CD34^(+)细胞数对单倍体造血干细胞移植治疗恶性血液病的影响

背景:单倍体造血干细胞移植与较高的植入功能不良相关,因此经常要求更高的CD34^(+)细胞数量,但现有研究关于异基因造血干细胞移植CD34+细胞剂量和研究终点关系的结论是有争议的。目的:探究CD34^(+)细胞数对单倍体造血干细胞移植治疗恶性血液疾病临床结果的影响。方法:纳入2019年1月至2021年12月期间于郑州大学第一附属医院造血干细胞移植中心行单倍体造血干细胞移植的恶性血液病患者,总计135例。结合既往研究结果及移植中心经验,以CD34+细胞数5.0×10^(6)/kg为截止点,将队列分为2组。评估两组的移植物植入情况、复发率及非复发死亡率、总生存期和无进展生存期等相关临床指标。结果与结论:①CD34+细胞剂量与血小板的植入相关,高剂量组血小板的植入时间早于低剂量组(14 d vs.16 d,P=0.013)。②两组患者3年总生存期无显著差异(67.5%vs.53.8%,P=0.257);两组间的无进展生存期也无显著性差异(65.6%vs.44.2%,P=0.106),但根据疾病风险指数(DRI)进行分层分析后发现低危患者高剂量组的3年无进展生存期较低剂量组升高(72.0%vs.49.3%,P=0.036)。③高剂量组3年累积复发率小于低剂量组(16.0%vs.33.5%,P=0.05)。④两组100 d内非复发死亡率高剂量组大于低剂量组,但无显著差异(17.3%vs.6.7%,P=0.070);进行分层分析发现,高危患者中高剂量组100 d内非复发死亡率明显高于低剂量组(20.0%vs.3.3%,P=0.046)。⑤综上所述,输注>5.0×10^(6)/kg的CD34^(+)细胞可促进血小板早期植入,可改善移植中低危风险患者的3年无进展生存期,并且降低移植后累积复发率;但在高危患者中,高剂量CD34+细胞导致移植后100 d内的非复发死亡率增高,考虑可能与移植后早期重度急性移植物抗宿主病的发生增多相关,因此考虑对回输高剂量CD34+细胞的患者应加强移植物抗宿主病的监测。

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。

Effect of Homoharringtonine on Bone Morrow CD34^+CD7^+ Cells in Chronic Granulocytic Leukemia

Objective： To explore the effect of homoharringtonine （HHT） on bone morrow CD34^＋CD7^＋ cells in chronic granulocytic leukemia （CGL）. Methods： The changes of bone morrow CD34^＋CD7^＋ cells were observed after the treatment of HHT in 23 cases with CGL. The proliferation and apoptosis of CD34^＋CD7^＋ cells treated with HHT in vitro were studied. Results： The proportion of CD34^＋CD7^＋ cells in CGL （0.145±0.021） was higher than that of normal control （0.052±0.013）. The proportion of CD34^＋CD7^＋ cells in patients who got cytogenetic responses to HHT （0.072±0.020） decreased remarkably, but not in those patients who did not got cytogenetic responses to HHT, （0.137±0.023）. the proliferation of CD34^＋ cells was inhibited and the proportion of CD34^＋CD7^＋ cells decreased after cultured with HHT （0.134 in 24 h, 0.126 in 48 h and 0.102 in 72）. The apoptosis rate of CD34^＋CD7^＋ cells was higher than that in CD34^＋CDT cells （35.39%±4.39% versus 24.57%±4.01%, P〈0.05） 72 h after culture with HHT. Conclusion： The proportion of CD34^＋CD7^＋ cells in CGL was higher than that of normal control and HHT may inhibit the proliferation and induce apoptosis of bone marrow CD34^＋CD7^＋ cells.

Effects of dendritic cells from cord blood CD34^+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCTD) mice.METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocarcinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect.RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%,47.92% vs 19.44%, P＜0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P＞0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80%vs 100%, P＜0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11 d vs 7 d, P＜0.01).CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

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.

