CHOP在三氧化二砷诱导SMMC-7721 凋亡中的作用

目的:探讨CHOP在三氧化二砷(Arsenic Trioxide,As_2O_3)诱导肝癌细胞系SMMC-7721凋亡中的作用。方法:通过MTT法观察不同浓度的As_2O_3对肝癌细胞系SMMC-7721增殖活性的影响;通过流式细胞术检测不同浓度的As_2O_3作用后,SMMC-7721细胞的凋亡率;以Western-blot方法检测As_2O_3处理SMMC-7721细胞后,内质网应激相关蛋白GRP78,CHOP的变化。通过CHOP siRNA沉默CHOP后,观察As_2O_3对SMMC-7721细胞凋亡的影响。结果:As_2O_3能显著抑制SMMC-7721细胞的增殖活性,并呈剂量依赖性诱导SMMC-7721细胞的凋亡;Western blot结果显示三氧化二砷诱导内质网应激相关蛋白的表达。CHOP siRNA沉默CHOP后能够显著抑制As_2O_3对SMMC-7721细胞凋亡的诱导。结论:As_2O_3诱导SMMC-7721细胞凋亡可能与其诱导CHOP表达有关。     

壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响

目的检测壳寡糖对人肝癌SMMC-7721细胞的抑制效果及对凋亡调控蛋白Bcl-2和Caspase-3的影响。方法采用噻唑蓝（MTT）法检测不同浓度壳寡糖对肝癌细胞SMMC-7721细胞增殖的抑制作用,并利用荧光Hoechst33258染色法检测细胞凋亡状况。最后通过免疫细胞化学方法研究壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响。结果壳寡糖能够抑制SMMC-7721细胞增殖,并且促进SMMC-7721细胞的凋亡,并且壳寡糖能够上调促凋亡蛋白Caspase-3的表达和降低抑制凋亡蛋白Bcl-2的表达。结论壳寡糖对人肝癌SMMC-7721细胞的增殖有抑制作用,此作用可能是通过促进Caspase-3的表达和抑制Bcl-2的表达来实现的。     

姜黄素通过MAPK信号通路诱导人肝癌SMMC-7721细胞凋亡

本文阐述了姜黄素(Curcumin)对体外培养的人肝癌SMMC-7721细胞增殖和凋亡的影响,并探讨了其诱导凋亡的信号转导机制。采用MTT法和细胞计数法检测不同浓度姜黄素对人肝癌细胞株SMMC-7721增殖的影响,利用流式细胞术检测姜黄素对人肝癌SMMC-7721细胞凋亡的影响,通过RT-PCR及Western blot检测姜黄素对人肝癌SMMC-7721细胞中凋亡相关蛋白Caspase-3、Survivin、Bcl-2和Bax表达的影响,最后通过检测MAPK的磷酸化水平分析姜黄素诱导SMMC-7721细胞凋亡的信号转导机制,通过MAPK抑制剂实验进一步证实诱导凋亡的分子机制。研究结果显示,姜黄素呈时间和剂量依赖性抑制人肝癌SMMC-7721细胞的增殖,其中40μmol/L姜黄素可明显诱导SMMC-7721细胞的凋亡,并呈时间依赖性上调促凋亡蛋白Caspase-3和Bax的表达、下调抗凋亡蛋白Survivin和Bcl-2的表达,姜黄素对凋亡相关蛋白表达的调节及诱导凋亡可以通过激活JNK、抑制ERK和p38 MAPK信号通路实现。表明姜黄素可诱导人肝癌SMMC-7721细胞凋亡,其机制与姜黄素激活JNK、抑制ERK和p38 MAPK信号通路从而上调Caspase-3和Bax的表达,下调Survivin和Bcl-2的表达有关。     

壳寡糖抑制肝癌细胞SMMC-7721的增殖及其机制探讨

探讨壳寡糖抑制人肝癌细胞株SMMC-7721的增殖作用及其分子机制.采用噻唑蓝(MTT)法检测壳寡糖的细胞增殖抑制作用;利用流式细胞仪检测肝癌细胞的凋亡情况;用RT-PCR和Western blot方法研究壳寡糖引起凋亡的原因.结果显示,0.8 mg/mL的壳寡糖能够抑制SMMC-7721细胞增殖,并且促进 SMMC-7721细胞的凋亡,其原因是壳寡糖能够上调促凋亡蛋白Bax的表达.     

