Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97

ALM To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97. andbiological characteristics of the target clones selected by in vivo screening were studied.``RESULTS Two clones with high MHCC97-H and IowMHCC9--L1 metastatic potential were isolated from theparent cell line. Compared with MHCC97-L. MHCC97-H hadsmaller cell size average cell diameter 43 um vs 50 μmand faster in vitro and in vivo growth rate tumor celldoubling time was 34.2 h vs 60.0 h. The main ranges ofchromosomes were 5.5 58 in MHCC97-H and 57 62 inMHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was 137.5 - 11 .0) cellsfield for MHC_C99--H vs 17.7 - 6.3) field for MHCC97-L.The proportions of cells in GO Gl phase. S phase, and G_ M phase for MHCC97-H MHCC97-L were 0.56 6.65.0.28 0.25 and 0.l6 0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5 wk after orthotopic implantation of tumor tissue were ( 24666 μg. L for MHCC97-H and (91- 66) μg' L 1 for MHCC97L. The pulmonary metastatic rate was 100% (10-10) vs40% 4- 10).``CONCLUSION Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis.

Hepatoprotective Effect of Guava (<i>Psidium guajava</i>L.) Leaf Extracts on Ethanol-Induced Injury on Clone 9 Rat Liver Cells

Guava (Psidium guajava L.), a tropical fruit, belongs to Myrtaceae family. Leaves and fruits of guava have been reported to have an anti-diarrheal, hypoglycemic, lipid lowering, anti-bacterial in addition to antioxidant activities. The aim of this study was to investigate several guava leaf extract cytotoxic effects on healthy clone 9 liver cells and its hepatoprotective effects on ethanol-induced heap-toxicity. It was discovered that when the clone 9 liver cells were treated with guava (Psidium guajava Linn.) extracts for 24 hours, there was no retardation of growth as well as when ethanol and acetone extracts at low concentrations 100 μg/mL and 50 μg/mL were administered however cytototoxic effects were detected at higher concentrations. Water and hot water extracts in concentrations lower than or equal to 500 μg/mL revealed no cytotoxic effects. Injury induction to healthy clone 9 liver cells using 5% alcohol concentration for 30 minutes revealed the hepatoprotective properties of guava (Psidium guajava Linn.) extracts. This was significant in concentrations of 100 μg/mL or lower for ethanol and all concentrations for hot water extracts. Hot water extracts showed higher hepatoprotective and lower cytotoxic properties than other extracts.

ESTABLISHMENT OF CELL CLONE OF LYMPHOMA AND A CELL LINE INFECTED WITH LEUKEMIA VIRUS AND STUDY ON ITS INDUCTED DIFFERENTIATION

Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS-82 cell line, SACIIB2, SACIIC3 cell clones and a cell line infected with SRS leukemia virus (SRSV/3T3) were obtained at vitro. The main results of study on the biology, virology and Its Induction of differentiation are summarily reported.

Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine

Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.

ELECTRON MICROSCOPIC OBSERVATION OF DIFFERENTIATION OF LEUKEMIC CELL CLONE AFTER CFU-MIX CULTURE

By scanning and transmission electron microscopy, leukemlc celb were obwrved after CFU-Mix culture. Even though granulocytlc growth factor, erythropoietin and lymphocytlc growth factor were added at vitor, acute leukemlc celb still showed defects In differentiation and maturation. These were characterized by abnormal colony which consisted of smooth cells, bizarre shape, nuclear-cytoplasmic asynchrony In development, and appearance of nuclear bleb. However, chronic myelogenous leukemlc celb were more nature than the acute ones, manifesting in normal colony with finger- like projections and ruffled membrane. Macrophages and eosinophils could be observed. It b suggested that there b a difference In differentiation between acute and chronic leukemia cells.

