婴幼儿血管瘤内皮细胞的分离、培养及鉴定

目的 建立一个稳定的婴幼儿血管瘤内皮细胞体外培养体系.方法 增生期血管瘤标本经胶原酶消化,CD31-免疫磁珠分选纯化细胞,接种于纤维蛋白铺层的培养皿;分别行细胞形态学观察、细胞生长曲线的测定、培养细胞免疫荧光鉴定、低密度脂蛋白吸收试验.结果 从血管瘤组织分离纯化出CD31+细胞,4 h开始贴壁,细胞呈梭形,7～10 d生长融合呈铺路石状,CD31-免疫磁珠黏附于细胞上.血管瘤内皮细胞CD31、vWF表达阳性,可摄取低密度脂蛋白.结论 我们建立的婴幼儿血管瘤内皮细胞的分离培养方法,可获得血管瘤内皮细胞,并为血管瘤增生消退机制的研究奠定了细胞学基础.     

大鼠CD4^＋CD25^＋调节性T细胞的分离及功能鉴定

目的：研究利用免疫磁珠分选法稳定分离正常大鼠脾脏CD4^＋CD25^＋调节性T细胞的方法。方法：采用免疫磁珠两步法分离大鼠脾组织CD4^＋CD25^＋T细胞。首先采用藻红蛋白（PE）标记的抗CD25抗体和抗PE多功能磁珠试剂盒阳性分选CD25^＋T细胞,再用抗异硫氰酸荧光素（FITC）标记抗体和抗IgG磁珠阳性分选获得CD4^＋CD25^＋T细胞。分离后的细胞经流式细胞仪检测分离纯度,台盼蓝染色检测细胞存活率,体外增殖实验检测其对CD4^＋CD25^-T细胞的免疫抑制作用。结果：两次阳性分选后获得的CD4^＋CD25^＋T细胞纯度为（90.4±1.6）%,细胞存活率为（92.6±2.4）%。体外增殖实验表明,CD4^＋CD25^＋T细胞能明显抑制CD4^＋CD25^-T细胞的增殖（P〈0.01）。结论：采用免疫磁珠法两次阳性分选,可稳定地获得纯度理想并有免疫抑制功能的大鼠CD4^＋CD25^＋T细胞。     

血管瘤血管内皮细胞体外分离纯化方法的优化

目的:寻找更高效的婴幼儿血管瘤内皮细胞培养方法,为婴幼儿血管瘤的研究提供基础的科研保证。方法:选取增生期婴幼儿血管瘤手术标本,采用组织块贴壁法与酶消化法结合进行原代培养;二代细胞进行流式分选,取CD31阳性的细胞继续培养。分别进行细胞形态学观察、细胞表面标记物免疫荧光鉴定、细胞表面标记物流式鉴定。结果:培养48～72h清晰可见组织块周围细胞游出,细胞核清晰,细胞呈长梭形及多边形,2周左右部分可形成"血管腔样"结构,20d可铺满平传代;细胞流式测定CD31阳性率达98.4%,VWF与CD31双阳率90.3%,免疫荧光染色CD31、VWF均呈阳性表达。结论:组织块与消化结合法经流式分选后可获得高纯度的内皮细胞,为下一步研究婴幼儿血管瘤增生消退机制奠定了基础。     

