AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Analysis of Genetic Alterations in TP53 Gene in Breast Cancer - A Secondary Publication

Tumor protein p53 (TP53) mediates DNA repair and cell proliferation in growing cells. The TP53 gene is a tumor suppressor that regulates the expression of target genes in response to multiple cellular stress factors. Key target genes are involved in crucial cellular events such as DNA repair, cell cycle regulation, apoptosis, metabolism, and senescence. TP53 genetic variants and the activity of the wild-type p53 protein (WT-p53) have been linked to a wide range of tumorigenesis. Various genetic and epigenetic alterations, including germline and somatic mutations, loss of heterozygosity, and DNA methylation, can alter TP53 activity, potentially resulting in cancer initiation and progression. This study was designed to screen three reported mutations in the DNA-binding domain of the p53 protein in breast cancer, to evaluate the relative susceptibility and risk associated with breast cancer in the local population. Genomic DNA was isolated from 30 breast tumor tissues along with controls. Tetra and Tri ARMS PCR were performed to detect mutations in the TP53 coding region. For SNPs c.637C>T and c.733C>T, all analyzed cases were homozygous for the wild-type allele ‘C,’ while for SNP c.745A>G, all cases were homozygous for the wild-type allele ‘A.’ These results indicate no relevance of these three SNPs to cancer progression in our study cohort. Additionally, the findings from whole exon sequencing will help to predict more precise outcomes and assess the importance of TP53 gene mutations in breast cancer patients.

乳腺癌组织miR-1247-5p表达及其靶基因生物信息学预测研究

目的:探讨微小核糖核酸-1247-5p(miR-1247-5p)在乳腺癌组织中的表达,并对其预测的靶基因进行生物信息学分析。方法:收集2019年6月至2020年12月本院手术治疗的104例乳腺癌患者癌组织及其癌旁组织。RT-PCR检测标本中miR-1247-5p的相对表达量;比较不同临床病理特征患者癌组织miR-1247-5p表达;预测miR-1247-5p的靶基因;采用DAVID数据库对miR-1247-5p靶基因进行功能和通路富集分析;String数据库对miR-1247-5p靶基因进行互作分析。结果:miR-1247-5p在乳腺癌组织中的表达水平较癌旁组织下调(P<0.05)。miR-1247-5p潜在靶基因38个,其靶基因功能主要富集于RNA聚合酶Ⅱ启动子的转录调控、基因表达/转录调控、质膜部分、突触小体、蛋白质结合、poly(A)RNA结合等;参与的通路主要富集于癌症通路、细胞因子与细胞因子受体的相互作用、磷脂酰肌醇3激酶(PI3K)-蛋白激酶B(Akt)信号通路等。磷酸酯酶与张力蛋白同源物(PTEN)、上皮生长因子受体(EGFR)、假尿苷酸合酶1(PUS1)的编码蛋白质在miR-1247-5p基因靶基因蛋白质互作网络(PPI)中关键节点位置。结论:miR-1247-5p在乳腺癌组织中的表达下调,其靶基因富集于多个生物学过程及与肿瘤相关的信号转导通路上,miR-1247-5p可能通过调控其靶基因的表达参与乳腺癌的发生发展过程。

Role of p53 suppression in the pathogenesis of hepatocellular carcinoma

In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrhosis.Among the most prevalent defects in a wide range of tumours,notably HCC,is the silencing of the p53 tumour suppressor gene.The control of the cell cycle and the preservation of gene function are both critically important functions of p53.In order to pinpoint the core mechanisms of HCC and find more efficient treatments,molecular research employing HCC tissues has been the main focus.Stimulated p53 triggers necessary reactions that achieve cell cycle arrest,genetic stability,DNA repair and the elimination of DNA-damaged cells’responses to biological stressors(like oncogenes or DNA damage).To the contrary hand,the oncogene protein of the murine double minute 2(MDM2)is a significant biological inhibitor of p53.MDM2 causes p53 protein degradation,which in turn adversely controls p53 function.Despite carrying wt-p53,the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway.High p53 in-vivo expression might have two clinical impacts on HCC:(1)Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways;and(2)Exogenous p53 makes HCC susceptible to various anticancer drugs.This review describes the functions and primary mechanisms of p53 in pathological mechanism,chemoresistance and therapeutic mechanisms of HCC.

