EFFECT OF TNF-a AND IFN-g ON THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE AND PROLIFERATION INHIBITION OF HUMAN COLON CANCER CELL LINE

Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes.

Hypoxia upregulates hypoxia inducible factor(HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

The role of the hypoxia-inducible factor(HIF)subunits 1α and 2α in response to hypoxia is well established in lungepithelial cells,whereas little is known about HIF-3α with respect to transcriptional and translational regulation by hy-poxia.HIF-3α and HIF-1α are two similar but distinct basic helix-loop-helix-PAS proteins,which have been postulatedto activate hypoxia responsive genes in response to hypoxia.Here,we used quantitative real time RT-PCR and immu-noblotting to determine the activation of HIF-3α vs.HIF-1α by hypoxia.HIF-3α was strongly induced by hypoxia(1%O_2)both at the level of protein and mRNA due to an increase in protein stability and transcriptional activation,whereasHIF-1α protein and mRNA levels enhanced transiently and then decreased because of a reduction in its mRNA stabilityin A549 cells,as measured on mRNA and protein levels.Interestingly,HIF-3α and HIF-1α exhibited strikingly similarresponses to a variety of activating or inhibitory pharmacological agents.These results demonstrate that HIF-3α is ex-pressed abundantly in lung epithelial cells,and that the transcriptional induction of HIF-3α plays an important role in theresponse to hypoxia in vitro.Our findings suggest that HIF-3α,as a member of the HIF system,is complementary ratherthan redundant to HIF-1α induction in protection against hypoxic damage in alveolar epithelial cells.

Regulated Gene Expression with Promoters Responding to Inducers

Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic research in molecular biology and biotechnological applications.Various chemical inducible systems based on activation and inactivation of the target gene had been described.The transfer of regulatory elements from prokaryotes,insects,and mammals has opened new avenues to construct chemically inducible promoters that differ in their ability to regulate the temporal and spatial expression patterns,and this will dramatically increase the application of transgenic technology.This review provides an overview on regulation of gene expression,promoter activating systems,promoter inactivation systems,inducible gene over expression,and inducible anti suppression.

Abnormal expression of hypoxia inducible factor-1α and clinical values of molecular-targeted interference in hepatocellular carcinoma

Objective:The aim of this study was to analyze the expression features of hypoxia inducible factor-1α (HIF-1α) in hepatocellular carcinoma (HCC) and effects of HIF-1α silencing on HepG2 cells.Methods:HIF-1α expression was analyzed in the self-control HCC specimens by immunohistochemistry.After HepG2 cells with miRNA transfection,the expression of HIF-1α was determined at mRNA or protein level by real-time polymerase chain reaction (PCR) or Western blotting.Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG-2) were determined by ELISA.Alterations of cell cycles and apoptosis of HepG2 cells were measured using a flow cytometer.Results:Positive HIF-1α was brown and granule-like in the cytoplasm or nucleus.Significant difference was found between HCC (80%) and its surrounding tissues (100%,χ2=22.35,P < 0.001) and HIF-1α expression related to tumor size.At 72 h after miRNA transfection,the expression of HIF-1α in HepG2 cells was down-regulated by 87% at mRNA or 65% at protein level,with VEGF and ANG-2 decreased to 54% and 36%,respectively.After RNA interference combined with anti-cancer drug,the apoptotic rate of HepG2 cells was increasing from 22.46% ± 0.61% to 36.99% ± 0.88%,with up-regulation of G1 phase (65.68% ± 0.91%) and down-regulation of S phase (19.47 ± 1.34 %).Conclusion:Abnormal expression of HIF-1α is associated with development of HCC,and HIF-1α gene silencing can effectively inhibit HepG2 cell proliferation.

Gene expression profiles associated with osteoblasts differentiated from bone marrow stromal cells

Objective:To study the changes of gene expression profiles associated with osteoblasts differentiated from rat bone marrow stromal cells in vitro by gene chip technique.Methods:rat Rone marrow stromal cells were isolated and cultured,and differentiation was induced by dexamethasone,β-glycerol phosphate and vitamin C.Cellular mRNA was extracted and reverse transcribed into cDNA,thus related genes expression differences were detected by gene expression profile chip.Results:Calcifying nodules were visible in the induced cells.There were27.7%genes expressed differentially,three times more than the normal and induced cells,and some genes were related to transcription,translation,glycosylation modification.Extracellular matrix,signal molecules and metabolism were up—regulated.Conclusions:The gene chip technique can be used to detect the multi-gene different expression in the differentiationinduceed rat BMSCs,and these differentially expressed genes are necessary genes related to rat BMSCs proliferation and induction of osteoblastic differentiation.

