依地福新抗spm1重组酵母生长作用研究

目的将spm1基因再转染至相应的粟酒裂殖酵母突变体spm1Δ中,观察依地福新对其生长的抵抗作用。方法抽提野生型粟酒裂殖酵母细胞的RNA;RT PCR制备粟酒裂殖酵母细胞的cDNA; PCR扩增spm1基因;将spm1基因克隆到pREP3X HA质粒中构建pREP3X HA spm1穿梭载体,电转化穿梭载体到spm1Δ细胞中;pREP3X HA spm1鉴定重组酵母及依地福新对其生长的抵抗作用。结果在35个酵母转化单菌落中,筛选到一个有表达活性的pREP3X HA spm1重组子,且重组子对依地福新有抵抗作用。结论依地福新的抗肿瘤作用可能与spm1基因有关。     

TOPO克隆构建SARS-CoV M蛋白基因裂殖酵母表达载体及其稳定性

目的 利用TOPO克隆法构建SARS-CoV M蛋白基因的裂殖酵母重组表达载体，并验证重组载体在宿主细胞中的稳定性。方法 运用逆转录-聚合酶链反应（RT-PCR）技术从SARS-CoV RNA中扩增出M蛋白基因,AT克隆构建出序列正确的pMD18-T-M重组载体。设计含Kozak序列的引物从pMD18-T-M载体上亚克隆出M蛋白基因，与裂殖酵母表达载体pNMT1-TOPO进行TOPO克隆,构建出重组表达载体pNMT1-M,转化TOP10感受态细胞，菌落PCR鉴定阳性转化子后进行测序鉴定。将序列正确的pNMT1-M重组载体电转化入裂殖酵母TCP1菌株中,在EMM培养基中诱导表达,连续传代100代，在EMM+T培养基中验证其稳定性。结果 RT-PCR获得666 bp的片段,pMD18-T-M重组载体经测序验证序列正确；重组表达载体pNMT1-M经菌落PCR和测序鉴定均正确;重组裂殖酵母菌经诱导后，SDS-PAGE检测出了表达条带;重组表达载体连续传代后,未发现丢失现象。结论 成功地构建出了SARS-CoV M蛋白基因的裂殖酵母表达载体，验证了其在裂殖酵母中能稳定地进行遗传,为下一步的表达优化、活性和功能研究垫定了基础.     

依地福新诱导异源表达人凋亡基因的酿酒酵母细胞的凋亡

酵母作为一种容易进行操作的生物模型,广泛应用于异基因表达和结构与功能的研究中[1]。尽管它们可能缺乏某些与高等生物相似的凋亡调节因子的同功异构体,但在某些生理情况下(如突变体、乙酸处理等)或在异源表达哺乳动物促凋亡基因时,可观察到细胞的凋亡。依地福新是目前所发现的第一个通过激活细胞内Fas/CD95死亡受体而起作用的抗癌药物,其抗癌机制涉及到膜阀(membrane raft)介导的过程[2]。在本实验中笔者将Fas、FADD、caspase-8酶原转入到酿酒酵母(Saccharomyces cerevi-siae,S cercvisiae)细胞中,然后用依地福新进行处理,以确定依地福新是否能激活Fas/FADD/caspase-8这一凋亡通路,从而进一步阐明依地福新促细胞凋亡的机制。1资料与方法1.1试验菌株试验中使用的酿酒酵母菌株为:BY4742,MAThis3Δleu2Δlys2Δura3Δ。pGILDA质粒和大肠埃希菌活化态细胞DH5α,均购自于Invitrogen Life Techologies(USA)。1.2主要试剂依地福新由华北生物制药公司(合肥,河北)生产;酵母膏(Yeast Extract...     

N-糖基化IL-24的表达及体外诱导肿瘤细胞凋亡的研究

目的构建IL-24基因的真核表达载体,在毕赤酵母GS115中高效表达,研究重组N-糖基化IL-24蛋白体外诱导肿瘤细胞凋亡的活性。方法借助过渡质粒α/pUC18,将IL-24基因插入到质粒pPIC9K的BamHⅠ和EcoRⅠ之间,构建重组质粒IL-24/pPIC9K,转化毕赤酵母GS115分泌表达,Tricine-SDS-PAGE和Western blot鉴定目的蛋白,ELISA检测蛋白表达量,糖苷酶PNGaseF分析IL-24糖基化形式和程度。MTT法和形态学分析重组IL-24诱导MCF-7乳腺癌细胞凋亡的活性。结果成功构建重组表达质粒IL-24/pPIC9K,IL-24在毕赤酵母最高表达量为(81.31±14.46)mg·L-1。约70%的IL-24发生了N-糖基化。重组IL-24诱导MCF-7乳腺癌细胞凋亡,对正常人肺成纤维细胞NHLF没有影响。N-糖基化IL-24对MCF-7抑制率约高于去糖基化IL-24。结论毕赤酵母分泌形式的表达和适度的糖基化修饰都有利于目的蛋白IL-24的生物学活性,为后续的研究提供基础。     

