utility of an autologous CD34 + and CD133 + stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases.METHODS:One hundred and forty patients with endstage liver diseases were randomized into two groups.Group 1,comprising 90 patients,received granulocyte colony stimulating factor for five days followed by autologous CD34 + and CD133 + stem cell infusion in the portal vein.Group 2,comprising 50 patients,received regular liver treatment only and served as a control group.RESULTS:Near normalization of liver enzymes and improvement in synthetic function were observed in 54.5% of the group 1 patients;13.6% of the patients showed stable states in the infused group.None of the patients in the control group showed improvement.No adverse effects were noted.CONCLUSION:Our data showed that a CD34 + and CD133 + stem cells infusion can be used as supportive treatment for end-stage liver disease with satisfactory tolerability.

Inhibitive effect of IL-24 gene on CD133^+ laryngeal cancer cells

Objective:To explore the inhihitive and apoptosis inductive effect of IL-24 genes on CD133^+laryngeal cancer cells in Hep-2 line.Methods:Human peripheral blood monocytes were isolated.The total RNA was extracted by using Trizol method and reverse transcripted into cDNA using RT-PCR method.Primers P1 and P2 was designed for the amplification of human IL-24 genes.After confirmation of agarose gel electrophoresis tests,TA was cloned into pMD19-T simple vector.Nhe Ⅰ and Xho Ⅰ double digesting human IL-24 and pIRES2-ZsGreen1 and eukaryotic expression vector were used to establish the pIRES2-ZsGreen1-hIL-24 vector,and detected by enzyme digestion and gene sequencing methods.Flow cytometry(FCM) was used to isolate CD133^+ cells from Hep-2 cells.CD133^+ cells were transfected with pIRES2-ZsGreen1-hIL-24 through liposome 2000.After detection,MTT and FCM were used to observe the effect of IL-24 gene on CD133^+ laryngeal cancer Hep-2 cells.Results:Lipotin mediated transfection of recombinant pIRES2-ZsGreen1-hIL-24 plasmid into CD133^+ Hep-2 could expressed IL-24 gene in cells stably.MTT results showed that IL-24 transfected group was significantly suppressed compared to empty vector group and control group(P<0.05);FCM results showed that the apoptosis rate of experimental group increased significantly compared to empty vector group and control group(P<0.05).Conclusions:IL-24 gene expressions can inhibit proliferation of CD133^+laryngeal cells in Hep-2 line and promote their apoptosis.

Angioarchitecture and CD133^+ tumor stem cell distribution in intracranial hemangiopericytoma A comparative study with meningioma

Angioarchitecture plays an important role in the malignant development of intracranial hemangiopericytoma. It remains poorly understood whether high frequency of hemorrhage during clinical surgery for intracranial hemangiopericytoma is associated with angioarchitecture. The present study utilized hematoxylin-eosin staining, and immunohistochemical staining with epithelial membrane antigen, vimentin, CD34, von Willebrand factor （vWF） and CD133 to observe characteristics of angioarchitecture. In addition, silver stains were used to demonstrate changes in reticular fibers in the wall of vessel channels in intracranial hemangiopericytoma and meningioma. Five patterns of angioarchitecture were identified in intracranial hemangiopericytoma, namely tumor cell islands, vasculogenic mimicry, mosaic blood vessels, sprouting angiogenesis, and intussusceptive angiogenesis. Several CD133＋ tumor cells were found to form tumor cell islands. A connection between vWF ^＋ and vWF channels was detected in the pattern of intussusceptive angiogenesis, and some vimentin^＋ tumor cells were embedded in the periodic acid-Schiff positive channel wall. Incomplete threads of reticular fibers formed the walls of larger pseudo-vascular channels and some tumor clumps or scattered tumor cells were detected ＂floating＂ in them. The angioarchitecture, specific markers and reticular fibers of intracranial hemangiopericytoma were significantly different from meningioma. Angioarchitecture provides a functional vascular network for vascular evolution in intracranial hemangiopericytoma and contributes to significant intra-operative bleeding.

