利用IL—3／GM—CSF融合蛋白（PIXY—321）扩增脐血造血细胞方…

利用单克隆抗体免疫磁珠吸附方法脐血ＣＤ３４＋细胞,并观察了ＩＬ－３／ＧＭ－ＣＳＦ融合蛋白（ＰＩＸＹ－３２１）对脐血ＣＤ３４＋细胞的刺激作用。ＰＩＸＹ－３２１对脐血ＣＤ３４＋细胞扩增作用大于ＩＬ－３和ＧＭ－ＣＳＦ单独及联合应用,在液体培养条件下,每毫升２０ｎｇＰＩＸＹ－３２１可有效地扩增脐血造血祖细胞,适宜扩增时间为５－８天,扩增后造血细胞的数量可达扩增前的８－１０倍,从而初步建立了一种简单可行的脐     

磷脂酰肌醇—3激酶／蛋白激酶B信号系统

质膜民分磷脂酰肌醇被其３位激酶（ＰＩ３Ｋ）活化后,可以使蛋白激酶Ｂ（ＰＫＢ）由细胞浆定位于胞膜,继而被磷酸化激活。ＰＫＢ是原癌基因ａｋｔ的产物,具有丝／苏氨酸蛋白激酶活性,介导细胞代谢、生长、增殖等效应。ＰＩ３／ＰＫＢ系统是近年来发现的一个生长因子信号转导通路。     

酪氨酸激酶Jak3与核小体组装蛋白Nap1的相互作用

在白细胞介素－２（ｉｎｔｅｒｌｅｕｋｉｎ－２,ＩＬ－２）的信号转导过程中,酪氨酸激酶Ｊａｋ３起着至关重要的作用。它不但参与Ｊａｋ－Ｓｔａｔ通路的传递,而且通过与一些未知的信号分子相互作用调节一些原癌基因的表达,如ｃ－ｆｏｓ,ｃ－ｍｙｃ等。由于目前越来越多的证据表明在信号转导过程中两个蛋白质之间的相互作用需要磷酸酪氨酸的存在,为此构建了酪氨酸磷酸化相关原酵母双杂交系统,以进一步阐释Ｊａｋ３在ＩＬ－２     

蛋白激酶B及其在磷脂酰肌醇3-激酶介导的信号转导中的作用

蛋白激酶Ｂ（ＰＫＢ）是原癌基因ｃ－ａｋｔ的表达产物,它参与由生长因子激活的经磷脂磷肌醇３－激酶（ＰＩ３Ｋ）介导的信号转导过程。与许多蛋白激酶相似,ＰＫＢ分子具有一特殊的ＡＨ／ＰＨ结构域（ＡＨ／ＰＨｄｏｍａｉｎ）,后者能介导信号分子间的相互作用。ＰＫＢ是ＰＩ３Ｋ直接的靶蛋白。ＰＩ３Ｋ产生的脂类第二信使ＰＩ－３,４,Ｐ２和ＰＩ－３,４,５－Ｐ３等均能与ＰＫＢ和磷酸肌醇依赖性蛋白激酶（ＰＤＫ）的ＡＨ／Ｐ     

IL—18信号转导途径研究进展

白细胞介素１８（ＩＬ－１８）是新近发现的细胞因子,其独特的少ＴＡＴＡ型启动子、特殊的ｍＲＮＡ结构及其前体蛋白需ＩＬ－１β转化酶（ＩＣＥ）加工成熟的特点,便得ＩＬ－１８基因可广泛表达于多种类型的细胞,ＩＬ－１８ＩＬ－１８受体结合组成受体复合物,受体复合物信号通过ＩＲＡＫ－ＴＲＡＦ６途径激活ＮＦ－кＢ,及通过酪氨酸蛋白激酶（ＰＴＫ）的ＬＣＫ－ＭＡＰＫ信号途径诱导ＴＨ１细胞产生ＩＦＮ－γ、ＩＬ－２等细胞     