Bcl-1 Rearrangement and Cyclin D1 Protein Expression in Multiple Myeloma Precursor Cells

The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL-1 rearrangement and cyclin D1 protein expression in 15 cases of MM were detected. By using hemi-nested polymerase chain reaction (PCR) the genomic DNA from fresh peripheral blood and bone marrow was amplified and the expression of cyclin D1 in the smears was detected by using immunohistochemical method. Ten volunteer with normal bone marrow served as control group. The results showed Bcl-1 rearrangement was detectable in 3/15 (20 %) MM patients and cyclin D, expression in 4/15 (27 % ) MM patients. BeLl-1 rearrangement and cyclin D1 protein expression were also detected in MM precursor cells. No overexpression of cyclin D1 or the rearrangement of the BeL-1 gene was found in the 10 volunteers. It was concluded that Bel-1 rearrangement and cyclin D1 protein overexpression were detected in MM precursor cells, speculating that overexpression of cyclin D1 protein may play an initial (critical) role in the pathogenesis of MM.

七彩神仙鱼催乳素基因的克隆、定位及表达分析

【目的】七彩神仙鱼(Symphysodon aequifasciatus)具有特殊的亲代抚育行为,克隆并定位七彩神仙鱼催乳素基因,分析其在亲代抚育中的表达模式,为理解催乳素在七彩神仙鱼亲代抚育中的作用提供依据。【方法】通过BLASTP对七彩神仙鱼全基因组进行分析,鉴定出1个催乳素基因,命名为dfprl。基于dfprl基因的CDS区,设计克隆和PCR引物,通过RACE技术克隆获得dfprl基因全长,并利用生物信息学对dfprl基因进行结构分析,对其氨基酸序列进行理化性质和进化分析。对不同抚育阶段七彩神仙鱼的脑、性腺和皮肤进行转录组分析,探究dfprl基因在亲代抚育中的表达特征,并利用原位杂交技术对dfprl基因在七彩神仙鱼皮肤中的表达进行定位。【结果】dfprl全长为1282 bp,cDNA序列开放阅读框长度为639 bp,共编码212个氨基酸,5'-UTR为309 bp,3'-UTR为334 bp。dfprl蛋白存在PRL家族的典型结构域Hormone_1,dfprl蛋白与慈鲷科其他鱼类PRL蛋白相似性较高,与尼加拉瓜湖始丽鱼(Archocentrus centrarchus)对应的氨基酸序列同源性最高、为96.70%。亲鱼进入抚育阶段后,dfprl在性腺和皮肤中的表达水平逐渐上升,抚育结束时表达水平下降。原位杂交结果显示,dfprl在皮肤粘液细胞中表达,且与七彩神仙鱼催乳素受体(dfprlr)表达位点重叠,在抚育早期阶段高表达。【结论】七彩神仙鱼dfprl基因高度保守,存在稳定的Hormone_1结构域。七彩神仙鱼dfprl基因在亲代抚育阶段的皮肤中高表达,且在亲代抚育阶段的皮肤粘液细胞中与dfprlr表达位点重合,表明dfprl可能通过作用于dfprlr进而促进七彩神仙鱼独特抚育行为的发生。

戊地昔布抑制clone 26肿瘤细胞增生及机制

目的探讨戊地昔布对小鼠肠癌clone26细胞的生长抑制作用及机制。方法用噻唑蓝（MTT）法检测戊地昔布对clone26细胞生长的作用。用流氏细胞仪检测clone26细胞凋亡率和细胞周期分布。用Western blot检测clone26细胞caspase-3，PCNA和COX-2的表达．结果①戊地昔布可抑制clone26细胞生长并呈时间和浓度依赖性。②50-400μmol·L^-1戊地昔布可明显提高clone26细胞的凋亡率，从对照的3．1％提高到4．4％～11．0％。给予戊地昔布后，细胞的增殖指数，S期的细胞比例和G2／M期细胞有下降趋势，但只有在400μmol·L^-1戊地昔布组时才有统计学意义。③给予戊地昔布后，细胞PCNA的表达降低，caspase-3的表达升高，COX-2的表达没有变化。结论戊地昔布通过诱导凋亡和细胞周期停滞而抑制clone26细胞生长，小剂量时主要与凋亡有关，大剂量时与诱导凋亡和细胞周期停滞有关、Caspase-3参与戊地昔布诱导的凋亡。