人脐血CD133+血管内皮祖细胞的培养、鉴定与分化研究

目的探讨从人脐血中分离、体外培养CD133^＋血管内皮祖细胞（EPCs）的方法及其生长特性和鉴定。方法采用密度梯度离心法结合MACS分选法提取人脐血中CD133^＋细胞,进行流式细胞仪检测纯度,予EBM-2培养液接种培养,观察其生长特性;利用Dil-LDL及FITC-Lectin摄取实验、细胞免疫组织化学等进行鉴定。结果经流式细胞仪检测CD133^＋细胞平均占单核细胞的（1．13±0．10）％,磁珠分选所得CD133^＋细胞平均纯度为（91．45±1．04）％;CD133^＋细胞贴壁生长,可分化为梭形血管内皮细胞及形成集落;CD133^＋细胞培养过程中Dil-LDL及FITC-Lectin摄取阳性,双染阳性率为（95．83±1．72）％;CD133^＋细胞培养1周后经免疫组织化学检测CD34、Ⅷ因子阳性率分别为（95．83±2．23）％和（95．92±1．43）％,与人脐静脉内皮细胞比较差异无统计学意义;CD133^＋细胞体外培养4d、1周可形成小血管结构。结论免疫磁珠分选法可获取较高纯度CD133^＋血管内皮祖细胞,在生长因子作用下诱导其分化为成熟血管内皮细胞。     

人真皮微淋巴管内皮细胞的分离培养和鉴定

目的建立人真皮来源淋巴管内皮细胞(LECs)分离和培养的方法。方法采用中性蛋白酶及胶原酶消化方法结合免疫磁珠从真皮组织分选CD34-/CD31 细胞。免疫细胞化学、免疫荧光及逆转录-聚合酶链反应(RT-PCR)方法检测LECs特异标志的表达,噻唑蓝(MTT)比色试验测定多种与内皮细胞增殖相关的细胞因子和低氧条件对其增殖的影响。结果CD34-/CD31 细胞在单层细胞培养时形成内皮细胞典型的铺路石样外观,而在胶原凝胶三维培养时形成管状结构。CD34-/CD31 细胞表达淋巴管内皮细胞特异性标志,包括LYVE-1,VEGFR-3和Prox-1。RT- PCR显示Proxl和VEGFR-3 mRNA在CD34-/CD31 细胞中特异性表达。特异作用于淋巴管内皮细胞的VEGF-C对LECs的促增殖作用明显(P<0.01)。低氧条件和Ang2在体外未显示对LECs有促进增殖的作用。结论应用中性蛋白酶及胶原酶消化方法结合免疫磁珠分选可以成功分离获取人真皮LECs。     

骨髓CD34+干细胞与单核细胞应用于组织工程小血管内皮化的比较

目的 探讨骨髓CD34+干细胞与骨髓单核细胞(MNCs)应用于组织工程小血管内皮化的效果,并作比较.方法 采集犬骨髓,经免疫磁珠分离出CD34+细胞,内皮细胞生长因子(VEGF)诱导分化为内皮细胞并扩增,行细胞免疫荧光、免疫组织化学和体外成血管实验鉴定;将培养后的细胞和未经分离的MNCs分别种植于人工小血管支架,扫描电镜观察,比较两组组织工程小血管的内皮化.结果 经流式细胞仪测定,分离后的细胞中CD34+细胞含量为(78.46±6.37)％.CD34+细胞培养2周后细胞基本铺满培养瓶底面,细胞呈“铺路石”状排列,CD31和Ⅷ因子免疫细胞化学染色均为阳性,体外成血管实验显示6h后CD34+细胞组出现较多毛细血管样网状结构.CD34+细胞组人工血管内膜化面积为(68.4±2.3)％,明显高于MNCs组人工血管内膜化面积(41.3±3.4)％,差异有统计学意义(P＜0.01).结论 通过免疫磁珠方法可分离得到高纯度的骨髓CD34+细胞,经体外培养VEGF诱导后可定向分化为内皮细胞,种植于人工血管内表面,较MNCs组内皮化效果理想.     

D2-40、CD31、CD34在脾脏脉管瘤中的表达及意义

目的 探讨脾脏脉管瘤的病理形态特征、形成原因及鉴别诊断.方法 对7例脾脏脉管瘤病例进行病理形态观察,EnVision法进行免疫组织化学染色.结果 7例脾脏脉管瘤中,2例为血管瘤,脉管内皮细胞CD31、CD34表达阳性;2例淋巴管瘤,脉管内皮细胞D2-40表达阳性;3例血管淋巴管瘤,D2-40在淋巴管内皮细胞表达阳性,CD31、CD34在血管内皮细胞表达阳性.结论 脾脏脉管瘤是少见肿瘤,免疫表型D2-40、CD31、CD34检查阳性,对脾脏脉管诊断有较高价值.     

