不同浓度肝细胞生长因子及表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化

背景：研究证实在多种微环境下可以将骨髓间充质干细胞诱导分化为成熟肝细胞,但诱导条件及诱导分化率尚无定论,选择合适的诱导剂及诱导剂浓度尤为重要。 目的：观察肝细胞生长因子和表皮细胞生长因子体外联合诱导兔骨髓间充质干细胞向肝细胞分化的最适浓度。 方法：不同浓度肝细胞生长因子、表皮细胞生长因子联合诱导培养原代兔骨髓间充质干细胞,观察细胞形态学变化,并检测肝细胞表面标志物甲胎蛋白、白蛋白表达及肝细胞合成功能。 结果与结论：随诱导时间延长,可观察到多极性的肝细胞样细胞。7 d时甲胎蛋白表达阳性,14 d达最大值后即开始下降(P < 0.05),此后各浓度组间表达无差别(P  > 0.05)。14 d时白蛋白表达阳性,随时间延长,阳性细胞数递增(P  < 0.05),且细胞生长因子浓度越高,阳性细胞数越高(P < 0.05)。诱导早期(9~15 d) 白蛋白上清液中白蛋白水平与诱导剂浓度成正比(P < 0.05)。18 d或21 d达高峰后下降,此时各浓度组间含量无差别(P  > 0.05)。结果显示高浓度的肝细胞生长因子及表皮细胞生长因子可提高骨髓间充质干细胞向肝细胞的分化率,诱导剂最适浓度为肝细胞生长因子60 μg/L、表皮细胞生长因子4.5 mg/L。     

老年初诊急性髓细胞白血病患者CD4+CD25+调节性T细胞的表达

背景：研究证实,很多恶性肿瘤患者体内CD4⁺CD25⁺调节性T细胞存在高表达,近期也有研究发现,急性髓细胞白血病患者外周血CD4⁺CD25⁺调节性T细胞同样表现出高比例表达。 目的：分析老年初诊急性髓细胞白血病患者CD4⁺CD25⁺调节性T细胞的表达特点。 方法：纳入初诊急性髓细胞白血病患者92例,将年龄在60岁以下者设为中青年组(n=22),年龄在60岁以上者设为老年观察组(n=70)。在老年观察组中,32例经规范化疗后完全缓解,设为完全缓解组;将余下38例设为老年组,依据FAB分型标准,分为M2 6例、M3 19例、M4 7例、M5 6例。另选择同期体检健康人群42名作为正常对照组。抽取受试者外周静脉血,检测CD4⁺CD25⁺调节性T细胞表达情况。 结果与结论：老年组、完全缓解组CD4⁺CD25^highFOXP3⁺调节性T细胞比例高于正常对照组(P < 0.01),并且老年组CD4⁺CD25^high FOXP3⁺调节性T细胞比例高于完全缓解组(P < 0.01)。老年组、完全缓解组CD4⁺FOXP3⁺T细胞比例高于正常对照组(P < 0.01),并且老年组CD4⁺ FOXP3⁺T细胞比例高于完全缓解组(P < 0.01)。老年组CD4⁺CD25^high FOXP3⁺调节性T细胞与CD4⁺ FOXP3⁺T细胞比例高于中青年组(P < 0.01)。老年组不同分型间CD4⁺CD25^high FOXP3⁺调节性T细胞和CD4⁺ FOXP3⁺T细胞比例比较差异均无显著性意义(P > 0.05)。Pearson相关性检验结果显示,老年初诊急性髓细胞白血病患者外周血CD4⁺CD25^high FOXP3⁺调节性T细胞比例和CD4⁺ FOXP3⁺T细胞比例呈正相关(r=0.87,P=0.019)。表明老年初诊急性髓细胞白血病患者CD4⁺CD25⁺调节性T细胞比例高于健康人群和中青年急性髓细胞白血病患者。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