雄激素受体调控的lncRNA ARLNC1对前列腺癌细胞增殖、克隆、迁移和细胞周期的影响

目的:探讨雄激素受体(androgen receptor,AR)调控的长链非编码RNA(long non-coding RNA,lncRNA)ARLNC1对前列腺癌细胞增殖、克隆、迁移和细胞周期的影响。方法:选取正常前列腺上皮细胞株WPMY-1和前列腺癌细胞株PC3、VCaP、22RV1、DU145和LNCaP为研究对象,qRT-PCR分析lncRNA ARLNC1的表达水平;双氢睾酮(DHT)以时间和浓度依赖的方式刺激LNCaP细胞,分析lncRNA ARLNC1的转录水平;采用数据库预测和双荧光素酶报告基因系统分析雄激素受体与lncRNA ARLNC1的关系;将LNCaP细胞分为sh-NC和sh-lncRNA ARLNC1组,采用CCK-8分析细胞增殖,检测细胞克隆和迁移能力;流式细胞仪分析细胞周期变化,Western blot分析细胞周期蛋白CyclinD1、CDK6、p27的表达水平。结果:与正常前列腺上皮细胞相比,lncRNA ARLNC1在雄激素依赖性前列腺癌细胞株(LNCaP、VCaP、22RV1)中的表达水平明显增加,而在雄激素非依赖性前列腺癌细胞株(PC3、DU145)中几乎不表达(P<0.05),其中LNCaP细胞变化最显著,所以下续选用LNCaP细胞作为实验株。100 nmol/L DHT刺激LNCaP细胞0、6、12、18、24 h后,lncRNA ARLNC1的转录水平相对于对照组以时间依赖的方式逐渐升高(P<0.05);不同浓度(0、0.1、1、10、100 nmol/L)DHT刺激LNCaP细胞24 h后,lncRNA ARLNC1转录水平随浓度不断增加(P<0.05);JASPAR数据库预测到lncRNA ARLNC1启动子区域有AR结合的位点,双荧光素酶报告基因系统表明AR能结合在lncRNA ARLNC1启动子区域,并激活其转录(P<0.05);与sh-NC组相比,sh-lncRNA ARLNC1组细胞增殖、迁移和克隆能力减弱,细胞周期阻滞在S、G 2期(P<0.05);sh-NC组相对于sh-lncRNA ARLNC1组,CyclinD1、CDK6蛋白水平增加,而p27蛋白水平降低(P<0.05)。结论:AR调控的lncRNA ARLNC1作为癌基因在雄激素受体阳性的前列腺癌细胞系中高表达,能促进前列腺癌细胞的增殖、克隆、迁移和细胞周期进展。

体细胞克隆技术扩繁超细型二狼山绒山羊的应用研究

【目的】通过体细胞克隆技术生产超细型二狼山绒山羊克隆个体,为该技术在地方优质羊品种资源扩繁及新品种培育中的应用提供理论依据。【方法】选择1只4岁龄羊绒细度为13.89μm的二狼山绒山羊(♂),采集耳组织并分离培养成纤维细胞,以其为供体细胞,利用体细胞克隆技术开展超细型二狼山绒山羊的扩繁研究,通过克隆羊体重、羊绒细度、繁殖性能等指标评估该技术的可行性。【结果】试验成功建立了稳定的山羊卵母细胞体外成熟培养及体细胞克隆技术体系,卵母细胞成熟率为44.8%,孤雌激活卵裂率、囊胚率分别为83.0%、22.4%;二狼山绒山羊克隆胚的卵裂率、囊胚率分别为77.8%、13.7%。获得5只克隆羊个体,其生长发育正常,二狼山绒山羊母羊自然交配共获得16只健康的F1代个体,具有正常繁殖能力。克隆个体羊绒细度极显著低于同龄对照个体(P<0.01)。【结论】本研究通过体细胞克隆技术成功克隆了超细型二狼山绒山羊个体,其具备正常生产性能和繁殖性能,并保留了超细羊绒的特性,为地方优良羊种质资源扩繁和新品种培育应用研究奠定了基础。