EPA诱导人肝癌细胞SMMC-7721凋亡及端粒酶活性调控机制研究

目的:探究二十碳五烯酸(Eicosa Pentaenoic Acid.EPA)对SMMC-7721人肝癌细胞的凋亡、端粒逆转录酶h TERT的调控作用及端粒酶表达活性的影响。方法:体外培养SMMC-7721人肝癌细胞,用不同浓度的EPA(0μM、25μM、50μM、100μM、200μM)作用于SMMC-7721肝癌细胞(24 h、48 h、72 h)后,显微镜下观察其形态学变化;应用MTT法检测SMMC-7721肝癌细胞细胞增殖变化情况;Western-blot法检测h TERT、Bax、Bcl-2蛋白表达水平变化;Real Time-PCR检测h TERTm RNA的表达变化;ELISA法检测SMMC-7721肝癌细胞端粒酶活性的表达水平。结果:EPA可诱导肝癌细胞SMMC-7721发生细胞凋亡,具有明显的时间计量依赖关系。在此过程中Bcl-2蛋白表达的降低和Bax蛋白表达上调,同时端粒酶逆转录酶h TERT蛋白及其m RNA的表达水平和端粒酶活性均明显降低。结论:抑制端粒酶逆转录酶基因(h TERTm RNA)表达而抑制端粒酶的活性、诱导癌细胞凋亡,可能是EPA的抗癌作用机制之一。     

抑癌基因p16对人肝癌细胞生长抑制的机制研究

目的：探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法：将p16cDNA亚克隆至pcDNA3．1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721：用MTT法和Western blot分析转染细胞的生长情况。结果：成功构建重组表达质粒pcDNA3．1-p16,转染pcDNA3．1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论：重组pcDNA3．1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

重楼皂苷Ⅰ对肝癌细胞的增殖及凋亡的影响

探讨重楼皂苷Ⅰ(paris saponinⅠ,PSⅠ)在体外对人肝癌SMMC-7721细胞株增殖和凋亡的影响及相关机制.MTT法检测PSⅠ对肝癌SMM-C7721细胞株的增殖抑制作用,Hoechst 33528染色法观察细胞核的形态学变化,流式细胞术Propidium iodide(PI)染色检测细胞周期及凋亡的情况,免疫印迹的方法检测Fas、Bcl-2、Bax、细胞周期素D1(cell cycle regulatory factor D1,Cyclin D1)和细胞周期素E(cell cycle regulatory factor E,Cy-clin E)的表达情况.PSⅠ能时间和浓度依赖性的抑制肝癌SMMC-7721细胞的增殖,与对照组比较,差异有统计学意义(P<0.05).干预后的SMMC-7721细胞染色质浓缩,核碎裂,凋亡小体形成,呈典型的凋亡变化.细胞周期阻滞于G1期,并且有凋亡峰形成,呈浓度依赖性.PSⅠ能浓度依赖性地上调Fas和Bax的表达,下调Bcl-2、Cyclin D1和Cyclin E蛋白的表达水平.PSⅠ可能是通过阻滞肿瘤细胞的生长及诱导细胞凋亡等机制,从而抑制肝癌细胞的增殖.     