微小扇头蜱P0基因的克隆及其编码蛋白的生物信息学分析

为探究微小扇头蜱P0基因序列特征,预测P0蛋白的理化性质和二、三级结构,筛选出P0蛋白的B、T优势抗原表位,本研究克隆了微小扇头蜱P0基因,运用Clustal X软件分析P0基因序列特征,用在线软件EXPASY、PRABI和SWISS-MODEL预测P0蛋白的理化性质和二、三级结构,用在线软件ABCpred Prediction、Scratch、IEDB和NetCTL筛选P0蛋白的B、T优势抗原表位。试验结果显示:微小扇头蜱P0基因全长957 bp,碱基A含量为24.0%,T含量为20.3%,G含量为27.5%,C含量为28.2%,A+T含量为44.3%,G+C含量为55.7%,共编码318个氨基酸;P0蛋白分子量为34 ku,理论等电点(pI)为5.86,平均亲水系数为-0.153,不稳定指数为38.15;P0蛋白的二级结构含163个α螺旋(占比51.25%),130个无规卷曲(占比40.88%),25个延伸链(占比7.86%),其中以α螺旋为主要结构;P0蛋白的三级结构以α螺旋的含量最高,该蛋白的全局模型质量评估(global model quality estimation, GMQE)、定性模型能量分析(qualitative model energy analysis, QMEAN)值分别为0.49和0.52±0.05,无信号肽和跨膜结构域,但存在40个磷酸化位点和1个糖基化位点;P0蛋白有13个B淋巴细胞优势抗原表位和6个T淋巴细胞优势抗原表位。综上所述,微小扇头蜱P0基因序列呈GC偏好,P0蛋白是以α螺旋为主要结构成分的亲水性酸蛋白,具有B、T淋巴细胞优势抗原表位,是今后研制防控微小扇头蜱疫苗的理想靶标。

Study of pathogenic genes in a pedigree with familial dilated cardiomyopathy

BACKGROUND Dilated cardiomyopathy(DCM)is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction.The substantial genetic heterogeneity evident in patients with DCM contributes to variable disease severity and complicates overall prognosis,which can be very poor.AIM To identify pathogenic genes in DCM through pedigree analysis.METHODS Our research team identified a patient with DCM in the clinic.Through invest-igation,we found that the family of this patient has a typical DCM pedigree.High-throughput sequencing technology,next-generation sequencing,was used to sequence the whole exomes of seven samples in the pedigree.RESULTS A novel and potentially pathogenic gene mutation-ANK2p.F3067L-was discovered.The mutation was completely consistent with the clinical information for this DCM pedigree.Sanger sequencing was used to further verify the locus of the mutation in pedigree samples.These results were consistent with those of high-throughput sequencing.CONCLUSIONS ANK2p.F3067L is considered a novel and potentially pathogenic gene mutation in DCM.

Exercise-induced modulation of miR-149-5p and MMP9 in LPS-triggered diabetic myoblast ER stress: licorice glycoside E as a potential therapeutic target

Background:This study explores the relationship between endoplasmic reticulum(ER)stress and diabetes,particularly focusing on the impact of physical exercise on ER stress mechanisms and identifying potential therapeutic drugs and targets for diabetes-related sepsis.The research also incorporates traditional physical therapy perspectives,emphasizing the genomic insights gained from exercise therapy in disease management and prevention.Methods:Gene analysis was conducted on the GSE168796 and GSE94717 datasets to identify ER stress-related genes.Gene interactions and immune cell correlations were mapped using GeneCard and STRING databases.A screening of 2,456 compounds from the TCMSP database was performed to identify potential therapeutic agents,with a focus on their docking potential.Techniques such as luciferase reporter gene assay and RNA interference were used to examine the interactions between microRNA-149-5p and MMP9.Results:The study identified 2,006 differentially expressed genes and 616 miRNAs.Key genes like MMP9,TNF-α,and IL1B were linked to an immunosuppressive state.Licorice glycoside E demonstrated high affinity for MMP9,suggesting its potential effectiveness in treating diabetes.The constructed miRNA network highlighted the regulatory roles of MMP9,IL1B,IFNG,and TNF-α.Experimental evidence confirmed the binding of microRNA-149-5p to MMP9,impacting apoptosis in diabetic cells.Conclusion:The findings highlight the regulatory role of microRNA-149-5p in managing MMP9,a crucial gene in diabetes pathophysiology.Licorice glycoside E emerges as a promising treatment option for diabetes,especially targeting MMP9 affected by ER stress.The study also underscores the significance of physical exercise in modulating ER stress pathways in diabetes management,bridging traditional physical therapy and modern scientific understanding.Our study has limitations.It focuses on the microRNA-149-5p-MMP9 network in sepsis,using cell-based methods without animal or clinical trials.Despite strong in vitro findings,in vivo studies are needed to confirm licorice glycoside E’s therapeutic potential and understand the microRNA-149-5p-MMP9 dynamics in real conditions.