免疫磁珠两步法分离大鼠脾脏CD4+CD25+调节性T细胞

目的探索利用磁性细胞分离(MACS)系统高效、快速地分离大鼠脾脏CD4+CD25+调节性T细胞.方法采用免疫磁珠两步法分离大鼠脾脏内的CD4+CD25+调节性T细胞, 首先采用"鸡尾酒"抗体和抗IgG磁珠阴性分选CD4+ T细胞,再用抗CD25-PE抗体和抗PE磁珠阳性分选获得CD4+CD25+T 细胞.分离后的细胞经流式细胞仪检测分离纯度; 台盼蓝染色检测细胞存活率; 体外增殖实验检测其对CD4+CD25-T细胞的免疫抑制作用.结果阴性分选获得的CD4+T细胞纯度为(83.6±2.5)%(79%～87%), 阳性分选后获得的CD4+CD25+T细胞纯度为(90.2±1.8)%(86%～93%), 细胞存活率为(92.8±3.4)%(92%～95%), 体外增殖实验表明,CD4+CD25+T细胞能明显抑制CD4+CD25-T 细胞的增殖(P＜0.01). 结论采用MACS系统阴性加阳性分选可以高效、快速地获得理想纯度和有免疫抑制功能的大鼠CD4+CD25+调节性T细胞.     

骨肉瘤细胞的仿血管发生

目的 探讨骨肉瘤细胞的仿血管发生现象,骨肉瘤细胞是否具有血管形成相关基因的表达.方法 应用Ⅰ型胶原蛋白凝胶和Matrigel的三维培养基对骨肉瘤细胞进行培养,并运用电镜、光镜观察这些类血管样结构的形态学构成;应用逆转录-聚合酶链反应(RT-PCR)技术检测骨肉瘤细胞血管形成相关基因(VEGF、Flt-1、KDR、Ang-1、Tie-2、Ang-2、Tie-1、EPHA2、CD31、Thrombin、VE-Cadherin)的表达.结果 电镜、光镜下观察到骨肉瘤细胞在Ⅰ型胶原蛋白凝胶和Matrigel的三维培养基中能够形成良好的类血管样的管道结构;骨肉瘤细胞类内皮细胞和内皮前驱细胞,具有血管形成相关基因(VEGF、Flt-1、KDR、Ang-1、Tie-2、EPHA2、CD31、Thrombin、VE-Cadherin)的表达.结论 骨肉瘤细胞具有仿血管发生的能力和血管形成相关基因的表达,三维培养是研究肿瘤细胞仿血管发生现象较好的工具.     

人肝癌组织原代巨噬细胞的提取与鉴定

目的研究人肝癌组织原代肿瘤相关性巨噬细胞（TAMs）的提取与鉴定方法。 方法收集原发性肝癌患者术中切取的肝癌组织标本15份,免疫组织化学染色方法检测肝癌组织中肿瘤相关巨噬细胞表面标志物CD68及CD206的表达。利用机械法和酶消化法结合分离TAMs,贴壁培养24 h后在倒置显微镜下进行细胞形态学观察,利用CD68和CD206作为标志物,流式细胞术鉴定细胞纯度。 结果癌巢和癌旁组织中均可见CD68、CD206阳性表达,阳性细胞表现为棕褐色;获得一定纯度的原代巨噬细胞,具备巨噬细胞的形态特征,其中CD68单阳性比例为36.29%,CD68和CD206双阳性比例为15.96%。 结论本研究采用的方法可有效提取和培养人肝癌组织来源的原代TAMs,对进一步研究人类肝癌组织TAMs特性及对肿瘤生物学行为的影响具有重要价值。     

