沙眼衣原体感染HeLa229细胞Bim表达及抑制凋亡研究

研究沙眼衣原体(D血清型)感染的HeLa229细胞中Bim蛋白质的表达及凋亡诱导剂作用后的凋亡情况。Western-blot检测沙眼衣原体感染和未感染的HeLa229细胞Bim蛋白质的表达水平。凋亡诱导剂etopo- side作用HeLa229细胞后,经Hoechst33258染色用荧光显微镜观察核浓缩和凋亡小体;流式细胞仪检测凋亡率。HeLa229细胞在未感染及感染沙眼衣原体6 h后可检测到Bim的表达;在感染24、48 h后均未检测到Bim的表达。经etoposide作用后,未感染的HeLa229细胞观察到明显的核浓缩和凋亡小体;流式细胞仪检测的凋亡率为90.64%。感染24 h的HeLa229细胞,未观察到核浓缩和凋亡小体;流式细胞仪检测的凋亡率为11.50%,与未感染的HeLa229细胞诱导后的凋亡率比较有统计学意义(P<0.05)。沙眼衣原体感染HeLa229细胞后可降低Bim蛋白质的表达;并能抑制etoposide诱导的细胞凋亡。     

CCCTC结合因子在HeLa细胞中调控X染色体连锁的凋亡抑制蛋白的表达

目的:确定HeLa细胞的CCCTC结合因子(CTCF)表达水平是否与细胞的抗凋亡能力相关,并研究其具体分子机制。方法:用顺铂和阿糖胞苷分别诱导HeLa细胞和CTCF敲降的HeLa-CTCF-II-11细胞凋亡,比较两者的凋亡率;用基因表达芯片检测CTCF敲降后HeLa细胞的表达谱变化,寻找并验证受CTCF调控的与凋亡相关的蛋白。结果:用顺铂和阿糖胞苷诱导后,HeLa-CTCF-II-11细胞的凋亡率显著高于HeLa细胞;HeLa细胞的CTCF敲降后,X染色体连锁的凋亡抑制蛋白(XIAP)的表达显著下降。结论:CTCF敲降使HeLa细胞的抗凋亡能力下降,CTCF对XIAP基因的表达调控在这个过程中起了重要作用。     

脂蟾毒配基诱导人肝癌细胞凋亡作用的研究

目的 通过体外脂蟾毒配基对人肝癌细胞（Bel7402）的凋亡诱导作用,为研究其抑制肿瘤细胞生长的作用机制提供依据。方法 通过应用流式细胞光度术检测细胞凋亡;采用细胞免疫细胞化学显色检测和Western blotting分析凋亡相关基因蛋白的表达来研究脂蟾毒配基对人肝癌细胞（Bel7402）凋亡的诱导作用。结果 表明脂蟾毒配基能够诱导Bel7402细胞发生凋亡,凋亡率大于50%;脂蟾毒配基提取液(浓度1.0 μm）作用于Bel7402细胞24小时后,bc1-2蛋白的表达下调,到48、72小时后下调更明显;而Bax蛋白的表达从24小时后开始上调,到48、72小时后表达上调明显。使用方差分析法与对照组相比较,P<0.05 ,统计学有显著意义。结论 提示诱导肿瘤细胞凋亡可能是脂蟾毒配基抑制、杀伤人肝癌细胞的机制之一。     

siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

天花粉蛋白对宫颈癌HeLa细胞TSLC1基因甲基化及其表达的影响

目的：检测人宫颈癌HeLa细胞中TSLC1基因甲基化的状况,研究在人宫颈癌HeLa细胞凋亡过程中TSLC1基因甲基化的变化情况,探讨肿瘤细胞凋亡与抑癌基因甲基化的相关性,并进一步证实天花粉蛋白（TCS）去甲基化作用是否存在普遍性,以促进天花粉蛋白的临床应用。方法：应用甲基化特异性PCR（MSP）法检测人宫颈癌HeLa细胞及其凋亡过程中TSLC1基因甲基化的状况;采用实时定量RT-PCR技术检测TCS处理前、后HeLa细胞TSLC1基因表达的变化。结果：肿瘤抑制基因TSLC1在人宫颈癌HeLa细胞中呈高度甲基化状态,经40μg／mL TCS处理48h后,TSLC1基因甲基化程度明显降低;RT-PCR检测结果显示,TCS处理组HeLa细胞中TSLC1 mRNA的表达量高于未处理组,提示TSLC1基因启动子区CpG岛甲基化是导致其低表达的重要机制。结论：肿瘤抑制基因TSLC1启动子甲基化在人宫颈癌癌变过程中可能是一种重要的分子调控机制;人宫颈癌HeLa细胞凋亡与抑癌基因的去甲基化之间可能存在某些密切的相关性;TCS对肿瘤抑制基因TSLC1有一定的去甲基化作用。     

