胚胎干细胞SM22α-EGFP表达克隆的建立及平滑肌细胞体外发育的动态观察

为探讨血管发育早期血管平滑肌细胞(VSMCs)募集和增殖特点,构建了含有SM22α启动子序列和增强型绿色荧光蛋白(EGFP)编码序列的质粒,建立了平滑肌特异性蛋白SM22α启动子控制下稳定表达EGFP的胚胎干细胞株(ESCs),以研究VSMCs的发育特点.实验发现,起源于SM22α-EGFPESCs形成的胚胎小体(EBs)在第11天开启SM22α启动子并表达EGFP.此后EGFP阳性细胞持续增加,在第30天达到高峰.VSMCs多起源于EBs中细胞密集处,应用免疫荧光染色及RT-PCR观察到EGFP阳性细胞表达多种平滑肌特异性标志物.在贴壁培养的胚胎小体中VSMCs形态可分为纺锤形及上皮样的多角形,慢速视频显微摄像测得纺锤形细胞迁移速度较上皮形细胞快.以上结果表明,SM22α-EGFPESCs分化形成的EBs可以模拟体内早期胚胎血管形成过程,从形态学上获得VSMCs募集分化的证据.     

PDGF-BB下调血管平滑肌标志物SM22α表达的机制

血管平滑肌细胞（vascular smooth muscle cell,VSMC）表型转化是血管重塑性疾病的细胞病理学基础,血小板源性生长因子（platelet-derived growth factor,PDGF）-BB抑制平滑肌分化标志基因表达、加速其降解,是VSMC表型转化的关键。该研究用PDGF-BB刺激VSMC诱导细胞发生表型转化,利用Western blot和免疫共沉淀等技术,检测PDGF-BB对早期分化相关基因平滑肌22 alpha（smooth muscle 22 alpha,SM22α）磷酸化与泛素化的影响。实验结果显示,PDGF-BB促进VSMC增殖;上调增殖相关蛋白PCNA的表达,下调分化相关蛋白SM22α与SMα-actin的表达;诱导SM22α发生磷酸化和泛素化,而且,该过程与SM22α水平下调具有时相相关性;抑制剂阻断分析证实,ERK和PKC参与介导了PDGF-BB诱导的SM22α磷酸化。以上结果提示,在VSMCs表型转化中,PDGF-BB可能是通过激活ERK-PKC信号通路,促进SM22α的磷酸化和泛素依赖的蛋白质降解。     

E1A激活基因阻遏子表达与小鼠胚胎血管发生的关系

目的探讨E1A激活基因阻遏子(CREG)蛋白表达与小鼠动脉形态学发生的关系,了解CREG蛋白在血管发育中的生物学作用。方法制备E9.5d-E18.5d胎鼠、新生1d、28d和2月成鼠不同脏器功能血管的石蜡组织切片,观察主动脉发生过程中的形态学变化;采用免疫组化染色法观察不同发育时相的血管细胞中CREG蛋白和血管平滑肌细胞(vascular smooth muscle cells,VSMCs)标记物肌动蛋白(SMα-actin)的表达变化。结果HE染色显示E9.5d的胚胎血管由单层血管内皮细胞构成,免疫组化显示CREG表达阳性,主要定位于血管壁的单层内皮细胞中,SMα-actin表达为阴性;E10.5d的胚胎血管内皮细胞周围开始出现少量SMα-actin表达阳性的原始VSMCs,同时CREG蛋白表达,定位与SMα-ac-tin蛋白一致。自E12.5d开始SMα-actin蛋白在血管中膜VSMCs中表达增强并持续至成年。CREG蛋白在三层结构的血管细胞中均为阳性表达,其表达强度在E15.5d达到最高,E18.5d表达下降并维持至成年。进一步分析CREG蛋白在成年鼠心、肺、脾和肾等多个脏器血管中的分布,发现所有脏器血管细胞均表达CREG,但表达丰度明显不同,在储存血管和分配血管中CREG蛋白呈高表达,在调节血管中低表达。结论CREG蛋白在小鼠胚胎血管发育早期、持续表达的特点及其在不同脏器功能血管中表达的差异,提示CREG蛋白可能通过调控并维持血管细胞,特别是VSMCs的分化,参与了胚胎血管发生的调控。     