镜像神经元疗法治疗中风后复杂性区域疼痛综合征Ⅰ期的疗效及对血清SP、CGRP水平的影响

目的镜像神经元疗法对中风后复杂性区域疼痛综合征(CRPS)Ⅰ期的临床疗效及作用机制的探讨。方法选择康复科收治的40例CRPSⅠ期患者为研究对象。在研究中采用随机法,将选择的40例患者分为对照组与镜像组,每组20例。均采取基础以及常规康复治疗,镜像组额外追加镜像治疗,两组每天上下午各治疗1次,5次/周,共治疗3周。采用休息状态视觉模拟评分(resting state visual analogue scale,R-VAS)与被动运动视觉模拟评分(passive movement state visual analogue scale,P-VAS)来评估上肢肩关节疼痛状况,选择运用排水法来评估手部肿胀状况,并且选择运用Fugl-Meyer量表,评估上肢运动功能,采用ELISA检测技术进行测定血清降钙素基因相关肽(CGRP)、P物质(SP)含量。结果镜像组患者治疗3周后的肩关节R-VAS、P-VAS评分及肿胀程度均低于对照组(P<0.05),Fugl-Meyer评分高于对照组(P<0.05),SP含量低于对照组(P<0.05),CGRP含量高于对照组(P<0.05)。结论镜像组能更有效地改善CRPSⅠ期患者肩关节疼痛、手部肿胀及上肢功能障碍,调节血清CGRP及SP细胞水平。

产气荚膜梭菌四环素耐药基因双重荧光定量PCR检测方法的建立

旨在开发一种快速、灵敏的针对产气荚膜梭菌四环素类耐药基因tetA(P)和tetB(P)的双重荧光定量PCR检测方法,为快速检测产气荚膜梭菌四环素类抗生素耐药性提供新策略。本研究针对tetA(P)和tetB(P)的保守序列设计引物和探针,构建重组质粒,对退火温度和体系进行优化,同时采用药敏纸片琼脂扩散法测定菌株对四环素类药物的敏感性加以验证。结果发现,优化后的反应程序为95℃30 s;95℃5 s,56℃30 s,共39个循环。使用该反应程序仅能扩增重组质粒,对照菌株无扩增曲线;组内变异系数小于1.3%,组间变异系数小于1.8%,重组质粒最低检测限度为102 copies·μL^(-1)。使用本方法对33份产气荚膜梭菌临床分离株样品进行耐药基因检测,同时进行药敏试验,发现本研究建立的检测方法对四环素耐药基因的检出率均为75.8%(25/33),且与药敏试验结果一致。综上,本研究建立了一种特异、敏感、重复性好的产气荚膜梭菌四环素类耐药基因tetA(P)和tetB(P)的双重荧光定量PCR检测方法。

机械振动对去卵巢大鼠骨质疏松性骨折愈合中CGRP和SP表达的影响

目的探讨机械振动对去卵巢后骨质疏松性骨折大鼠骨折端愈合过程中神经肽降钙素基因相关肽(calcitonin gene-related peptide,CGRP)和P物质(substance P,SP)表达的影响。方法选取3月龄的雌性Wistar大鼠30只,按照随机法分为对照组(Sham)、去卵巢组(OVX)、振动组(OVX-V),每组10只。摘除去卵巢组和振动组大鼠的卵巢以建立骨质疏松模型,对照组则去除同质量脂肪组织构建假去卵巢骨质疏松模型,同时对振动组大鼠进行频率35 Hz、振幅2 mm、加速度0.5 g的振动干预,持续20 min,其他组放于关闭的振动台上自由活动20 min,每周5 d。在干预2周和6周后,拍摄大鼠骨折端X线片观察对比3组的愈合情况,采用酶联免疫吸附测定(ELISA)技术来测定骨折端CGRP和SP的表达水平。结果对照组与振动组在干预2周后大鼠骨折愈合率较去卵巢组高,振动组在干预6周后大鼠骨折愈合率高于其他两组(P<0.05)。在干预2周后,振动组大鼠骨组织CGRP含量[0.464±0.018]和SP含量[0.450±0.019]均高于去卵巢组但明显低于对照组(P<0.05)。在干预6周后,振动组大鼠骨组织中CGRP含量[0.632±0.016]和SP含量[0.636±0.017]呈上升趋势且明显高于去卵巢组,但仍低于对照组(P<0.05)。结论机械振动有助于提高去卵巢骨质疏松骨折大鼠骨组织中神经肽CGRP和SP的表达水平,从而增加成骨细胞活性并抑制骨吸收,加速骨折愈合。