Inheritance and Expression of Potato Proteinase Inhibitor Gene Ⅱ （pinⅡ） in Transgenic Rice

The inheritance and expression of bar gene and pinII gene were studied in three transgenic rice lines and their F2 hybrid populations, which were created through hybridization with a PGMS line, ZAU11S. By Basta painting, PCR analysis and determining of the inhibitory trypsin activity, the results show that bar gene and pinII gene in rice F2 population fit the simple Mendel's low of inheritance and close linkage, but a few plants in F2 have not sufficiently expressed. The wound inducible pin II gene has an expression regulated spatially and temporally, and the signal transduction pathway is not only upward, but also downward. The inducible expression of pin II in different rice transgenic lines is not completely coincident.

Expression of a Carrot Antifreeze Protein Gene in Escherichia coli

The recombinant expression vector pET43.1b-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol稬-1 IPTG (isopropyl--D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein. The analysis of product solubility revealed that pET43.1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.

Factors Influencing Induction of Agrobacterium tumefaciens Virulence Genes

Agrobacterium species are routinely employed for plant genetic modification due to the relatively simple procedures, cost-competitiveness, low copy num- ber, independence to vector DNAs, and targeted integration into transcriptionally active regions of plant chromosomes with defined T-DNA. However, to date, there are still a great number of plant species reluctant to Agrobacterium-mediated transformation. Evidence suggests that the infection capability of Agrobacterium is deter- mined by virulence （vir） genes of Ti plasmid outside ofA. tumefaciens chromosome. Among all v/r genes, virA and virG are constitutively expressed, while the ex- pression of other vir genes is induced by phenolic compounds. In addition, carbohydrates can enhance vir induction mediated by phenolic compounds, while low phosphate and acidic pH conditions may also enhance the induction of vir genes. To improve Agrobacterium-mediated transformation efficiency for potential applica- tions in research and industry, molecular mechanisms of vir induction by factors such as phenolic compounds, carbohydrates, low phosphate, acidic pH and incuba- tion temperature are discussed in this review.

IL-7的诱导表达增强靶向GPC3 CAR-T细胞的增殖及体外抗肿瘤活性

目的:探讨IL-7的诱导表达对靶向磷脂酰肌醇蛋白聚糖3(GPC3)嵌合抗原受体基因修饰T淋巴细胞(CAR-T细胞)的增殖和体外抗肿瘤活性的影响。方法:通过无缝克隆将GPC3 CAR序列片段插入GV400载体的Bam HⅠ/Eco RⅠ位置,构建第二代CAR慢病毒载体GPC3-BBZ及GPC3-BBZ-NFAT-IL-7,以293T细胞包装相应的慢病毒载体后,感染人T细胞制备CAR-T细胞。实验分为未转导T细胞(NT)组、GPC3-BBZ CAR-T细胞组、GPC3-BBZ-NFAT-IL-7 CAR-T细胞组。采用流式细胞术检测各组CAR-T细胞中CAR的表达水平,qPCR法检测经GPC3蛋白激活的CAR-T细胞中IL-7 m RNA的表达水平,细胞计数法检测CAR-T细胞在GPC3抗原刺激下的增殖能力,ELISA检测CAR-T细胞在受到肿瘤细胞刺激后IL-7、IFN-γ和TNF-α的分泌水平。应用实时细胞分析(RTCA)技术检测CAR-T细胞对人肝癌Huh-7细胞的杀伤作用。结果:成功构建慢病毒载体GPC3-BBZ和GPC3-BBZ-NFAT-IL-7,制备出靶向GPC3的CAR-T细胞。经GPC3抗原激活后,GPC3-BBZ-NFAT-IL-7 CAR-T细胞可有效表达IL-7 mRNA(P<0.01),其表现出更强的增殖能力(P<0.05)。与GPC3-BBZ CAR-T细胞相比,GPC3-BBZ-NFAT-IL-7 CAR-T细胞与GPC3阳性靶细胞Huh-7细胞共培养后,分泌更高水平的IL-7、IFN-γ和TNF-α(P<0.01或P<0.001)。RTCA结果显示,GPC3-BBZ-NFAT-IL-7 CAR-T细胞对GPC3阳性Huh-7细胞的杀伤活性显著高于GPC3-BBZ CAR-T细胞(P<0.05)。结论:成功制备可诱导表达IL-7的靶向GPC3的CAR-T细胞,IL-7的诱导表达增强靶向GPC3 CAR-T细胞的免疫活性,在体外展现出较强的肿瘤细胞杀伤能力。

