抗体滴定在血液细胞流式细胞术中的应用

流式细胞术（flow cytometry）可以实现高速、逐一的细胞定量分析和分选，是研究和诊断血液病的重要手段之一。但是由于不同实验所用细胞和实验条件不同，经常存在抗原阴性细胞非特异染色等问题。利用抗体滴定法，可通过计算、比较染色指数，得到使抗原阳性细胞群和阴性细胞群达到最佳分离效果的实验条件。为了优化血液细胞流式细胞术中荧光抗体染色的实验条件，以小鼠骨髓细胞为被标记细胞，选择利用非串联荧光染料FITC标记的大鼠抗小鼠CD11b抗体（FITC Rat Anti-Mouse CD11b）和串联荧光染料APC-eFluor780标记的大鼠抗小鼠CD11b抗体（APC-eFluor780 Rat Anti-Mouse CD11b）进行标记。通过计算不同浓度抗体标记小鼠骨髓细胞的染色指数进行抗体滴定，确定合适的抗体浓度区间，进而分析细胞数量、染色时间及固定步骤对抗体染色指数的影响，探究影响血液细胞抗体染色的关键因素。结果显示，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b的浓度分别在0.156~2.500 μg·mL-1和0.25~1.00 μg·mL-1范围内染色指数较高，但是超出这个范围的抗体浓度会使染色指数降低；抗体浓度、染色时间一定时，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b分别在细胞数量为1.56×105~5.00×106 cells·管-1和1.56×105~3.12×105 cells·管-1范围内染色指数较高，但是超出这个范围的细胞数量会使染色指数降低；抗体浓度、细胞数量一定时，对于FITC Rat Anti-Mouse CD11b，随着染色时间的延长，染色指数降低，而APC-eFluor780 Rat Anti-Mouse CD11b与之相反；通过比较固定前后染色指数的高低发现，FITC Rat Anti-Mouse CD11b和APC-eFluor780 Rat Anti-Mouse CD11b在固定后染色指数均显著下降（P<0.01和P<0.05）。研究结果提供了一种通过抗体滴定优化流式分析血液细胞的方法，并指出在特定实验中根据抗体滴定结果选择合适的抗体浓度、细胞数量、染色时间和固定步骤对标记血液细胞进行流式检测的研究至关重要。     

基于c-Kit~+显微形态标记及流式分选分离心脏Telocytes

目的:建立基于显微形态标记及流式分选分离大鼠心脏Telocytes(CTs)的方法。方法:采用抗体c-Kit免疫磁珠法获得原代心脏Telocytes,显微注射Di I标记具"Telopode"典型形态的细胞,使用流式分离及回收单个Di I+细胞;使用免疫荧光技术和RT-PCR方法对经回收的单个细胞来源的细胞进行表型鉴定。结果:显微注射Di I能较好地标记具Telocytes典型形态的细胞,结合流式分选及单细胞回收,能有效回收经标记的Di I+细胞,经回收的Di I标记阳性Telocytes的贴壁率为14.9%,增殖率为5.6%,呈克隆样生长率为2.4%,该呈克隆样生长的细胞能通过消化传代。免疫荧光染色证明,该回收Telocytes表达其相对特异性表面标记物c-Kit和CD34,RT-PCR的结果也证明:经回收Telocytes表达其相对特异基因c-Kit、CD34、Vimentin和PDGFR-β。结论:研究所建立的方法能有效分离及单细胞回收高纯度的心脏Telocytes,经回收的心脏Telocytes具有增殖及传代能力,且能维持其特异表型。     

磁性细胞分选技术的应用与生物学评价

磁性细胞分选技术是一种利用超顺磁性纳米复合材料进行细胞分选的细胞高度特异性快速分选技术,在免疫学、干细胞学、肿瘤学和临床医学等领域应用广泛。本文综合阐述了磁性细胞分选技术的分类和应用,讨论了近几年出现的几项基于磁性细胞分选的新技术和面临的挑战。重点分析了磁性细胞分选产品生物学评价的必要性,并提出了10项与磁性细胞分选产品相关的生物学评价技术参数：得率、纯度、无菌、细胞毒性、细胞形态、活率、细胞的光散射特性、细胞的荧光抗体标记能力、细胞活化、细胞增殖,该评价技术参数的提出对磁性细胞分选规范化应用具有重要的推动作用。     