粒细胞—巨噬细胞集落刺激因子／白细胞介素—3融合蛋白的表达研究

利用ＰＣＲ扩增得到粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）、白细胞介素－３（ＩＬ－３）完整基因片段,将其分别克隆ｐＧＥＭ－Ｔ构建成ＧＭ－ＣＳＦ／ＩＬ－３融合蛋白基因,ＤＮＡ序列与设计预期一致。将得到的融合蛋白基因克隆对７２ＲＮＡ聚合酶表达载体ｐＴ７ｚｚ,得到表达质粒ｐＦｕ,经转化至表达宿主Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）,在ＩＰＴＧ诱导下获得融合蛋白目的产物的直接表达。经ＳＤＳ－ＰＡＧＥ电泳鉴     

IFN-γ/STAT通路抑制TGF-β/SMAD信号转导

ＴＧＦ－β和ＩＦＮ－γ对各种细胞功能有相反的作用,但是此拮抗作用的基础不清楚。ＴＧＦ－β信号转导是通过受体丝氨酸激酶磷酸化并激活转录因子Ｓｍａｄ２和Ｓｍａｄ３实现的,而ＩＦＮ－γ受体及其结合的蛋白质酪氨酸激酶Ｊａｋ１能介导Ｓｔａｔ１的磷酸化和活化。最...     

764－3对血管平滑肌细胞增殖的影响

为寻找新型有效的ＡⅡ拮抗剂,本实验应用细胞计数、￣３Ｈ－ＴｄＲ掺入、逆转录─聚合酶链反应及放射免疫实验方法,研究了中药单体７６４－３对卒中易感型自发性高血压大鼠（ＳＨＲｓｐ）及其对照（ＷＫＹ大鼠）主动脉平滑肌细胞（ＡｓＭＣ）增殖的影响及其作用机制。结果表明：７６４－３可显著抑制ＡＳＭＣ增殖,并有剂量依赖性。２０ｍｇ／Ｌ的７６４－３可极显著地抑制ＡＳＭＣ上ＡⅡ受体（ＡＴ＿１）的ｍＫＮＡ表达（ＳＨＲｓｐ下降７６．３％,ＷＫＹ大鼠下降６１．６％）,并可降低ＳＨＲｓｐＡＳＭＣ内ＡⅡ含量（下降２０％）。推测７６４－３对ＡＳＭＣ增殖的抑制作用可能部分地是由于降低了ＡＴ＿１受体的基固表达引起。比较其对两种大鼠ＡＳＭＣ的抑制效率,发现它对ＳＨＲｓｐ的ＡＳＭＣ的作用更强。     

脑啡肽对大鼠海马神经细胞IL-6基因表达的影响

本文研究了大鼠海马内微量注射甲硫－脑啡肽（Ｍ－ＥＮＫ）对海马细胞的白细胞介素－６（Ｉｎｔｅｒｌｅｕｋｉｎ－６,ＩＬ－６）基因表达的影响。大鼠双侧海马内微量注射细菌内毒素脂多糖各１μｌ（ＬＰＳ,浓度：５０ｎｇ／ｍｌ）,于９０ｍｉｎ后用原位杂交技术检测到海马结构ＩＬ－６的基因表达,以齿状回颗粒细胞层显著。当海马内预先注射Ｍ－ＥＮＫ（每侧１μｌ,浓度：１０μｇ／μｌ）,３０ｍｉｎ后再给予脂多糖则未见ＩＬ－６基因表达。结果表明Ｍ－ＥＮＫ及ＬＰＳ可影响脑内ＩＬ－６的基因表达,在中枢调节机体免疫功能中,ＩＬ－６可能具有重要作用。     

