树鼩视网膜微血管内皮细胞永生化细胞株的建立及鉴定

目的 建立树鼩视网膜来源的微血管内皮细胞的体外分离培养技术以及永生化细胞株，为体外利用树鼩视网膜微血管内皮细胞开展相关研究提供新的实验材料。方法 利用Ⅱ型胶原酶、分散酶和DNaseⅠ酶消化法分离培养出原代视网膜微血管内皮细胞，利用差速消化法纯化内皮细胞，然后利用携带SV40T基因的慢病毒转染细胞，再挑取单克隆后进行传代培养。对传至50代以上的细胞进行形态学观察、免疫荧光鉴定以及核型鉴定。结果 采用混合酶消化法能够分离获得微血管内皮细胞，纯化后的细胞呈不规则多角形和梭形。经慢病毒转染后的细胞传代培养后细胞形态一致，到第50代时细胞形态结构仍较好。细胞免疫荧光结果显示标志性蛋白VWF、CD34、Claudin1、ZO-1和永生化SV40T表达阳性。生长曲线结果显示：细胞生长旺盛，第2～4天时处于对数生长期，第4天进入平台期。细胞核型结果显示：染色体数与树鼩染色体相同，即所获取的细胞为永生化的树鼩视网膜微血管内皮细胞。结论 成功建立的树鼩视网膜微血管内皮细胞永生化细胞株具有较好的形态结构与功能，为视网膜病变及眼科相关疾病的研究提供了新的实验材料。     

成人宫颈上皮细胞的原代培养及鉴定

目的:探索采用无血清培养基原代培养成人宫颈上皮细胞的方法。方法:以成人的宫颈上皮组织为研究对象,采用胰蛋白酶-EDTA消化法获得宫颈上皮细胞悬液,于上皮细胞专用无血清培养基中培养,采用免疫细胞化学法测定细胞中角蛋白及波形蛋白的表达,对细胞纯度进行鉴定。结果:原代培养10-15天细胞融合达60%,传代至4-6代,细胞出现生长衰退。早期细胞生长状态良好,细胞纯度在90%以上。结论:采用酶消化法及K-SFM无血清培养基培养可获得纯度高的成人宫颈上皮细胞。     

周细胞的原代培养

目的:探讨Wistar大鼠视网膜毛细血管周细胞(pericyte,PC)的原代培养和鉴定方法。方法:结合视网膜微血管的消化分离,采用含10%胎牛血清的DMEM培养基选择性培养PC,通过活细胞观察原代PC的形态、生长特性以及与血管碎片之间的关系,同时应用免疫细胞化学染色来鉴定PC。结果:选择性培养获得的PC的纯度达到95%以上,并能连续传代。该细胞呈长梭形或星芒状,漩涡或栅栏状生长,无接触性抑制,单核,偶见双核,核卵圆形,细胞浆丰富,α-SMA、PDGFR-β染色阳性。结论:通过对视网膜微血管的消化分离能够获得较为纯净的PC。     

人正常食管鳞状上皮不同原代培养方法的比较

该研究通过比较人正常食管鳞状上皮不同的原代培养方法,以期为不同的实验目的提供不同的培养方法。实验用到的正常食管粘膜上皮来源于食管癌患者手术切除的标本,采用组织块法和酶消化法,分别用DMEM/F12混合培养基和K-SFM无血清培养基进行培养。通过直接观察、细胞形态学观察和免疫细胞化学方法观察细胞的生长情况、细胞形态学特征及鉴定所得到的细胞,比较不同方法与不同培养基组合中原代培养细胞的生长状况。用组织块法,在DMEM/F12混合培养基中人正常食管上皮细胞生长较好,细胞融合较快,成纤维细胞污染较少,15～17天上皮细胞铺满瓶底的70%～80%,获得的细胞数量大,但细胞传代后成纤维细胞污染严重。用酶消化法,在K-SFM无血清培养基中人正常食管上皮细胞生长好,细胞融合快,成纤维细胞污染基本消除,细胞纯度高,10～12天细胞便可以铺满瓶底的70%～80%,这种方法培养的细胞可以冻存、复苏和传代。其余各种培养方法所得细胞无论在生长状态、培养周期、成纤维细胞污染和传代方面均较前两种方法差。以上各种方法培养的细胞经免疫细胞化学染色鉴定证实细胞呈广谱细胞角蛋白阳性,确定是食管上皮来源的细胞。酶消化法加K-SFM无血清培养基是本实...     

小鼠主动脉平滑肌细胞的分离培养及鉴定

目的:建立分离培养小鼠原代主动脉血管平滑肌细胞(VSMC)的方法并检测其生长特性。方法:剥离小鼠主动脉中膜层,分别采用组织块培养法及胶原酶消化法分离培养小鼠主动脉来源的原代VSMC,免疫荧光法检测细胞的纯度和分化状态;3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑蓝(MTT)法测定小鼠主动脉VSMC传代细胞的生长、增殖特性。结果:组织块培养法培养组织块8d后,细胞从组织块边缘爬出,18 d后细胞汇合度达到80%以上后传代;胶原酶消化法分离培养的细胞生长7 d后,汇合度可达80%,此时进行传代;2种方法获得的细胞进行免疫荧光染色,结果显示细胞传至第3代时纯度在95%以上,传至第8代时分化状态并没有改变;MTT法显示细胞生长3~5 d时处于指数生长期。结论:本研究建立了2种可靠稳定的分离和培养小鼠主动脉VSMC的方法,VSMC纯度高,多次传代后细胞特征稳定。     