肉桂醛抗人宫颈癌相关机制的研究

目的探讨不同浓度的肉桂醛对HeLa细胞P21、CDK4蛋白表达的影响及意义。方法不同浓度的经过纯度鉴定的肉桂醛处理体外培养的HeLa细胞,培养24 h后免疫组织化学和Western blotting法检测HeLa细胞P21、CDK4蛋白表达的变化。结果肉桂醛纯度〉96.24%;肉桂醛能显著增高P21和降低CDK4蛋白在HeLa细胞中的表达,各浓度肉桂醛处理组的P21、CDK4蛋白表达与溶剂对照组相比差异均有统计学意义（P〈0.01,P〈0.05）。结论肉桂醛能上调宫颈癌HeLa细胞P21蛋白表达和下调CDK4蛋白表达,可能是促进HeLa细胞凋亡的机制之一。     

FAS/ActD通过线粒体途径引起细胞阻滞诱导人宫颈癌HeLa细胞凋亡

为了探讨FAS抗体与放线菌素D(actinomycin D,ActD)联合作用诱导人宫颈癌HeLa细胞凋亡的分子机制,通过MTT法检测细胞活力,利用流式细胞仪检测细胞凋亡和细胞周期,从而研究FAS/ActD抑制细胞增殖的作用. 结果表明,FAS/ActD能明显降低HeLa细胞的活力,并且通过G1/G0期阻滞和S期阻滞诱导HeLa细胞凋亡. 此外,Western印迹分析进一步显示,FAS/ActD还能引起Bcl-2蛋白表达降低, Bax蛋白表达增加,Bid蛋白发生断裂激活,导致细胞质中Cyto-c释放的增加,并激活在细胞凋亡的执行过程中起着关键作用的caspase 9和caspase 3. 以上结果提示,FAS抗体与ActD的联合作用可能经线粒体途径引起细胞周期阻滞,从而诱导HeLa细胞凋亡. 该研究为宫颈癌的免疫治疗提供了新的思路.     

DNA损伤诱导的长链非编码RNA-UCA1促进HeLa细胞增殖

尿路上皮癌抗原1 (UCA1)是一种长链非编码RNA,在多种肿瘤内高表达.然而,其在宫颈癌细胞和组织中的表达报告颇不一致,且功能尚未确定.本文探索UCA1在宫颈癌HeLa细胞中的生物学功能.实时定量PCR（qRT-PCR）结果显示,UCA1、p21和p53 mRNA在阿霉素（doxorubicin,DOX）或γ射线照射的HeLa细胞中表达上调|相反,敲减p53表达则可抑制DOX诱导的UCA1上调.表明DNA损伤诱导的UCA1可能与p53有关.转染结合CCK8检测HeLa细胞增殖活力结果显示,与对照比较,过表达UCA1促进HeLa细胞增殖,干扰UCA1表达则减缓细胞增殖.此外,流式细胞术结果显示,过表达UCA1导致阿霉素诱导的凋亡率下降;siRNA抑制UCA1表达后引起细胞G2/M期比例上升,S期下降,且阿霉素诱导的细胞凋亡率上升.上述结果说明,DNA损伤诱导的UCA1可促进HeLa细胞增殖,减少细胞凋亡.然而,是否DNA损伤诱导的UCA1上调依赖p53尚需进一步实验证明.     

野生型rLj-RGD3基因重组突变体rLj-112分子克隆及对HeLa细胞活性影响

为研究突变体rLj-112蛋白的抗肿瘤活性,人工合成七鳃鳗野生型rLj-RGD3蛋白和突变型rLj-112蛋白,通过比较两种蛋白质的抗增殖、迁移和促凋亡的活性,确定突变体rLj-112蛋白的生物学意义及地位。采用MTT方法检测不同浓度的rLj-112蛋白对HeLa细胞增殖的抑制作用。结果表明,rLj-112蛋白能显著抑制HeLa细胞的增殖,且IC50为4.3 μmol/L。使用Transwell细胞培养板对bFGF诱导的HeLa细胞迁移实验表明,rLj-112蛋白能抑制HeLa细胞的迁移。rLj-112蛋白作用后,HeLa细胞经Hoechst33258和AnnexinV-FITC染色结果显示,细胞均凋亡。流式细胞仪进一步证明,rLj-112蛋白能诱导HeLa细胞发生凋亡,且呈剂量依赖性。由此可见,与野生型rLj-RGD3蛋白比较,突变型rLj-112蛋白有较高的细胞毒性作用,具有抗肿瘤的功能,有望应用于抗肿瘤基因工程药物的开发,具有重要的生物学意义。     