人肝癌血管内皮细胞分离培养、鉴定及比较

目的 建立人肝癌血管内皮细胞(TEC)分离培养方法并鉴定比较.方法 采用CD105、CD31抗体交联免疫磁珠方法优化分选TEC并传代培养;通过形态观察、功能实验、流式检测及实时聚合酶链反应(real-time PCR)等验证比较.结果 CD105+TEC呈细长"纤维条索状",CD31+TEC则呈"梭形";流式检测TEC表达CD68和α-平滑肌肌动蛋白(α-SMA)低于5%,排除巨噬细胞和成纤维细胞污染,PCR检测CD105+TEC、CD31+TEC甲胎蛋白mRNA显著低于肿瘤细胞HepG2(P＜0.01),表达水平低于HepG2的1/8,排除肿瘤细胞污染;95%以上阳性分选细胞呈乙酰化低密度脂蛋白摄取实验阳性;增殖实验检测CD105+TEC和CD31+TEC生长48 h的吸光度值分别为0.45±0.04和0.35±0.02(P＜0.05),CD105+TEC增殖能力强于CD31+TEC;TEC在Matrigel胶中可形成毛细管网状结构,CD31+TEC强于CD105+TEC.结论 CD105、CD31抗体交联免疫磁珠分选法均可分离得到高纯度人肝癌TEC.

Abstract：

Objective To isolate, identify and compare vascular endothelial cells derived from human hepatocellular carcinoma (HCC). Methods Modified immunomagnefic methods using magnetic beads conjugated with anti-CD105 and anti-CD31 antibody were used to isolate tumor-derived endothelial cells (TEC), namely CD31 +TEC and CD105 +TEC. CD31 +TEC and CD105 +TEC were cultured in vitro,identified and compared by morphological appearance, functional characterization, flow cytometry and realtime polymerase chain reaction (PCR) analyses. Results The isolated CD105 + TEC presented "fibrous bands" appearance while CD31 + TEC presented "fusiformis" appearance. Macrophages, fibrocytes, and tumor cells contamination was excluded. Surface CD68 and α-SMA expression was less than 5%. Alpha fetoprotein expression was significantly lower in CD31 + TEC and CD105 + TEC than that in HepG2 cells ( P ＜0. 01 ), and the expression level was less than 1/8 folds of HepG2 cells. Internalization of acetylated lowdensity lipoprotein was positive in more than 95% of isolated cells. The absorbance (A) values of CD105 +TEC and CD31 + TEC grown for 48 h were 0. 45 ± 0. 04 and 0. 35 ± 0. 02 respectively ( P ＜ 0. 05 ). Proliferation of CD105 + TEC was significantly more active than CD31 + TEC when cultured in the serum supplemented with medium for 24, 48, and 72 h. The isolated cells could form capillary-like tubes on Matrigel matrix, stronger capability of CD31 + TEC than CD105 + TEC. Conclusion CD31 + TEC and CD105 + TEC can be conveniently purified by modified immunomagnetic methods.     

核转录因子-κB通路在CD40-CD154诱导血管内皮细胞活化中的作用

目的 探讨血管内皮细胞(VEC)内核转录因子-κB(NF-κB)通路在CD40-CD154相互作用诱导VEC活化中的作用.方法 应用重组人肿瘤坏死因子α(rhTNF-α)和重组人γ干扰素(rhIFN-γ)刺激人主动脉VEC,通过流式细胞仪(FACS)检测CD40和粘附因子表达水平;通过表达CD154的细胞株D1.1和VEC共同培养后,观察VEC粘附因子的表达水平;通过抗CD154单克隆抗体阻断CD40-CD154间的反应;应用NF-κB阻断剂BAY11-7082观察阻断NF-κB通路后对CD40-CD154诱导VEC活化的影响.结果 未活化的VEC膜表达低水平的CD40、CD54和CD106,但不表达CD62E,经白细胞介素刺激后可上调这些蛋白分子的表达水平.D1.1细胞株和VEC共同培养后可诱导VEC活化并上调CD62E、CD54和CD106的表达水平;抗CD154抗体可阻断CD40-CD154间的反应所诱导的VEC活化;NF-κB阻断剂可阻断rhTNF-α诱导的VEC活化以及CD40-CD154间的反应所诱导的VEC活化.结论 VEC中CD40和T淋巴细胞中CD154之间的相互作用在诱导VEC活化过程中起重要作用;CD40-CD154相互作用并通过NF-κB通路诱导VEC活化;NF-κB通路阻断剂可抑制VEC活化.     