RhoA和血清反应因子(SRF)介导E1A激活基因阻遏子诱导人血管平滑肌细胞分化

为探讨E1A激活基因阻遏子(CREG)对人血管平滑肌细胞(VSMCs)分化的调控机制,应用重组逆转录病毒表达载体pLNCX2( )/CREG及pLXSN(-)/CREG制备稳定感染HITASY细胞模型,观察CREG蛋白过表达及表达抑制对人VSMCs分化的影响并探讨其调控机制.结果显示:pLNCX2( )/CREG稳定感染细胞呈分化表型,细胞细长变成组织样聚集生长趋势,细胞中CREG蛋白和平滑肌分化标志蛋白平滑肌α-肌动蛋白(SMα-actin)表达显著增加,同时SMα-actin相关调控因子——血清反应因子(SRF)入核转位,RhoA总蛋白表达上调,以Rho激酶特异性抑制剂Y-27632作用后,CREG诱导的SMα-actin表达下调的同时SRF出核转位;pLXSN(-)/CREG稳定感染的细胞体积变大,细胞极性消失,呈无序生长,细胞中CREG和SMα-actin蛋白表达显著降低,同时伴有SRF出核转位及RhoA总蛋白表达下调.免疫共沉淀分析发现,CREG蛋白能被分泌到VSMCs培养基中表达,并可与细胞膜受体6-磷酸甘露糖/胰岛素样生长因子Ⅱ型受体(M6P/IGF2R)发生直接相互作用.用蛋白磷酸酶PP2A特异性抑制剂okadaicacid减少M6P/IGF2R在细胞膜表面分布,可明显抑制CREG过表达引起的RhoA、SRF和SMα-actin表达.上述结果提示,在体外培养的人VSMCs中,CREG可能作为一种分泌型蛋白质通过与细胞膜受体M6P/IGF2R相互作用,依次激活SMα-actin蛋白相关调控因子RhoA和SRF引起SMα-actin表达增加,促进VSMCs向分化表型转换.     

E1A激活基因阻遏子过表达诱导体外培养大鼠平滑肌细胞分化

为探讨E1A激活基因阻遏子蛋白(CREG)对血管平滑肌细胞表型转换的调控机制,应用pRC/CMV-hCREG真核表达载体转染体外培养的大鼠血管平滑肌细胞(VSMCs),观察了转染前后细胞的表型改变.结果发现,pRC/CMV-hCREG质粒转染后,大鼠平滑肌细胞增殖受抑,细胞分化标志蛋白SM α-actin表达显著增加.免疫共沉淀分析发现,CREG与血清反应因子(SRF)发生相互作用形成复合体,在CREG基因过表达时,与CREG结合的SRF蛋白增加.凝胶迁移阻滞分析(GSMA)和抗体凝胶迁移阻滞分析(supershift)显示,过表达的CREG蛋白与SRF蛋白共同结合到 SM α-actin基因启动子区CArG位点上,可能参与 SM α-actin表达的调控. 构建SM α-actin promoter- pCAT^® 3报告基因载体并与pRC/CMV-hCREG质粒共转染VSMCs也证实,CREG蛋白过表达可增强SM α-actin基因表达.上述研究提示,CREG蛋白可能是调控VSMCs表型转换的重要分子.     

血清后人血管平滑肌细胞表型转化与microRNAs表达间关系的研究

目的:研究去血清诱导人血管平滑肌细胞发生表型转化与microRNAs表达间的关系。方法:采取人血管平滑肌细胞克隆株HITASY,培养人血管平滑肌细胞(VSMCs)。用去血清的方法处理人血管平滑肌细胞,使VSMCs由去分化表型向分化表型转化,通过蛋白印迹法观察平滑肌细胞中分化标记物蛋白的变化,同时用实时定量PCR法检测细胞中相关microRNA的表达。结果:①去血清处理组与无去血清处理组比较,平滑肌细胞分化标记物(SM-α-actin、calponin)表达显著性增加,而通过SM-α-actin、calponin的蛋白表达量显著增加,提示血管平滑肌细胞向分化表型转化(P0.05);②同时去血清处理组与无去血清处理组比较,Mir-649、Mir-944表达量显著增加,Mir-140、Mir-361表达量显著减少(P0.05)。结论:Mir-649、Mir-944、Mir-140、Mir-361在血管平滑肌细胞表型转化中有关键性作用。     

SM22α:血管平滑肌细胞分化的分子标志

SM22α是一种分化型血管平滑肌细胞(VSMC)的标志基因,编码一种22kDa的收缩调节蛋白。由于SM22α基因结构短小,表达具有VSMC特异性、调控机制较为清楚,因而被广泛用于VSMC发育分化的研究。利用该基因的表达调控特征,设计可在VSMC中高表达目的蛋白的人工启动子,是心血管病基因治疗的新策略。     

