视网膜母细胞瘤基因产物部分性质的研究

研究Ｒｂ基因产物的代谢动力学和功能调节，发现整个细胞周期中都有Ｒｂ蛋白合成，其含量随细胞周期进行而变化，蛋白代谢的半衰期约为１１—１２小时，有非磷酸化和多种程度磷酸化形式。Ｒｂ蛋白的磷酸化水平是随细胞周期过程和细胞生长状态而变化的，在Ｇ０、Ｇ１期蛋白处于低磷酸化状态，到达Ｇ１／Ｓ界限，细胞获得磷酸化Ｒｂ蛋白的能力．     

视网膜母细胞瘤基因产物磷酸化水平与细胞生长状态的关系

随着无血清培养细胞时间的延长，红胞合成Ｒｂ蛋白量和磷酸化水平逐渐降低，当细胞生长停止时，Ｒｂ蛋白处于低磷外化状态．正丁酸钠可诱导ＨＬ６０终端分化为单核－巨噬细胞；视黄酸、二甲基亚砜使ＨＬ６０分化为粒细胞．尽管分化途径不同．但是终端分化的细胞都只能合成低磷酸化的Ｒｂ蛋白．结果表明当细胞处于生长停滞状态，细胞都只能合成低磷酸化的Ｒｂ蛋白．     

人生长激素诱导体外培养的大肠癌细胞株细胞周期动？…

研究人生长激素（ｈＧＨ）对大肠癌细胞增殖和分化的影响，收集人大肠癌细胞株Ｌｏｖｏ和ＬＳ－１７４－Ｔ（简称１７４），以顺铂和／或不同浓度的ｈＧＨ体外培养细胞，２４ｈ后以流式细胞仪测定细胞增殖周期等指标。结果显示ｈＧＨ能诱导１７４细胞Ｓ期数率非常显著地增高（Ｐ〈０．０１）；Ｌｏｖｏ细胞１００ｎｇ／ｍＬ组Ｇ０－Ｇ１期参数下降（Ｐ〈０．０５），Ｓ期参数增加（Ｐ〈０．０１），２００ｎｇ／ｍＬ组Ｇ２－Ｍ期百分     

生长调节物质对丹参叶片脱分化及根芽分化的效应

生长调节物质对丹参叶片脱分化及根芽分化效应的研究结果表明：（１）０．５～５．０ｍｇ／Ｌ２，４－Ｄ，０．１～０．５ｍｇ／ＬＮＡＡ０．５～１．５ｍｇ／ＬＩＢＡ或０．２～１．０ｍｇ／Ｌ４ＰＵ－３０诱导叶片的脱分化率高达９７％～１０００％。（２）０．１～０．５ｍｇ／ＬＮＡＡ或０．５～２．０ｍｇ／ＩＢＡ诱导芽化率为９３％～１００％，３种细胞分裂素中，４ＰＵ－３０对叶片芽化最有效，６－ＢＡ和ＫＴ次之。（３）随     

HMBA和亚硒酸钠对人胃腺癌细胞的诱导分化

用５×１０（－３）ｍｏｌ／Ｌ环六亚甲基双乙酰胺和３×１０（－６）ｍｏｌ／Ｌ亚硒酸钠诱导剂组合处理人胃腺癌ＭＧｃ８０－３细胞，结果细胞的群体增殖和ＤＮＡ合成均受到显著抑制，生长抑制率最高达７２．８％，ＤＮＡ合成的抑制率达８４．１％，液门测定的ＣＰＭ值也较诱导处理之前降低了５．２６倍；同时，ＭＧＣ８０－３细胞的表型由典型恶性特征向相应正常细胞类似的形态结构特征转变，细胞对裸鼠的致瘤性也明显降低（Ｐ值＜０．０５）·表明ＨＭＢＡ和亚硒酸钠的组合能够有效地诱导人胃腺癌细胞的分化，而且这种诱导分化作用比单独使用其中一种诱导物所产生的效果更显著．     

辣椒（Capsicum annuum L.）下胚轴离体培养的研究

将辣椒无菌苗下胚轴切段接种在ＭＳ培养基（附加不同的植物激素）上，诱导产生愈伤组织。最适的诱导培养基为ＭＳ＋２ｍｇ／Ｌ ＢＡ，０．５ｍｇ／Ｌ ＩＡＡ，在此培养基上产生的愈伤组织具有较强的分化能力。将愈伤组织转移到分化培养基，光照培养，芽分化率可达２５．０％。不定芽在含１ｍｇ／Ｌ ＧＡ３和２ｍｇ／Ｌ ＢＡ的ＭＳ培养基上伸长成苗，再移入生根培养基上诱导生根，长成完整植株。     