巢式RT-PCR检测荷人肝癌裸鼠周围静脉血液中AFPmRNA

目的应用巢式RT－PCR检测荷人肝癌裸鼠周围静脉血液中AFPmRNA并探讨其意义。方法抽取20只荷人肝癌裸鼠周围静脉血液各1ml,应用巢式RT－PCR检测其周围静脉血液中AFPmRNA。结果20只荷瘤裸鼠中有6只血液中被检测到AFPmRNA（30.0％）,其中有4只发现了肺脏、肝脏或肾脏转移（66.67％）。结论AFPmRNA可以作为肝癌血液转移的敏感性指标,也可作为肝癌远处转移的预测性指标之一。     

抑制黏附分子CD44S阻断胃癌腹膜转移的实验研究

目的：评价细胞表面黏附分子CD44被单克隆抗体封闭后,胃癌细胞黏附、迁移能力及其腹膜种植潜能的变化,为将阻断CD44作为抑制胃癌腹膜转移的治疗策略提供实验依据.方法：用细胞黏附试验及迁移试验（Boyden小室法）评价经抗CD44S单克隆抗体作用后,体外培养的MKN45胃癌细胞与间皮细胞发生黏附的能力及其穿越间皮细胞发生迁移的能力变化.通过建立裸鼠腹膜种植瘤模型,评价抗CD44S抗体处理对胃癌细胞腹膜肿瘤生成能力及荷瘤裸鼠生存时间的影响.结果：经抗CD44S抗体作用后,胃癌细胞MKN45与间皮细胞间的黏附能力明显减弱;经较高浓度抗体处理后,MKN45细胞的迁移能力也有明显下降.体内实验结果提示,接种经抗CD44S抗体处理MKN45胃癌细胞的裸鼠生存时间明显长于对照组（P＜0.01）.结论：黏附分子CD44参与胃癌腹膜转移过程,与肿瘤细胞-间皮细胞间的黏附和肿瘤细胞迁移等步骤密切相关;阻断CD44可作为抑制胃癌腹膜转移的潜在治疗策略.     

Isolation, culture, and characterization of endothelial cells from mouse glomeruli

BACKGROUND: Cloned glomerular endothelial cells (GENC) have many potential uses and applications in immunologic and physiologic studies. Propagation of GENC has been difficult and available homogeneous GENC, particularly from mice, are limited. Herein we report isolation, cloning, propagation, and characterization of GENC from mice. METHODS: tsA58 immorto mice were used to isolate glomerular cells. Glomeruli were isolated by differential sieving, and decapsulated explants were cultured in permissive and optimal conditions for endothelial cells. The primary cells from glomerular outgrowths were expanded, taking advantage of the temperature-sensitive tsA58 gene, and then the cells were allowed to undergo spontaneous transformation. The cells were then sorted using anti-CD31 antibodies and their capacity to uptake acetylated-low-density lipoprotein (LDL). Individual subclones isolated by patch cloning were characterized using multiple markers. RESULTS: One of the homogeneous clones was morphologically endothelial-like, positive for CD31, CD106, CD62E, CD54, and acetylated-LDL uptake, formed tubes, and was negative for epithelial and mesangial cell markers. The functional properties of this GENC clone appeared to be intact, and signaling pathway was not altered. Two of the clones displayed the characteristics of either visceral epithelial or mesangial cells. CONCLUSION: The identified clones should have utility in multiple areas of investigation.     

Resistance of established porcine islet xenografts to humoral rejection by hyperimmune sera.