人IL-24基因在CHO细胞中的表达及其抗肿瘤效应

目的构建人IL-24基因真核表达载体,在CHO细胞中进行稳定表达,并检测重组表达蛋白rhIL-24的抗肿瘤活性。方法将测序验证的人IL-24基因亚克隆至真核表达载体pcDNA3,构建重组真核表达载体pcDNA3-hIL-24,转染CHO细胞进行稳定表达,经RT-PCR鉴定后用MTT法、Ho-echst染色和流式细胞术检测CHO细胞表达的rhIL-24诱导A549人肺腺癌细胞凋亡的抗肿瘤效应,用ELISA检测其刺激免疫细胞分泌IL-6和IFN-γ的功能。结果经双酶切和PCR鉴定,重组真核表达载体构建正确,人IL-24在CHO细胞中获得稳定表达,且所表达的人IL-24具有较强的诱导A549人肺腺癌细胞凋亡及上调免疫细胞表达IL-6和IFN-γ的免疫刺激活性。结论人IL-24基因的稳定表达和抗肿瘤效应的实验研究,为进一步研究人IL-24抗肿瘤的分子机制及潜在的应用奠定了基础。     

人源抗HBsAg单链抗体与α干扰素融合蛋白酵母表达载体的构建

目的构建以人HBsAg单链抗体(HBScFv)为导向作用、α干扰素为治疗作用的融合蛋白酵母表达载体pPICZ-αB/ScFv-IFN-α。方法通过重叠PCR构建目的蛋白质基因,再亚克隆到酵母表达载体pPICZαB中,DNA测序后,转化巴氏毕赤酵母宿主菌GS115 ,菌落PCR、高浓度Zeocin抗性筛选鉴定重组酵母,重组酵母经甲醇诱导后,通过SDS PAGE分析鉴定表达产物。结果DNA测序结果显示构建的目的基因片段(HBScFv IFN-α)的序列与原设计序列相符合;菌落PCR结果显示目的基因已整合到酵母菌中;诱导表达后,SDS PAGE结果显示在相对分子质量约4 4 0 0 0处可见目的蛋白质表达带。结论成功构建了抗HBsAg单链抗体与α干扰素融合蛋白酵母表达载体     

T4溶菌酶在毕赤酵母中的诱导表达及抑菌活性测定

目的利用毕赤酵母诱导表达T4溶菌酶蛋白并测定其抗菌活性。方法T4溶菌酶（T4lysozyme）基因以N端融合的方式被准确插入到质粒pPIC9K的EcoR I-Not I位点内,得到分泌型重组表达载体 pPIC9K-T4L。该载体首先经限制性内切酶Sal I酶切,进而采用电击方法将线性化的重组质粒DNA导入到毕赤酵母中。经过梯度筛选得到多个单拷贝和多拷贝转化重组子。随机挑取部分重组子PCR扩增阳性克隆,经菌体培养和甲醇诱导后获得了分泌表达,表达产物存在于培养上清液中。结果表达蛋白经琼脂孔扩散抗菌实验显示抑菌圈明显;重组蛋白对金黄色葡萄球菌与肺炎链球菌均具有显著抑制作用;多拷贝与单拷贝重组表达子没有抗菌活性差异与蛋白表达量的差异;加热煮沸对于T4溶菌酶蛋白的抗菌活性无明显影响。结论T4溶菌酶在毕赤酵母中得以成功诱导与表达;表达产物不受拷贝数影响并具热稳定性。     

