转基因骨髓基质细胞系对人脐血CD34+细胞的体外扩增作用

为探讨转染FL和/或TPO基因骨髓基质细胞系对脐血CD34+细胞的体外扩增效应, 建立了转基因骨髓基质细胞系共培养体系. 采用免疫磁珠法分离人脐血CD34+细胞, 在CD34+细胞不同体外培养体系中取样测试细胞总数、CD34+细胞百分率和CFC(包括CFU-GM 和BFU-E). 结果表明, 在8种不同组合的培养体系中, 转基因基质细胞共培养体系较无基质液体培养体系对细胞总数, CFC, CD34+细胞均具有明显的扩增效应, 其中以SCF + IL-3 + HFT扩增效果最好, 分别扩增了(893.3±52.1), (74.5±5.2)和15.7倍. CFU-GM和BFU-E在第2周时达扩增高峰, 扩增倍数分别为(78.1±5.5)和(57.0±19.7). LTC-IC测定结果显示, 只有SCF + IL-3 + FL + TPO和SCF + IL-3 + HFT组有LTC-IC的存在, 统计学检验无显著性差异. 上述结果提示, 转基因骨髓基质细胞系可通过细胞间的接触协同其他细胞因子增强对脐血CD34+细胞的体外扩增作用.     

小鼠胎肝造血基质细胞对CFU—E,BFU—E生长的体外调控作用

造血基质细胞是造血微环境的重要成分,它对造血干细胞和祖细胞增殖分化的影响已引起人们重视。本室建立的小鼠胎肝造血基质细胞系(MFLSC)为研究其在调控造血中的意义提供了方便条件。 以往研究证明,MFLSC可向培养上清液中释放多种造血活性物质,将此培养上清代替外源性刺激因子加入到半固体培养体系中可支持红系、粒系或由红、粒、巨噬细胞组成的集落(CFU—EGM)生长。MFLSC本身对CFU-GM生长的调控作用已有报道。本工作观察MFLSC对小鼠骨髓红系造血祖细胞生长的影响,旨在进一步认识小鼠胎肝基质细胞在调控造血中的作用机理。     

造血微环境细胞组成及功能的研究进展

造血微环境是造血干细胞(HSCs)居住的场所,对于维持HSCs自我更新、分化与稳态有着重要的调控作用。伴随着胚胎发育,造血主要分为卵黄囊造血、主动脉-性腺-中肾造血、胎肝造血及骨髓造血4个时期,因而研究造血发育时期的微环境对体外HSCs的扩增及诱导分化有着重要的指导意义。现对4个造血时期的造血微环境细胞组成及功能进行综述,阐明不同时期造血微环境调控作用的异同,为实现HSCs在体外的扩增与分化打下基础。     

Wnt3a 转基因基质细胞的建立及其对脐血CD34+ 造血干/祖细胞扩增作用的研究

Wnt 信号通路在造血干/祖细胞自我更新的过程中发挥至关重要的作用 . 纯化的 Wnt3a 蛋白可以实现造血干/祖细胞的扩增 . 通过病毒转染原代小鼠骨髓基质细胞,建立转基因滋养层细胞 . 通过共培养对转基因滋养层细胞扩增 CD34+ 造血干/祖细胞的作用进行了研究 . 实验结果显示 , 与普通滋养层加细胞因子组相比,经转基因滋养层加细胞因子组培养的 CD34+造血干/祖细胞集落形成能力 (CFC) 是其 (1.55±0.06) 倍;混合集落形成能力是其 (1.95±0.26) 倍;高增殖潜能集落形成能力 (HPP-CFC) 是其 (1.45±0.40) 倍; LTC-IC 活性是其 (3.83±0.86) 倍 . 结果表明,转基因滋养层细胞通过分泌具有天然活性的 Wnt3a 蛋白能在体外有效地扩增造血干/祖细胞的数量 .     

内皮和造血系统特异性敲除Rictor基因对胎肝造血的影响

目的:研究Rictor基因对胚胎发育过程中胎肝造血的影响。方法:利用Cre-LoxP基因敲除系统,特异性在小鼠内皮(VEC-Cre)和造血(Vav1-Cre)系统敲除Rictor基因;通过流式细胞术分析特异敲除Rictor基因后小鼠胚胎第15 d胎肝中的各系细胞比例的变化,并进一步分析造血干细胞的变化。结果:利用VEC-Cre和Vav1-Cre小鼠敲除Rictor基因后,胎肝中各系细胞比例均有所减少,B细胞比例的减少较为明显,造血干细胞比例也明显减少。结论:Rictor基因敲除损害胎肝组织中造血干细胞的产生和各系细胞的分化。     