溶血素 E基因原核表达载体的构建与蛋白诱导表达

目的构建大肠杆菌(Escherichia Coli,E.coli)溶血素E(Hemolysin E)基因的重组质粒pCZN1-hlyE,诱导表达目的蛋白。方法通过NCBI数据库检索获取E.coli Hemolysin E基因序列(NC_000913.3),采用基于PAS(PCR-based Accurate Synthesis)的全基因合成法,设计全长拼接引物,合成目的基因,并成功构建重组质粒pCZN1-hlyE。将构建好的pCZN1-hlyE重组质粒转入大肠杆菌TOP10菌株中,并用异丙基-β-D-硫代半乳糖苷(IPTG)诱导Hemolysin E基因的表达,生成Hemolysin E毒力蛋白。结果经SDS-PAGE凝胶电泳,Western blot分析鉴定目的蛋白Hemolysin E分子量为28.72 kDa,与Hemolysin E基因核酸序列经DNAMAN软件分析所得蛋白分子量一致。结论被诱导表达的Hemolysin E蛋白存在于菌体裂解液上清中,并纯化制备重组蛋白。

Isolation and Expression Analysis of Two Cold-Inducible Genes Encoding Putative CBF Transcription Factors from Chinese Cabbage （Brassica pekinensis Rupr）

Two homologous genes of the Arabidopsis C-repeat/dehydration-responsive element binding factors （CBF/ DREB1） transcriptional activator were isolated by RT-PCR from Chinese cabbage （Brassica pekinensis Rupr. cv. Qinbai 5） and were designated as BcCBF1 and BcCBF2. Each encodes a putative CBF/DREB1 protein with an AP2 （Apetal2） DNA-bindlng domain, a putative nuclear localization signal, and a possible acidic activation domain. Deduced amino acid sequences show that BcCBF1 is very similar to the Arabidopsis CBF1, whereas BcCBF2 Is different in that it contains two extra regions of 24 and 20 amino acids in the acidic domain. The mRNA accumulation profiles indicated that the expression of BcCBF1 and BcCBF2 is strongly induced by cold treatment, but does not respond similarly to dehydration or abscisic acid （ABA） treatment. However, the cold-induced accumulation of BcCBF2 mRNA was rapid but short-lived compared with that of BcCBFI. The mRNA levels of both BcCBF1 and BcCBF2 were higher in leaves than in roots when plants were exposed to cold, whereas, salt stress caused higher accumulation of BcCBF2 mRNA in roots than in leaves, suggesting that the organ specificity of the gene expression of the BcCBFs is probably stress dependent. In addition, the accumulation of BcCBF1 and BcCBF2 mRNAs was greatly enhanced by light compared with darkness when seedlings were exposed to cold. It is concluded that the two BcCBF proteins may be involved in the process of plant response to cold stress through an ABA-independent pathway and that there is also a cross-talk between the light signaling conduction pathway and the cold response pathway in B. pekinensis as in Arabidopsis.

Gene expression of inducible nitric oxide synthase in injured spinal cord tissue

Objective: To investigate gene expression of inducible nitric oxide synthase (iNOS) in injured spinal cord tissue of rats. Methods: Thirty-six adult Sprague-Dawley rats were divided randomly into six groups: a normal group and five injury groups, six animals in each group. Animals in the injury groups were killed at 2, 6, 12, 24, 48 hours after injury, respectively. A compression injury model of spinal cord was established according to Nystrom B et al, and gene expression of iNOS in spinal cord tissue was examined by means of reverse transcription polymerase chain reaction (RT-PCR). Results: Gene expression of iNOS was not detectable in normal spinal cord tissue but was seen in the injury groups. The expression was gradually up-regulated, reaching the maximum at 24 hours. The expression at 48 hours began to decrease but was still significantly higher than that at 2 hours. Conclusions: iNOS is not involved in the normal physiological activities of spinal cord. Expression of iNOS is up-regulated in spinal cord tissue in response to injury and the up-regulation exists mainly in the late stage after injury. Over-expression of iNOS may contribute to the late injury of spinal cord.