靶向IRE1a干扰质粒构建及对ERS时细胞增殖及凋亡的影响

目的构建靶向人IRE1a的shRNA干扰质粒（pSUPER-IRE1a）并观察其对人HeLa细胞和HepG2细胞增殖及凋亡的影响。方法设计并合成靶向IRE1a基因的两条shRNA,分别克隆至真核表达载体pSUPER构建重组质粒pS1、pS2,依次转染入HeLa细胞和HepG2细胞中。采用RT—PCR检测pS1、pS2转染前后IRE1a在HeLa细胞和HepG2细胞中的mRNA水平,免疫印迹检测pS1、pS2转染前后IRE1a蛋白的表达;MTT比色法、BrdU／DAPI双免疫荧光法及流式细胞仪分别检测各重组质粒对HeLa细胞和HepG2细胞增殖及凋亡的影响。结果干扰质粒（pSUPER-IRE1a）能有效抑制HeLa细胞和HepG2细胞中IRE1a基因的表达;成功转染后,细胞处于内质网应激（ER stress）状态时,各实验组细胞增殖率及凋亡率与对照组比较,差异均具有统计学意义P〈0．05）。结论成功构建靶向人IRE1a的shRNA真核表达载体pS1、pS2,有效抑制了HeLa细胞和HepG2细胞中IRE1a的表达;细胞处于ERS状态时,IRE1a-shRNA有效促进HeLa、HepG2两种肿瘤细胞的增殖;抑制HeLa和HepG2细胞的凋亡。     

Isolation of recombinant interleukin-3 produced by E. coli]

A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer. Under constitutive biosynthesis conditions in E. coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein. An effective procedure of the hIL3 isolation is suggested. The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step. The protein's yield was 34 mg/g wet cells. The isolated hIL3 showed a specific biological activity.     

人白细胞介素－3基因翻译起始区的改造提高其在大肠杆菌中的表达水平

为了提高人白细胞介素－3(bhIL-3)在大肠杆菌中的表达,在计算机辅助下,设计合成了PCR突变引物,用于改造起始密码AUG上下游序列,并在不改变5’端氨基酸编码的前提下,尽可能选用大肠杆菌高频使用的密码子。将经改造后的Hil-3cDNA和翻译起始区置于PL启动子之下,转入大肠杆菌Tapl06,经42℃热诱导后．获得表达产物,提高表达水平近一倍,表达量达到菌体总蛋白量的30％左右。表达产物经Western blot验证,经PVDF膜转移后进行N端顺序分析,证明前15个氨基酸正确,产物经包涵体纯化后,纯度提高至80％以上,初步复性后能明显促进Hil－3依赖细胞的生长。     

人白细胞介素18的基因克隆与表达

人白细胞介素１８（ＩＬ－１８）是新近发现的细胞因子之一．研究表明它参与Ｔ１辅助细胞介导的细胞免疫．利用ＲＴ－ＰＣＲ技术从人外周血细胞扩增得到了ＩＬ－１８的ｃＤＮＡ并测定其核酸序列．利用基因重组技术构建ＩＬ－１８的表达载体,并在大肠杆菌中进行了表达．这为以后进一步研究ＩＬ－１８的功能奠定了基础．     

Recombinant protein yield in rice seed is enhanced by specific suppression of endogenous seed proteins at the same deposit site

Human IL‐10 (hIL‐10) is a therapeutic treatment candidate for inflammatory allergy and autoimmune diseases. Rice seed‐produced IL‐10 can be effectively delivered directly to gut‐associated lymphoreticular tissue (GALT) via bio‐encapsulation. Previously, the codon‐optimized hIL‐10 gene was expressed in transgenic rice with the signal peptide and endoplasmic reticulum (ER) retention signal (KDEL) at its 5′ and 3′ ends, respectively, under the control of the endosperm‐specific glutelin GluB‐1 promoter. The resulting purified hIL‐10 was biologically active. In this study, the yield of hIL‐10 in transgenic rice seed was improved. This protein accumulated at the intended deposition sites, which had been made vacant through the selective reduction, via RNA interference, of the endogenous seed storage proteins prolamins or glutelins. Upon suppression of prolamins that were sequestered into ER‐derived protein bodies (PB‐I), hIL‐10 accumulation increased approximately 3‐fold as compared to rice seed with no such suppression and reached 219 μg/grain. In contrast, reducing the majority of the glutelins stored in protein‐storage vacuoles (PB‐II) did not significantly affect the accumulation of hIL‐10. Considering that hIL‐10 is synthesized in the ER lumen and subsequently buds off in ER‐derived granules called IL‐10 granules in a manner similar to PB‐Is, these results indicate that increases in the available deposition space for the desired recombinant proteins may be crucial for improvements in yield. Furthermore, efficient dimeric intermolecular formation of hIL‐10 by inhibiting interaction with Cys‐rich prolamins also contributed to the enhanced formation of IL‐10 bodies. Higher yield of hIL‐10 produced in rice seeds is expected to have broad application in the future.     