^(18)F-FDG-PET/CT糖代谢参数与B7-H3、CD8^(+)T细胞在结直肠癌中的相关性研究

背景:糖酵解功能与结直肠癌的增殖、转移、耐药性等具有明显相关性,对如何量化评估糖酵解水平还缺乏相应指标。目的:探讨术前^(18)F-FDG-PET/CT糖代谢参数与结直肠癌患者临床病理特征、B7-H3、CD8^(+)T细胞表达的相关性。方法:收集2021年4月—2022年7月苏州大学附属第一医院确诊的40例结直肠腺癌患者,所有患者在术前2周内行全身^(18)F-FDG-PET/CT检查,获取最大标准化摄取值(SUVmax)、平均标准化摄取值(SUVmean)、肿瘤代谢体积(MTV)、病灶糖酵解总量(TLG)。采用免疫组化染色检测B7-H3、CD8^(+)T细胞表达,并分析其与临床病理特征、^(18)F-FDG-PET/CT糖代谢参数的相关性。结果:MTV、TLG与结直肠癌患者的T分期、淋巴结转移相关(P<0.05),而SUVmax、SUVmean、MTV、TLG与其他临床病理特征无关(P>0.05)。与癌旁组织相比,结直肠癌组织中B7-H3表达显著升高(P<0.001),CD8^(+)T细胞表达显著降低(P<0.001)。结直肠癌组织中B7-H3表达与CD8^(+)T细胞表达无关(P>0.05);B7-H3表达与T分期、淋巴结转移相关(P<0.05);CD8^(+)T细胞表达与肿瘤大小、分化程度相关(P<0.05)。结直肠癌组织中B7-H3表达与SUVmax、SUVmean、MTV和TLG相关(P<0.05),CD8^(+)T细胞与MTV、TLG相关(P<0.01)。结论:^(18)F-FDG-PET/CT糖代谢参数中的MTV和TLG能更有效地反映结直肠癌病理特征和转移情况,可能具有更高的评估价值。在结直肠癌组织中,B7-H3明显高表达、CD8^(+)T细胞明显低表达,两者与PET/CT糖代谢参数均存在相关性。联合^(18)F-FDG-PET/CT和B7-H3、CD8^(+)T细胞的表达情况可能为结直肠癌临床判断分期、转移以及评估糖酵解水平等提供更为可靠的参考价值。

Expression of Caspase－3 in Cord Blood CD34^＋ Cells during Culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34^+ cells from cord blood (CB) during culture in vitro with different growth factors. Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34^+ CB cells during culture in vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34^+ cells. The expression of caspase-3 mRNA and protein was upregulated when these cellswere first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34^+ cells as well as during the first 3 days expansion with cytokines. With longer culture time in vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34^+ cells during expansion in vitro.