胚胎干细胞分化形成囊状胚胎小体的形态学变化特征

目的观察胚胎干细胞R1(ESs)体外分化形成囊状胚胎小体(CEBs)及其血管内皮细胞和平滑肌细胞的时相规律和结构特点,探讨其在血管发育生物学研究中的作用和意义。方法体外悬浮培养ESs形成CEBs,采用HE染色观察CEBs的结构特点。用免疫荧光染色鉴定不同培养时间的CEBs中CASPASE-3的表达。加入全反式维甲酸(atRA)及联丁酰基cAMP(DBcAMP)定向诱导ESs分化,其后行平滑肌特异性标志物α-肌动蛋白(SMα-actin)及内皮细胞特异性标志物PECAM-1免疫荧光染色。结果典型的CEBs在第13d左右可以形成。第6d就可见CEBs中心部位出现调亡细胞,坏死腔开始形成,到第13d时中心坏死腔完全形成,凋亡细胞明显增多。将第6d的CEBs接种于培养皿后用atRA及DBcAMP处理,3d后可见SMα-actin染色阳性细胞。而未经处理的CEBs贴壁后,在第10d可见PECAM-1的表达。结论CEBs能够模拟体内血管发生形成的过程,可作为研究血管发育生物学的较好模型。     

SM-22α、α-SMA的表达与下肢静脉曲张具有相关性

探讨SM-22α、α-SMA水平变化与下肢静脉曲张的相关性及其意义。采用间苯二酚品红染色法观察血管平滑肌细胞(VSMCs)结构,通过免疫组织化学染色观察正常大隐静脉及曲张大隐静脉切片SM-22α和α-SMA表达情况;利用RT-PCR检测正常大隐静脉及曲张大隐静脉中SM-22α和α-SMA m RNA表达水平,并用Western blotting检测正常大隐静脉及曲张大隐静脉SM-22α和α-SMA蛋白表达水平。结果显示,曲张静脉管壁血管平滑肌细胞SM-22α和α-SMA阳性表达率较正常大隐静脉均明显降低,与正常组比较,静脉曲张患者血清中SM-22α和α-SMA m RNA表达水平均明显降低,血管管壁平滑肌细胞SM-22α和α-SMA蛋白表达水平均明显降低(p0.05或p0.01)。曲张静脉VSMCs有明显向内膜增殖"迁移"现象,且收缩型VSMCs中SM-22α和α-SMA m RNA及蛋白表达水平均随VSMCs去分化程度增加而减少,这可能是静脉曲张发生、发展的机制之一。     

胚胎干细胞起源的探讨

目前胚胎干细胞(ESCs)建系的取材来源包括桑椹胚的卵裂球、囊胚的内细胞团(ICM)、上胚层细胞和原始生殖细胞(PGCs),甚至从新生鼠睾丸细胞也分离得到类ES样细胞系。这就提出了一个问题,什么是ESCs最接近的体内细胞来源。传统观念常常把ESCs等同于ICM细胞,也有学者认为ESCs更象上胚层细胞,而在已知的分子标记基因方面,ESCs所具有的特征更接近体内早期生殖细胞。不清楚ESCs最接近的体内细胞来源,可能是制约许多品系小鼠和大多哺乳类动物建系成功率提高的原因之一。ESCs系与EG细胞系的分离条件不同表明,加强对ESCs多能性维持基因调控研究具有重要意义。本文从ESCs的经典概念及其发展,早期胚胎细胞和生殖细胞发育规律,早期胚胎细胞、早期生殖细胞和ESCs的关系等方面进行综合分析,认为ESCs可能有多种接近的体内细胞来源。进一步应通过对ESCs建系不同的取材细胞和不同品系的ESCs间进行比较研究,以便弄清ESCs的来源和转化机制,为提高不同物种ESCs建系效率提供理论支持。     

Fine needle aspiration (FNA) cytology of pilomatrixoma

FNA smears from five histologically confirmed cases of pilomatrixoma were reviewed to delineate the cytological features helpful in diagnosis. A combination of basaloid cells, ghost cells and foreign body giant cells appeared to be necessary in FNA smear for a confident cytodiagnosis of pilomatrixoma. Presence of naked nuclei, nucleated squamous cells and calcification were additional features in favour of the diagnosis. Another 10 cases with initial cytodiagnosis of pilomatrixoma or benign skin appendage tumour were reviewed. Using the above criteria, diagnosis of pilomatrixoma was easy in five cases. One case was problematical due to presence of atypical squamous cells. Initially the cytological features were most commonly confused with epidermal inclusion cyst, giant cell lesion or a squamous cell carcinoma. The main reasons for erroneous diagnosis were lack of awareness of cytological features, predominance of one component over the others, and non‐representative FNA smears. Atypia in nucleated squamous cells, and misinterpretation of basaloid cells as malignant can lead to diagnostic dilemma. Adequate clinical data are also necessary.     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.     