新西兰白兔BMP 15基因的真核表达载体构建、表达模式及其在卵巢组织的表达

旨在获得新西兰白兔BMP 15基因序列及其组织表达规律,构建其真核表达载体,预测BMP15的生物学功能。本研究选择180日龄性成熟的健康新西兰白兔作为研究对象,采用RT-PCR技术扩增BMP 15基因CDS区序列,利用T4连接方法将目的片段连接至线性化的Pmcherry-N1和pCMV-Myc空载体,构建真核表达载体,利用生物信息学分析其编码蛋白的性质及结构。采用实时荧光定量PCR(qRT-PCR)技术与Western blotting技术检测pCMV-Myc-BMP 15过表达情况。接下来,使用qRT-PCR检测BMP 15基因在不同组织中的表达水平。激光共聚焦方法检测BMP 15基因的亚细胞定位。利用免疫荧光技术检测内源BMP15在新西兰白兔卵巢组织定位情况。结果表明:克隆得到新西兰白兔BMP 15基因CDS区序列长1182 bp,PCR及测序结果表明pCMV-Myc-BMP 15和Pmcherry-N1-BMP 15真核表达载体构建成功。生物信息学分析显示,BMP 15基因编码393个氨基酸;BMP15蛋白不稳定系数为55.32,等电点大小为9.69,是一种稳定的碱性蛋白质。BMP15蛋白共有26个磷酸化位点,15个糖基化位点,存在信号肽,不存在跨膜结构域。系统进化树表明,新西兰白兔与猪亲缘关系最近,与鸡的亲缘关系最远。二级结构和三级结构分析结果表明,BMP15蛋白主要由α-螺旋(38.68%)、无规则卷曲(37.4%)、延伸链(15.52%)、β-转角(8.40%)组成,为混合型蛋白。蛋白互作关系预测发现,BMP15蛋白与FSHR、FIGLA、BMPR1B、AMHR2、NOBOX等卵巢生长发育的相关蛋白之间存在相互作用。不同组织表达分析显示,BMP 15基因在卵巢组织中特异性表达;激光共聚焦结果显示,BMP15主要存在于在细胞质中。将pCMV-Myc-BMP 15转染至HEK293T细胞内,BMP15的mRNA水平和蛋白水平均显著上调。卵巢组织免疫荧光检测结果显示,BMP15主要定位在卵巢颗粒细胞的细胞质中。本研究成功构建了BMP15的真核表达载体,对BMP 15基因及其编码的蛋白的理化性质和生物学特性进行了预测分析,在HEK293T细胞内成功过表达,并得到了该基因的亚细胞定位情况、组织表达情况及在卵巢组织的分布情况。为后续开展BMP 15基因在卵巢生长发育中的功能及机制研究提供了理论依据。

