体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34⁺富集细胞在SCF+IL-3+IL-6+FL+Tpo细胞因子组合下的体外扩增特性,发现：CD34⁺富集细胞具有很高的扩增潜力,在本实验条件下其总细胞持续扩增了8周,扩增倍数达31270.9±8640.5倍;而MNC在培养至第4周扩增就已呈现下降趋势,最大仅扩增了53.3±6.2倍。对比集落和CD34⁺细胞的扩增发现,MNC的集落密度和CD34⁺细胞含量由第0天至第7天有一个上升的过程,而CD34⁺富集细胞在培养过程中,集落密度和CD34⁺细胞含量却始终呈下降趋势。在体外培养过程中,CD34⁺富集细胞的CFU-GM和CD34⁺细胞最大分别扩增了185.7±14.1和191.7±188.8倍,明显高于MNC的12.4±3.2和50.6±33.2倍;而CD34⁺富集细胞和MNC的BFU-E则只实现了少量扩增,分别为7.2±5.2和10.1±3.4倍。结果显示,从CD34⁺富集细胞出发扩增造血干/祖细胞,可以得到更多的CD34⁺细胞和CFU-GM集落形成细胞。      

脐血造血细胞的体外扩增：1.生长规律的研究

考察了在添加细胞因子和未添加细胞因子培养条件下的造血细胞群体的生长和代谢,研究了长期培养条件下造血祖细胞生长规律。在静态培养条件下,脐血造血细胞群体的比生长速率为0.34d^-1,倍增时间为2d。培养后期,造血细胞消耗了大部分葡萄糖,乳酸浓度可达40 mmol/L。在造血细胞长期培养条件下,CFU-GM产出最高峰在培养第2周与第3周之间。BFU-E产出最高峰在培养第1周。每天换50%培养液,造血细胞总数扩增了14倍,CFU-GM扩增了13倍,BFU-E扩增了5倍。     

具有维持造血干/祖细胞能力的新型豆类凝集素的分离、纯化及功能鉴定

一种分离自豆类的新型凝集素不仅具有凝集活性,还具有体外长期维持造血干/祖细胞的能力.由眉豆（Dolichos lablab）中分离得到了这种多亚基的凝集素——FRIL（Flt3 receptor-interacting lectin）,并对它进行了核酸和蛋白质序列分析.免疫细胞分析显示,它的受体是CD34⁺造血干/祖细胞所特有的.在培养基中添加这种凝集素可长期维持CD34⁺细胞存活和增殖能力.以Flt3配基（FL）作为对照,在28天的培养时间内,相对于FL,FRIL可维持细胞较高的G0/G1期比例（80%以上G0/G1期）和长期培养中（14天以上）1.5倍以上的集落形成量.可见FRIL通过滞留造血干/祖细胞于G0/G1期而维持它们的自我更新潜能.     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34 +富集细胞在SCF +IL 3+IL 6 +FL +Tpo细胞因子组合下的体外扩增特性 ,发现 :CD34 +富集细胞具有很高的扩增潜力 ,在本实验条件下其总细胞持续扩增了 8周 ,扩增倍数达 312 70 9± 86 40 5倍 ;而MNC在培养至第 4周扩增就已呈现下降趋势 ,最大仅扩增了 5 3 3± 6 2倍。对比集落和CD34 +细胞的扩增发现 ,MNC的集落密度和CD34 +细胞含量由第 0天至第 7天有一个上升的过程 ,而CD34 +富集细胞在培养过程中 ,集落密度和CD34 +细胞含量却始终呈下降趋势。在体外培养过程中 ,CD34 +富集细胞的CFU GM和CD34 +细胞最大分别扩增了 185 7± 14 1和 191 7± 188 8倍 ,明显高于MNC的 12 4± 3 2和 5 0 6± 33 2倍 ;而CD34 +富集细胞和MNC的BFU E则只实现了少量扩增 ,分别为 7 2± 5 2和 10 1± 3 4倍。结果显示 ,从CD34 +富集细胞出发扩增造血干 祖细胞 ,可以得到更多的CD34 +细胞和CFU GM集落形成细胞     

两种免疫磁珠分离法分选小鼠骨髓粒-单核祖细胞的效率比较

摘要 目的：提取小鼠骨髓细胞（bone marrow cell, BMC）,用两种不同的免疫磁珠分离（magnetic activated cell sorting, MACS）试剂盒从小鼠BMC中分选提纯粒-单核祖细胞（granulocyte-monocyte progenitor, GMP）,比较这两种免疫磁珠的分选效率。方法：从小鼠股骨和胫骨中提取BMC,通过两种不同的MACS试剂盒,即Lineage阳性细胞清除试剂盒和CD117阳性细胞分选试剂盒,分别得到Lineage-细胞群和CD117⁺细胞群,用代表GMP细胞表面标志物的荧光抗体标记,孵育后通过流式细胞荧光分选技术得到GMP细胞,并且对比得到GMP细胞的效率。结果：每2只野生型C57BL/6J小鼠可共收集骨髓细胞（7.02±1.24）×10⁷个,细胞活力为（91.86±5.24）%。经过Lineage阳性细胞清除试剂盒得到的细胞数量为（5.71±2.86）×10⁶个;经过CD117阳性细胞分选试剂盒得到的细胞数量为（2.70±0.56）×10⁶个。Lineage磁珠分选纯化得到的GMP细胞数占总细胞数的比例为（10.90±1.37）%,CD117磁珠分选纯化得到的GMP细胞数占总细胞数的比例为（4.83±2.08）%。结论：Lineage阳性细胞清除试剂盒能更有效分选小鼠骨髓细胞中的粒-单核祖细胞。     