m^(6)A相关基因在激素性股骨头坏死中的生物信息学分析

背景:m^(6)A修饰与股骨头坏死的发生发展相关,但在激素性股骨头坏死中的作用尚不清楚。目的:基于GEO数据库,采用生物信息学方法分析激素性股骨头坏死中表达差异的m^(6)A基因及互作miRNAs,探寻其潜在发病机制。方法:在GEO数据库中检索并下载与激素性股骨头坏死相关的mRNA表达谱数据集(GSE123568),通过R软件对数据集进行差异基因筛选及GO功能、KEGG通路富集分析。识别差异基因中的m^(6)A差异表达基因(m^(6)A-DEGs)并对其进行GO功能与KEGG通路富集分析,比较m^(6)A-DEGs的表达量并分析它们之间的相关性。最后通过Cytoscape构建m^(6)A-DEGs的PPI互作网络及筛选核心基因。使用TargetScan,miRTarBase和miRBD数据库预测m^(6)A-DEGs相关的潜在miRNAs,同时使用ChIPBase及hTFtarget数据库预测7个核心基因潜在转录因子,然后分别构建m^(6)A-miRNA与转录因子m^(6)A调控网络。最后使用数据集GSE74089验证7个核心m^(6)A-DEGs的表达水平。结果与结论:①从数据集中共筛选出2460个差异表达的基因,其中1455个上调,1005个下调。②从数据集中筛选出了14个m^(6)A-DEGs,包括3个下调和11个上调基因,m^(6)A-DEGs在激素性股骨头坏死中的表达具有显著差异(P<0.05),Spearman分析表明它们之间具有一定相关性。③m^(6)A-DEGs的GO和KEGG富集分析主要集中在骨髓细胞分化与发育、免疫受体与细胞因子受体活性、破骨细胞分化、AMPK与白细胞介素17信号通路。④m^(6)A-DEGs前7个核心基因包括YTHDF3,YTHDF1,YTHDF2,ALKBH5,METTL3,HNRNPA2B1及HNRNPC,它们在miRTarBase,miRDB和TargetScan数据库中共有44个miRNA重叠,在ChIPBase及hTFtarget数据库中共有79个重叠转录因子。⑤在GSE74089数据集中有6个核心m^(6)A-DEGs的表达水平与GSE123568数据集一致。⑥结果证实,根据生物信息学方法筛选的7个m^(6)A-DEGs可能通过调控多个miRNA、转录因子和AMPK及白细胞介素17信号通路表达,进而影响激素性股骨头坏死中骨髓细胞分化发育与破骨细胞分化,为进一步深入研究激素性股骨头坏死的发病机制和靶向治疗提供了数据支持和研究方向。

Dexamethasone-Inducible Green Fluorescent Protein Gene Expression in Transgenic Plant Cells

Genomic research has made a large number of sequences of novel genes orexpressed sequence tags available. To investigate functions of these genes, a system for conditionalcontrol of gene expression would be a useful tool. Inducible trans-gene expression that uses greenfluorescent protein gene (gfp) as a reporter gene has been investigated in transgenic cell lines ofcotton (COT; Gossypium hirsutum L.), Fraser fir [FRA; Abies fraseri (Pursh) Poir], Nordmann fir(NOR; Abies nord-manniana Lk.), and rice (RIC; Oryza sativa L. cv. Radon). Transgenic cell lineswere used to test the function of the chemical inducer dexamethasone. Inducible transgene expressionwas observed with fluorescence and confocal microscopy, and was confirmed by northern blotanalyses. Dexamethasone at 5 mg/L induced gfp expression to the nearly highest level 48 h aftertreatment in COT, FRA, NOR, and RIC. Dexamethasone at 10 mg/L inhibited the growth of transgeniccells in FRA and NOR, but not COT and RIC. These results demonstrated that concentrations of inducerfor optimum inducible gene expression system varied among transgenic cell lines. The inducible geneexpression system described here was very effective and could be valuable in evaluating thefunction of novel gene.