小鼠胎肝和骨髓造血基质细胞贴壁层对红系祖细胞生长的影响

本实验以Dexter培养体系作小鼠胎肝和骨髓造血基质细胞贴壁培养。在所获的基质细胞贴壁层上作红系造血祖细胞集落培养,观察两种来源造血基质细胞对红系集落生长的影响。实验结果表明,胎肝造血基质细胞贴壁层能明显促进早期红系造血祖细胞(BFU-E)形成集落,却不明显影响晚期红系造血祖细胞(CFU-E)的生长。成年小鼠骨髓造血基质细胞贴壁层对BFU-E和CFU-E均有刺激生长的作用;但对前者生长的刺激性影响较胎肝造血基质细胞贴壁层为弱。造血基质细胞贴壁层对红系集落生长的促进作用主要是通过体液因子实现的,细胞间短距离调节的影响亦不能除外。     

小鼠胎肝造血基质细胞系对CFU—GM生长的调控作用

利用本实验室建立的小鼠胎肝造血基质细胞系(MFLSC)研究了造血基质细胞对粒-巨噬系造血祖细胞(CFU-GM)生长的影响。实验结果表明,MFLSC对CFUGM集落的生成有双向调控作用,即既有刺激性作用,又有抑制性影响。这种调控有体液因子之中介,亦有细胞与细胞间近距离的效应。文中讨论了MFLSC双向调控CFUGM生长的意义及其机理。     

小鼠胎肝红系细胞在分化过程中形态学观察及时序性表达分析

小鼠胎肝是小鼠发育早期主要的造血器官,红系细胞在胎肝造血过程中形态特征和组成成分等方面发生了明显变化。根据红系细胞体积的变化,利用Countstar细胞计数仪对小鼠E12．5-E17．5胎肝中直径8-14岬细胞进行数量统计,再结合观测到的红系细胞的形态特征和血红蛋白表达量的不同,将E9．5．E17．5胎肝中的细胞分为10类。统计结果显示,随着胎肝造血系统的发育,哺乳类红系细胞在终末分化时出现细胞体积减小、细胞核固缩、排核和血红蛋白表达量增加等时序性变化。红系细胞表面特异标志Terll9和CD71在EryD中高表达而在成体骨髓细胞和外周血细胞中表达较低的结果表明胎肝中红系细胞具有较高的分化能力。这些数据为研究红系分化、克隆红系分化相关基因及探讨红白血病发生的机制提供了理论依据。     

小鼠胎肝基质细胞系MFLC自发分泌多种细胞因子

在无外源刺激条件下,我室所建小鼠胎肝基质细胞系MFLC可自发分泌多种类型细胞因子,其中IL-6及化学趋化因子水平较高,GM-CSF水平较低,但未检测到IL-3及IL-7活性。此细胞上清对小鼠骨髓造血干细胞有明显的促集落形成效应,并呈现剂量依赖关系,所形成的集落以CFU-GMM及CFU-GM为主;此细胞上清还促进5-Fu耐受小鼠骨髓造血干细胞的集落形成,提示上清中存在SCF样活性成份。上述结果表明,MFLC的建立有利于分析干细胞在胎肝内如何向pro-T细胞分化发育的机理并有利于阐明细胞因子网络调节在其中的作用。     

逆转录病毒介导的稳定表达E4orf1和绿色荧光蛋白的胎肝窦内皮细胞株的建立

目的:为构建优化的内皮细胞用于造血干细胞(HSCs)的支持培养,通过逆转录病毒载体系统建立稳定表达腺病毒E4区开放读框1(E4orf1)和绿色荧光蛋白(GFP)的人胎肝窦内皮细胞(HFLSECs)株。方法:利用Plat-A细胞将质粒MSCV-N E4orf1和p MX-GFP分别包装为逆转录病毒,共同感染HFLSECs,将原代培养的HFLSECs和转基因的HFLSECs分别在含有0.5、1、2、4μg/m L嘌呤霉素的EGM-2培养基中培养,以测试最适药物筛选浓度,药筛1周后通过流式细胞分选获得稳定转染E4orf1的GFP~+HFLSECs(E4orf1-GFP/HFLSECs)。通过密度梯度离心法从脐带血中分离单个核细胞,利用免疫磁柱分选获得CD34~+造血干/祖细胞(HSPCs),以E4orf1-GFP/HFLSECs作为饲养层联合SCF、TPO、Flt-3L等3种基础造血相关因子进行体外扩增和半固体造血细胞集落培养。结果:0.5μg/m L嘌呤霉素在5 d内即能完全杀死HFLSECs,而转基因的HFLSECs可以正常存活,以此药物浓度加压筛选1周,后续通过流式分选GFP阳性细胞,阳性率为90.5%,在E4orf1-GFP/HFLSECs饲养层支持下,人脐带血来源的CD34~+细胞15 d扩增了360倍,是单纯细胞因子悬浮培养组的2.2倍,且扩增后的HSPCs在体外仍具有多种造血集落形成能力。结论:该细胞株的建立将为构建造血干细胞体外培养体系及研究造血细胞的发育分化提供适宜的微环境。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.