流式细胞仪分选人外周血T淋巴细胞的方法建立与评价

目的:利用流式细胞仪同时分离外人周血单个核细胞中T淋巴细胞并检测其分离纯度及存活率。方法:本文采用流式细胞仪同时分选人外周血CD4~+、CD8~+T淋巴细胞为例,推而广之,采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞,采用流式细胞仪同时分选CD4~+、CD8~+T淋巴细胞,分离细胞再通过流式细胞仪回测其分离纯度并通过台盼蓝染色检测分离细胞的存活率。结果:采用此方法能有效人外周血细胞CD4~+、CD8~+T淋巴细胞,分选前CD4~+淋巴细胞纯度为(50.5±11.5)%、CD8~+T淋巴细胞纯度为纯度为(15.4±7.1)%;分选后CD4~+T淋巴细胞纯度为(94.3±1.3)%、CD8~+T淋巴细胞纯度为(93.6±1.6)%;分选后CD4~+T淋巴细胞存活率为(95.3±1.8)%,CD8~+T淋巴细胞存活率为(94.8±1.5)%,细胞的形态完整。结论:采用人外周血淋巴细胞分离液梯度离心法制备外周血单个核细胞后利用流式细胞仪分选的方法能够高效、快速的分离人外周血CD4~+、CD8~+T淋巴细胞,且存活率高,为进一步研究其功能提供了保证。采用不同的荧光抗体标记其他淋巴细胞亚群,也能高效、快速的分离出细胞。     

一个用于转染细胞分选的双顺反子载体的构建及其应用

为简化转染细胞的分选过程,构建了一个含有细胞表面标志 CD34 基因的双顺反子载体 p3.1-IRES-CD34. 利用来源于脑心肌炎病毒 (EMCV) 的内部核糖体进入位点 (IRES) ,实现目的基因与 CD34 基因的共同表达 . 将绿色荧光蛋白 (EGFP) 作为目的基因插入载体的多克隆位点,然后转染 NIH-3T3 细胞,通过免疫磁珠分选 (MACS) 方法来分选细胞 . 结果表明：对于转染细胞,均可实现快速分选 ( 瞬时转染细胞约 48 h ,稳定转染 10~15 天 ) ,并且获得较高纯度 (95% 以上 ) 的表达目的基因细胞 .     

免疫磁珠纯化小鼠精原干细胞的研究

精原干细胞（spermatogonial stem cells,SSCs）富集纯化是利用SSCs进行基因修饰新方法等研究的前提基础。采用免疫磁珠分选法,使用干细胞抗体CD90.2进行小鼠SSCs的纯化富集,并采用流式细胞分析法和定量PCR验证了磁珠分选效率。流式细胞分析结果：免疫磁珠分选后SSCs纯度为50.11%。荧光定量PCR检测结果：磁珠分选后支持细胞特异表达基因 GATA4 显著下调（6倍）、SSCs表达基因 GFRα-1 上调（6.5倍）、生殖干细胞特异表达基因 OCT4 极显著上调（5.9倍）,3个基因相对表达量的变化说明,免疫磁珠分选效率为6倍。流式细胞分析法所产生的偏差可能是受到了未解离磁珠及SSCs本身转基因荧光的影响。     

稳定转染结合流式分选技术筛选猪GBP1,GBP2基因高表达PK-15细胞

猪的GBP1,GBP2基因是重要的抗病候选基因,建立其高表达细胞模型可为深入研究基因的抗病能力及机理提供良好的素材。利用pEGFP载体上的Neor抗性筛选标记,采用G418药物筛选方法,结合利用GFP荧光标记,采用流式细胞分选技术,获得了超表达猪GBP1和GBP2基因的PK-15细胞,并通过定量PCR方法对筛选后细胞的超表达效果进行验证。结果显示猪GBP1和GBP2基因在转录水平的表达量相对于正常的PK-15细胞分别升高了近40倍和60倍,表明药物筛选结合流式分选是获得目的基因稳定高表达细胞株的快速便捷的方法。     

小鼠骨片间充质干细胞分离培养及鉴定

目的建立小鼠骨片间充质干细胞（MSC）分离培养及扩增的方法。方法取小鼠胫骨和股骨,洗去骨髓后,用胶原酶I消化疏松骨密质,利用MSC具有迁徙和贴壁生长的能力进行分离。并对获取的细胞进行流式鉴定和诱导分化。结果培养2d小鼠骨片边缘爬出成纤维样细胞,呈克隆和鱼群样生长,并可以进行持续传代培养。流式鉴定结果显示这群细胞表达MSC标志Scall（92．7％）,CD29（98．4％）,CD90（91．6％）,不表达造血细胞标志CD34（1．57％）,CD45（3．99％）,CD11b（0．63％）,并可成功诱导分化成骨细胞和脂肪细胞。结论成功建立从小鼠骨片中获得MSC的方法,为实验研究提供可靠的细晌实源．     