重组人可溶性TRAIL在毕赤酵母中表达与纯化及其抗肿瘤细胞活性

目的研究重组人可溶性TRAIL(sTRAIL)蛋白的表达和纯化,以及对HepG2细胞增殖与凋亡的影响。方法将人sTRAIL基因插入毕赤酵母表达载体pPIC9中,构建甲醇酵母分泌型表达载体pPIC9-sTRAIL,电击转化至GS115(his4),MD平板筛选出阳性菌株。对工程菌株发酵培养基成分、pH、甲醇浓度、诱导表达时间等条件进行优化,并初步分析重组sTRAIL蛋白的体外抗肿瘤活性。结果工程菌株在BMMY培养基(pH6.0)、1%甲醇条件下诱导表达48h目的蛋白浓度最高可达(58.7±2.4)mg/L。重组人sTRAIL蛋白能抑制HepG2细胞的增殖、诱导HepG2细胞的凋亡。结论优化了人sTRAIL的表达和纯化工艺条件,所生产的sTRAIL可用于抗肝癌新药的开发。     

核糖体蛋白RPS3a腺病毒载体的构建和体外表达

目的 构建核糖体蛋白RPS3a的腺病毒表达载体,并进一步验证其在细胞中的表达。方法 应用RT-PCR从人胚肾HEK293细胞的总RNA中反转录并扩增出RPS3a基因,并将RPS3a亚克隆到穿梭质粒pAdTrack-CMV中,酶切及测序鉴定后,再将含RPS3a基因的重组穿梭质粒pAdTrack-CMV-RPS3a进行Pme I酶线性化处理,转化到含有骨架质粒的BJ5183感受态菌,进行同源重组。鉴定后,经Pac I线性化转染HEK293细胞,包装重组腺病毒。病毒感染NIH3T3细胞,Western blot 分析RPS3a蛋白的表达。结果 证实pAdTrack-CMV-RPS3a及pAdEas-RPS3a质粒构建正确;Western blot检测病毒感染的NIH3T3细胞总蛋白,与pAd-GFP阴性对照组相比,RPS3a蛋白表达量显著提高。结论 成功构建了能表达RPS3a基因的重组腺病毒载体,为进一步研究其生物学功能奠定基础。     

5-氟尿嘧啶诱导Jurkat细胞凋亡及Fas、FasL的表达

目的探讨5-氟尿嘧啶（5-FU）诱导Jurkat细胞株的凋亡和Fas、FasL的表达。方法不同浓度的5-FU分别处理Jurkat细胞12h和24h后,流式细胞仪检测细胞表达CD95、CD95L的百分率。结果与对照组相比,5-FU在10、20、40μg.mL-1浓度下明显促进Jurkat细胞表达Fas、FasL;且5-FU在24h比处理12h有更强的诱导效果。结论 5-FU可显著提高Jurkat细胞凋亡率,并增强Fas、FasL的表达率。     

Mitochondrial-derived ROS in edelfosine-induced apoptosis in yeasts and tumor cells

Aim： To investigate whether a similar process mediates cytotoxicity of 1-O- octadecyl-2-O-methyl-rac-glycero-3-phosphocholine （ET- 18-OCH3, edelfosine） in both yeasts and human tumor cells. Methods： A modified version of a previously described assay for the intracellular conversion of nitro blue tetrazolium to formazan by superoxide anion was used to measure the generation of reactive oxygen species （ROS）. Apoptotic yeast cells were detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay. DNA fragmentation and the generation of ROS were measured by cytofluorimetric analysis in Jurkat cells. Results： Edelfosine induced apoptosis in Saccharomyces cerevisiae, as assessed by TUNEL assay. Meanwhile, edelfosine induced a time- and concentration-dependent generation of ROS in yeasts. Rotenone, an inhibitor of the mitochondrial electron transport chain, prevented ROS generation and apoptosis in response to edelfosine in S cerevisiae. α-Tocopherol abrogated the edelfosineinduced generation of intracellular ROS and apoptosis. Edelfosine also induced an increase of ROS in human leukemic ceils that preceded apoptosis. The overexpression of Bcl-2 by gene transfer abrogated both ROS generation and apoptosis induced by edelfosine in leukemic cells. Changes in the relative mito- chondrial membrane potential were detected in both yeasts and Jurkat cells. Conclusion： These results indicate that edelfosine induces apoptosis in yeasts in addition to human tumor cells, and this apoptotic process involves mitochondria, likely through mitochondrial-derived ROS. These data also suggest that yeasts can be used as a suitable cell model in elucidating the antitumor mechanism of action of edelfosine.     