In situ RT-PCR detection of inducible nitric oxide synthetase gene expression in lung during endotoxemia in rabbits

To detect the location of inducible nitric oxide synthetase (iNOS) protein and mRNA in lung during endotoxemia in rabbits Methods Northern blotting was performed before, 1 hour and 5 hours after the intravenous administration of lipopolysaccharide (LPS) in rabbits Immuno^histochemical analysis (IA), in situ hybridization and in situ reverse transcription polymerase chain reaction (in situ RT PCR) were also performed in lung sections Results iNOS mRNA expression was found using Northern blotting in lung 5 hours after LPS injection, while it was not found in control The positive stain was found only in macrophages in lung 5 hours after LPS injection by standard hybridization and IA; while by in situ RT PCR, the amplification products were found in macrophages, airway epithelial cells, vascular endothelial cells, smooth muscle cells and leukocytes, in addition to macrophages distributed abundantly throughout the lung The signal was absent in control or samples Conclusions Using an in situ RT PCR technique, iNOS expression was not only observed in macrophages but also in many other kinds of cells in lung during endotoxemia in rabbits This suggests that in situ RT PCR is much more sensitive than in situ hybridization, and can be used to examine genes with low expression

卷丹百合LlARF12基因的克隆、表达及功能分析

【目的】探究卷丹百合珠芽发生前细胞和组织学层面的变化,讨论卷丹百合珠芽发生的内在机制。克隆生长素响应因子基因LlARF12,为解析LlARF12基因在卷丹百合珠芽发生过程中的作用机制提供参考。【方法】利用石蜡切片的方法对卷丹百合珠芽器官形成前的叶腋部位进行了组织学观测,从卷丹珠芽形成过程的转录组中筛选得到参与珠芽发生的关键基因LlARF12,克隆得到其编码区(CDS)序列,利用生物信息学软件对其进行分析预测,通过qRT-PCR对不同时期、不同种与品种的百合组织中的LlARF12基因进行表达模式分析,并通过VIGS技术对卷丹种球进行LlARF12基因沉默,从而验证其基因功能。【结果】克隆获得LlARF12基因长度为2346 bp,编码781个氨基酸;qRT-PCR结果显示,LlARF12在珠芽发生的小白点时期表达量显著低于未发生珠芽时期、在S1时期植株茎秆上部表达量显著低于茎秆下部、在能够自然发生珠芽的百合品种中表达量显著低于其他百合品种;沉默LlARF12基因导致卷丹珠芽发生时间提前,且发生的珠芽数量增多。【结论】在卷丹百合珠芽发生的S1时期之前,腋生分生组织已经开始启动,此处靠近表皮的薄壁细胞层数增加、细胞核堆积,并且逐渐产生成熟的维管组织。LlARF12在卷丹百合不能发生珠芽的组织部位和时期的表达量普遍更高,推测其在珠芽发生过程中可能发挥转录抑制作用,通过VIGS技术进行的基因功能验证证明了LlARF12对珠芽发生的抑制作用。

烟草NtPRR37基因克隆及功能分析

【目的】伪答应基因家族(pseudo response regulators,PRRs)是高等植物调控开花途径的重要基因。克隆烟草NtPRR37基因并分析其对不同光周期的应答及对开花的影响,为烟草开花调控提供靶标基因。【方法】利用同源克隆方法从普通烟草(Nicotiana tabacum L.)中克隆得到NtPRR37基因,并对其进行生物信息学分析,利用实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)分析其在不同组织中的表达及不同光照时长处理的表达模式。同时利用病毒诱导基因沉默(virus induced gene silence,VIGS)技术降低NtPRR37表达水平并观察表型变化及检测开花相关基因表达变化。【结果】NtPRR37基因全长2472 bp,编码823个氨基酸,相对分子质量90.16 kD,含有PRRs基因家族的典型保守结构域(REC和CCT结构域)。通过同源进化分析发现,烟草NtPRR37与绒毛烟草(Nicotiana tomentosiformis)、林烟草(Nicotiana sylvestris)及本氏烟草(Nicotiana benthamiana)的PRR37在进化上属于同一分支。采用RT-qPCR分析发现,该基因在盛花期烟草各个组织的表达特征存在差异性,在雌蕊中的表达量最高,在侧根的表达量最低;在不同光照时长处理下,NtPRR37随着光照时间的增加表达量呈上升趋势,全黑暗处理下表达量最低,且具有生物节律性;NtPRR37沉默植株中NtPRR37表达量明显下调且沉默植株开花期提前,这可能与诱导开花相关基因(NtFT4、NtAP1、NtCO、NtSOC1)表达量显著上调有关。【结论】NtPRR37的表达受到光周期的调控,且在烟草开花过程中NtPRR37作为开花抑制因子存在。

