PP2R1A 逆转录病毒的构建及对细胞周期的影响

目的:构建重组PP2R1A基因的逆转录病毒感染HEKTER细胞,观察其定位,验证表达,研究过表达PP2R1A对细胞生长及周期的影响。方法:逆转录病毒载体pMIG-Flag-PP2R1A-IRES-GFP与Pcl10A1瞬时共转染293T细胞,收集病毒感染HEKTER细胞,在荧光显微镜下观察定位,标记荧光单克隆。挑取不同表达强度单克隆做western验证PP2R1A蛋白表达。运用流式细胞分析、体外创伤试验及生长曲线试验研究单克隆细胞的增殖及周期。结果:获得了过表达PP2R1A的单克隆细胞株,PP2R1A在细胞内广泛表达,结合western及细胞试验证实PP2R1A高表达阻滞细胞周期并减慢细胞生长。结论:PP2R1A是丝苏氨酸蛋白磷酸酶PP2A的结构A亚基的a亚型,在细胞内广泛表达。本文成功构建了表达PP2R1A的细胞株,研究发现PP2R1A高表达会影响细胞生长及细胞周期,减缓了细胞增殖。为进一步深入研究PP2R1A对PP2A全酶活性及功能、细胞转化的影响奠定了重要的实验基础。     

基于逆转录病毒载体构建稳定表达人CD19和荧光蛋白的SW620细胞株

摘要 目的：构建表达人CD19和增强型绿色荧光蛋白（EGFP）和荧光素酶（LUC）的SW620细胞株。方法：构建MFG-CD19质粒,进行逆转录病毒载体包装,与MFG-EGFP-P2A-LUC逆转录病毒载体共同转导SW620细胞,筛选出稳定且强表达的单克隆细胞株SW620-CD19-EGFP-LUC并扩大培养。通过流式细胞术与qPCR技术检测细胞CD19的表达、通过荧光素酶法对细胞系表面荧光素酶的表达和细胞系功能进行鉴定。结果：流式检测构建完成的细胞株中CD19阳性的细胞占比为99.9%,EGFP阳性的细胞占比为99.2%;荧光显微镜下能够明显观察到细胞的绿色荧光;qPCR检测结果表明细胞株中CD19的表达水平相比原始细胞株明显上调,并且能够激活CD19 CAR-T细胞对其进行杀伤。结论：成功构建了能够稳定表达人CD19和荧光蛋白的SW620细胞株。     

蛋白磷酸酶2A的结构、功能和活性调节

蛋白磷酸酶 2A(proteinphosphatase 2A ,PP2A)是主要的丝 /苏氨酸蛋白磷酸酶 ,拥有众多不同基因编码的亚基 ,分别组成多种不同的PP2A全酶 ,参与细胞周期、DNA复制、信号转导、细胞分化和细胞恶性转化等多种细胞生物学事件 ,并和神经退行性疾病、肿瘤等多种疾病的发生、发展有关。PP2A调节亚基的组织特异性表达和细胞内定位 ,催化亚基羧基末端的磷酸化和甲基化 ,第二信使神经酰胺 (ceramide)、天然小分子抑制剂等都能够调节PP2A的活性。     

沉默DEPDC1基因表达对鼻咽癌细胞系HNE-1生长和细胞周期的影响

为探讨沉默DEPDC,基因表达对鼻咽癌细胞系HNE．1生长和细胞周期的影响,该实验设计合成靶向DEPDCl的小分子干扰RNA（smallinterferingRNA,siRNA）转染人鼻咽癌HNE-1细胞。转染后,采用荧光定量PCR、免疫印迹、MTT及流式细胞术方法检测细胞内DEPDCl的表达量以及细胞周期、生长增殖、凋亡的变化及其可能机制。结果显示,转染DEPDClsiRNA后,DEPDC1基因在mRNA及蛋白水平的表达量明显降低;大量细胞被阻滞于G2／M期,生长增殖减慢,凋亡增加。荧光定量PCR结果表明,抑制NF—KB激活的A20基因表达量明显上调,受NF-κB调控的肿瘤相关靶基因的表达量下降,包括C-MYC、MMP9、ICAM-1、BCL-2基因。由此说日月,沉默DEPDC1基因可以影响HNE-1细胞的周期,抑制其生长增殖,促进凋亡,其机制可能与抑制NF-κB通路有关。     