家蚕glial cell missing(BmGcm)基因鉴定、表达、亚细胞定位和功能

【目的】鉴定、克隆家蚕(Bombyx mori)glial cell missing(Bm Gcm)基因,分析其m RNA表达及亚细胞定位特征。制备多克隆抗体,同时在细胞水平进行过表达,检测Gcm对细胞增殖和周期的影响,为探究Bm Gcm功能打下基础。【方法】利用RACE方法克隆获得Bm Gcm全长c DNA序列,利用ORF Finder和SMART等在线工具对Bm Gcm基本序列特征和结构信息进行分析,运用Clustalx和MEGA 6.0等软件对多物种Gcm蛋白进行同源序列比对和进化分析。采用RT-PCR和q RT-PCR方法检测Bm Gcm的表达情况。利用原核表达系统获得重组蛋白,通过蛋白纯化和免疫小鼠制备多克隆抗体,运用Western blot对抗体进行检测。构建Bm Gcm表达载体,转染家蚕胚胎细胞系,分析其亚细胞定位情况,同时利用EDU细胞增殖标记和流式细胞仪对其功能进行探索。【结果】Bm Gcm(BGIBMGA006182)定位于4号染色体的nscaf2847上,其基因全长4 046 bp,包含4个外显子和3个内含子。其c DNA全长1 734 bp,包含166 bp的5′UTR、227 bp的3′UTR和1 341 bp的完整开放阅读框(ORF)。该基因编码446个氨基酸残基,预测蛋白分子量为50.61 k D,等电点5.557,含有典型的GCM结构域。多重比对结果显示GCM结构域在不同物种间具有高度的保守性,进化分析显示昆虫Gcm蛋白单独聚为一支,其中Bm Gcm蛋白与帝王蝶同源蛋白亲缘关系最为接近。表达分析结果显示Bm Gcm在胚胎发育第4天表达达到峰值,随后表达水平逐渐下调,而在幼虫阶段,Bm Gcm主要表达于中肠、精巢和卵巢。将Bm Gcm完整的开放阅读框序列构建至原核表达系统,经IPTG诱导和亲和层析纯化获得高纯度重组蛋白,通过免疫小鼠获得了多克隆抗体,Western blot检测该抗体可以特异性识别重组蛋白。在家蚕细胞系中过表达Bm Gcm蛋白,结果显示其定位于细胞核。在细胞水平,过表达Bm Gcm会明显抑制细胞增殖,将细胞周期阻滞于G1/S期。【结论】克隆鉴定得到Bmgcm全长序列,获得其表达和亚细胞定位信息。通过原核表达、蛋白纯化和免疫小鼠制备了可用的多克隆抗体。细胞实验发现Bm Gcm可以显著抑制增殖和影响正常的细胞周期进程。

Stem cell pluripotency and transcription factor Oct4

Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 218t has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarizere the structure and function of Oct4 and address to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cels.

Establishment of customized mouse stem cell lines by sequential nuclear transfer

治疗学的克隆，胚胎的干细胞(转换字符) 由此从原子转移(NT ) 被导出胚胎，可以在再生药的新时代起一个主要作用。Inthis 学习我们建立了四十原子转移 -- 从不同施主房间类型或段落的 NTembryos 被导出的转换字符(NT 转换字符) 线。我们发现 NT 转换字符能够形成胚胎植物或动物身体。另外， NT 转换字符表示了 pluripotency 干细胞标记试管内并且能区分进胚胎的纸巾体内。从施主房间能形成完整的术语的早经过 R1 的 NT 胚胎开发了小狗，而那些为为实时出生是必要的 reprogramming 从迟了的经过 R1 ES 施主房间失去了潜力。我们随后建立了近来从 NT 胚囊被开发的 sequentialNT-R1-ESC 线经过 R1 转换字符施主。然而，当在他们的早段落用作原子转移施主时，这些 NT-R1-ESC 排队，没能导致实时小狗。这显示用顺序的 NT 转换字符的治疗学的克隆过程不能救居住在以前的施主代的发展缺乏。

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation.

Complete Genomic Sequence of Transmissible Gastroenteritis Virus TS and 3' End Sequence Characterization Following Cell Culture

The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.

Activating interactions of sulfanilamides with T cell receptors

Activation and expansion of drug reactive T cells are key features in drug hypersensitivity reactions. Drugs may interact directly with immune receptors such as the human leukocyte antigens (HLA) or the T-cell receptors (TCR) itself, the pharmacological interaction [p-i] concept. To analyze whether the drug sulfamethoxazole (SMX) interacts directly with the TCR and thereby contributing to signaling and T cell activation, we analyze two SMX specific T cell clones (TCC “1.3”and “H13”). Proliferation to SMX and 11 related sulfanilamides, Ca++ influx in drug stimulated T-cells and the inhibitory effect of non-reactive sulfanilamides on SMX stimulation were analyzed. In silico docking of SMX and related sulfanilamide to the TCR were used to identify possible drug binding sites, and correlated to in vitro data to find the correct docking. In Ca++ influx assays, reactions occurred as early as 14 sec after adding SMX to TCC and APC. The broadly reactive clone (“H13”) was stimulated by 5 additional sulfanilamide, while one TCC (“1.3”) was reactive exclusively with SMX but not other sulfanilamides. Competition experiments with sulfanilamide inhibited SMX induced Ca++ influx and proliferation of the TCC1.3 ina dose dependent way. Docking experiments with SMX and related sulfanilamides confirmed and explained the in vitro data as docking localized binding sites for SMX and the 5 stimulating sulfanilamides on the CDR2β domain of the clone H13, while the 6 non-stimulatory SA failed to bind. In TCC 1.3, SMX could be docked on the CDR3α of the TCR. The other, non-stimulatory but inhibitory SA could also be docked to the same site. The combined analysis of in vitro and in silico data show that sulfanilamide can bind directly to TCRs. It shows that TCR, like other receptors, appear to be reamenable to manipulations by small molecules.