慢性阻塞性肺疾病患者血清VEGF水平、外周血CD34^(+)细胞数量与骨骼肌功能障碍的关系

目的检测慢性阻塞性肺疾病(COPD)患者血清血管内皮生长因子(VEGF)水平、外周血CD34^(+)细胞数量,并分析二者与骨骼肌功能障碍的关系。方法选取北京市门头沟区医院2021年7月至2022年12月收治的96例COPD患者,依据COPD患者骨骼肌功能障碍诊断标准将患者分为骨骼肌功能障碍组52例和无骨骼肌功能障碍组44例;选择同期在医院体检的健康者96例为对照组。测定所有受试者肺功能指标[用力肺活量(FVC)、第一秒用力呼气容积占预计值的百分比(FEV1%)、第一秒用力呼气容积/用力肺活量(FEV1/FVC)],骨骼肌功能[握力、4m步速、5次起坐试验时间、6min步行距离(6MWD)],血清C-反应蛋白(CRP)、白细胞计数(WBC)水平;酶联免疫吸附法、流式细胞仪分别检测血清VEGF水平、CD34^(+)细胞数量;分析COPD患者血清VEGF、CD34^(+)细胞数量、骨骼肌功能的相关性,COPD患者发生骨骼肌功能障碍的影响因素,血清VEGF、CD34^(+)细胞数量预测COPD患者发生骨骼肌功能障碍的价值。结果与对照组相比,无骨骼肌功能障碍组FVC、FEV1%、FEV1/FVC、握力、4m步速、6MWD降低(P<0.05),5次起坐试验时间较长、CRP、WBC水平升高(P<0.05),骨骼肌功能障碍组BMI、FVC、FEV1%、FEV1/FVC、握力、4m步速、6MWD降低(P<0.05),5次起坐试验时间较长、CRP、WBC水平升高(P<0.05);与无骨骼肌功能障碍组相比,骨骼肌功能障碍组BMI、握力、4m步速、6MWD降低(P<0.05),5次起坐试验时间较长(P<0.05)。对照组、无骨骼肌功能障碍组、骨骼肌功能障碍组血清VEGF水平依次升高,CD34^(+)细胞数量依次降低(P<0.05)。COPD患者血清VEGF水平与CD34^(+)细胞数量呈负相关(P<0.05);血清VEGF水平与握力、4m步速、6MWD呈负相关(P<0.05),与5次起坐试验时间呈正相关(P<0.05);CD34^(+)细胞数量与握力、4m步速、6MWD呈正相关(P<0.05),与5次起坐试验时间呈负相关(P<0.05)。BMI低水平、VEGF高水平、CD34^(+)细胞数量低水平是COPD患者发生骨骼肌功能障碍的危险因素(P<0.05)。血清VEGF、CD34^(+)细胞数量、两者联合预测COPD患者发生骨骼肌功能障碍的曲线下面积分别为0.900、0.835、0.935。结论COPD患者血清VEGF水平明显增加,外周血CD34^(+)细胞数量明显下降,二者水平变化与骨骼肌功能障碍的发生具有相关性。

组蛋白H3K27甲基化抑制剂EPZ005687对U937细胞和正常骨髓CD34^+细胞凋亡、增殖及细胞周期的影响

本研究目的在于探讨组蛋白H3K27甲基化抑制剂新药EPZ005687对白血病细胞系U937细胞和正常骨髓CD34+细胞的凋亡、增殖抑制和细胞周期的影响。以不同浓度的EPZ005687作用于U937细胞,在不同时间点采用Annexin V/PI法检测细胞凋亡,WST-1法检测细胞增殖,7-AAD流式细胞术检测法检测细胞周期,免疫化学法检测H3K27组蛋白甲基化活性。结果表明:EPZ005687显著诱导U937细胞的凋亡,在0.5、1、5和10μmol/L浓度下作用于U937细胞48 h后,其凋亡率分别为3.96%±0.79%、5.74%±0.73%、13.34%±1.77%和25.24%±2.55%,而EPZ005687对正常骨髓CD34+细胞的凋亡影响较小;在0.5、1、5和10μmol/L浓度下CD34+细胞凋亡率分别为3.64%±0.62%、4.28%±0.99%、6.18%±1.19%和7.56%±1.34%;0.5、1、5和10μmol/L浓度的EPZ005687分别作用于U937细胞12 h至96 h,作用CD34+细胞1至5 d,明显观察到EPZ005687显著抑制U937细胞的增殖且呈剂量依赖性,而对正常CD34+细胞的增殖抑制并不明显。细胞周期分析显示,1μmol/L EPZ005687作用72 h可使U937细胞明显阻滞于G1期(64.18%±13.27%vs 49.43%±12.54%),S期细胞比例明显下降低(9.67%±2.61%vs 15.26%±5.58%),而正常CD34+细胞因多数细胞位于G1期,S期细胞较少而不受其影响。进一步的H3K27组蛋白甲基化检测分析显示,EZP005687可明显地降低U937细胞的H3K27组蛋白甲基化,而不降低正常CD34+细胞的H3K27组蛋白甲基化。结论:组蛋白H3K27甲基化抑制剂EPZ005687明显抑制U937细胞的增殖,诱导细胞凋亡和细胞周期阻滞,但对正常造血细胞CD34+影响较小,可作为一种潜在的血液肿瘤治疗药物应用于临床。