rBTI通过上调PTEN的表达抑制Hep G2细胞增殖

磷酸酶及张力蛋白的同源基因(PTEN) 是一种抑癌基因,可以调控细胞的增殖,与癌症的发生和发展息息相关。本研究采用MTT法和流式细胞术分别检测了重组荞麦胰蛋白酶抑制剂(rBTI)对人肝癌细胞株Hep G2细胞的增殖以及周期的影响。免疫荧光及Western印迹法检测了PTEN和p PTEN的亚细胞定位及蛋白表达的变化。采用qRT-PCR及Western印迹法检测了周期相关蛋白的表达。旨在探究PTEN和p PTEN在rBTI抑制Hep G2细胞增殖和周期阻滞中的作用。结果表明,rBTI能显著抑制Hep G2细胞增殖,将细胞周期阻滞在G0/G1期,并呈时间和剂量依赖性;rBTI作用于Hep G2后,可显著上调PTEN和p-PTEN的表达。同时发现,p-PTEN主要分布于细胞核中,能与核仁发生共定位;周期相关蛋白检测表明,细胞内p53、p21转录水平和蛋白水平均增加。综上所述,rBTI通过上调PTEN的表达,使得细胞周期阻滞于G0/G1期,进而抑制Hep G2细胞的增殖。     

PP2A通过Akt/Skp2通路调控p27~(Kip1)的表达并抑制肺癌细胞的致瘤能力

蛋白磷酸酶2A(PP2A)是由36 k Da的催化亚基C(PP2Ac)和65 k Da的结构亚基A(PP2Aα/β)一起组成PP2A的核心酶,并且和各种不同的调节亚基B形成具有不同功能的PP2A全酶复合体。在细胞中PP2A发挥着重要作用,特别是在抑制肿瘤的形成当中,编码PP2Aα/β基因的突变将导致肿瘤的形成和其他疾病。当非小细胞肺癌细胞H1299中过表达PP2A-Aα时,细胞生长被抑制,细胞周期停留在G0/G1期,致瘤能力也同时被抑制。进一步研究证明当PP2A-Aα过表达时,Akt被去磷酸化失活使Skp2的表达下调,从而导致细胞周期抑制因子p27kip1的表达上调。肿瘤细胞软琼脂克隆形成实验的结果表明过表达PP2A-Aα之后H1299细胞的锚定非依赖性生长能力明显的降低,形成的克隆细胞团也较小,这些结果和裸鼠成瘤实验的结果是一致的。     

小干扰RNA抑制LRP16基因表达限制了MCF-7乳腺癌细胞增殖

雌激素雌二醇上调人乳腺癌细胞MCF 7中LRP16基因表达 ,该基因过表达促进MCF 7细胞增殖 .为进一步探讨LRP16基因不同表达水平对MCF 7细胞增殖的影响以及对雌激素的反应性增殖能力 ,采用针对LRP16基因特异的小干扰RNA策略 ,通过逆转录病毒介导及抗性筛选构建了LRP16基因被稳定抑制的 2个MCF 7细胞系 ,针对绿色荧光蛋白的干扰序列作为阴性对照 .Northern印迹实验检测了LRP16基因在各个细胞株中mNRA的水平 ,与对照组细胞比较 ,针对LRP16基因不同位置的 2个小干扰RNA可分别将该基因抑制 90 %和 6 0 % .细胞增殖试验结果显示 ,MCF 7细胞中LRP16基因表达抑制率越高 ,细胞增殖速率减慢越显著 (P <0 0 5 ) ;软琼脂集落形成试验结果显示 ,抑制LRP16基因在MCF 7细胞中表达 ,限制了细胞锚定非依赖性生长 ;细胞周期分析结果表明 ,LRP16基因抑表达使MCF 7细胞G1 S周期转换受抑 ;Western印迹结果表明 ,LRP16基因表达抑制的细胞中细胞周期蛋白E及细胞周期蛋白D1蛋白水平显著下调 ,但未检测到P5 3及Rb蛋白表达水平的影响 .雌二醇刺激的增殖实验结果显示 ,抑制LRP16基因表达没有消除MCF 7细胞的反应性增殖特征 .上述结果表明 ,LRP16基因表达量与MCF 7细胞增殖能力密切相关 ,抑制其表达可有效限制MCF 7细胞的增殖能力 ,提     

