下调microRNA-34a增强对人脂肪干细胞成骨诱导分化作用的初步探索

目的:颅颌面临界骨缺损是整复外科常见疾患,常常给患者的身心带来障碍和压力。为解决骨形成不足这一难题,我们拟通过表观遗传修饰的手段,调节microRNA-34a在人脂肪干细胞中的表达,来探讨microRNA-34a对人脂肪干细胞成骨诱导分化的影响。方法:分离培养原代人脂肪干细胞,以慢病毒为转染载体,分三组对其进行上调、下调microRNA-34a的表达及阴性对照。然后对其进行成骨诱导培养,于诱导的第7天行碱性磷酸酶(ALP)染色,第14天行茜素红(Alizarin Red)染色,定性比较转染后人脂肪干细胞成骨诱导分化的效果。结果:成功分离和培养原代人脂肪干细胞,并以慢病毒高效率转染调控microRNA-34a的表达。对比碱性磷酸酶和茜素红染色可见下调microRNA-34a组成骨效率最高,其次是阴性对照组,上调组效率最低。结论:1.人脂肪干细胞通过慢病毒转染获得高效率的表观遗传修饰;2.下调microRNA-34a的表达对人脂肪干细胞成骨的诱导分化有一定的促进作用。     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

miR-26a抑制转化生长因子β诱导的韧带成纤维细胞增殖

目的:研究miR-26a对转化生长因子-β(Transforming growth factor-β,TGF-β)诱导的韧带成纤维细胞增殖的影响。方法:原代分离大鼠肩周韧带成纤维细胞,通过免疫荧光鉴定细胞纯度,应用脂质体细胞转染miRNA模拟物的方式过表达miR-26a,定量PCR的方法检测miR-26a模拟物的过表达效率,CCK8法检测细胞增殖能力的变化,生物信息学预测的方法分析miR-26a可能的作用靶基因。结果:免疫荧光检测发现原代分离细胞中,Vimentin阳性细胞在90%以上;转染miR-26a模拟物后,细胞内miR-26a水平显著高于阴性对照组(P0.05);TGF-β刺激明显促进韧带成纤维细胞生长(P0.01),而miR-26a则对TGF-β诱导的韧带成纤维细胞增殖具有显著的抑制作用(P0.01);生物信息学预测显示SMAD1/4、EIF4G2、PTEN和MARK1可能是miR-26a影响细胞增殖的作用靶基因。结论:miR-26a可抑制TGF-β诱导的韧带成纤维细胞增殖。     

miR-26a抑制转化生长因子beta诱导的韧带成纤维细胞增殖

目的:研究miR-26a对转化生长因子-β(Transforming growth factor-β,TGF-β)诱导的韧带成纤维细胞增殖的影响。方法:原代分离大鼠肩周韧带成纤维细胞,通过免疫荧光鉴定细胞纯度,应用脂质体细胞转染miRNA模拟物的方式过表达miR-26a,定量PCR的方法检测miR-26a模拟物的过表达效率,CCK8法检测细胞增殖能力的变化,生物信息学预测的方法分析miR-26a可能的作用靶基因。结果:免疫荧光检测发现原代分离细胞中,Vimentin阳性细胞在90%以上;转染miR-26a模拟物后,细胞内miR-26a水平显著高于阴性对照组(P<0.05);TGF-β刺激明显促进韧带成纤维细胞生长(P<0.01),而miR-26a则对TGF-β诱导的韧带成纤维细胞增殖具有显著的抑制作用(P<0.01);生物信息学预测显示SMAD1/4、EIF4G2、PTEN和MARK1可能是miR-26a影响细胞增殖的作用靶基因。结论:miR-26a可抑制TGF-β诱导的韧带成纤维细胞增殖。     

人参皂苷Rg1促进人牙周膜干细胞增殖及骨向分化

人参皂苷Rg1 (GS Rg1) 是人参的主要药理活性成分. GS Rg1有刺激造血干细胞的形成和促进骨髓间充质干细胞增殖和分化的作用.而人牙周膜干细胞（human periodontal ligament stem cells, hPDLSCs）具有自我更新和多向分化的干细胞特性.但目前关于GS Rg1能否促进牙周膜干细胞增殖和分化的研究尚不多见.本研究证明,1×10-5 mol/L GS Rg1作用于hPDLSCs后,能明显促进牙周膜干细胞的增殖与分化.MTT检测细胞增殖显示,培养液中加入了GS Rg1实验组在第2,3,4,5 d增殖情况明显高于对照组,提示GS Rg1成分促进了牙周膜干细胞的增殖. 检测ALP表达量,RUNX2,Collagen I,OPN,OCN表达水平,加药实验组表达水平均高于未加药对照组,它们的表达量的增高提示成骨分化的增强. 牙周膜干细胞与纳米羟基磷灰石支架结合的电镜图片及其支架植入小鼠体内后的免疫组化检测显示,含有GS Rg1成分的细胞支架能促进形成更多的骨样组织.因此,GS Rg1能促进hPDLSCs体内体外的增殖及骨向分化,并在替代传统生长因子应用于牙周组织工程方面有较好前景.     