The Cross-talk between ROS and p38MAPKα in the Ex Vivo Expanded Human Umbilical Cord Blood CD133^+ Cells

This study investigated the correlation between and compared the effects of reactive oxygen species（ROS） and p38 mitogen-activated protein kinase α（p38MAPKα） in the ex vivo expanded umbilical cord blood（hUCB） CD133＋ cells.hUCB CD133＋ cells were cultured in the hematopoietic stem cells（HSCs） culture medium with N-acetylcysteine（NAC,an anti-oxidant）,p38MAPKα-specific inhibitor（SB203580） or their combination.The levels of ROS and expression of phosphorylated p38MAPKα（p-p38） in CD133＋ cells were flow cytometrically detected.The efficacy of ex vivo expansion was evaluated by the density of CD133＋ cell sub-group colony-forming cells（CFC） and cobblestone area-forming cells（CAFC） assay.Our results showed decreased ROS levels in NAC,SB203580,and their combination treatment groups were almost 37%,48%,and 85%,respectively.Furthermore,SB203580 abrogated the activation of p38MAPKα more obviously than NAC.Moreover,the CD133＋ cells in SB203580 treatment group had a 21.93±1.36-fold increase,and 14.50±1.19-fold increase in NAC treatment group,but only 10.13±0.57-fold increase in control group.In addition,SB203580 treatment led a higher level increase in the number of CFU and CAFC than NAC did.These findings suggested that,in expanded CD133＋ cells,ROS activates p38MAPKα,which,in turn,induces ROS production,and p38MAPKα might be the most suitable regulator in ROS-p38MAPKα pathway for the promotion of HSCs ex vivo expansion.

CD133^+ gallbladder carcinoma cells exhibit self-renewal ability and tumorigenicity

AIM:To identify cancer stem cells(CSCs) in human gallbladder carcinomas(GBCs).METHODS:Primary GBC cells were cultured under serum-free conditions to produce floating spheres.The stem-cell properties of the sphere-forming cells,including self-renewal,differentiation potential,chemoresistance and tumorigenicity,were determined in vitro or in vivo.Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry.The sphere-colony-formation ability and tumorigenicity of CD133+ cells were assayed.RESULTS:In vitro culture experiments revealed thatfloating spheroids were generated from primary GBC cells,and these sphere-forming cells could generate new progeny spheroids in serum-free media.Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4,Nanog,and nestin(P < 0.05).The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells(P < 0.05).Spheroid cells were more resistant to chemotherapeutic reagents than the congenetic adherent cells(P < 0.05).Flow cytometry showed enriched CD133+ population in sphereforming cells(P < 0.05).CD133+ cells possessed high colony-formation ability than the CD133-population(P < 0.01).CD133+ cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts(P < 0.05).CONCLUSION:CD133 may be a cell surface marker for CSCs in GBC.

Influence of CD133^+ expression on patients' survival and resistance of CD133^+ cells to anti-tumor reagents in gastric cancer

Objective:To investigate the influence of CD133+expression on patients'survival and resistance of CD133+cells to anti-tumor agents in gastric cancer(GC).Methods:Influence of CD133 expression on prognosis was analyzed employing samples from patients with GC.GC cell lines were utilized to separate CD133+and CD133-subpopulations by immunomagnetic separation and to analyze the biological features of two subpopulations in vitro and in vivo,especially in resistant to anti-tumor reagents and its apoptotic mechanism.Results:The lower CD133+group showed a significantly better survival compared with the higher CD133+group.The highest content of CD133+subpopulations for KATO-III cells had stronger proliferative ability than CD133-subpopulations.A single CD133+cell was capable of generating new cell colony and the tumorigenicity rate in nude mice was100%for CD133+clonal spheres or for CD133+cells,but 0%for CD133-cells.Furthermore,the higher expression levels of Oct-4,Sox-2,Musashi-1 and ABCG2 in CD133+clonal spheres were identified compared with CD133+cells or CD133-cells.Under the treatment of anti-tumor reagents,CD133+cells had lower suppression rates compared with CD133-cells while lower level of Bcl-2 and higher level of Bax were found in CD133+cells compared with CD133-cells.Conclusions:The patients with lower CD133+expression had a better survival.Enriched CD133+cells in clonal sphere shared the ability to be self-renewable,proliferative,tumorigenic and resistant to anti-tumor agents as probably regulated by Bcl-2 and Bax.