共表达口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因牛肾细胞株的筛选及稳定性

[目的]筛选稳定表达口蹄疫病毒衣壳蛋白的牛肾细胞(Madin-Darby bovinekidney,MDBK)株.[方法]采用聚合酶链式反应(Polymerase chain reaction,PCR)方法从重组质粒pMD-P1-2A和pMD-3C中分别扩增口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因,将两基因依次插入逆转录病毒载体pBABE-puro.重组逆转录病毒载体pBABE-puro/P1-2A-3C和pVSV-G质粒载体用脂质体介导共转染GP2-293包装细胞.产生的重组逆转录病毒感染MDBK细胞后使用嘌呤霉素筛选抗性细胞.利用克隆环套取法得到单克隆细胞.经间接免疫荧光和酶联免疫吸附测定(Enzyme-linkedimmunosorbent assay,ELISA)方法检测MDBK细胞中衣壳蛋白的表达,并在电镜下观察口蹄疫病毒空衣壳.[结果]成功筛选到稳定表达口蹄疫病毒衣壳蛋白的MDBK细胞株,衣壳前体蛋白P1-2A在蛋白酶3C裂解作用下正确组装成空衣壳.[结论]该研究为口蹄疫亚单位疫苗的研制提供了实验材料.     

人ABCB6表达载体的构建及稳定表达细胞株A375的筛选

目的构建表达载体pIRES2-ZsGreen1-ABCB6,在转染的人黑素瘤细胞系株A375中筛选其稳定表达的细胞株。方法抽取健康人外周血,分离外周血单个核细胞,提取总RNA,逆转录获取cDNA序列,加入特异性引物经PCR扩增获得ABCB6cDNA双链,再经过BglII、EcoRI双酶切PCR产物及质粒载体pIRES2-ZsGreen1,酶切产物经回收、T4DNA连接酶连接,产物转化到大肠杆菌DH5α,挑取阳性克隆经菌落PCR鉴定、酶切鉴定和测序分析,以确定构建质粒正确。转染人黑素瘤细胞株A375,G418筛选稳定表达ABCB6的单克隆细胞株,应用荧光显微镜鉴定ABCB6蛋白的表达情况。结果 pIRES2-ZsGreen1-ABCB6质粒经菌落PCR、酶切、测序鉴定正确,经过G418筛选后获得稳定细胞株,在荧光显微镜下可观察到绿色荧光蛋白在A375细胞中的表达。结论表达载体pIRES2-ZsGreen1-ABCB6构建正确,并成功筛选出稳定表达ABCB6的A375细胞株,为进一步研究ABCB6的生物学功能奠定了良好基础。     

稳定表达呼吸道合胞病毒非结构蛋白NS1细胞的构建及其生物特性研究

获得稳定表达RSV病毒NS1基因的HEp-2-NS1细胞株并对其生物学特性进行初步研究。采用RT-PCR方法从RSV病毒中获得NS1全长基因,克隆到逆转录病毒载体pBABE-puro中,重组载体与包装质粒PIK通过磷酸钙共沉淀法转染包装细胞293FT细胞,产生的逆转录病毒颗粒感染HEp-2细胞,经嘌呤霉素筛选后得到稳定表达细胞株,用QPCR、细胞病变染色法、RT-PCR和间接免疫荧光法检测细胞中NS1 mRNA和蛋白表达情况。结果表明重组逆转录病毒载体pBABE-NS1经双酶切及测序鉴定正确;筛选获得5株阳性单克隆细胞株,QPCR结果显示HEp-2-NS1细胞有NS1基因扩增,其中d株的相对表达量是正常细胞对照组的8 483倍;在外源干扰素作用下,HEp-2-NS1细胞仍对VSV病毒保持敏感,细胞感染病毒48h后染色结果显示全部死亡;对第3代及第30代细胞的RT-PCR和间接免疫荧光法检测结果显示,导入的NS1基因在HEp-2细胞中不仅能转录成相应的mRNA而且能成功稳定地表达。成功构建了稳定表达RSV病毒非结构蛋白NS1的HEp-2-NS1改造细胞株,为建立适用于临床分离的转基因细胞提供良好的基础。     

Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A.

The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.     

A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression

Activity of protein phosphatase 2A (PP2A) is tightly regulated and performs a diverse repertoire of cellular functions. Previously we isolated a dominant-negative active site mutant of the PP2A catalytic (C) subunit using a yeast complementation assay. We have established stable fibroblastic cell lines expressing epitope-tagged versions of the wild-type and H118N mutant C subunits and have used these cells to investigate mechanisms that regulate PP2A activity. Cells expressing the mutant C subunit exhibit a decreased growth rate and a prolonged G1 cell cycle phase. The mutant protein is enzymatically inactive, but extracts made from cells expressing the H118N C subunit show normal levels of total PP2A activity in vitro. The H118N mutant shows reduced binding to the regulatory A subunit, but binds normally to the alpha4 protein, a non-canonical regulator of PP2A. Expression of the H118N mutant interferes with the normal control of C subunit abundance, causing accumulation of the endogenous wild-type protein as well as the mutant transgene product. Our results indicate that the H118N mutant isoform retards C subunit turnover and suggest that PP2A C subunit turnover may be important for normal cell cycle progression.     

IPP5, a novel protein inhibitor of protein phosphatase 1, promotes G1/S progression in a Thr-40-dependent manner

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. Here we describe the characterization of a novel inhibitory molecule for PP1, human inhibitor-5 of protein phosphatase 1 (IPP5). We find that IPP5, containing the PP1 inhibitory subunits, specifically interacts with the PP1 catalytic subunit and inhibits PP1 phosphatase activity. Furthermore, the mutation of Thr-40 within the inhibitory subunit of IPP5 into Ala eliminates the phosphorylation of IPP5 by protein kinase A and its inhibitor activity to PP1, whereas the mutation of Thr-40 within a truncated form of IPP5 into Asp can serve as a dominant active form of IPP5 in inhibiting PP1 activity. In IPP5-negative SW480 and IPP5-highly positive SW620 human colon cancer cells, we find that overexpression of IPP5 promotes the growth and accelerates the G(1)-S transition of SW480 cells in a Thr-40-dependent manner, which could be reversed by downregulation of the PP1 expression. Moreover, silencing of IPP5 inhibits the growth of SW620 cells both in vitro and in nude mice possibly by inducing G(0)/G(1) arrest but not by promoting apoptosis. According to its role in the promotion of cell cycle progression and cell growth, IPP5 up-regulates the expression of cyclin E and the phosphorylated form of retinoblastoma protein. Our findings suggest that IPP5, by acting as an inhibitory molecule for PP1, can promote tumor cell growth and cell cycle progression, and may be a promising target in cancer therapeutics in IPP5-highly expressing tumor cells.     

Recent progress on the structure of Ser/Thr protein phosphatases

PP1, PP2A and PP2B, belonging to the PPP family of Ser/Thr protein phosphatases, participate in regulating many important physiological processes, such as cell cycle control, regulation of cell growth and division regulation, etc. The sequence homology between them is relatively high, and ter- tiary structure is conserved. Because of the complexity of the structure of PP2A and the diversity of its regulatory subunits, its structure is less well known than those of PP1 and PP2B. The PP2A holoen- zyme consists of a heterodimeric core enzyme, comprising a scaffolding subunit and a catalytic sub- unit, as well as a variable regulatory subunit. In this study, the subunit compositions, similarities and differences between the Ser/Thr protein phsphatases structures are summarized.     

Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.