慢性粒细胞性白血病患者骨髓源bcr/abl融合基因阳性Flk1^+CD31^-CD34^-干细胞体外抗STI571机制的初步研究

为进一步了解STI571对慢性粒细胞性白血病(CML)患者体内bcr/abl融合基因阳性的原始及定向白血病干/祖细胞分化及增殖的影响,更深入阐明部分CML患者在经历一段血液学甚至是细胞遗传学水平上的完全缓解后复发及对STI571的耐药机制,利用从CML患者骨髓分离到的具有血管母细胞特性、bcr/abl融合基因阳性、免疫表型为Flk1+CD31-CD34-细胞,体外检测了STI571对其在造血集落培养基中的分化及处于分化阶段时的增殖抑制作用。结果显示:浓度为5μmol/LSTI571,维持作用96小时(病人体内维持96小时的STI571浓度只可能达到1-2μmol/L),即可有效抑制定向造血祖细胞的增殖。没有充分的证据显示,相对原始的bcr/abl融合基因阳性、免疫表型为Flk1+CD31-CD34-细胞的分化及增殖受到明显的抑制作用。结论:CML患者体内的原始白血病干/祖细胞对STI571具有一定的抗性,临床上所观察到的CML患者在运用STI571一段时间后出现正常的造血恢复现象,可能仅仅是因为STI571杀死或抑制了定向恶性白血病祖细胞的增殖,但随着时间的推移,在经历了短暂的血液学甚至是细胞遗传学水平上的缓解后,对STI571耐药的原始白血病干/祖细胞终究会再次导致CML的复发。

In vitro study of the influence of GST-π gene transfer on drug-resistance of human cord blood CD34^+ cells

Objective:To investigate the influence of GST-π gene transfer on drug-resistance of human cord blood CD34 + cells.Methods:CD34 + cells were purified from cord blood from normal full-term pregnancy.Gene transduction into human cord blood CD34 + cells was carried out using GST-π gene containing retrovirus vector.The GST-π gene expression in transduced CD34 + cell was confirmed by RT-PCR.After confirmation of GST-π gene transfer,the transfected CD34 + cells were cultured by colony assay in the presence of carboplatin.Results:GST-π mRNA was detected in 30% of CFU-GM derived from GST-π gene transduced CD34 + cells.In vitro drug resistance test showed that the number of CFU-GM formed was significantly higher (2～3 fold) in GST-π gene transduced CD34 + cells than untransduced CD34 +cells.Conclusion:GST-π gene transfer can confer resistance to hematopoietic progenitors against carboplatin in vitro.

C57BL/6j小鼠骨髓CD34^+造血干细胞来源的iDC的体外培养与鉴定

目的:探讨以恰当的体外定向诱导和培养小鼠骨髓CD34+造血干细胞以获得足够数量的未成熟树突状细胞(immatureden driticcells,iDC)的方法。方法:采用自健康C57BL/6j小鼠骨髓中分离CD34+造血干细胞,进而于含rmGM-CSF和rmIL-4的RPMI-1640完全培养液中定向诱导和扩增,收集悬浮生长细胞后,予以流式细胞学检测其CD80、CD86和CD11c等表面分子的表达,扫描电镜观察悬浮细胞的形态。结果:去除红细胞的骨髓细胞培养4h后,10%左右细胞贴壁生长。加rmGM-CSF和rmIL-4培养,逐渐呈集落样悬浮生长,有树枝状突起。第6天时,细胞集落呈密集的葡萄串状,细胞体积增大,毛刺状或根须状突起更明显。流式细胞学显示:体外培养第7天的iDC的表面分子CD11c的表达率为75%~81%[(78.4±8.36)%],表面分子CD80的表达率为32%~51%[(38.26±8.77)%],而表面分子CD86的表达率为18%~53%[(27.5±8.30)%]。扫描电镜见细胞有典型的树枝状突起。结论:联合使用rmGM-CSF和rmIL-4定向诱导培养骨髓CD34+造血干细胞,可大量扩增出iDC。