树突状细胞与CIK细胞共培养诱生的DCCIK细胞群对肿瘤的杀伤作用

由树突状细胞(DC)与细胞因子诱导的同源杀伤细胞(CIK)的共培养诱生的细胞群(DCCIK)对肿瘤细胞的细胞毒活性的研究。DCCIK细胞体外杀伤肿瘤靶细胞A549(MTT法),效靶比为10∶1、5∶1时杀伤率分别为61%、52%。DCCIK细胞诱导培养3周后,效靶比为10∶1、5∶1时杀伤率分别为64%和56%。数据亦表明DCCIK细胞对靶细胞的杀伤优于CIK细胞。动物体内实验分荷瘤A549、BEL7404和A375三组,每组分(A)DCCIK 化疗、(B)单用化疗。治疗20天、35天后测量各组肿瘤消失率。结果显示:DCCIK 化疗的抑瘤效果明显好于单纯化疗。提示DCCIK细胞有临床应用前景。     

Resistance and augmentation of innate immunity in mice exposed to starvation

Mice were exposed to starvation for 3 days. Body temperature and various parameters were examined. By starvation, body temperature, blood glucose and ACTH decreased, especially on days 2 and 3. The level of corticosterone increased at this time. On the other hand, the number of lymphocytes yielded by the liver, spleen and thymus decreased from day 1 to 3. The change of the distribution of lymphocyte subsets was unique because NK, NKT and extrathymic T cells were stress-resistant in the liver. Conventional T and B cells were stress-sensitive. Reflecting the increased proportion of NK and NKT cells, NK and NKT activities were augmented. The increased proportion of NKT cells produced both IFNγ and IL-4 (Th0-type profile). The proportion and some functions of granulocytes and macrophages increased on Day 1 after starvation. These results suggest that starvation has a potential to increase the functions of unconventional lymphocytes and myeloid cells.     

Ultrastructure of the surface of the iris in the rat eye

Summary The surface structure of the iris in the rat eye was studied by light and electron microscopy.The anterior surface of the rat iris is covered with a discontinuous layer of large, polygonal endothelial cells with microvilli on their surface. Crypts and holes between adjacent endothelial cells extend into the stroma and form there a complicated network of interconnected spaces occupying about one half or more of the volume of the pupillary part of the stroma. The crypts are occasionally partly covered with endothelial cells. The posterior surface is covered with a continuous layer of polyhedronal epithelial cells. These are covered with many folds and processes, partly masked by an amorphous coat. The sphincter pupillae and dilatator muscles are possible to recognize on the scanning electron micrographs as well as blood vessels and nerve fibers in the iris stroma.The endothelial cells show many structural similarities with the endothelial cells on the cornea, probably reflecting their common origin. The results obtained, especially those from the scanning electron microscopic studies, are discussed and interpreted in relation to previous studies. The advantages in using different light and electron microscopic techniques are stressed.Supported by grants from Magnus Bergwall's Stiftelse and the Swedish Medical Research Council (B71-12X-2543-03).     

猪多能干细胞的研究进展

多能干细胞,如胚胎干细胞（embryonic stem cells,ESCs）、诱导多能干细胞（induced pluripotent stem cells,iPSCs）和成体干细胞（adultstemcells,ASCs）,是一类具有巨大潜能的独特细胞。猪作为试验材料,在遗传、代谢、生理生化及基因序列等方面较小鼠更接近于人类,正逐渐成为人类异种移植和再生医学研究的理想生物学模型。然而,目前对猪多能干细胞种类、来源、特征及机制的有限认识直接阻碍了其相关应用。该文将分别对猪ASCs的研究现状、猪类ESCs的分离培养、猪iPSCs的研究进展、多能干细胞间的联系和展望进行论述,以期为从事该领域研究的科研人员提供参考。     

Enhancement of BDNF production by co-cultivation of human neuroblastoma and fibroblast cells

It has been proved that co-cultivation of human neuroblastoma cells and human fibrolast cells can enhance nerve cell growth and the production of BDNF in perfusion cultivation. In batch co-cultivation, maximum cell density was increased up to 1.76×10⁶ viable cells/mL from 9×10⁵ viable cells/mL of only neuroblastoma cell culture. The growth of neuroblastoma cells was greatly improved by culturing both nerve and fibroblast cells in a perfusion process, maintaining 1.5×10⁶ viable cells/mL, which was much higher than that from fed-batch cultivation. The nerve cell growth was greatly enhanced in both fed-batch and perfusion cultivations while the growth of fibroblast cells was not. It strongly implies that the factors secreted from, human fibroblast cells and/or the environments of co-culture system can enhance both cell growth and BDNF secretion. Specific BDNF production rate was not enhanced in co-cultures; however, the production period was increased as the cell growth was lengthened in the co-culture case. Competitive growth between nerve cells and fibroblast cells was not observed in all cases, showing no changes of fibroblast cell growth and only enhancement of the neuroblastoma cell growth and overall BDNF production. It was also found that the perfusion cultivation was the most appropriate process for cultivating two cell lines simultaneously in a bioreactor.