过表达miR-455促进肝癌细胞诱导的管型形成

目的:构建miR-455前体的真核表达载体,探讨其对肝癌细胞诱导管型形成的影响。方法:采用PCR扩增miR-455前体序列,通过双酶切将其连接到真核表达载体pcDNA3中。连接产物转化入大肠杆菌内进行扩增,采用菌落PCR、双酶切和测序鉴定重组子。将构建的质粒转染SMMC-7721细胞,通过real-time PCR检测成熟miR-455的表达,采用ELISA方法检测培养液上清中VEGF的表达。用该上清处理人脐静脉内皮细胞HUVEC后,检测管型形成的情况。结果:本实验成功构建了miR-455前体的真核表达载体,将其瞬时转染SMMC-7721细胞后,real-time PCR检测结果显示miR-455表达水平显著升高(P0.01)。过表达miR-455后,SMMC-7721细胞上清VEGF表达水平呈时间依赖性升高(24 h P0.05,48 h和72 h P0.01)。人脐静脉内皮细胞分别用转染pcDNA3和pcDNA3-pre-455的肝癌细胞培养液上清(72 h)重悬,接种于基质胶Matrigel上,转染pcDNA3-pre-455组形成典型的微管,管样结构明显完整。结论:过表达miR-455可促进肝癌细胞诱导的微管形成。     

抗病毒固有免疫分子TRIM22 C端SPRY结构域对其转录、翻译及亚细胞定位的影响

本文旨在探讨TRIM22 C端SRPY结构域对其转录、翻译及亚细胞定位的影响。以野生型TRIM22真核表达质粒为模板,扩增出C端SPRY结构域缺失的TRIM22基因片段,并将其克隆入真核表达质粒pcDNA3.1。将野生型和突变型TRIM22真核表达载体分别转染高分化人肝癌细胞株HepG2。通过反转录-聚合酶链反应检测其mRNA表达水平,蛋白质免疫印迹法(Western blot)检测其蛋白表达水平,并通过间接免疫荧光法检测其亚细胞内定位。结果成功构建了C端SPRY结构域缺失的TRIM22真核表达质粒。转染HepG2细胞后,TRIM22 SPRY结构域缺失突变体在转录和翻译水平上均与野生型TRIM22无明显差异,但TRIM22 SPRY结构域缺失突变体完全丧失了核定位能力,这为进一步研究SPRY结构域在TRIM22抗病毒过程中所发挥的作用及机制提供了有益的启示。     

乙型肝炎病毒X基因真核表达载体的构建及表达

目的:构建含有天然完整的乙型肝炎病毒(HBV)X基因序列的真核表达载体,观察其在肝癌细胞株中的表达。方法:设计并合成HBV X基因的引物,用PCR方法从含完整HBV全基因的HepG2细胞中扩增X基因序列,并将其连接到真核表达载体pVAX-1上,酶切、PCR鉴定;用Triton X-114去除质粒内毒素后,采用电穿孔法将重组质粒pVAX-HBV X和空质粒pVAX-1分别转染SMMC-7721细胞,RT-PCR法检测HBV X基因mRNA的表达,Western印迹鉴定HBV X蛋白(HBx)的表达。结果:酶切和PCR鉴定证实pVAX-HBV X载体中包含完整的HBVX基因片段,该重组质粒转染的SMMC-7721细胞中HBV X基因mRNA及HBx蛋白的表达稳定。结论:构建了HBV X基因的真核表达载体,为X基因及其编码蛋白的生物学功能的研究提供了可靠的基因材料。     

The In Vivo Association of BiP with Newly Synthesized Proteins Is Dependent on the Rate and Stability of Folding and Not Simply on the Presence of Sequences That Can Bind to BiP

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.     

In vivo analysis of the lumenal binding protein (BiP) reveals multiple functions of its ATPase domain

The endoplasmic reticulum (ER) chaperone binding protein (BiP) binds exposed hydrophobic regions of misfolded proteins. Cycles of ATP hydrolysis and nucleotide exchange on the ATPase domain were shown to regulate the function of the ligand-binding domain in vitro. Here we show that ATPase mutants of BiP with defective ATP-hydrolysis (T46G) or ATP-binding (G235D) caused permanent association with a model ligand, but also interfered with the production of secretory, but not cytosolic, proteins in vivo. Furthermore, the negative effect of BiP(T46G) on secretory protein synthesis was rescued by increased levels of wild-type BiP, whereas the G235D mutation was dominant. Unexpectedly, expression of a mutant BiP with impaired ligand binding also interfered with secretory protein production. Although mutant BiP lacking its ATPase domain had no detrimental effect on ER function, expression of an isolated ATPase domain interfered with secretory protein synthesis. Interestingly, the inhibitory effect of the isolated ATPase was alleviated by the T46G mutation and aggravated by the G235D mutation. We propose that in addition to its role in ligand release, the ATPase domain can interact with other components of the protein translocation and folding machinery to influence secretory protein synthesis.     