人脐血CD133^+细胞体外短期培养中生物学特性的变化

为了解脐血CD133+细胞的生物学特性及其在体外短期扩增培养中的变化 ,探讨其体外扩增的可行性 ,初步观察了脐血CD133+细胞的免疫表型、细胞周期、端粒酶活性以及粘附分子的表达等生物学特性及其在体外扩增中的动态变化并与CD34+细胞进行比较。结果显示 ,新鲜脐血CD133+和CD34+细胞的含量分别为 ( 1.0 5±0 73) %和 ( 1.4 0± 0 .5 6 ) % ,CD34+细胞中 79.6 2 %为CD133+CD34+细胞 ,而CD133+细胞中 97%以上为CD133+CD34+细胞。短期扩增培养结果显示 ,CD133+细胞组扩增第 10天 ,CD133+,CD133+CD34+和CD34+CD38-细胞以及第 6天的CFU mix ,HPP CFC和CD34+CD38-细胞的扩增倍数要高于CD34+细胞组 ( P <0 .0 5 ) ;扩增中 ,CD133+CD34+细胞的比例逐渐下降 ,而CD133-CD34+和CD133-CD34-细胞的比例则逐渐上升。新鲜脐血CD133+和CD34+细胞的端粒酶活性较低 ,但高于CD34-细胞。扩增 1周后 ,端粒酶活性明显上调 ,15天以后又逐渐下降 ;90 %以上脐血CD133+细胞表达CD11a ,CD4 9d和CD5 4 ,约 5 0 %表达CD6 2L。扩增早期 ,CD4 9d表达上调 ,CD11a表达无明显变化 ,而CD5 4和CD6 2L则有下调趋势 ,随着扩增时间的延长 ,各种粘附分子的表达均有不同程度的下调。在整个扩增过程中 ,大部分CD34+细胞仍然表达CD11a,CD4 9d和CD5 4?

分离、鉴定CD133^+人肝癌干细胞以及^(131)I-CD133mAb对其生物学效应的影响

目的分离、鉴定CD133+人肝癌干细胞以及131I-CD133mAb对其生物学效应的影响。方法为选择最适的细胞系,流式细胞仪检测Huh-7和HepG2细胞中CD133表达率;磁珠分选Huh-7细胞,流式细胞仪检测分选后CD133表达率;体外成球、克隆形成实验及体内成瘤实验验证干细胞特性;氯胺T法制备131I标记CD133单克隆抗体(131ICD133mAb)并鉴定;取分选后的CD133+-Huh-7细胞、Huh-7细胞和HepG2细胞进行实验,将每种细胞分成4组(131ICD133mAb组、131I组、CD133mAb组、131I+CD133mAb组);MTT法检测各组对CD133+-Huh-7细胞生长抑制的最适剂量和各组在最适剂量作用后24、48、72 h对3组细胞生长抑制率;流式细胞仪检测131I-CD133mAb作用3组细胞72 h时细胞凋亡和细胞周期的变化。结果 Huh-7和HepG2细胞系CD133表达率分别为18.8%和5.2%,故选用Huh-7细胞进行分选;磁珠分选后CD133+-Huh-7细胞CD133的表达率为99.28%,与分选前比较差异有统计学意义(P<0.01)。CD133+-Huh-7细胞相对于CD133--Huh-7细胞具有更强的体外成球能力、克隆形成能力和体内成瘤能力。131I-CD133mAb标记率为87.92%,放射化学纯度为97.54%,具有较好的稳定性和免疫活性。当131I 4.8 MBq/100μL、CD133mAb 4.8μg/100μL时对CD133+-Huh-7细胞抑制率最高(P<0.05),取此放射性浓度进行实验。24、48、72 h时各组对CD133+-Huh-7细胞的抑制作用明显高于Huh-7和HepG2细胞组,131I-CD133mAb组对3种细胞生长抑制率明显高于其余各组,抑制率随时间的增加而升高(P<0.01)。131I-CD133mAb作用CD133+-Huh-7细胞72 h后凋亡率为31.21%,明显高于其余2组(P<0.05)。细胞周期检测结果显示CD133+-Huh-7细胞G0/G1期减少54.77%,明显高于其余2组(P<0.05)。结论 CD133+细胞具有肿瘤干细胞特性,131I-CD133mAb能特异性结合CD133+-Huh-7细胞,调控细胞周期和诱导细胞凋亡。