妊娠及围产因素对子代脐带血CD34^(+)造血干细胞的影响分析

目的分析妊娠及围产因素对子代CD34^(+)造血干细胞数量和脐带血有核细胞总数的影响。方法选择乌鲁木齐市妇幼保健院2021年5月至2022年5月收集的120例健康产妇的新生儿脐带血标本,其中男性60例,女性60例;新生儿体质量2500~4000 g。采用XE-2100全自动血液分析仪对子代脐带血中的有核细胞进行计数。采用流式细胞分析仪分析CD34^(+)造血干细胞。收集新生儿及相应产妇的临床资料,主要包括孕母年龄、孕母血型、孕母身体质量指数(BMI)、孕母过敏史、妊娠期糖尿病、妊娠期高血压、分娩方式、产次、孕周、新生儿性别、新生儿体质量、胎盘质量、是否胎膜早破、是否羊水污染、是否脐带绕颈等信息,分析其对脐带血CD34^(+)造血干细胞影响。结果妊娠期因素主要有孕母年龄、孕母血型、孕母BMI、孕母过敏史、妊娠期糖尿病、妊娠期高血压、分娩方式、产次和孕周。围产期因素主要包括新生儿性别、新生儿体质量、胎盘质量、胎膜早破、羊水污染、脐带绕颈。根据临床信息分组进行组间比较发现,孕母血型、孕母BMI、孕母过敏史、妊娠期糖尿病、妊娠期高血压、分娩方式、产次、新生儿性别、新生儿体质量、胎盘质量、是否胎膜早破、是否羊水污染、是否脐带绕颈等因素与脐带血有核细胞总数和CD34^(+)造血干细胞数量无相关性(P>0.05),而孕母年龄和孕周均与CD34^(+)造血干细胞数量呈正相关(P<0.05)。结论子代脐带血中CD34^(+)造血干细胞数量与孕母年龄和孕周密切相关,孕母的年龄越大或孕周越大则CD34^(+)造血干细胞数量越多,这为脐血库筛选脐带血入库时提供了更多的理论依据。

监测外周血CD34^(+)细胞计数预测普乐沙福联合G-CSF自体干细胞动员的效果

目的探讨外周血CD34阳性(CD34^(+))细胞计数对普乐沙福自体干细胞动员效果的预测价值。方法回顾性分析2021年5月—2023年7月中山大学附属第七医院使用人粒细胞集落刺激因子(G-CSF)联合普乐沙福进行自体干细胞动员的13例患者临床资料,分析普乐沙福动员前后外周血CD34^(+)细胞计数的变化及干细胞采集情况。结果共有13例患者纳入研究,包括淋巴瘤10例和多发性骨髓瘤3例。多发性骨髓瘤患者中1例为新诊断,另2例为复发患者;淋巴瘤患者中3例为套细胞淋巴瘤,6例为弥漫大B细胞淋巴瘤(包括1例复发),1例为B细胞淋巴瘤(不能明确类型)。本研究纳入的患者均使用G-CSF动员,在使用普乐沙福后CD34^(+)细胞计数均升高,使用普乐沙福前中位CD34^(+)细胞计数为13.3(2.5~76.1)/μL,使用普乐沙福后中位CD34^(+)细胞计数为73.6(10.4~208.70)/μL,升高4.18(1.99~13.60)倍。13例患者中有2例患者在使用普乐沙福前外周血CD34^(+)细胞计数<5/μL,均动员失败。Spearman相关分析结果显示,使用普乐沙福后CD34^(+)细胞计数与使用普乐沙福前CD34^(+)细胞数呈正相关(rs=0.769,P=0.003)。多元线性回归分析显示,使用普乐沙福后CD34^(+)细胞计数能较好地预测采集结果(P=0.004)。结论监测外周血CD34^(+)细胞计数可预测普乐沙福自体干细胞动员效果,使用普乐沙福后CD34^(+)细胞计数越多,CD34^(+)细胞采集量越大。