银杏愈伤组织的诱导与激素优化组合研究

通过实验,筛选出银杏愈组织的最佳培养基为Ｎ６＋ＮＡＡ３．０ｍｇ／Ｌ＋ＢＡ１．０ｍｇ／Ｌ,其次为ＭＳ＋ＮＡＡ３．０ｍｇ／Ｌ＋ＢＡ１．０ｍｇ／Ｌ,分别对ＮＡＡ、２,４－Ｄ、ＧＡ、ＢＡ和ＫＴ等在培养过程中的作用与浓度进行了探索,结果表明ＮＡＡ、ＢＡ结合使用,既适合诱导又利于继代培养,２,４－Ｄ适合诱导,ＧＡ不利于诱导,ＫＴ有利于分化。     

无花果的组织培养

对影响无花果外植体不定芽诱导的四个因子进行了研究：不同器官、培养基的组成、外植体的预处理和不同的品种。细胞分裂素ＢＡ的效果优于ＫＴ。茎尖和茎段不定芽诱导培养基为ｍｇ·Ｌ￣（－１）（ＭＳ＋ＢＡ），叶片的分化以浓度８ｍｇ·Ｌ￣（－１）的ＢＡ效果最好。赤霉素（ＧＡ＿３）有助于芽的萌发和生长，浓度在０．５～１ｍｇ·Ｌ￣（－１）为宜。割取嫩枝插入１／２ＭＳ，附加１ＢＡ或ＮＡＡ１．０～２．０ｍｇ·Ｌ￣（－１）的生根培养基中，根原基诱导率９５％以上，移栽成活率９０％以上。     

太白米愈伤组织的诱导

把太白米小鳞茎基部０．１－０．５ｍｍ处切口，接种于ＭＳ＋ＮＡＡ １ｍｇ／Ｌ＋２．４－Ｄ１ｍｌ／Ｌ＋ＺＴ０．１ｍｇ／Ｌ和ＭＳ＋ＮＡＡ０．５ｍｇ／Ｌ＋ＺＴ０．１ｍｇ／Ｌ培养基上，置黑暗中培养，小鳞茎能脱分化产生大量愈伤组织，诱导率在９５％以上，且生长迅速。     

植物激素对药用植物山丹离体子叶愈伤组织诱导、器官分化及植株再生的影响

ＭＳ培养基附加ＢＡ１，０～２．２／ＮＡＡ０．０１～０．１ｍｇ／Ｌ、ＫＴ１．０～２．０／ＮＡＡ０．０１～０．１ｍｇ／Ｌ、ＢＡ０．５＋ＫＴ０．５／ＮＡＡ０．１ｍｇ／Ｌ及ＢＡ１．０＋ＫＴ１．０／ＮＡＡ０．１ｍｇ／Ｌ的不同激素组合，处理山丹离体子叶，均能诱导其产生愈伤组织和分化芽，除３Ａ１．０／ＮＡＡ０．０１和ＢＡ１．０／ＮＡＡ０．１外，各组合还均能诱发生根和使植株再生。     

Abrogation by c-myc of G1 phase arrest induced by RB protein but not by p53.

Inactivating mutations of the retinoblastoma gene (RB) are found in a wide variety of tumour cells. Replacement of wild-type RB can suppress the tumorigenicity of some of these cells, suggesting that the RB protein (Rb) may negatively regulate cell growth. As activation of c-myc expression promotes cell proliferation and blocks differentiation, it may positively regulate cell growth. The c-myc protein is localized in the nucleus and can physically associate with RB protein in vitro, hence c-myc may functionally antagonize RB function. Microinjection of Rb in G1 phase reversibly arrests cell-cycle progression. Here we co-inject RB protein with c-myc, EJ-ras, c-fos or c-jun protein. Co-injection of c-myc, but not EJ-ras, c-fos or c-jun, inhibits the ability of Rb to arrest the cell cycle. The c-myc does not inhibit the activity of another tumour supressor, p53 (ref. 12). Thus, c-myc and RB specifically antagonize one another in the cell.     