胃肠安及四藤方对人结肠癌细胞株干细胞CD133^+的影响

目的观察胃肠安和四藤方对结肠癌细胞株HCT-116、SW480、HT-29及其干细胞CD133^+的抑制作用。方法人结肠癌细胞株(HCT-116、SW480、HT-29)随机分为对照组和不同浓度胃肠安(500 mg/L和1 000mg/L)、四藤方(100 mg/L和200 mg/L)干预组,干预24 h后,CCK-8法测定细胞活力,倒置显微镜观察细胞形态;流式细胞仪分析胃肠安和四藤方对HCT-116、SW480、HT-29中CD133^+表达的影响。结果 CCK-8法检测结果显示,胃肠安及四藤方干预HCT-116、SW480、HT-29细胞后,细胞活力均下降且呈剂量依赖性;倒置显微镜观察发现:胃肠安及四藤方干预24 h,HCT-116、SW480、HT-29的细胞数量减少,细胞体积缩小,遮光性差,细胞悬浮、脱落而死亡。流式细胞术分析结果显示:对照组HCT-116、SW480、HT-29中CD133^+细胞的比例分别为12.52%、10.98%、2.33%;胃肠安方1 000 mg/L干预24 h后,CD133^+细胞比例分别下降为7.49%、5.36%、0.11%;四藤方组200 mg/L干预24 h后,CD133^+细胞比例分别下降为7.83%、6.45%、0.18%。结论不同结肠癌细胞株中CD133^+细胞比例有差异。胃肠安及四藤方均具有抑制结肠癌细胞株HCT-116、SW480、HT-29增殖的作用;但在同样的干预时间内,较高浓度胃肠安(1 000 mg/L)及四藤方(200 mg/L)才具有抑制3种细胞株干细胞CD133^+表达的作用。

骨髓基质细胞联合细胞因子对脐血CD133^+细胞体外扩增作用的研究

为了探讨胚胎骨髓基质细胞(FBMSC)联合细胞因子对脐血单个核细胞(MNC)中CD133+细胞的体外扩增作用,将新鲜脐血(CB)中分离出来的MNC接种于无血清培养体系中培养14天。实验分为4组:C组为空白对照组,不含基质细胞和细胞因子;S组为单用基质细胞组;F组为单用细胞因子组;SF组为联合使用基质细胞和细胞因子组。在第0,6,10及14天检测有核细胞总数、CD133+细胞数及集落形成单位(CFU)数。结果表明:各时间点SF组有核细胞总数的扩增倍数均高于其它组;除了第14天外,SF组在第6、10天时CD133+细胞数、CFU数的扩增倍数均高于其它组。结论:胚胎骨髓基质细胞对延缓造血细胞的分化具有重要的作用,基质细胞联合细胞因子可以有效的扩增脐血单个核细胞及其中的CD133+细胞,这是一种比较接近于临床移植要求的造血细胞体外扩增方法。

^(131)I标记CD133单链抗体对人肝癌CD133^+ HepG2干细胞的抑制作用

目的:研究131I标记CD133单链抗体(single chain rariable fragment,ScFv)在体外对人肝癌CD133+HepG2干细胞的抑制作用。方法:免疫磁珠分选HepG2细胞,流式细胞术检测分选前后HepG2细胞的CD133表达率,克隆形成实验及体内成瘤实验验证CD133+HepG2细胞的"干性"。氯胺T法131I标记CD133 ScFv并测定标记率、比活度、放射性浓度。将分选出的CD133+HepG2细胞分为131I-CD133抗体治疗组、131I治疗组、CD133抗体治疗组和131I+CD133抗体治疗组,MTT法检测各组中对CD133+HepG2细胞生长抑制的最适剂量和不同药物在12、48、72 h三个时间点对CD133+HepG2干细胞的生长抑制作用,流式细胞术检测各组细胞周期的变化。结果:分选的HepG2细胞的CD133表达率显著高于未分选细胞[(97.71±1.13)%vs(1.52±0.78)%,t=1.13、P=0.000]。CD133+HepG2细胞相对于CD133-HepG2细胞具有更强的体外成球、克隆形成能力[(45.03±1.35)%vs(7.4±0.54)%;t=3.92,P=0.000]和体内成瘤能力。131I-CD133 ScFv的标记率为88.92%,放射化学纯度为98.63%。当131I为3.7 MBq/100μl、CD133抗体为1μg/100μl时,对CD133+HepG2细胞的抑制率最高,达(89.58±0.74)%;在此剂量下131I-CD133 ScFv治疗组对CD133+HepG2细胞生长抑制率显著高于其余各实验组,且呈时间依赖性。131I-CD133 ScFv治疗组G0/G1期细胞比例为(27.50±1.12)%,较其余各组均明显减少(P<0.05)。结论:成功制备的131I-CD133 ScFv在体外能有效抑制人肝癌CD133+HepG2干细胞的生长。