Production of an active anti‐CD20‐hIL‐2 immunocytokine in Nicotiana benthamiana

Anti‐CD20 murine or chimeric antibodies (Abs) have been used to treat non‐Hodgkin lymphomas (NHLs) and other diseases characterized by overactive or dysfunctional B cells. Anti‐CD20 Abs demonstrated to be effective in inducing regression of B‐cell lymphomas, although in many cases patients relapse following treatment. A promising approach to improve the outcome of mAb therapy is the use of anti‐CD20 antibodies to deliver cytokines to the tumour microenvironment. In particular, IL‐2‐based immunocytokines have shown enhanced antitumour activity in several preclinical studies. Here, we report on the engineering of an anti‐CD20‐human interleukin‐2 (hIL‐2) immunocytokine (2B8‐Fc‐hIL2) based on the C2B8 mAb (Rituximab) and the resulting ectopic expression in Nicotiana benthamiana. The scFv‐Fc‐engineered immunocytokine is fully assembled in plants with minor degradation products as assessed by SDS‐PAGE and gel filtration. Purification yields using protein‐A affinity chromatography were in the range of 15–20 mg/kg of fresh leaf weight (FW). Glycopeptide analysis confirmed the presence of a highly homogeneous plant‐type glycosylation. 2B8‐Fc‐hIL2 and the cognate 2B8‐Fc antibody, devoid of hIL‐2, were assayed by flow cytometry on Daudi cells revealing a CD20 binding activity comparable to that of Rituximab and were effective in eliciting antibody‐dependent cell‐mediated cytotoxicity of human PBMC versus Daudi cells, demonstrating their functional integrity. In 2B8‐Fc‐hIL2, IL‐2 accessibility and biological activity were verified by flow cytometry and cell proliferation assay. To our knowledge, this is the first example of a recombinant immunocytokine based on the therapeutic Rituximab antibody scaffold, whose expression in plants may be a valuable tool for NHLs treatment.     

Expression, purification and characterization of recombinant human angiogenin in Pichia pastoris

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.     

重组可溶性TRAIL的表达与生物学活性

研究重组可溶性TRAIL的抗肿瘤生物学活性。采用基因分子生物学方法构建重组可溶性TRAIL的表达载体,建立大肠杆菌表达菌株,采用柱层析等方法获得纯化的重组可溶性TRAIL;采用流式细胞术、细胞活性测定法和体内药效学实验分析重组可溶性TRAIL杀伤肿瘤细胞的生物学活性。结果表明重组可溶性TRAIL在体外可诱导人白血病细胞和肝癌细胞凋亡,凋亡率达50%以上。中枢神经系统的肿瘤细胞对重组可溶性TRAIL不敏感。重组可溶性TRAIL在体内能显著抑制人非小细胞肺癌细胞在小鼠体内生长,抑制率达70%以上。结论为研究制备的重组可溶性TRAIL能在体内外杀死多种肿瘤细胞,具有显著的临床应用前景。     

白介素24的抗肿瘤机理

白介素24(interleukin 24,IL-24)是利用消减杂交技术,从重组的纤维母细胞干扰素和密执毒素共同作用的黑色素瘤细胞株中筛选得到的一种高表达基因。由于IL-24能选择性地诱导肿瘤细胞凋亡,而对正常细胞没有细胞毒作用,因此在肿瘤治疗研究中受到人们广泛的重视。IL-24诱导肿瘤细胞凋亡的机理涉及多种信号途径,有些信号途径目前还不十分清楚。本文就IL-24通过启动不同的信号途径,诱导广泛的肿瘤细胞凋亡的机理作一综述,从而为IL-24抗肿瘤机理研究提供一些有用的信息。     

Expression,Purification and Characterization of Recombinant Human Angiogenin in Pichia pastoris

The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.