造血细胞体外悬浮培养和生物反应器开发

为解决造血细胞的静态培养中由浓度梯度引起的培养不稳定、环境不均一、难放大等问题,首先采用转瓶对脐血单个核细胞进行了悬浮培养研究,结果表明,悬浮培养中总细胞、集落和CD34^＋细胞的扩增都高于静态的方瓶培养。在测试了所用材料生物相容性的基础上,开发了可以控制溶氧和pH的生物反应器,并将其应用到造血细胞的批培养中,结果表明反应器的培养环境均一,可实现较高密度的培养,而且总细胞、集落和CD34^＋细胞的扩增都优于静态培养。大规模的反应器培养有利于解决临床应用中细胞数量不足的问题。     

人树突状细胞的大量培养及鉴定

摘要 目的：探讨人树突状细胞体外大量培养及鉴定方法。方法：采用免疫磁珠法分离纯化CD34⁺干细胞;采用含有TPO、SCF、Flt3L和IL-3的扩增培养基培养1周,以及含有SCF、Flt3L、GM-CSF和IL-4的分化培养基培养2-3周,获得CD34⁺细胞来源树突状细胞。采用普通光学显微镜观察细胞形态,牛鲍氏血细胞计数板进行细胞计数,荧光抗体标记、流式细胞仪检测细胞纯度和细胞表面共刺激分子的表达情况。结果：以含有TPO、SCF、Flt3L和IL-3的培养基扩展培养一周,及含有SCF、Flt3L、GM-CSF和IL-4的培养基诱导分化3周,可获得大量悬浮细胞;细胞数目扩增倍数约达50倍;普通光学显微镜下可见悬浮细胞有明显的树突状凸起;流式细胞术检测结果显示悬浮细胞中CD141和CD11c双阳性细胞（等同于单核细胞来源树突状细胞）比例达30%,此群细胞高表达HLA-DR和CD209,低表达共刺激分子CD80和CD86;细胞寿命较短,40天时培养体系中悬浮细胞和CD34⁺细胞来源树突状细胞数目急剧减少。结论：采用多细胞因子联合刺激可获得大量的树突状细胞,为树突状细胞的特性及功能学研究奠定了基础。     

葡萄糖的添加对昆虫细胞Sf21悬浮生长的影响

添加葡萄糖对昆虫细胞Sf21(Spodoptera frugiperda)悬浮生长的影响,发现补加糖量在lg／L时·能明显提高细胞生长的速度和最高密度,细胞最高密度由2．5×10⁴／ml增加到4.9×10⁶／ml; 朴加糖量在2g／L时,则有显著的抑制作用,即使增加接种密度．细胞生长的最高密度也只有2．1×10⁶／ml。当采取流加葡萄糖方法来培养细胞时,则其生长的最大密度可提高到5．2×10⁶／ml。     

人核糖核酸抑制因子基因在人脐血干细胞中的转染表达及对小鼠黑色素瘤基因治疗的研究

探讨人核糖核酸抑制因子 (hRI)基因在人脐血干细胞中的转染及表达情况 ,及转染后对小鼠B16黑色素瘤生长的影响。用免疫磁珠分离系统 (MACS)分离纯化人脐血CD34⁺ 细胞后 ,用制备的含hRI基因的逆转录病毒上清转染脐血CD34⁺ 细胞 ,采用克隆形成法和PCR法检测转染效率 ,Western blot和免疫荧光法检测基因表达 ,同时观察RI对荷瘤C57BL小鼠B16黑色素瘤生长的影响。应用MACS能高度纯化人脐血CD34⁺ 细胞 ,使分选后的脐血CD34⁺ 细胞纯度平均达96.15%。hRI基因能够转染到脐血CD34⁺ 细胞上 ,转染效率达 35% ,Western blot和免疫荧光检测转染后CD34⁺ 细胞hRI基因有阳性表达。经转hRICD34⁺ 细胞治疗 ,使小鼠B16黑色素瘤的生长速度减慢 ,成瘤率和瘤重降低 ,成瘤潜伏期延长。     

杂交瘤细胞培养的优化

根据杂交瘤细胞培养中单克隆抗体生产的动力学原理,采用灌注培养方式、添加醋酸钾和丰富营养物培养的途径,对反应器放大培养过程进行了优化。每天灌注l／2反应器工作体积,与分批培养相比,细胞密度由4 5×10⁵／ml提高到l1×10⁵／ml,单抗浓度由19mg／L提高到28mg/L;添加1g／L醋酸钾,细胞密度基本保持不变．但单抗浓度增加到38μg／ml;用丰富营养物培养后,细胞密度和单抗浓度分别进一步提高到42×10⁵／ml和94mg/L。抗B型红细胞单抗的血凝滴度,由分批培养的1：32．最终提高到l：256     

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.