兔骨髓间充质干细胞体外向肝样细胞的诱导分化

背景：研究表明,应用四氯化碳建造的重型肝衰竭小鼠模型中移植骨髓间充质干细胞后能够明显改善肝功能,促进肝再生。 目的：观察骨髓间充质干细胞移植后动物模型肝功能及肝硬化程度等指标的变化情况。 方法：采用密度梯度离心法和贴壁培养法获取兔骨髓间充质干细胞。取传代培养至3代的兔骨髓间充质干细胞,添加肝细胞生长因子和表皮生长因子,诱导21 d后进行BrdU标记。建立肝硬化兔模型,实验组兔将收集的BrdU标记过的肝样细胞悬液注入门静脉,对照组注入相同体积生理盐水。细胞移植后21 d麻醉下处死试验兔,观察肝硬化兔肝脏内分布及肝功能与对照组变化情况。 结果与结论：兔骨髓间充质干细胞在体外经肝细胞生长因子、表皮生长因子的诱导条件下,于21 d发现细胞呈类圆形,细胞染色显示,甲胎蛋白、血清白蛋白和糖原表达均呈阳性表达。肝样细胞经门静脉移植后,实验组兔肝生化指标与对照组相比较显著改善(P < 0.05)。BrdU免疫组织化学染色显示：在汇管区和肝索肝窦内均可见BrdU阳性细胞分布。说明兔骨髓间充质干细胞能够向肝样细胞分化,经门静脉移植诱导后的肝样细胞可以改善肝硬化兔的肝功能,促进兔肝细胞再生。     

骨髓间充质干细胞对肾病综合征大鼠CD4+CD25+调节性T细胞的影响

背景：CD4⁺CD25⁺调节性T细胞功能降低、数量下降已被公认为是肾病综合征患儿免疫失调的重要表现。骨髓间充质干细胞具有免疫调节功能,可上调CD4⁺CD25⁺调节性T细胞,抑制淋巴细胞增殖,并已在许多免疫性疾病方面成功应用。 目的：观察骨髓间充质干细胞移植对原发性肾病综合征大鼠外周血CD4⁺CD25⁺调节性T细胞的影响。 方法：自SD大鼠分离骨髓间充质干细胞,经体外传代培养及鉴定后制备细胞悬液。30只SD大鼠随机均分为3组,生理盐水组和干细胞移植组尾静脉注射阿霉素建立阿霉素肾病大鼠模型,干细胞移植组注射阿霉素当日尾静脉射干细胞1×10⁷;正常组不做处理。 结果与结论：①与正常组比较,生理盐水组大鼠均出现肾病综合征表现,以腹水、大量蛋白尿、低蛋白血症、高胆固醇血症为特征;干细胞移植组较生理盐水移植组则有明显改善(P < 0.05)。②造模第28天,干细胞移植组和生理盐水组大鼠造模后外周血CD4⁺CD25⁺Treg/ CD4⁺ Treg均明显高于正常组(P < 0.05);干细胞移植组与生理盐水组比较差异无显著性意义(P > 0.05)。③造模第28天,干细胞移植组大鼠外周血单个核细胞FoxP3mRNA的表达显著高于生理盐水组和正常组(P < 0.05)。提示骨髓间充质干细胞移植对阿霉素肾病综合征模型有一定的治疗作用,其机制可能与上调FoxP3在组织局部的表达而诱导CD4⁺CD25⁺Treg产生有关。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

