MDCK单克隆细胞库的建立及检定

目的:建立甲型H1N1流感病毒疫苗株高适应MDCK单克隆细胞库,用于培养生产流感病毒疫苗,为细胞代替鸡胚制备流感病毒疫苗提供保证。方法:用有限稀释法将MDCK细胞进行单克隆化,通过血凝和TCID50筛选甲型H1N1流感病毒疫苗株的高适应性单克隆化细胞株,扩大培养建立细胞库并进行检定。结果:共制备了97株单克隆化MDCK细胞,筛选到甲型H1N1流感病毒疫苗株高适应性MDCK单克隆细胞株,扩大培养建立了细胞库,经检定符合《中华人民共和国药典.三部》(2010版)对细胞库的要求。结论:建立了甲型H1N1流感病毒疫苗株高适应性MDCK单克隆细胞库,为细胞培养生产流感病毒疫苗奠定了基础。     

单克隆人胰腺干细胞分离培养体系的建立

本研究探讨了建立单克隆人胰腺干细胞分离培养体系及单克隆人胰腺干细胞系．对一些影响干细胞增殖的因素进行了分析。无菌取人流产胎儿胰腺组织,切碎至1mm3,0．1％ Ⅳ型胶原酶消化,低糖DMEM、10％FBS、3．7g／LNaHCO3、0．08g／L青霉素及0．1g/L链霉素培养液贴壁培养细胞,2．5g／L胰蛋白酶＋0．4g／LEDTA消化传代。克隆环筛选单克隆干细胞,培养液中添加10ng／mLEGF．扩增单克隆干细胞。采用核型分析法检测干细胞染色体,MTT法测定干细胞生长曲线。胶原酶消化胰腺组织．获得单个细胞和细胞团。贴壁培养．原代上皮样胰腺干细胞克隆性生长。胰蛋白酶消化传代,上皮样胰腺干细胞逐渐被纯化。克隆环筛选,获得单克隆人胰腺干细胞。扩增培养,1例来源于4月龄男性流产胎儿胰腺组织干细胞建系,传50代。染色体核型分析．该干细胞系为正常的二倍体细胞。细胞生长曲线显示培养1—4d,干细胞生长缓慢,5-6d,进入倍增期。培养液中添加15％FBS,干细胞增殖较快。再添加15ng／mLEGF或10ng／mL IGF—Ⅱ．干细胞增殖更快。研究结果表明应用本实验建立的细胞分离培养体系获得了单克隆人胰腺干细胞系。     

单克隆卵黄抗体技术研究进展

单克隆抗体具有特异性强、表达较稳定等优势,而鸡IgY抗体因其种属距离等特点而具备一系列特殊优势,诸如交叉反应小、产生IgY的鸡免疫系统对哺乳动物保守的生物分子反应明显等,如能将这两种抗体的优势结合起来,开发单克隆卵黄抗体则有望大大拓展抗体的研究模式和应用领域.针对这一新兴领域,从单克隆卵黄抗体的理论背景、研发与技术现状和可能的应用前景三个角度综述了国外的研究现状.     

单克隆人胰腺干细胞分化特性的研究

研究1例来源于4月龄男性流产胎儿胰腺组织的单克隆人胰腺干细胞（monoclonal human pancreatic stem cell,mhPSC）系的体内外分化特性。将mhPSCs接种在铺有0．1％明胶的培养皿内,扩增培养3d后,加高糖DMEM诱导液诱导培养25d。相差显微镜下．观察细胞生长状况。采用双硫腙染色法、RT—PCR及葡萄糖刺激释放胰岛素和C肽实验．对体外定向诱导mhPSCs分化为功能性胰岛进行检测。将mhPSCs悬液注射在成年雄性裸鼠腹股沟皮下．注射30d时,取出移植物,采用SP法进行免疫组织化学反应,以检测mhPSCs的体内自然分化潜能。体外扩增培养,mhPSCs贴壁生长,呈多角形上皮样。生长至单层．呈“铺路石”状。体外定向诱导,细胞逐渐由多角形变成圆形,并聚集成类胰岛。诱导培养15d时．形成的类胰岛中少数细胞分化为B细胞,双硫腙染色阳性。诱导培养25d时,多数细胞分化为8细胞,双硫腙染色阳性,转录表达胰岛素的mRNA。用不同浓度葡萄糖刺激．诱导胰岛不仅释放胰岛素和C肽,而且其释放量随糖刺激浓度升高显著增加（0．01〈P〈0．05）。体内分化实验显示,mhPSCs在裸鼠背部形成类畸胎瘤。类畸胎瘤易与裸鼠分离,色白,血管丰富。显著表达pdx1、胰岛素、胰高血糖素、CK、MBP及NF蛋白。该研究结果证实单克隆人胰腺干细胞系体外定向诱导分化为包含大量β细胞的功能性类胰岛,在体内自然分化为胰岛、上皮及神经组织细胞。     