优化CD115稳定性因素以精确检测小鼠单核细胞及其亚群

单核细胞是机体防御系统的重要组成部分,单核细胞数量发生改变一定程度上可反映机体炎症或其他疾病状态。应用流式细胞术对单核细胞表面抗原进行检测,可了解单核细胞占比变化,有助于揭示单核细胞在疾病发生和转归中的作用。鉴于小鼠单核细胞特异性抗原CD115的稳定性较差,在流式检测过程中,对其影响因素进行优化,以确保检测结果的准确性。首先,探讨CD115抗原-抗体结合前后温度和放置时间对其影响及多聚甲醛（paraformalclehyde,PFA）固定对CD115稳定性的保持作用。在此基础上,通过组合流式抗体CD11c、 CD49b、 Ly6G、 Ter119、 CD3e 和 B220选定阴性细胞群,再用CD11b和CD115圈门得到双阳性的单核细胞,最后根据Ly6C表达强弱区分单细胞亚群。结果显示,温度和放置时间对小鼠单核细胞CD115的稳定性影响显著。将小鼠样本在冰上放置6 h时,CD11b和CD115双阳性细胞比例仍保持稳定,在室温放置1 h即明显下降,在37 ℃放置0.5 h则大部分消失。该结果在抗体结合前后趋势一致。PFA固定后,在4 ℃条件下避光保存, CD11b和CD115双阳性细胞比例可稳定保持3 d。在此优化条件下,应用组合抗体可较好地分析小鼠单核细胞及其亚群,为精准检测小鼠单核细胞提供了技术支持,并为进一步研究单核细胞生物学特性及其在疾病中的作用提供了可能。     

CD47-siRNA通过调控ROS诱导食管癌细胞SEG-1凋亡

目的:探讨CD47-siRNA对食管癌细胞SEG-1凋亡的影响及其机制研究。方法:Western blot法检测食管癌细胞SEG-1中CD47蛋白、促凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达;CCK-8法检测食管癌细胞SEG-1细胞活力;DCFDA细胞ROS检测试剂盒检测食管癌细胞SEG-1中ROS水平。结果:CD47-siRNA转染组食管癌细胞SEG-1中CD47蛋白明显低于空载对照组(P0.05);CD47-siRNA转染组食管癌细胞SEG-1细胞活力显著低于空载对照组(P0.05);CD47-siRNA转染组食管癌细胞SEG-1中促凋亡蛋白Bax表达显著高于空载对照组(P0.05),抗凋亡蛋白Bcl-2表达低于空载对照组(P0.05);CD47-siRNA转染食管癌细胞SEG-1组ROS水平明显高于空载对照组(P0.05);与CD47-siRNA转染组比较,CAT (ROS抑制剂,5 m M/L, 6 h)预处理增加了细胞活力;与CD47-siRNA转染组比较,CAT (ROS抑制剂,5 m M/L, 6 h)预处理显著降低食管癌细胞SEG-1中Bax蛋白表达以及增加抗凋亡蛋白Bcl-2表达。结论:CD47-siRNA转染通过上调食管癌细胞SEG-1内ROS水平促进食管癌细胞SEG-1凋亡。     

Gr-1 Ab Administered after Bone Marrow Transplantation plus Thymus Transplantation Suppresses Tumor Growth by Depleting Granulocytic Myeloid-Derived Suppressor Cells

It has been shown that allogeneic intra-bone marrow–bone marrow transplantation (IBM-BMT) plus thymus transplantation (TT) is effective in treating recipients with malignant tumors. Although TT increases the percentage of T cells in the early term after BMT, the myeloid-derived suppressor cells (MDSCs) are still the dominant population. We used the Gr-1 Ab to deplete the granulocytic MDSCs (G-MDSCs) in tumor-bearing mice that had received BMT+TT. Two weeks after the BMT, the mice injected with Gr-1 Ab showed smaller tumors than those in the control group. In addition, Gr-1 Ab significantly increased the percentages and numbers of CD4⁺ and CD8⁺ T cells, and decreased the percentages and numbers of MDSCs and G-MDSCs. No side effects of the Gr-1 Ab on recipient or donor thymus were observed. These findings indicate that Gr-1 Ab administered after BMT+TT may enhance the effectiveness of tumor suppression.     