血红素加氧酶1在间充质干细胞上调哮喘患者外周血调节性T细胞中的作用

背景：上调CD4⁺CD25⁺CD127^low/-调节性T细胞是目前治疗哮喘的新靶点。骨髓间充质干细胞在体外可上调正常人外周血的调节性T细胞,但机制尚未明确。 目的：观察血红素加氧酶1对间充质干细胞上调哮喘患者外周血CD4⁺CD25⁺CD127^low/-调节性T细胞的影响。 方法：分别加入0,15,30,45,60 μmol/L氯化高铁血红素和0,5,10,15,20 μmol/L锌-原卟啉对骨髓间充质干细胞进行预处理,荧光定量PCR检测各组间充质干细胞中血红素加氧酶1 mRNA的表达量。密度梯度离心法分离哮喘急性发作期患者和健康对照者的外周血单个核细胞,分别与经氯化高铁血红素预处理、经锌-原卟啉预处理、不经预处理的间充质干细胞共孵育,加入植物血凝素反应72 h,流式细胞仪检测各组CD4⁺CD25⁺CD127^low/-调节性T细胞占CD4⁺T细胞的比例。 结果与结论：人骨髓间充质干细胞中有血红素加氧酶1表达,其表达在体外可被诱导和抑制。间充质干细胞中血红素加氧酶1 mRNA的表达量随氯化高铁血红素浓度增加而增加(P < 0.05),随锌-原卟啉浓度增加而减少(P < 0.05),具有剂量依赖性。间充质干细胞在体外可提高哮喘患者和健康对照者CD4⁺CD25⁺CD127^low/-调节性T细胞的水平(P < 0.01),诱导间充质干细胞中血红素加氧酶1的表达可使共孵育后CD4⁺CD25⁺CD127^low/-调节性T细胞水平提高(P < 0.01),抑制间充质干细胞中血红素加氧酶1的表达可使共孵育后CD4⁺CD25⁺CD127^low/-调节性T细胞的水平降低(P < 0.01),但仍高于对照组(P < 0.01)。提示骨髓间充质干细胞部分通过血红素加氧酶1上调哮喘患者外周血CD4⁺CD25⁺CD127^low/-调节性T细胞。     

酸性成纤维细胞生长因子与胚胎干细胞的造血分化

背景：有研究表明,在卵黄囊造血、胎肝造血和胚胎干细胞向造血干细胞分化过程中,酸性成纤维细胞生长因子可强烈表达。 目的：探讨酸性成纤维细胞生长因子对小鼠胚胎干细胞造血分化的作用,在拟胚体培养阶段施加酸性成纤维细胞生长因子,验证酸性成纤维细胞生长因子对造血形成细胞产生的调控作用。 方法：培养小鼠胚胎干细胞,将饲养层上生长状态良好的小鼠胚胎干细胞用胰酶消化成单个细胞后,利用悬滴法制备拟胚体,拟胚体继续悬浮培养,以1,2和5 μg/L酸性成纤维细胞生长因子分别培养3,5,7,9 d,通过免疫荧光法检测胚胎干细胞与拟胚体中酸性成纤维细胞生长因子的表达,流式细胞术检测Flk-1⁺与CD133⁺阳性细胞率。 结果与结论：酸性成纤维细胞生长因子在拟胚体中呈阳性表达。在5 μg/L酸性成纤维细胞生长因子作用下,Flk-1⁺细胞随时间增加表达增加,CD133⁺细胞表达模式与Flk-1⁺细胞类似。在1 μg/L时,Flk-1⁺细胞在7 d时表达达到高峰,随后下降。CD133⁺细胞表达结果类似。而在2 μg/L时,5 d时Flk-1⁺细胞表达升高,随后下降,在9 d时表达又增高。而CD133⁺细胞则总体呈现升高趋势。酸性成纤维细胞生长因子可促进Flk-1⁺与CD133⁺细胞的产生,证明酸性成纤维细胞生长因子能够有效促进拟胚体的扩增以及成血管血液干细胞的产生与增殖。     

肝细胞生长因子对过氧化氢诱导肝细胞凋亡的影响

背景：肝细胞生长因子对多种细胞具有保护作用。 目的：观察肝细胞生长因子对过氧化氢诱导肝细胞凋亡的保护作用及其机制。 方法：采用人LO2肝细胞系,随机分成3组：正常对照组为正常培养的LO2细胞;模型组加入100 mmol/L过氧化氢作用LO2细胞4 h;肝细胞生长因子组加入50 mg/L 肝细胞生长因子预处理LO2细胞24 h,再加入100 mmol/L过氧化氢继续培养4 h后处理细胞。 结果与结论：体外培养的LO2细胞经100 mmol/L过氧化氢作用4 h后,LO2细胞可出现明显的凋亡现象,表现为细胞存活率降低(P < 0.01),Caspase-3蛋白表达增加(P < 0.01),Bcl-2蛋白表达降低(P < 0.01)。给予质量浓度50 mg/L 肝细胞生长因子预处理24 h后再加入100 mmol/L过氧化氢继续培养4 h,LO2细胞的凋亡被显著抑制(P < 0.01),说明肝细胞生长因子可通过增加LO2细胞Bcl-2的表达来抑制过氧化氢诱导的LO2细胞凋亡。     