Edelfosine抑制S.pombe细胞分裂作用机制的研究

目的研究edelfosine(ET-18-OCH3,依地福新)抑制schizosaccharom yces pombe(S.pombe,粟酒裂殖酵母)细胞分裂的作用机制。方法应用胞质分裂抑制试验,平行生长抑制试验,观察edelfosine对S.pombe细胞分裂和生长的抑制作用;应用酵母基因再转染试验,探索其作用机制。结果edelfosine在低剂量浓度时,抑制S.pombe细胞分裂;高剂量时抑制其生长。平行生长抑制试验显示,删除了mid2、spm1和pmp1基因的细胞株(mid2△、spm1△和pmp1△),对高剂量edelfosine有抵抗作用;再转染了相应的基因后,细胞重新恢复了对edelfosine的敏感性。Spm1、Mid2和Pmp1相互作用的试验研究显示,Mid2介导磷酸化Spm1的生成,而Pmp1抑制磷酸化Spm1的生成。结论edelfosine可能通过影响Spm1、Mid2和Pmp1蛋白磷酸化而产生胞质抑制和生长抑制作用。     

Lipid rafts and Fas/CD95 signaling in cancer chemotherapy

Cholesterol- and sphingolipid-rich membrane domains, termed lipid rafts, have been recently involved in the triggering of death receptor-mediated apoptosis. The alkyl-lysophospholipid analogue edelfosine was the first antitumor drug reported to induce apoptosis in cancer cells through co-clustering of lipid rafts and Fas/CD95 death receptor. Recruitment and aggregation of Fas/CD95 in lipid raft clusters was independent of its cognate ligand FasL/CD95L, and could be pharmacologically modulated. The adaptor molecule Fas-associated death domain protein (FADD) and procaspase-8 were also recruited into lipid rafts following edelfosine treatment, forming the death-inducing signaling complex (DISC), and hence these membrane microdomains can act as scaffolds for Fas/CD95 death signaling. Edelfosine accumulated in lipid rafts of cancer cells, altering raft protein and lipid composition. Subsequently, an increasing number of antitumor drugs have been found to induce apoptosis through recruitment of Fas/CD95 into membrane rafts, and some of these compounds accumulated in raft membrane domains. Additional downstream apoptotic signaling molecules have also been reported to be recruited into rafts following treatment of cancer cells with antitumor agents, thus facilitating protein-protein interactions and conveying apoptotic signals. On these grounds, lipid rafts have become an appealing and promising target for therapeutic intervention in cancer chemotherapy. Co-clustering of lipid rafts and Fas/CD95 signaling provides a new insight in the regulation of death receptor-mediated apoptosis, opening a new avenue in cancer therapy. In this regard, an increasing number of patents are dealing with the above insights in order to improve cancer treatment.     

Effects of specific antisense oligonucleotide inhibition of Fas expression on T cell apoptosis induced by Fas ligand

To investigate the effect of specific antisense oligodeoxynucleotide (ASODN) inhibition of Fas expression on T cell apoptosis induced by hepatocarcinoma cells. Fas receptor (Fas) and Fas ligand (FasL) expressed by the hepatocarcinoma cell line HepG2.2.15 and Jurkat T cells were detected by flow cytometry (FCM) and the ability of FasL-inducing T cell apoptosis was tested by co-culture assay in vitro with HepG2.2.15 cells and Jurkat T cells. The Jurkat cells were transfected with Fas-ASODN using lipofectin, and the effects of Fas-ASODN on Fas mRNA level, Fas expression on T cells surface, and apoptosis were investigated by RT-PCR, FCM and co-culture assay, respectively. It was found that HepG2.2.15 cells expressing functional FasL could induce the apoptosis of Jurkat cells as demonstrated by co-culture assays. After the Jurkat cells were transfected with Fas ASODN, the level of Fas mRNA, the expression rate of Fas and the apoptotic rate induced by hepatocarcinoma cells were all decreased. As a conclusion, it is evident that hepatocarcinoma cells expressing FasL can induce apoptosis in Fas-expressing T cells, indicating that transfection of Fas ASODN can partially convert the immune inhibitory condition induced by hepatocarcinoma cells.     

Fluorescent phenylpolyene analogues of the ether phospholipid edelfosine for the selective labeling of cancer cells

Edelfosine (ET-18-OCH3), a synthetic antitumor ether lipid, is taken up by malignant but not by normal cells, triggering apoptosis in a large variety of human tumor cells. The synthesis of the first fluorescent edelfosine analogue (6), with apoptotic activity comparable to that of the parent drug, is described. Fluorescence microscopy experiments show that 6 selectively labels human cancer cells, accumulating into specific domains of the plasma membrane.     