Bone marrow cells are differentiated into MDSCs by BCC-Ex through down-regulating the expression of CXCR4 and activating STAT3 signalling pathway

Studies showed that the increase of myeloid-derived suppressor cells (MDSCs) in tumour microenvironment is closely related to the resistant treatment and poor prognosis of metastatic breast cancer. However, the effect of tumour-derived exosomes on MDSCs and its mechanism are not clear. Here, we reported that breast cancer cells (4T1)-secreted exosomes (BCC-Ex) were able to differentiate bone marrow cells into MDSCs and significantly inhibited the proliferation of T lymphocytes to provide an immunosuppressive microenvironment for cancer cells in vivo and in vitro. The number of MDSCs in bone marrow and spleen of 4T1 tumour-bearing mice and BCC-Ex infused mice was significantly higher than that of normal mice, whereas the number of T lymphocytes in spleen was significantly decreased. In addition, BCC-Ex markedly promoted the differentiation of MDSCs from bone marrow cells or bone marrow cells derived macrophages, seen as the increased expressions of MDSCs-related functional proteins Arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS). Furthermore, BCC-Ex significantly down-regulated the expressions of chemokine receptor CXCR4 and markedly up-regulated the levels of inflammatory cytokines IL-6 and IL-10 in bone marrow cells and macrophages and remarkably inhibited the division and proliferation of T cells. Importantly, CXCR4 agonist, CXCL12, could reverse the function of BCC-Ex, indicating that BCC-Ex-induced MDSCs might be dependent on the down-regulation of CXCR4. Western blot showed that BCC-Ex significantly promoted the phosphorylation of STAT3 in bone marrow cells, resulting in the inhibitions of the proliferation and apoptosis of bone marrow cells, and the aggravation of the differentiation of bone marrow cells into MDSCs.     

Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential

Muscle-derived stem cells (MDSCs) can differentiate into multiple lineages, including haematopoietic lineages. However, it is unknown whether MDSCs preserve their myogenic potential after differentiation into other lineages. To address this issue, we isolated from dystrophic muscle a population of MDSCs that express stem-cell markers and can differentiate into various lineages. After systemic delivery of three MDSC clones into lethally irradiated mice, we found that differentiation of the donor cells into various lineages of the haematopoietic system resulted in repopulation of the recipients' bone marrow. Donor-derived bone-marrow cells, isolated from these recipients by fluorescence-activated cell sorting (FACS), also repopulated the bone marrow of secondary, lethally irradiated, recipients and differentiated into myogenic cells both in vitro and in vivo in normal mdx mice. These findings demonstrate that MDSC clones retain their myogenic potential after haematopoietic differentiation.     

A novel role of hematopoietic CCL5 in promoting triple-negative mammary tumor progression by regulating generation of myeloid-derived suppressor cells

CCL5 is a member of the CC chemokine family expressed in a wide array of immune and non-immune cells in response to stress signals. CCL5 expression correlates with advanced human breast cancer. However, its functional significance and mode of action have not been established. Here, we show that CCL5-deficient mice are resistant to highly aggressive, triple-negative mammary tumor growth. Hematopoietic CCL5 is dominant in this phenotype. The absence of hematopoietic CCL5 causes aberrant generation of CD11b⁺/Gr-1⁺, myeloid-derived suppressor cells (MDSCs) in the bone marrow in response to tumor growth by accumulating Ly6C^hi and Ly6G⁺ MDSCs with impaired capacity to suppress cytotoxicity of CD8⁺ T cells. These properties of CCL5 are observed in both orthotopic and spontaneous mammary tumors. Antibody-mediated systemic blockade of CCL5 inhibits tumor progression and enhances the efficacy of therapeutic vaccination against non-immunogenic tumors. CCL5 also helps maintain the immunosuppressive capacity of human MDSCs. Our study uncovers a novel, chemokine-independent activity of the hematopoietically derived CCL5 that promotes mammary tumor progression via generating MDSCs in the bone marrow in cooperation with tumor-derived colony-stimulating factors. The study sheds considerable light on the interplay between the hematopoietic compartment and tumor niche. Because of the apparent dispensable nature of this molecule in normal physiology, CCL5 may represent an excellent therapeutic target in immunotherapy for breast cancer as well as a broad range of solid tumors that have significant amounts of MDSC infiltration.     