转染外源性Fas配体基因的正常骨髓CD34^+细胞诱导U937细胞凋亡的实验研究

目的:观察转染外源性FasL的CD34+细胞对白血病细胞系U937的凋亡诱导作用,探讨通过向CD34+细胞转染FasL以增强移植后GVL效应的可能性。方法:免疫磁珠法分选骨髓CD34+细胞并进行体外扩增培养;FasL基因逆转录病毒途径转染CD34+细胞,并与白血病细胞U937混合培养,加入或不加入柔红霉素、阿糖胞苷,借助TUNEL、流式细胞仪检测细胞的凋亡率。结果:逆转录病毒上清转染后48～72h,流式细胞仪检测24.2%±2.4%CD34+细胞表达FasL(P<0.01)(未转染的CD34+细胞FasL阳性率为7.6%±1.1%)。未转染或转染外源FasL的CD34+细胞与白血病细胞U937以1:1的比例混合培养18h后,细胞凋亡率分别为5.0%±1.3%、10.8%±0.6%(P<0.01);FasL+CD34++DNR或FasL+CD34++Arac作用后,细胞凋亡率分别为13.4%±1.0%(P<0.05)、17.95%±1.3%(P<0.01)。结论:表达外源性FasL的骨髓CD34+细胞可诱导白血病细胞U937凋亡。提示植入转染外源性FasL基因的骨髓CD34+细胞可增强BMT后的GVL效应。

血清sB7-H3、CCSP、CD4^(+)/CD8^(+)水平与MPP患儿病情程度的关系

目的:探讨血清共刺激分子可溶性B7-H3(Soluble costimulatory molecule B7-H3,sB7-H3)、Clara细胞分泌蛋白(Clara cell secretory protein,CCSP)、CD4^(+)/CD8^(+)水平与肺炎支原体肺炎(Mycoplasma pneumoniae pneumonia,MPP)患儿病情程度的关系。方法:选取我院2020年5月至2022年5月收治的80例MPP患儿为研究组,另选同期80例体检健康儿童为对照组。比较不同组别入院时血清sB7-H3、CCSP、CD4^(+)/CD8^(+)水平、肺功能指标及临床肺部感染评分(Clinical pulmonary infection score,CPIS)评分,并分析入院时血清sB7-H3、CCSP、CD4^(+)/CD8^(+)水平与CPIS评分及肺功能指标相关性及危险因素。结果:与对照组相比,入院时研究组血清sB7-H3水平较高,CCSP、CD4^(+)/CD8^(+)水平较低(P<0.05);重症患儿入院时血清sB7-H3水平、呼吸频率、CPIS评分高于轻症患儿,CCSP、CD4^(+)/CD8^(+)水平、潮气量、达峰时间/呼气时间、达峰容积/潮气量低于轻症患儿(P<0.05);入院时血清sB7-H3水平与潮气量、达峰时间/呼气时间、达峰容积/潮气量呈负相关,与呼吸频率、CPIS评分呈正相关(P<0.05);血清CCSP水平、血清CD4^(+)/CD8^(+)水平与潮气量、达峰时间/呼气时间、达峰容积/潮气量呈正相关,与呼吸频率、CPIS评分呈负相关(P<0.05);Logistic回归分析显示入院时血清sB7-H3水平(>7.72μg·L^(-1))、CCSP水平(≤8.38μg·L^(-1))、CD4^(+)/CD8^(+)水平(≤1.32)是重症MPP发生的危险因素(P<0.05)。结论:血清sB7-H3水平于MPP患者中呈高表达,血清CD4^(+)/CD8^(+)、CCSP水平呈低表达,动态监测血清变化有助于了解病情变化,为临床制定治疗方案起到指导作用。

Interleukin-15 Promotes the Commitment of Cord Blood CD34^+ Stem Cells into NK Cells

To explore the effect of rhIL-15 on CB-CD34 + stem cells committing to NK cells, CD34 + stem cells were obtained from cord blood (CB) by magnetic-assisted cell sorting (MACS) method. CD3, CD16 and CD56 molecules expressed on cell surface were detected by flow cytometer. MTT method was used to test the cytotoxicity of NK cells. The results were that stem cell factor (SCF) alone has no effect on CD34 + stem cells. IL-15 stimulated CD34 + stem cells commit to NK cells, and SCF showed strong synergistic effect with IL-15. It was concluded that IL-15 and SCF played different roles during NK cell development, IL-15 promoted CD34 + stem cells differentiate to NK cell precursor and SCF improved the effects of IL-15 on NK cell differentiation.