不同时间移植人脐血CD34+细胞可影响心肌梗死大鼠心功能及血管内皮细胞生长因子的分泌

背景：干细胞移植可以改善心脏功能,改善预后。 目的：观察不同时间经静脉移植人脐血CD34+细胞对心肌梗死大鼠心功能及细胞因子分泌的影响。 方法：结扎冠状动脉左前降支制备Wistar大鼠心肌梗死模型,于梗死后1,5,10 ,30 d经尾静脉注入0.5 mL人脐血CD34+细胞(实验组)或磷酸盐缓冲溶液(对照组)。 结果与结论：与对照组相比,梗死后5,10 d实验组大鼠左室射血分数明显升高(P < 0.05),左室收缩末内径明显减小(P < 0.05),左室后壁增厚率更高(P < 0.05),毛细血管密度明显增加(P < 0.05),且以梗死后10 d移植大鼠心功能改善效果最明显(P < 0.05)。梗死后10 d实验组心肌局部血管内皮细胞生长因子最高(P < 0.05)。说明大鼠心肌梗死后5,10 d经静脉移植脐血CD34⁺细胞可明显改善心功能,梗死后10 d移植血管内皮细胞生长因子分泌更多,血管生成更多,对心功能的改善更明显;同时说明脐血单个核细胞移植可能是通过增加血管内皮细胞生长因子分泌,提高毛细血管密度来改善心功能的。     

注射用七叶皂苷钠联合脐带间充质干细胞移植改善脑梗死大鼠神经功能

背景：单纯的脐带间充质干细胞移植修复受损脑组织的作用并不十分理想。 目的：观察脐带间充质干细胞移植联合注射用七叶皂苷钠治疗大鼠脑梗死的效果。 方法：应用线栓法建立大鼠大脑中动脉阻塞模型,随机分为对照组、细胞移植组、七叶皂苷钠+ 细胞移植组,分别尾静脉注射细胞培养液、1×10¹⁰ L^-1脐带间充质干细胞悬液、尾静脉1×10¹⁰ L^-1脐带间充质干细胞悬液同时经腹腔注射七叶皂苷钠 5 mg/(kg•d),连续5 d。 结果与结论：移植后1周,七叶皂苷钠+细胞移植组大鼠神经功能障碍评分低于细胞移植组及对照组(P < 0.05);七叶皂苷钠+细胞移植组大鼠脑梗死周围组织AQP9 及AQP4 mRNA的表达低于细胞移植组,却高于对照组(P < 0.05);七叶皂苷钠+细胞移植组CM-Dil阳性细胞和神经元数量多于细胞移植组及对照组(P < 0.05)。提示脐带间充质干细胞移植联合注射用七叶皂苷钠治疗大鼠脑梗死可明显改善大鼠的神经功能。     

鼻饲热休克蛋白65诱导免疫耐受降低动脉粥样硬化的形成及其机制

背景：大量研究表明CD4⁺CD25⁺Foxp3⁺ 调节性T细胞与口服耐受和动脉粥样硬化抑制有关,然而有关经鼻耐受是否也有同样效应的报道较少。 目的：分析热休克蛋白65经鼻诱导免疫耐受对动脉粥样硬化的影响及其机制。 方法：6周龄ApoE基因敲除小鼠经鼻给予热休克蛋白65(实验组)或磷酸盐缓冲液(对照组),待小鼠16周龄时行冰冻切片测定小鼠主动脉根部粥样硬化斑块面积;流式细胞学检测小鼠体内CD4⁺CD25⁺Foxp3⁺  调节性T细胞水平;ELISA检测转化生长因子β水平。 结果与结论：经鼻给药后8周,实验组小鼠主动脉根部斑块面积较对照组明显减少,下降约32.7%(P < 0.01);经鼻给药14 d后,实验组小鼠CD4⁺CD25⁺Foxp3⁺ 调节性T细胞较对照组明显增加(P < 0 .01);经鼻给药第4,14天和8周,实验组转化生长因子β表达显著高于对照组。表明鼻饲热休克蛋白65通过诱导依赖抗炎因子转化生长因子β作用的调节性T细胞的产生建立免疫耐受,进一步抑制动脉粥样硬化形成,推测热休克蛋白65经鼻诱导免疫耐受是口服诱导免疫耐受之外另一种有效的抑制动脉粥样硬化的方法。     