人胎盘CD133^＋细胞具有高增殖潜能集落形成细胞特性

目的通过对人胎盘CD133+细胞群中高增殖潜能集落形成细胞(HPP-CFC)检测与生物学特性的分析,证明人胎盘存在早期造血干/祖细胞(HSPC)。方法采用机械法制备人胎盘组织(PT)单细胞悬液,用Histopaque-1007分离出单个核细胞(MNC),经磁式分选(MACS)富集CD133+细胞,培养28d后观察HPP-CFC集落形成能力,用流式细胞仪(FCM)对分选的细胞组份和HPP-CFC进行表型分析,实验全程用脐带血(UCB)作平行比较分析。结果培养28d后,PT-CD133+与UCB-CD133+细胞组份分别扩增了266和362倍,前者低于后者(P<0.01);PT-CD133+与UCB-CD133+细胞中HPP-CFC分别为(32.4±11.2)/5×103、(17.7±5.7)/5×103,前者形成的HPP-CFC数量明显高于后者(P<0.01);PT-CD133+、UCB-CD133+细胞培养至28d时,除UCB-CD133+组的CD133+CD34-亚群比例无明显改变外,CD133+CD34+、CD133-CD34+和CD133+CD34-(PT-CD133+组)亚型均比培养前减少。结论人胎盘组织CD133+细胞中存在HPP-CFC,说明胎盘CD133+细胞群中存在早期HSPC。

CD133^+胶质瘤干细胞化疗耐受机制分析

目的:探讨ABC超家族转运体蛋白在CD133+胶质瘤干细胞多药耐药性中的作用机制。方法:选取30例人脑胶质瘤标本,利用免疫磁珠法分选标本中的CD133+胶质瘤干细胞(悬浮细胞)和CD133-肿瘤细胞(黏附细胞),并进行分选细胞的体外扩增培养、传代与鉴定,应用免疫组化和RT-PCR法分别检测细胞中MDR1和MRP1的蛋白及其活性表达情况。结果:3例胶质母细胞瘤来源的CD133+细胞能在含血清培养基中形成肿瘤干细胞球,并进行1～3次传代;MDR1与MRP1耐药蛋白在CD133+细胞中高度表达,阳性细胞比例范围分别为18%～67%和23%～73%;MDR1与MRP1在CD133+细胞中的表达活性分别为在CD133-细胞中的16.1倍和19.6倍;多药耐药蛋白的阳性细胞比例和其表达活性与肿瘤的恶性程度呈正相关,而表达阳性率与肿瘤的病理级别无关;MDR1与MRP1在CD133+细胞中的表达有明显的相关性。结论:神经上皮肿瘤中仅有小部分细胞亚型(CD133+胶质瘤干细胞)具有内在耐药性(天然耐药性),而ABC超家族转运体蛋白MDR1与MRP1在CD133+胶质瘤干细胞中的共同过度表达是胶质瘤多药耐药的主要原因之一;CD133+细胞是胶质瘤化疗的关键性治疗标靶。