STI571、三氧化二砷和Velcade对bcr/abl^+-CD34^+细胞增殖、凋亡的影响

目的研究STI571、三氧化二砷(As2O3)和Velcade在单独及联合应用时对bcr/abl+-CD34+细胞增殖、凋亡的影响。方法提取慢性粒细胞白血病患者骨髓的bcr/abl+-CD34+细胞,用STI571、As2O3、Velcade单独及联合处理96h,通过CCK-8分析及流式细胞术分别检测细胞增殖、凋亡的变化,并用Hoechst33342染色及荧光显微镜技术观察凋亡细胞形态学改变。同时检测上述方案对正常骨髓CD34+细胞的抑制作用。结果STI571、As2O3及Velcade对bcr/abl+-CD34+细胞的作用呈剂量依赖性,其中在低浓度时以抑制增殖作用为主,促凋亡作用不明显。当0.25～2μmol/LSTI571分别与2.5μmol/LAs2O3及15nmol/LVelcade联合后,抑制率和凋亡率均显著提高,呈相加或协同作用,且对正常CD34+细胞的抑制作用无进一步增强。结论As2O3及Velcade与STI571联合能够增强对BCR/ABL+-CD34+细胞的作用,具有一定临床意义。

rhIL-17对小鼠造血前体细胞和人脐血CD34^+干细胞分化发育的影响

目的:探讨重组人白细胞介素-17(Interleukin-17,IL-17)对小鼠骨髓造血前体细胞和人脐血来源的CD34^+干细胞生长发育的影响.方法:采用常规方法采集小鼠造血前体细胞;采用Mini-MACS分离技术,从正常人脐血分离人CD34^+干细胞.体外加入IL-17和/或GM-CSF、IL-4培养分离的前体细胞,应用流式细胞仪检测其表型,采用ELISA法检测了其分泌的IL-12水平,通过[~3H]-TdR掺入法测定其刺激同种异体T淋巴细胞增殖的能力.结果:IL-17促进了小鼠骨髓来源的未成熟DC表达Ia,B7-2等免疫分子,促使其分泌较高水平的IL-12,该细胞也能刺激同种异体T细胞有效增殖,表现出了成熟DC的特征.IL-17单独培养9d促使人脐血CD34^+干细胞扩增了2倍,部分细胞高表达CD1a及B7-2,低表达HLA-DR,未检测到CD83的表达.该细胞能促使同种异体T细胞增殖,但作用较弱;而rhIL-17与GM-CSF联合培养后扩增了14倍,培养细胞中CD1a、B7-2阳性细胞的比例明显升高,且此细胞刺激同种异体T细胞增殖的能力较强.结论:IL-17体外可促进小鼠骨髓造血前体细胞来源的DC成熟;与GM-CSF联合培养既能促进CD34^+干细胞增殖,又能使之获得DC特征,初步提示IL-17与GM-CSF联合作用可促进CD34^+干细胞向DC分化.

An Association between Immunosenescence and CD4^+CD25^+ Regulatory T Cells: A Systematic Review

Objective Age-related increment of the prevalence of CD4^＋CD25^＋ regulatory T （Treg） cells were described controversially, and whether such changes explain immune dysfunction in the elderly is still unclear. The aim of this systematic review is to evaluate the role of the Tregs in immunosenescence. Methods Medline and manual searches were performed to identify all published epidemiological and animal studies investigating the efficacy of the association between immunosenescence and Treg cells. Results It was founded that the frequency, phenotypic characteristics, and number/function of Tregs were altered significantly with aging. Medical conditions in individuals with advanced ageas well as apoptosis intensity of Treg cells had an impact on the accumulation of Tregs which in turn could deteriorate cytotoxic activity of CD8＋ T and NK cells and production of IL-2. The range of immune cells that could be suppressed by Treg cells was quite wide and covered CD4^＋CD25^＋ T cells, NK cells, dendritic cells and even monocytes. These changes were observed both in humans and experimental animals. Besides, it was believed that frequency of Tregs increased with age and was accompanied by intensified suppressive activity for Tregs in patients, for example, with Alzheimer disease （AD） and Parkinson disease （PD）. The impaired condition of CD4＋ T cells, so-called immunosenescence, rendered transplant recipients less responsive to an allogeneic kidney graft, an effect that was limited to transplant recipients who were aged over 60 years. Conclusions Treg cells are associated with immunosenescence. All these changes contribute to the aging-related decline of immune responses and lead to the higher risk of immune-mediated diseases, cancer or infections in aged individuals.