人鼻咽癌单克隆细胞株在传代移植的裸鼠体内转移与HLA表达定量的关系

为了研究人鼻咽癌单克隆细胞裸鼠体内传代转移特性及 HL A的表达变化 ,我们以人鼻咽癌单克隆细胞株 (CNE-2 Z- H5 )为对象 ,将各单克隆细胞株移植于裸鼠 ,待肿瘤发生转移后 ,取其淋巴结和肺转移灶癌细胞再次移植裸鼠 ,如此移植 2 - 4代 ,观察每代裸鼠的转移特性及鼻咽癌细胞 HL A表达的免疫组织化学定量变化。结果显示随着鼻咽癌单克隆细胞的传代移植 ,每代裸鼠肿瘤的转移率增高 (P<0 .0 1) ,转移途径更为单一。淋巴道转移的克隆细胞其 HL A 类抗原的表达随体内传代次数的增加而呈明显降低的趋势 ,与原代比较有显著性差异 (P<0 .0 1) ,但第 4代又急刷升高 (P<0 .0 1) ;HL A 类抗原的表达则呈波浪样改变 ;各代间表达平均强度变化不规则。肺转移灶癌细胞的 HL A 、 HL A 类抗原表达明显降低 ,两者与原代癌细胞比较都有显著性差异 (P<0 .0 1)。表明鼻咽癌细胞演进中转移能力和细胞本身特性、体内的环境选择关系密切 ,且可能和癌细胞 HL A的表达改变有关     

单克隆制造

《单克隆制造:抗细菌和病毒单克隆抗体生产和鉴定者具体指南》(Making monoclonais: A practical beginner′s guide to the production and characterisation ofmonoclonal antibodies against bacteria and viruses)由Diane G.Newell等人著,1988年Public Health Laboratory Service出版, 94页。很多实验室发现有必要针对传染因子特殊抗原去生产单克隆抗体。这种“设计”抗体最近成为必要的研究工具,可以作为免疫诊断试剂,具有一定商业价值。     

胰腺导管单克隆上皮样干细胞的蛋白表达特征

该文研究1例源于成年大鼠的胰腺导管单克隆上皮样干细胞系的蛋白表达特征、染色体核型及致瘤性。RPMI-1640有血清扩增培养源于成年大鼠的胰腺导管单克隆上皮样干细胞至单层,分别采用流式细胞术或免疫荧光反应检测干细胞蛋白水平的表达特征。常规法制备染色体标本,采用Adobe Photoshop CS6软件进行核型分析。将干细胞移植在裸鼠体内,观察其致瘤性。结果显示,该源于成年大鼠的胰腺导管单克隆上皮样干细胞在蛋白水平表达CK19、Neuro D2、Oct4、PCNA及Nanog,不表达Pdx1、Pax4、Pax6、Maf A、Ptf1a、Ngn3、nestin、Sox2、CD34、CD45、insulin、glucagon、ghrelin、somatostatin及secretin。细胞是正常的二倍体细胞(2n=42),无致瘤性。这表明,该源于成年大鼠的胰腺导管单克隆上皮样干细胞系共表达CK19、Neuro D2、Oct4、PCNA及Nanog蛋白。     

同源肿瘤克隆细胞蛋白表达和侵袭能力的异质性研究

摘要 目的：比较同源肿瘤细胞来源的不同单克隆表型差异。方法：采用极限稀释法,在悬浮培养条件下获取HCT116结肠癌细胞系的单个细胞,对每孔含单个的细胞进行扩增培养,获得子代单克隆,并以同样方法继续挑取单克隆,连续获得子三代克隆。根据单克隆形态特点,选取第三代的三株代表性的单克隆,采用Western blot和免疫荧光法比较其SOX2、EpCAM和Vimentin蛋白表达差异。采用放疗观察三株单克隆的Vimentin蛋白的动态变化,研究其放疗干预的时间异质性,Transwell体外侵袭实验比较克隆侵袭力的差异。结果：三株由单细胞扩增培养的同源第三代子克隆依然存在明显生物学差异。形态有明显区别的球形与不规则的克隆形态。不规则形态克隆更表现为SOX2低表达及Vimentin的高表达。并且在单个细胞水平上,同个单克隆群体内也存在个体细胞间蛋白的表达差异（Vimentin; EpCAM）。通过观察放疗前后Vimentin蛋白在不同时间点上的荧光强度,发现肿瘤单克隆细胞存在时间异质性。Transwell体外侵袭实验也显示三个同源克隆间存在明显的差异性。结论：同源的、连续单细胞扩增获得的第三代单克隆依然存在明显生物学差异,提示肿瘤内部异质性是其固有特征,并且在治疗干预下,也会引起肿瘤时间异质性的产生。     

哺乳动物细胞培养生产药用蛋白的关键环节

近年来,应用哺乳动物工程细胞系统生产药用蛋白显示了越业越重要的地位,本重点介绍了当前如何在生产药用蛋白的哺乳动物工程细胞表达系统,培养、生产技术,认证,生产过程组织等关键环节上进行优化,从而达到高效,安全、经济生产模式的核心技术和策略。     

抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究

用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分（integral viable cell concentration,IVCC）,提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl－xL的工程细胞的IVCC提高了50％,最终目的蛋白表达增加超过200％,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。     

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.