人胎盘CD133^+细胞具有LTC-IC集落启动细胞特性

本研究从功能上评价人胎盘CD133+细胞是否具有长期造血重建功能。采用免疫磁珠激活分选系统(MACS)富集胎盘CD133+细胞作为测试细胞,采用有限稀释法(LDA)设置4种不同浓度的测试细胞,用胎儿原代骨髓基质细胞制备的饲养层细胞共培养于长期培养体系中,以检测测试细胞中长期培养启动细胞(LTC-IC)的发生率及其增殖分化能力。结果表明:人胎盘CD133+细胞群中含有LTC-IC,其发生率为1/645;LTC-IC具有增殖分化为粒-单集落形成单位(CFU-GM)和混合集落形成单位(CFU-Mix)的能力;在所有LTC-IC阳性孔中,产生CFU-GM的孔占总阳性孔的71.4%,产生CFU-GM和CFU-Mix的孔占总阳性孔的28.6%。结论:人胎盘组织CD133+细胞群中存在LTC-IC,并具有造血早期祖细胞集落形成能力。

Lenti-TK介导间充质干细胞对鼻咽癌CD133^+干细胞的靶向迁移及杀伤作用

目的:观察慢病毒-胸苷激酶(lentivirus-thymidine kinase,Lenti-TK)/间充质干细胞(mesenchymal stem cell,MSC)对鼻咽癌CD133+干细胞的靶向迁移及杀伤作用。方法:构建包含TK基因的重组慢病毒表达载体Lenti-TK,感染MSC后得到Lenti-TK-MSC,RT-PCR及Western blotting检测Lenti-TK-MSC中HA-TK的表达。免疫磁珠法从鼻咽癌5-8F细胞中分选CD133+细胞;Transwell小室迁移实验检测Lenti-TK-MSC对CD133+5-8F细胞的趋向性;Lenti-TK-MSC联合更昔洛韦(ganciclovir,Lenti-TK-MSC/GCV)与CD133+5-8F细胞共培养,CCK-8试剂盒检测其对细胞的杀伤作用和旁观者效应。结果:成功构建重组慢病毒载体Lenti-TK,其滴度为1×108UT/ml,Lenti-TK(MOI=50)感染MSC 72 h时,感染效率达(95.1±0.1)%。Lenti-TK-MSC迁移至CD133+5-8F细胞组的细胞数明显多于CD133-5-8F细胞组、未分选5-8F细胞组[(83.0±8.7)vs(29.6±5.3)、(38.3±5.2),P=0.000]。Lenti-TK-MSC/GCV处理组与单独GCV处理组、Lenti-TK-MSC/GCV条件培养液(即Lenti-TK-MSC加入1 mg/L GCV培养48 h的培养上清)处理组相比,CD133+5-8F细胞的存活率明显降低[(37.2±2.3)%vs(98.5±3.1)%、(83.8±3.4)%,P=0.000]。Lenti-TK-MSC数量达到混合细胞总数(Lenti-TK-MSC和CD133+5-8F细胞)的20%时,CD133+5-8F细胞存活率为(68.2±2.3)%,表现出明显的旁观者杀伤效应。结论:Lenti-TK感染后MSC对鼻咽癌CD133+5-8F细胞具有靶向迁移及杀伤作用。

CD133^+骨肉瘤细胞的获取及其干细胞特性的初步鉴定

背景与目的:骨肉瘤是一种具有高度侵袭性的恶性肿瘤,目前应用化疗联合手术的治疗策略5年生存率仅为40%~60%,近期多项研究认为骨肉瘤细胞株中可能存在肿瘤干细胞。本研究旨在分离和鉴定骨肉瘤细胞株MG-63中的骨肉瘤干细胞样细胞。方法:用免疫磁珠分选法分离人骨肉瘤细胞株MG-63中的CD133+细胞,应用流式细胞学分析、蛋白质印迹法(Western blot)检测和免疫荧光染色测定CD133+细胞的纯度,应用CCK-8法、平板集落形成试验、划痕试验及Transwell侵袭小室试验,研究CD133+细胞的生物学特性。结果:本研究成功地从MG-63中获得CD133+细胞。流式细胞学分析、免疫荧光染色和Western blot分析显示该细胞群中高表达CD133(纯度约90%～95%);CD133+细胞的增殖、平板集落形成能力、迁移能力及侵袭能力均强于CD133-细胞(P<0.05)。结论:人骨肉瘤MG-63细胞株中存在肿瘤干细胞样细胞,CD133可能是骨肉瘤的干细胞标志物之一。