火龙果HubHLH基因家族的全基因组分析及其对冬季补光诱导开花的表达响应

为了获得较完整的候选基因,探讨HubHLH基因在火龙果(Hylocereus undatus)冬季补光诱导开花过程的表达响应,对火龙果HubHLH基因家族进行全基因组分析。鉴定出153个HubHLH基因;这些基因的编码蛋白含有176~687个氨基酸,分子量大小为19.28~74.44 kDa,等电点(pI)为4.81~9.88,均为亲水蛋白,亚细胞定位预测大多定位到细胞核。为鉴定HubHLH基因家族的同源性,本研究将153个火龙果HubHLH和120个拟南芥AtbHLH蛋白进行系统发育比较分析。系统发育比较分析结果:火龙果HubHLH基因家族成员被分为12个组,25个亚族;对HubHLH基因家族的保守motif、基因结构及在染色体的位置分布的分析结果表明,同一亚族的基因具有相似的基序组成和基因结构。对HubHLH基因家族的内部复制事件的分析结果表明,有78条片段复制被鉴定为片段重复基因,说明片段复制是HubHLH基因家族的主要扩张力量。此外,基于已测定的关于火龙果冬季补光诱导开花的4个时期转录组数据,筛选到59个HubHLH基因在冬季补光诱导成花过程中有差异表达,随后对这59个HubHLH基因进行GO功能富集,发现它们在红光或远红光的反应、对光刺激的反应、有性生殖功能、对辐射的反应等功能上均有富集,说明HubHLH基因家族可能在冬季补光诱导火龙果成花过程中起到了调控作用。

基于蛋白质组学的难治性高血压潜在生物标志物的筛选

目的 基于蛋白质组学和复杂网络分析挖掘难治性高血压的潜在生物标志物,探索其关键生物通路。方法 招募2022年1—12月于山东中医药大学附属医院、济南市第五人民医院住院治疗的10例难治性高血压患者作为高血压组,另外招募同时期10例健康人作为健康组。运用蛋白质组学技术分析2组受试者的血液样本,筛选难治性高血压的临床标志物,并结合加权基因共表达网络分析(WGCNA)各个标志物的潜在临床价值。结果 蛋白质组学分析发现,高血压组中共鉴定出60个差异蛋白,主要富集在磷脂酰肌醇3-激酶(PI3K)/蛋白激酶(Akt)和缺氧诱导因子-1(HIF-1)等关键信号传导通路上;蛋白互作结果分析发现,COL6A3、CSF1R、HSPG2、ITGA2、PDGFB、THBS1和vWF是参与难治性高血压发生发展的标志物,这些指标可以通过调节炎症反应、氧化应激、细胞自噬等参与难治性高血压发生发展的病理生理过程。结论 难治性高血压的发病和转归与PI3K/Akt和HIF-1通路中的潜在标志物密切相关,并诱导下游炎症反应,出现COL6A3、CSF1R、HSPG2、ITGA2、PDGFB、THBS1、vWF 7个异常表达的蛋白,这些发现为难治性高血压的诊断和治疗提供了潜在的蛋白靶点。

Tet-off诱导表达Egr-1HEK293稳定细胞株的建立和鉴定

目的:利用大肠埃希菌Tet-off诱导表达调控系统构建多西环素调控的稳定细胞株。方法:以pEGFPEgr-1质粒为模板,扩增含早期生长反应蛋白(early growth response-1,Egr-1)完整开放阅读框的DNA序列,经酶切后插入含四环素反应元件(tetracycline responsive element,TRE)序列的表达载体pTRE2hyg,获得重组质粒pTRE2hyg-Egr-1,将其转染293Tet-off细胞株,并用G418和多西环素筛选稳定表达Egr-1的细胞株。蛋白质印迹法检测多西环素诱导表达Egr-1情况;流式细胞术检测细胞增殖能力。结果:蛋白质印迹结果显示,外源性Egr-1蛋白表达受细胞培养液中多西环素调控,当从细胞培养液中去除多西环素后,Egr-1表达量明显升高。流式细胞检测结果显示Egr-1高表达细胞的增殖能力明显高于Egr-1低表达细胞。结论:利用Tet-off体系成功构建Egr-1诱导表达的细胞株,细胞的增殖能力受Egr-1表达水平的影响。