BACKGROUND: Although preformed natural antibodies cause hyperacute rejection of primarily vascularized xenografts, tissue grafts such as skin or islets are revascularized by in-growth of host capillaries and therefore might be resistant to circulating antibodies. We examined the effect of hyperimmune serum and primed T cells on the survival of long-term porcine islet xenografts in diabetic nude mice. METHODS: Porcine islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic BALB/c athymic mice. Hyperimmune serum and sensitized splenocytes were prepared by repeated immunization of BALB/c mice with porcine lymph node cells. Splenic T cells were enriched by nylon wool column separation. Tissues were examined by immunohistology using murine- and porcine-specific monoclonal antibodies. RESULTS: Porcine islets survived in nude mice for > 100 days with high levels of circulating porcine C-peptide and maintenance of normoglycemia. Injection of the hyperimmune sera (IgG) into normoglycemic nude mice bearing porcine islets for > 70 days failed to induce rejection despite the continued presence of circulating anti-porcine cytotoxic antibody. Injection of sensitized T cells caused acute rejection of long-term (>140 days) porcine islets, whereas injection of naive T cells had no effect. Histologically, porcine islets removed from mice treated with hyperimmune serum showed no staining for IgG. Long-surviving porcine islet grafts showed strong staining for interleukin (IL)-10 and a lesser amount of IL-4 but no staining for IL-2 or interferon-gamma. Although fresh porcine islets were positive for swine leukocyte antigen class 1 antigen and intercellular adhesion molecule (ICAM)-1 but negative for mouse platelet endothelial cell adhesion molecule and ICAM-2, long-surviving porcine islets showed positive endothelial staining for mouse platelet endothelial cell adhesion molecule and ICAM-2. CONCLUSIONS: Established islet xenografts are resistant to hyperimmune serum as a result of a lack of target endothelial antigens, whereas they remain susceptible to rejection caused by primed T cells. Local production of Th2 cytokines may explain the inability of long-surviving islet xenografts to activate injected naive T cells.     

丙戊酸钠诱导人脑胶质瘤细胞分化的靶细胞与靶分子研究

目的探讨丙戊酸钠诱导人脑胶质瘤细胞分化时作用的靶细胞和靶分子。方法丙戊酸钠治疗位于裸小鼠皮下的可移植性人脑胶质瘤,半定量RT-PCR检测移植瘤中HDAC1和Tob的表达;CD133免疫磁珠分离胶质瘤组织中的CD133^＋细胞,在分别含血清或不含血清加生长因子培养的条件下,加丙戊酸钠作用21d,流式细胞仪检测和共聚焦显微镜观察分化标志物表达。结果丙戊酸钠能明显抑制位于裸小鼠皮下移植瘤的增殖（P〈0．05）,使HDAC1表达下降（P〈0．01）,Tob表达升高（P〈0．05）。流式细胞仪检测丙戊酸钠作用21d的细胞,在含血清条件下,GFAP或β-tubulinⅢ阳性细胞数显著增加（P〈0．01）,而无血清加生长因子条件下两标志物表达均无显著差异（P〉0．05）;共聚焦显微镜观察显示上述的GFAP^＋或β-tubulinⅢ^＋细胞共表达Nestin。结论丙戊酸钠逆转人脑胶质瘤细胞分化抑制的靶分子有HDAC1和Tob,靶细胞为处于分化过程中的脑肿瘤干细胞。     

p14ARF基因对胰腺癌血管生成的影响

目的探讨外源性p14ARF基因对胰腺癌PC-3细胞血管生成的影响。方法将转染了外源性p14ARF基因的人胰腺癌PC-3细胞株接种裸鼠形成实体瘤，用RT-PCR和Westernblot法观察VEGFmRNA及VEGF蛋白的表达，并对实体肿瘤的微血管密度进行检测。结果转染p14ARF基因后的PC-3细胞实体瘤中VEGFmRNA及VEGF蛋白的表达下调，血管密度降低。结论p14ARF基因能下调内源性VEGF的表达，有着抗血管生成的治疗意义。