依地福新对Hela细胞生长的抑制作用及其机制研究

目的:观察依地福新对人宫颈癌Hela细胞的体外生长抑制作用,并进一步探讨其作用机制。方法:细胞中分别加入不同浓度的依地福新(0.5、1.0、5.0、10.0μmol.L-1)处理96h,并设立未加依地福新的对照组。应用MTT法检测细胞增殖活性;流式细胞仪检测细胞周期分布;染色法检测细胞凋亡率。结果:与对照组比较,不同浓度依地福新处理Hela细胞24～96h后,细胞增殖显著受到抑制,并呈浓度及时间依赖性;1.0、5.0、10.0μmol.L-1浓度依地福新处理72h后,Hela细胞G0/G1期细胞数量显著增加,S期细胞数量显著降低(P<0.01);各浓度组细胞凋亡率均显著增加(P<0.01)。结论:依地福新对Hela细胞具有生长抑制作用,其机制可能与阻滞细胞周期及诱导凋亡有关。     

Evidence that genetic deletion of the TNF receptor p60 or p80 inhibits Fas mediated apoptosis in macrophages

Almost 19 members of the tumor necrosis factor (TNF) superfamily have been identified that interact with 29 different receptors. Whether these receptors communicate with each other is not understood. Recently, we have shown that receptor activator of NF-kappaB ligand signaling is modulated by genetic deletion of the TNF receptor. In the current report, we investigated the possibility of a cross-talk between Fas and TNF-alpha signaling pathway in macrophage cell lines derived from wild-type (WT) mice and from mice with genetic deletion of the type 1 TNF receptor (p60(-/-)), the type 2 TNF receptor (p80(-/-)), or both receptors (p60(-/-)p80(-/-)). We found that the macrophages expressing TNF receptors were highly sensitive to apoptosis induced by anti-Fas. The genetic deletion of TNF receptors, however, made the cells resistance to anti-Fas-induced apoptosis. Anti-Fas induced activation of caspase-3 and PARP cleavage in WT cells but not in TNF receptor-deleted cells. This difference was found to be independent of the expression of Fas, Fas-associated protein with death domain (FADD) or TNF receptor-associated death domain (TRADD). We found that anti-Fas induced recruitment of TNFR1 into Fas-complex. We also found that TRADD, which mediates TNF signaling, was constitutively bound to Fas receptor in TNF receptor-deleted cells but not in wild-type cells. Transient transfection of TNFR1 in TNFR1-deleted cells sensitized them to anti-Fas-induced apoptosis. Overall our results demonstrate that Fas signaling is modulated by the TNF receptors and thus provide the evidence of cross-talk between the receptors of two cytokines.     

The SK3/K(Ca)2.3 potassium channel is a new cellular target for edelfosine

BACKGROUND AND PURPOSE

The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (edelfosine) is an ether-linked phospholipid with promising anti-cancer properties but some side effects that preclude its full clinical therapeutic exploitation. We hypothesized that this lipid could interact with plasma membrane ion channels and modulate their function.

EXPERIMENTAL APPROACH

Using cell migration-proliferation assays, patch clamp, spectrofluorimetry and ¹²⁵I-Apamin binding experiments, we studied the effects of edelfosine on the migration of breast cancer MDA-MB-435s cells, mediated by the small conductance Ca²⁺-activated K⁺ channel, SK3/K_Ca2.3.

KEY RESULTS

Edelfosine (1 µM) caused plasma membrane depolarization by substantially inhibiting activity of SK3/K_Ca2.3 channels, which we had previously demonstrated to play an important role in cancer cell migration. Edelfosine did not inhibit ¹²⁵I-Apamin binding to this SK_Ca channel; rather, it reduced the calcium sensitivity of SK3/K_Ca2.3 channel and dramatically decreased intracellular Ca²⁺ concentration, probably by insertion in the plasma membrane, as suggested by proteinase K experiments. Edelfosine reduced cell migration to the same extent as known SK_Ca channel blockers. In contrast, K+ channel openers prevented edelfosine-induced anti-migratory effects. SK3 protein knockdown decreased cell migration and totally abolished the effect of edelfosine on MDA-MB-435s cell migration. In contrast, transient expression of SK3/K_Ca2.3 protein in a SK3/K_Ca2.3-deficient cell line increased cell migration and made these cells responsive to edelfosine.

CONCLUSIONS AND IMPLICATIONS

Our data clearly establish edelfosine as an inhibitor of cancer cell migration by acting on SK3/K_Ca2.3 channels and provide insights into the future development of a new class of migration-targeted, anti-cancer agents.