小鼠胸树突状细胞系自发分泌多种类型细胞因子

建成小鼠胸腺基质细胞系，命名为ＭＴＳＣ４，经鉴定分析认为是ＤＣ来源。在无外来刺激条件下，ＭＴＳＣ４细胞可自发分泌多种类型细胞因子，已检测到的有ＩＬ－１，ＩＬ－６，ＩＬ－７，ＣＳＦ，ＩＦＮ及趋化因子（ＣＦ）等。其中ＩＬ－６，ＩＦＮ为较高水平分泌量，ＩＬ－１、ＣＦ为中等水平，ＩＬ－７、ＣＳＦ为较低水平分泌量。ＭＴＳＣ４不能自发分泌ＩＬ－３及ＴＮＦａ。ＭＴＳＣ４的建立有利于分析胸腺选择特别是阴性选择的机     

Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth

We have quantitated by autoradiography the binding of [¹²⁵I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G₁ phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high scrum) also reduce growth inhibitory activity. [¹²⁵I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.     

Differential expression and accumulation of 14-3-3 paralogs in 3T3-L1 preadipocytes and differentiated cells

The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3β, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.     

Type 3 IP3 receptors driving oncogenesis

Type 3 Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R3s) have been identified as anti-oncogenic channels by propelling pro-apoptotic Ca²⁺ signals to mitochondria. Yet, recent studies (Rezuchova et al, Cell Death Dis, 2019; Ueasilamongkol et al, Hepathology, 2019; Guerra et al, Gut, 2019) revealed that IP₃R3 upregulation drives oncogenesis via ER-mitochondrial Ca²⁺ crosstalk, adding complexity to IP₃R3’s role in cancer.     

Lnc-RAP3对小鼠3T3-L1前脂肪细胞分化的影响

长链非编码RNA (long non-coding RNA, lncRNA)是一类长度大于200nt、没有长开放阅读框架但往往具有mRNA结构特征的RNA,可以在转录及转录后水平参与基因的表达调控。近年来,有研究证实lncRNA对脂肪生成具有重要作用。Lnc-RAP3位于小鼠(Mus musculus)17号染色体,其表达量在小鼠脂肪细胞分化前后呈现显著差异,但其具体的生物学功能尚不清楚。为探讨lnc-RAP3在小鼠3T3-L1前脂肪细胞成脂分化中的作用,本文首先构建了lnc-RAP3的真核表达载体pcDNA3.1-RAP3,利用脂质体将pcDNA3.1-RAP3和人工合成的lnc-RAP3的siRNAs分别转染3T3-L1前脂肪细胞,并对转染后的细胞进行诱导分化,并通过油红O染色、qRT-PCR检测成脂分化相关基因表达等方法比较过表达和敲降lnc-RAP3对3T3-L1前脂肪细胞成脂分化的影响。结果显示,过表达lnc-RAP3后,细胞内脂滴聚集显著减少(P<0.05),在诱导分化第0 d、2 d和4 d时C/EBPα、Glut4、PPARγ、LPL和FAS的表达水平均呈显著(P<0.05)或极显著(P<0.01)下降;敲降lnc-RAP3后,细胞内脂滴聚集显著增多(P<0.05),同时在诱导分化第0 d、2 d时PPARγ、LPL、C/EBPα、FAS和Glut4的表达水平呈显著(P<0.05)或极显著(P<0.01)升高。本研究结果表明,lnc-RAP3可能通过影响成脂分化相关基因的表达来抑制3T3-L1前脂肪细胞的成脂分化。     

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells

Summary— The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-I and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.     

EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。     

Synthesis of 3-methyl-3-hydroxy-6-oxo-androstane derivatives

3alpha,17beta-Dihydroxy-3beta-methyl-5alpha-androstan-6-one (1) and 3beta,17beta-dihydroxy-3alpha-methyl-5alpha-androstan-6-one (13) were prepared by the reaction of methylmagnesium bromide with the 3-ketosteroids. Structures and configurations in position 3 were determined by NMR spectra. Substitution in the position 6 influences the ratio of the products.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.