Forced expression of theOct-4 gene influences differentiation of embryonic stem cells

By transfecting an Oct-4 expression plasmid into embryonic stem cells (ES cells), the ES-O cell line was constructed, which sustained the expression of Oct-4 gene when induced by retinoic acid. Forced expression of Oct-4 gene could not sustain the stem property of ES-O cells without the differentiation inhibiting factor LIF, but if LIF exists, forced expression of Oct-4 gene could enhance the ability to sustain the undifferentiation state and inhibit cell differentiation induced by retinoic acid. It was indicated that Oct-4 must cooperate with LIF to sustain the undifferentiation state of ES cells. During the cell differentiation, ES-O cells tend to differentiate into neural cells, suggesting that forced expression of Oct-4 gene may be in relation with the differentiation of neuroderm.     

Identification of pathways regulating cell size and cell-cycle progression by RNAi

Many high-throughput loss-of-function analyses of the eukaryotic cell cycle have relied on the unicellular yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. In multicellular organisms, however, additional control mechanisms regulate the cell cycle to specify the size of the organism and its constituent organs. To identify such genes, here we analysed the effect of the loss of function of 70% of Drosophila genes (including 90% of genes conserved in human) on cell-cycle progression of S2 cells using flow cytometry. To address redundancy, we also targeted genes involved in protein phosphorylation simultaneously with their homologues. We identify genes that control cell size, cytokinesis, cell death and/or apoptosis, and the G1 and G2/M phases of the cell cycle. Classification of the genes into pathways by unsupervised hierarchical clustering on the basis of these phenotypes shows that, in addition to classical regulatory mechanisms such as Myc/Max, Cyclin/Cdk and E2F, cell-cycle progression in S2 cells is controlled by vesicular and nuclear transport proteins, COP9 signalosome activity and four extracellular-signal-regulated pathways (Wnt, p38betaMAPK, FRAP/TOR and JAK/STAT). In addition, by simultaneously analysing several phenotypes, we identify a translational regulator, eIF-3p66, that specifically affects the Cyclin/Cdk pathway activity.     

Partial inhibition of CDK4 gene expression leads to the extension of G1 phase of V79-8 cells

V79-8 is an abnormal cell line which does not have detectable G1 and G2 phases in its cell cycle. This cell line is derived from V79 cell line which has Gl phase but lacks G2 phase. By using an anti-sense approach, CDK4 gene expression was partially inhibited to find whether CDK4 might contribute to the lack of Gl phase in V79-8 cells. Anti-CDK4 anti-sense plasmid was constructed and used to transfect V79-8 cells. Clones of transfected cells (V79-8-asCDK4) were examined, in comparison with V79-8 cells, to determine its growth curve, cell doubling-time (GT), the level of CDK4 gene expression and the levels of expression of some other growth related genes. V79-8-asCDK4 cells showed a slower growth rate with a doubling time 2.5-h longer than that of V79-8 cells. A flow cytometry (FCM) analysis demonstrated that the 2.5 h increase of the doubling time of V79-8-asCDK4 cells was mainly due to the appearance of Gl phase because its G2 + M phase was not significantly different from that of V79-8 cells. The decrease of CDK4 gene expression in V79-8-asCDK4 cells was shown by Northern-blot. Changes in the expression levels of the growth-related genes TGF-β, cyclin D1 and Rb were also detected in V79-8-asCDK4 cells. CDK4 functions mainly in G1 and at the transition between G1 and S phases. Expression of an anti-sense CDK4 gene fragment reduces the levels of endogenous CDK4, CDK4/cyclinD kinase activity and the phosphorylation of Rb. These events may postpone the inactivation of the check-point leading to the delay of entry into S phase and the reappearance of G1 phase in V79-8-asCDK4 cells.     

Pin1的细胞定位调控Rb蛋白的磷酸化状态

为了研究Pin1和Rb蛋白的细胞定位、Pin1调控Rb磷酸化的相关机制,本研究构建了慢病毒载体pLVX-Flag-HA-Pin1和Plko.1-shRNA,包装病毒感染人非小细胞肺癌(H1299),免疫印迹检测Pin1蛋白表达水平,采用免疫荧光染色、免疫组化技术研究蛋白的定位.结果表明,沉默Pin1可降低Rb的磷酸化并抑制细胞的增殖;培养的肿瘤细胞和肿瘤组织中,Pin1可定位到细胞质中,而胞质定位的Pin1也可增加Rb的磷酸化.