miR-155通过下调BMP9/Smad信号通路抑制间充质干细胞C3H10T1/2成骨分化

该文研究了在诱导小鼠间充质干细胞C3H10T1/2细胞成骨分化过程中miR-155的作用及其是否是通过调控BMP9/Smad(bonemorphogenetic protein 9/drosophila mothers against de-capentaplegic)信号通路发挥作用。在诱导C3H10T1/2细胞成骨分化过程中,采用实时定量PCR(Real-time quantitative PCR,q RT-PCR)检测miR-155水平的变化。转染miR-155模拟剂(miR-155)至C3H10T1/2细胞后,miR-155水平显著增高(P0.001),而转染其抑制剂(anti-miR-155)至C3H10T1/2细胞后,miR-155水平显著降低(P0.001)。转染后成骨诱导培养基诱导成骨7 d,碱性磷酸酶(alkaline phosphatase,ALP)活性及染色结果显示,miR-155能显著降低C3H10T1/2细胞成骨分化过程中的ALP活性(P0.01)、减弱ALP染色,而anti-miR-155则能逆转其作用。成骨诱导14 d茜素红S染色结果显示,miR-155组钙盐结节较对照组少,下调miR-155的水平后,钙盐沉积结节增多。q RT-RCR检测结果显示,miR-155显著降低BMP9 m RNA水平(P0.001),且miR-155组成骨基因Runx2和ALP表达均显著低于对照NC组(P0.05、P0.01)。Western blot检测BMP9、Runx2和p-Smad1/5/8蛋白质水平,结果显示,miR-155组蛋白质水平均显著降低(P0.01、P0.05、P0.001)。该研究结果提示,miR-155对C3H10T1/2细胞成骨分化的抑制作用可能是通过抑制BMP9/Smad信号通路发挥作用的。     

两种不同诱导液对人牙周膜干细胞膜片性能的影响

目的:对比分析发育期根端复合体(Dental Apical Complex,DAC)诱导液和成骨诱导液对人牙周膜干细胞(Periodontal ligam-ent Stem Cells,PDLSCs)膜片形成和分化能力的影响。方法:收集临床十名12-16岁的青少年因正畸需要拔除的健康、无龋的新鲜牙(前磨牙),刮取根面残留牙周膜进行牙周膜干细胞的分离培养,制备成牙周膜干细胞膜片,分别采用DAC诱导液和成骨诱导液对膜片进行诱导,对比分析两种诱导液对牙周膜干细胞膜片的影响。结果:DAC诱导液处理过的PDLSCs细胞膜片,细胞形态接近牙源性间充质细胞,DAC诱导液提供了良好的成牙骨质-牙周样结构诱导环境,可促进PDLSCs膜片向功能性成牙骨质细胞谱系分化,而成骨诱导液提供了良好的成骨诱导环境,可促进PDLSCs膜片向功能性成骨细胞谱系分化。结论:DAC诱导液较之于成骨诱导液更利于PDLSCs细胞膜片的成牙周样分化,利于牙周缺损修复和局部再生。     

不同干预时间正弦电磁场对大鼠骨髓基质干细胞成骨性分化的影响

目的:研究不同治疗时间正弦电磁场(50 Hz,1.8mT)对体外培养大鼠骨髓间充质干细胞成骨性分化的影响,筛选出最佳临床治疗时间.方法:采用贴壁筛选法培养原代大鼠骨髓间充质干细胞,每天在频率为50 Hz,强度为1.8 mT的磁场环境中处理0.5 h、1.0h、1.5 h、2.0h和2.5h;同时设立未经电磁场处理的细胞作为对照组.于处理后的第3 d、6d、9d和12 d分别测定细胞碱性磷酸酶活性、骨钙素分泌量、钙化结节数以及Ⅰ型胶原表达量,并比较各组间差异;于处理后12 h提取细胞总RNA,用RT Real-Time PCR法检测成骨性分化基因Osterix表达情况.结果:正弦电磁场干预1.0h能明显促进骨髓间充质干细胞的成骨性分化,表现在该组的碱性磷酸酶活性、骨钙素分泌量、钙化结节数、Ⅰ型胶原表达量以及成骨性分化基因的表达量最高,亦显著高于对照组(P＜0.05).结论:50Hz、1.8mT强度的正弦电磁场能促进骨髓间充质干细胞的成骨性分化,以作用1.0h成骨效果最为明显.     

MicroRNA-146a抑制胶质瘤细胞增殖的研究

目的:检测胶质瘤中miR-146a的表达水平,并研究miR-146a对胶质瘤细胞增殖的影响。方法:应用实时定量PCR的方法检测胶质瘤组织和癌旁组织中miR-146a的表达水平,采用脂质体细胞转染miRNA模拟物的方式过表达miR-146a,MTT法检测转染后细胞的增殖率,利用在线软件targetScan预测miRNA可能的靶基因。结果:miR-146a在胶质瘤组织中表达明显降低(P<0.01),相对表达水平为癌旁组织的35%,细胞转染miR-146a模拟物后,miR-146a表达明显增加,癌细胞增殖率明显降低(P<0.01),仅为原细胞的47%。Notch1基因是miR-146a影响胶质瘤细胞增殖活力的可能靶基因。结论:miR-146a可能通过抑制Notch1基因的表达调控胶质瘤细胞的增殖。     

在海藻酸钠凝胶上诱导骨髓间充质干细胞分化为成骨细胞

通过在海藻酸钠凝胶上诱导bMSCs向成骨细胞分化,探讨其对骨髓间充质干细胞（bone mesenchymal stem cells, bMSCs）的生物学效应。采用MTT、甲苯胺蓝染色、von Kossa染色和RT-PCR分别检测细胞的增殖、生长形态、诱导后细胞的钙化结节和成骨相关基因的表达。实验组bMSCs生长状况良好、细胞增殖迅速,与对照组的增殖无差异;bMSCs成集落样生长明显,集落中央细胞重叠生长形成钙化结节;培养至12d,实验组和对照组的成骨相关基因,包括碱性磷酸酶、I型胶原和骨钙素,均为阳性表达,但实验组的表达量高于对照组。海藻酸钠凝胶能够促进bMSCs向成骨细胞的分化,是良好的骨组织工程支架材料。     

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.