基于NF-κB信号通路探究木犀草素对宫颈癌细胞增殖和凋亡的调控作用

目的:探讨木犀草素对宫颈癌细胞增殖和凋亡的影响及其对核转录因子-κB(NF-κB)信号通路的调控作用。方法:将HeLa细胞分为对照组、5μmol/L木犀草素组、10μmol/L木犀草素组、20μmol/L木犀草素组和抑制剂组。5、10、20μmol/L木犀草素组HeLa细胞分别经不同浓度(5、10和20μmol/L)木犀草素处理24 h,对照组HeLa细胞不做处理。筛选出10μmol/L为木犀草素最适浓度,然后将细胞分为对照组、10μmol/L木犀草素组和抑制剂组[10μmol/L木犀草素+2.5μmol/L BAY 11-7082(NF-κB信号通路抑制剂)]。采用5-乙炔基-2'脱氧尿嘧啶核苷(EdU)测定细胞增殖情况,平板克隆测定细胞克隆情况,Hoechst 33258测定细胞凋亡情况,实时荧光定量PCR(RT-qPCR测定半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)mRNA和细胞周期素D1(Cyclin D1)mRNA表达,蛋白免疫印迹(Western blotting)法测定Caspase-3、Cyclin D1、NF-κB和p-NF-κB蛋白表达。结果:10、20μmol/L木犀草素组细胞增殖率、克隆形成率、Cyclin D1 mRNA和Cyclin D1蛋白相对表达量低于对照组,差异均有统计学意义(P<0.05);10、20μmol/L木犀草素组细胞凋亡数、Caspase-3 mRNA和Caspase-3蛋白相对表达量均高于对照组,差异均有统计学意义(P<0.05)。因此本实验选择低浓度(10μmol/L)木犀草素加入NF-κB通路抑制剂作为抑制剂组进行后续验证实验。抑制剂组细胞增殖率和克隆形成率低于10μmol/L木犀草素组,差异有统计学意义(P<0.05)。10μmol/L木犀草素组Cyclin D1 mRNA、Cyclin D1蛋白、p-NF-κB蛋白相对表达量低于对照组,Caspase-3 mRNA和Caspase-3蛋白相对表达量高于对照组,差异均有统计学意义(P<0.05);抑制剂组Cyclin D1 mRNA、Cyclin D1蛋白、p-NF-κB蛋白相对表达量低于10μmol/L木犀草素组,Caspase-3 mRNA和Caspase-3蛋白相对表达量高于10μmol/L木犀草素组,差异均有统计学意义(P<0.05)。结论:木犀草素可能通过抑制NF-κB信号通路诱导HeLa细胞的凋亡并减弱其增殖能力。

Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells

AIM: To clone the cDNA of UGT1 A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGTI A9.METHODS: cDNA of UGT1A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E. CoIl DH5α. The inserted fragment, verified by DNA sequencing, wes subcloned into the Hind III/Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9- UGT1A9, and selected by G418 (400 mg. L-1) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples for UGT1 A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC.RESULTS: The sequence of the cDNA segment cloned,which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9- UGT1 A9, contains the entire coding region, along with 18bp of the 5' and 55 bp of the 3′ untranslated region of the UGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1 A9, and the enzyme activity towards propranolol in S9 protein was found to be 101 ± 24pmol.min-1 .mg-1 protein (n = 3), but was not detectable in parental CHL cells.CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL ceils. The CHL-UGT1A9 cell lines established efficiently expressed the protein of UGT1A9 for the further enzyme study of drug glucuronidation.