鼠脑微血管内皮细胞的分离与长期培养

采用胶原酶消化、差速离心、尼龙网滤过技术分离和获取鼠脑微血管内皮细胞,接种后4h换液使获得的内皮细胞纯化,体外进行长期培养。细胞在体外生长176天,传至30代,细胞初期成活率为92％,纯度近90％。经形态学、超微结构和免疫组化鉴定,培养细胞为血管内皮细胞。培养至第30代的细胞仍能合成和分泌PGI2、ACE等,ⅧF:Ag阳性表达,染色体为二倍体(2n=42),基本保持着细胞的主要特征。该分离和培养方法的建立,将为研究与脑血管相关疾病提供有用工具。     

大鼠肺微血管内皮细胞培养及鉴定

取大鼠周边肺组织进行肺微血管内皮细胞培养。将肺组织切成小块,用含有２０％新生牛血清、肝素９０μｇ／ｍｌ、Ｌ－谷胺酰胺４ｍｍｏｌ、青霉素１００Ｕ／ｍｌ和链霉素１００μｇ／ｍｌ的ＲＰＭＩ－１６４０培养基培养。血细胞立即从肺组织周围游出。继而是肺微血管内皮细胞。成纤维细胞及其他细胞７２ｈ才游出。培养６０ｈ后取出肺组织块,培养瓶中只有微血管内皮细胞和血细胞。后者可通过传代除去。获得的肺微血管内皮细胞具有规律的鹅卵石样形态和对异植物血凝素结合试验及八因子相关抗原免疫荧光染色均阳性。     

低氧对血管内皮细胞肾上腺髓质素基因转录的影响

为研究低氧条件对肾上腺髓质素(adrenomedullin,ADM)基因表达的影响,本文观察了低氧引起血管内皮细胞ADM基因转录的变化,并探讨ADM在缺血缺氧性疾病机制中的作用.1 材料和方法(1)HUVEC细胞的培养人脐静脉内皮细胞株HUVEC(美国芝加哥大学Pritzker医学院Gewertz BL教授惠赠),用含20%小牛血清的M199培养基置于37℃,5%CO2培养箱内贴壁生长,待长满培养瓶培养面后用PBS冲洗2次,加入0.25%胰蛋白酶消化、传代.     

大鼠肺微血管周细胞的体外培养及鉴定

目的:探讨SD大鼠肺微血管周细胞(rat pulmonarymicrovessel pericytes,RPMPC)的分离、培养与鉴定方法。方法:采用机械剪切、Ⅰ型胶原酶消化法和微孔过滤结合超高速离心法分离大鼠肺微血管片段,用含15%胎牛血清(FBS)的高糖培养基(DMEM)进行培养,倒置显微镜观察原代RPMPC的形态及生长特性。免疫荧光法检测神经元-胶质抗原2(neuron-glial antigen 2,NG2),α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、结蛋白(desmin)和CD31相关抗原的表达,同时应用免疫细胞化学法检测血小板源性生长因子受体β(PDGFR-β)的表达。CCK-8测定周细胞生长曲线。通过周细胞-内皮细胞共培养成管实验检测细胞成管能力。结果:本方法培养获取的RPMPC纯度较高,并能连续传代。细胞48 h后爬出,呈长梭形、三角形等不规则形,8~10 d细胞汇合,呈栅栏或旋涡状生长,无接触性抑制,前期可见有少量内皮细胞伴随生长,单核偶见双核,核呈卵圆形,胞浆丰富。PDGFR-β、α-SMA、NG2、desmin染色阳性,CD31阴性;周细胞与内皮细胞共培养形成管腔结构。结论:通过本方法能够获得纯度较高的肺微血管周细胞,且所获细胞具有周细胞的特性及功能。     

兔骨髓基质干细胞向血管内皮样细胞的诱导分化

目的:研究血管内皮细胞生长因子(VEGF)联合碱性成纤维细胞生长因子(bFGF)促进兔骨髓基质干细胞向血管内皮样细胞的定向诱导分化,为血管化组织工程骨研究提供实验基础.方法:采集2周龄兔后肢长骨骨髓,用全骨骨髓贴壁法进行原代培养,将获得的第2代骨髓基质干细胞以1× 105/mL密度接种于内皮细胞条件培养基(含10 μg/L VEGF,10 μg/L bFGF,10％胎牛血清的DMEM/F12培养液)进行体外诱导培养,对诱导2周的细胞进行细胞形态观察和表型、功能鉴定.结果:经血管内皮细胞条件培养基诱导2周后的细胞呈扁平形,多边形,表达血管内皮细胞特异性标志CD31、VWF因子,细胞具有吞噬DiI-Ac-LDL和摄取FITC-UEA-1的功能,诱导的细胞可在BD基质胶内形成管腔样结构.结论:血管内皮细胞生长因子联合碱性成纤维细胞生长因子可以成功诱导兔骨髓基质干细胞为血管内皮样细胞,有希望作为组织工程骨的血管化的种子细胞.     

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.