Cramp过表达对小鼠骨髓造血干细胞功能的影响

目的研究Cramp蛋白过表达对小鼠骨髓造血干细胞自我更新和分化能力的影响。方法应用流式细胞仪分析Cramp过表达转基因小鼠及同龄野生型小鼠的骨髓、脾脏、胸腺等组织器官中各种细胞的比例;分选骨髓造血干细胞,体外培养,观察其克隆形成能力。结果与野生型小鼠相比,Cramp过表达转基因小鼠的骨髓、脾脏、胸腺等组织器官中各种细胞的比例、骨髓造血干细胞的克隆形成能力等均无明显变化。结论本研究中,Cramp过表达转基因小鼠骨髓造血干细胞的分化能力、克隆形成能力无明显变化。     

Myeloid-specific expression of human lysosomal acid lipase corrects malformation and malfunction of myeloid-derived suppressor cells in lal-/- mice

Lysosomal acid lipase (LAL) cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. LAL deficiency causes expansion of CD11b(+)Gr-1(+) immature myeloid cells, loss of T cells, and impairment of T cell function. To test how myeloid cell LAL controls myelopoiesis and lymphopoiesis, a myeloid-specific doxycycline-inducible transgenic system was used to reintroduce human lysosomal acid lipase (hLAL) expression into LAL gene knockout (lal(-/-)) mice. Expression of hLAL in myeloid cells of lal(-/-) mice reversed abnormal myelopoiesis in the bone marrow starting at the granulocyte-monocyte progenitor stage and reduced systemic expansion of myeloid-derived suppressor cells (MDSCs). Myeloid hLAL expression inhibited reactive oxygen species production and arginase expression in CD11b(+)Gr-1(+) cells of lal(-/-) mice. Structural organization of the thymus and spleen was partially restored in association with reduced infiltration of CD11b(+)Gr-1(+) cells in these mice. In the thymus, reconstitution of myeloid cell LAL restored development of thymocytes at the double-negative DN3 stage. Myeloid cell LAL expression improved the proliferation and function of peripheral T cells. In vitro coculture experiments showed that myeloid hLAL expression in lal(-/-) mice reversed CD11b(+)Gr-1(+) myeloid cell suppression of CD4(+) T cell proliferation, T cell signaling activation, and lymphokine secretion. Blocking stat3 and NF-κB p65 signaling by small-molecule inhibitors in MDSCs achieved a similar effect. Injection of anti-Gr-1 Ab into lal(-/-) mice to deplete MDSCs restored T cell proliferation. These studies demonstrate that LAL in myeloid cells plays a critical role in maintaining normal hematopoietic cell development and balancing immunosuppression and inflammation.     

SD大鼠骨髓间充质干细胞体外原代培养及鉴定

目的：探讨骨髓间充质干细胞（BMSCs）体外分离培养以及扩增的方法并鉴定。方法：取100g左右雄性SD大鼠后肢股骨、胫骨骨髓,原代全骨髓培养法,多次传代纯化,体外扩增后,观察细胞形态,并免疫组化及流式细胞仪检测cd34、cd90、cd105细胞因子,鉴定是否为BMSCs。结果：所获取的细胞呈长梭形,呈现特征性的漩涡状生长,CD34阴性,CD90、CD105阳性。结论：利用全骨髓培养法成功分离骨髓间充质干细胞,10代以内的细胞纯度高,活性好。全骨髓培养较为简便、易行。     

Swertianolin ameliorates immune dysfunction in sepsis via blocking the immunosuppressive function of myeloid-derived suppressor cells

In this study, we studied the long-term proliferation trajectory of myeloid-derived suppressor cells (MDSCs) in murine sepsis model and investigated whether swertianolin could modulate the immunosuppressive function of MDSCs. A murine sepsis model was established by cecal ligation and perforation (CLP), according to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. The bone marrow and spleen of the mice were collected at 24 h, 72 h, 7 and 15 d after sepsis induction. The proportions of monocytic- MDSCs (M-MDSCs; CD11b⁺LY6G^–LY6C^hi) and granulocytic-MDSCs (G-MDSC, CD11b⁺ Ly6G⁺ Ly6C^low) were analyzed by flow cytometry. Then, we have investigated whether swertianolin could modulate the immunosuppressive function of MDSCs in in vitro experiments. G-MDSCs and M-MDSCs increased acutely after sepsis with high levels sustained over a long period of time. G-MDSCs were the main subtype identified in the murine model of sepsis with polymicrobial peritonitis. Furthermore, it was found that swertianolin reduced significantly interleukin-10 (IL-10), nitric oxide (NO), reactive oxygen species (ROS), and arginase production in MDSCs, while reducing MDSC proliferation and promoting MDSC differentiation into dendritic cells. Swertianolin also improved T-cell activity by blocking the immunosuppressive effect of MDSCs. Both subsets of MDSCs significantly increased in the bone marrow and spleen of the mice with sepsis, with GMDSCs being the main subtype identified. Swertianolin effectively regulated the functions of MDSCs and reduced immune suppression.Key words: Sepsis, myeloid-derived suppressor cells (MDSCs), immunosuppression, swertianolin