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.     

Specific cellular proteins associate with angiotensin-converting enzyme and regulate its intracellular transport and cleavage-secretion

Angiotensin-converting enzyme (ACE) is an extensively glycosylated type I ectoprotein anchored in the plasma membrane by a hydrophobic transmembrane domain. In tissue culture as well as in vivo, the extracellular domain of ACE is released into the culture medium by a regulated proteolytic cleavage. To identify the cellular proteins that regulate ACE processing and cleavage-secretion, ACE-bound proteins were purified by affinity chromatography and characterized by microsequencing and Western blotting. One protein was identified as ribophorin and another as immunoglobulin-binding protein (BiP), a chaperone. Metabolic labeling and immunoprecipitation of ACE confirmed its interaction with BiP. Overexpression of BiP inhibited ACE secretion, an effect accentuated by the expression of an enzymatically inactive mutant BiP. This inhibition was caused by the retention of ACE precursors by BiP in the endoplasmic reticulum, as revealed by immunoprecipitation and immunofluorescence experiments. However, treatment with a phorbol ester, phorbol 12-myristate 13-acetate, enhanced ACE secretion even from cells overexpressing BiP. Western blot analysis of ACE-associated proteins with antibodies to protein kinase C (PKC) revealed the presence of its specific isozymes. Treatment with phorbol 12-myristate 13-acetate caused marked reduction in ACE association of selective PKC species. Thus, our studies have identified PKC and BiP as two proteins that directly interact with ACE and modulate its cell-surface expression and cleavage-secretion.     

In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.     

Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein

To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a panel of well characterized mutant G proteins and immunoprecipitation with anti-BiP antibodies to determine if BiP interacted with newly synthesized G protein and/or mutant G proteins retained in the endoplasmic reticulum. We made three major observations: 1) BiP bound transiently to folding intermediates of wild-type G protein which were incompletely disulfide-bonded; 2) BiP did not bind stably to all mutant G proteins which remain in the endoplasmic reticulum; and 3) BiP bound stably only to mutant G proteins which do not form correct intrachain disulfide bonds.     

The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae

We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae. A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP. This domain, purified as a fusion protein (63Jp) with glutathione S–transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP. The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein. GST alone was inactive in these assays. Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP. Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein. We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.     

ERdj3, a stress-inducible endoplasmic reticulum DnaJ homologue, serves as a cofactor for BiP's interactions with unfolded substrates

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.     

Interaction of murine BiP/GRP78 with the DnaJ homologue MTJ1

The activity of Hsp70 proteins is regulated by accessory proteins, among which the most studied are the members of the DnaJ-like protein family. BiP/GRP78 chaperones the translocation and maturation of secreted and membrane proteins in the endoplasmic reticulum. No DnaJ-like partner has been described so far to regulate the function of mammalian BiP/GRP78. We show here that murine BiP/GRP78 interacts with the lumenal J domain of the murine transmembrane protein MTJ1 (J-MTJ1). J-MTJ1 stimulates the ATPase activity of BiP/GRP78 at stoichiometric concentrations. The C-terminal tail of BiP/GRP78 is not required for the interaction with J-MTJ1, leaving the function of this portion of the molecule still unclear. Physical interactions between J-MTJ1 and BiP/GRP78 are stable and can be abolished by a single histidine --> glutamine substitution in the highly conserved HPD motif shared by all DnaJ-like proteins. The J-MTJ1 fragment, but not the mutant J-MTJ1:H89Q fragment, stimulates the ATPase activity of Escherichia coli DnaK, although at a higher concentration than its genuine partner DnaJ. Full-length DnaJ does not stimulate BiP over the range of concentrations investigated. These results indicate that the J domain of MTJ1 is sufficient for its interaction with BiP/GRP78 and cannot be substituted by E. coli DnaJ.