bFGF在人胚胎干细胞中自我更新的研究进展

人胚胎干细胞具有自我更新和多向分化的独特生物学特性。维持人和小鼠胚胎干细胞增殖的生长因子不同,白血病抑制因子（LIF）不能维持人胚胎干细胞的生长。目前已经确定了数种维持人胚胎干细胞（hESCs）自我更新的生长因子,其中碱性成纤维细胞生长因子（bFGF）信号系统是人胚胎干细胞自我更新中最重要的调节因素之一。将从bFGF及其受体在人胚胎干细胞中的表达和作用的最新进展进行综述。     

Phenotypical changes of embryonic chick adrenal medullary cells in vitro induced by nerve growth factor and ciliary neuronotrophic factor

This study investigates the survival properties and changes in the morphological phenotype of adrenal medullary (chromaffin and neuronal) cells cultured from embryonic chicks at different developmental ages (embryonic days E8 to E16) in response to nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF). The 4-day survival of medullary cells from all embryonic ages except E8 was about 80% of the seeded cells and was only slightly enhanced by the addition of saturating doses of CNTF (10 ng/ml). With no factors, after 4 days 10-30% of the surviving medullary cells extended neurites. NGF (100 ng/ml) and, even more, CNTF (10 ng/ml) and their combination substantially increased the proportions of neurite-bearing cells (up to 70%). The effect of the factors were maximal at E10 and E12 and declined at older developmental ages. Neurite growth was virtually unaffected by NGF and CNTF at E8. These results show that in vitro survival and neurite growth of chick adrenal medullary cells in response to trophic factors is developmentally regulated.     

Platelet-derived growth factors induced lymphangiogenesis: evidence,unanswered questions and upcoming challenges

Crosstalk between angiogenesis and lymphangiogenesis in embryonic development continues during postnatal life and has specific mechanisms involving factors that initiate activation of the intracellular cascade for their specific receptors. Platelet-derived growth factors (PDGFs) and their corresponding receptors (PDGFRs) are known as important regulators of blood vessel development in both normal and pathologic angiogenesis. Despite some recent papers which reported a potential role of the PDGF/PDGFR axis in lymphatic spread of tumor cells, a few papers have suggested the potential role of PDGFs in tumor lymphangiogenesis development. The present paper summarizes the potential lymphangiogenic role of the PDGF/PDGFR axis, underlying upcoming challenges in the field.     

Exploitation of receptor tyrosine kinases by viral-encoded growth factors

Receptor tyrosine kinases (RTKs) are essential components of cell communication pathways utilized from the embryonic to adult stages of life. These transmembrane receptors bind polypeptide ligands, such as growth factors, inducing signalling cascades that control cellular processes such as proliferation, survival, differentiation, motility and inflammation. Many viruses have acquired homologs of growth factors encoded by the hosts that they infect. Production of growth factors during infection allows viruses to exploit RTKs for entry and replication in cells, as well as for host and environmental dissemination. This review describes the genetic diversity amongst virus-derived growth factors and the mechanisms by which RTK exploitation enhances virus survival, then highlights how viral ligands can be used to further understanding of RTK signalling and function during embryogenesis, homeostasis and disease scenarios.     

Immunolocalisation of epidermal growth factor (EGF), EGF receptor and transforming growth factor alpha (TGFα) during murine palatogenesis in vivo and in vitro

Summary The distribution of epidermal growth factor, the epidermal growth factor receptor and transforming growth factor alpha during murine palatogenesis was investigated immunocytochemically. On embryonic day 12 staining for transforming growth factor alpha was present throughout the palatal mesenchyme, with little in the epithelia. On embryonic day 13 staining increased in the palatal epithelia and in the mesenchyme at the tip of the palate. As the palatal shelves fused together (embryonic day 14.5) intense staining for transforming growth factor alpha was seen in the midline epithelial seam and in the subjacent mesenchyme. On embryonic day 15 there was a generalised increase in palatal epithelial staining; this was most marked in the remnants of the degenerating epithelial seam. Mesenchymal staining was, however, uniform. Whilst palatal staining for epidermal growth factor was sparse, at all stages, staining for its receptor was present throughout the palatal epithelia and mesenchyme. This was most intense in the palatal medial edge epithelia at the time of midline epithelial seam degeneration. The regional and temporal differences in staining for the epidermal growth factor receptor and transforming growth factor alpha suggested that these molecules may play an important role in normal palate development in vivo, particularly in degeneration of the midline epithelial seam.     

HLF细胞高氧暴露后miR-15b及miR-16上调并抑制VEGF表达

目的探讨高氧暴露下人胚肺成纤维细胞(HLF)中miR-15b及miR-16对VEGF蛋白的调控作用。方法用转染技术分别上调及下调HLF细胞miR-15b和miR-16水平;用Western blot技术检测细胞中VEGF蛋白;同时,用体外细胞培养方法,观察高氧暴露下HLF细胞miR-15b、miR-16及VEGF蛋白水平的影响。结果上调miR-15b和miR-16可使HLF细胞中VEGF蛋白降低,反之可使其升高;高氧暴露可诱导HLF细胞miR-15b和miR-16上调,而VEGF表达则下降。结论高氧暴露可诱导HLF细胞中miR-15b及miR-16上调并抑制VEGF的表达,可能参与支气管肺发育不良(BPD)的发生过程。     

Human mast cells express functional TrkA and are a source of nerve growth factor

Mast cells are the principal effector cells in IgE-dependent hypersensitivity reactions. Despite reports that rodent mast cells proliferate in the presence of nerve growth factor (NGF), human mast cells reportedly do not respond to this factor. To determine if human mast cells express the NGF receptors, TrkA tyrosine receptor and the low affinity NGF receptor (LNGFR), we first analyzed the mRNA expression by RT-PCR of TrkA and LNGFR in a human mast cell line (HMC-1) and in human mast cells cultured in the presence of stem cell factor. Both HMC-1 and cultured human mast cells were found to express TrkA but not LNGFR. TrkA protein was demonstrated by Western blot analysis of HMC-1 lysates. Using flow cytometric analysis and mast cell tryptase as a mast cell marker, both HMC-1 cells and cultured human mast cells were shown to co-express tryptase and TrkA. Treatment of mast cells with NGF resulted in phosphorylation of TrkA on tyrosine residues as detected by immunoblotting with an antiphosphotyrosine antibody. Furthermore, NGF induced the immediate early gene c-fos in HMC-1 cells. HMC-1 cells and cultured human mast cells were also found to express NGF mRNA, and conditioned medium from HMC-1 cells stimulated neurite outgrowth from chicken embryonic sensory ganglia in culture. This effect was blocked by anti-NGF. Thus, mast cells express functional TrkA and synthesize NGF, suggesting a mechanism by which NGF may act as an autocrine factor for human mast cells, and by which mast cells and nerves may interact.     

碱性成纤维细胞生长因子对胚鼠海马神经前体细胞的增殖和分化作用

为观察碱性成纤维细胞生长因子对体外培养的神经前体细胞的增殖和分化作用 ,本实验取胚胎 18d大鼠海马神经细胞 ,加入 2 5 ng/ m l碱性成纤维细胞生长因子置无血清培养基中进行培养。于培养第 4d和第 8d,用四唑盐比色法测定细胞活性 ,并用免疫组化方法定性、定量分析神经前体细胞的分裂和分化为神经元及神经胶质细胞的状态。结果显示 ,培养第 4d和第 8d,实验组的 OD值均增高 ,分别是对照组的 1.5倍和 1.8倍。免疫组化细胞分类计数显示 :培养第 4d,实验组神经前体细胞、神经元和少突胶质细胞数均明显增加 ,约是对照组的 2倍 ;但星形胶质细胞数无明显的变化。培养第 8d,实验组四类细胞均增加 ,约是对照组的 1.7倍。本实验提示 ,碱性成纤维细胞生长因子既能促进 E18的神经前体细胞的存活和分裂又能促进其向神经元和神经胶质细胞分化。并提示如拟在体外获得较大量和较高纯度的神经前体细胞 ,E18不是最理想的胚龄 ,需考虑选取胎龄更小的脑组织和加进足量的碱性成纤维细胞生长因子并进行传代培养。     

不同生长因子对人胚胎脑海马区神经干细胞体外生长的影响

本研究比较了不同生长因子对人胚胎脑海马区神经干细胞(NSCs)体外生长的影响。取胎龄8 ~12周的人胚胎脑海马,按基础培养液中生长因子的不同将培养细胞分为:h EGF组、h -bFGF组、h- EGF+h -bFGF组、h -EGF+h LIF组、h- bFGF+h LIF组、h -EGF+h bFGF+h- LIF组,将机械分离的单细胞悬液以2×106 个细胞/ml接种到上述组别,观察各生长因子的作用。结果发现,原代细胞培养6h时,各组细胞情形区别不大;随培养时间的延长,h -EGF+h- bFGF组和h- -EGF+h -bFGF+h- LIF组先形成许多小细胞簇, 7d后,这两组的细胞球数量进一步增加;而h- bFGF+h -LIF组和h- EGF+h -LIF组有较少的细胞球,h bFGF组偶见小细胞球,h- EGF组细胞几乎全部死亡。将培养的h -EGF+h -bFGF+h -LIF组的神经干细胞用1%FBS诱导分化后,行免疫细胞化学鉴定。结果表明,诱导分化12h时,细胞呈RNA 结合蛋白(Musashi1)阳性, 7d时,细胞大部分呈βⅢ管蛋白(βⅢ- tu- bulin)和胶质原纤维酸性蛋白(GFAP)阳性,极少数为半乳糖脑苷脂(Galc)阳性。以上结果提示,人胚胎脑海马区NSCs的体外存活增殖必须依赖于h -EGF和h- bFGF的共同作用,而h- LIF对早期培养的人NSCs的影响不明显。     

An immunohistochemical study of tumour vessels in metastatic liver cancers and the surrounding liver tissue

To elucidate the phenotype of the blood vessels and the expression of the growth factors involved in angiogenesis in metastatic liver cancers, we carried out an immunohistochemical study of 57 surgically resected livers with metastatic cancer. Blood vessels in the metastatic liver cancers frequently expressed von Willebrand factor (vWF), Ulex europaeus agglutinin I (UEA I)-binding sites, α-smooth muscle actin (α-SMA), type IV collagen and laminin. Sinusoidal endothelial cells around the metastatic liver cancers were positive for vWF in 33.3% of the specimens examined and for UEA I in 28.1%. α-SMA-positive perisinusoidal cells accumulated in the vicinity of the metastatic liver cancers in 68.4% of the specimens. Type IV collagen was detected in the perisinusoidal space close to the metastatic cancers as well as distant from them (91.2%). Laminin was detected in the perisinusoidal space in only one specimen (1.8%). Tumour cells of the metastatic liver cancers were positive for vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and acidic fibroblast growth factor (aFGF) in 78.9%, 38.4% and 7.0% of the specimens, respectively. Hepatocytes close to the metastatic liver cancers expressed bFGF more strongly than those distant from the metastatic liver cancers, and their expression of bFGF was more intense than that in the tumour cells. These results suggest that: (1) tumour vessels in metastatic liver cancers consist of endothelium, basement membrane and pericytes, (2) the sinusoids adjacent to tumours undergo capillarization, and (3) vascular endothelial growth factor may contribute to angiogenesis in metastatic liver cancer. Basic fibroblast growth factor may be responsible for the sinusoidal capillarization and the peritumoral fibrosis.