Allotetraploidization event of Coptis chinensis shared by all Ranunculales

Coptis chinensis Franch.,also named Chinese goldthread is a member of Ranunculaceae in the order Ranunculales and represents an important lineage of early eudicots with traditional medicinal value.In our study,by using syntenic analysis combined with phylogenomic analysis of C.chinensis and four other representative genomes from basal and core eudicots,we confirmed that the WGD event in C.chinensis was shared by Aquilegia coerulea and Papaver somniferum L.and quickly occurred after Ranunculales diverged from other eudicots,likely a Ranunculales common tetraploidization(RCT).The synonymous nucleotide substitutions at synonymous sites distribution of syntenic blocks across these genomes showed that the evolutionary rate of the P.somniferum genome is faster than that of the C.chinensis genome by approximately 13.7%,possibly due to Papaveraceaes having an additional special tetraploidization event(PST).After Ks correction,the RCT dated to 115—130 million years ago(MYA),which was close to the divergence of Ranunculaceaes and Papaveraceaes approximately115.45—130.51 MYA.Moreover,we identified homologous genes related to polyploidization and speciation and constructed multiple sequence alignments with different reference genomes.Notably,the event-related subgenomes in the basal genomes all showed genomic fractionation bias,suggesting a likely allopolyploid nature of the RCT,PST and T-Alpha and T-Beta events in Tetracentron sinense.In addition,we detected that the sixteen P450 subfamilies were markedly expanded in the genomes of Ranunculales,and most of them were related to the RCT and PST events.We constructed a new platform for Early Eudicot Comparative Genomic Research(http://www.cgrpoee.top/index.html)to store more information.In summary,our findings support the WGD of C.chinensis shared by Ranunculales,which is likely an allotetraploidization event.This present effort offered new insights into the evolution of key polyploidization events and the genes related to secondary metabolites during the diversification of early eudicots.

ExosomalmicroRNA let-7c-5p enhances cell malignant characteristics by inhibiting TAGLN in oral cancer

Background:Oral cancer,a malignancy that is prevalent worldwide,is often diagnosed at an advanced stage.MicroRNAs(miRNAs)in circulating exosomes have emerged as promising cancer biomarkers.The role of miRNA let-7c-5p in oral cancer remains underexplored,and its potential involvement in tumorigenesis warrants comprehensive investigation.Methods:Serum samples from 30 patients with oral cancer and 20 healthy controls were used to isolate exosomes and quantify their RNA content.Isolation of the exosomes was confirmed through transmission electron microscopy.Quantitative PCR was used to assess the miRNA profiles.The effects of let-7c-5p and TAGLN overexpression on oral cancer cell viability,migration,and invasion were analyzed via CCK-8 and Transwell assays.Moreover,we conducted mRNA sequencing of exosomal RNA from exosomes overexpressing let-7c-5p to delineate the gene expression profile and identify potential let-7c-5p target genes.Results:let-7c-5p was upregulated in serumderived exosomes of patients with oral cancer.Overexpression of let-7c-5p in the TCA8113 and CAL-27 cell lines enhanced their proliferative,migratory,and invasive capacities,and overexpression of let-7c-5p cell-derived exosomes promoted oral cancer cell invasiveness.Exosomal mRNA sequencing revealed 2,551 differentially expressed genes between control cell-derived exosomes and overexpressed let-7c-5p cell-derived exosomes.We further identified TAGLN as a direct target of let-7c-5p,which has been implicated in modulating the oncogenic potential of oral cancer cells.Overexpression of TAGLN reverses the promoting role of let-7c-5p on oral cancer cells.Conclusion:Our findings highlight the role of exosomal let-7c-5p in enhancing oral cancer cell aggressiveness by downregulating TAGLN expression,highlighting its potential as a diagnostic and therapeutic strategy.

Integrated Effects of Phosphate Rock and Chemical Fertilizers on the Dynamics of Soil Bacterial in Acidic Rice Paddy Soils of Man (Ivory Coast)

In agricultural soils, phosphorus is often limited, leading farmers to employ artificial supplementation through both inorganic and organic fertilization methods due to its restricted availability. Soil fertilization has the potential to augment both the abundance and diversity of bacterial communities. Our study aimed to assess the effects of phosphate amendments, derived from natural phosphate rock, and chemical fertilizers (TSP, NPK), on the density and diversity of bacterial communities within the study plots. We developed and applied eight phosphate amendments during the initial cultivation cycle. Soil samples were collected post 1st and 2nd cultivation cycles, and the quantification of both total and cultivable phosphate-solubilizing bacteria (PSB) was conducted. Additionally, we analyzed bacterial community structure, α-diversity (Shannon Diversity Index, Evenness Index, Chao1 Index). The combination of natural phosphate rock (PR) and chemical fertilizers (TSP, NPK) significantly increased (p 7 bacteria/g dry soil) and phosphate-solubilizing bacteria (0.01 to 6.8 × 107 PSB/g dry soil) in comparison to unamended control soils. The diversity of bacterial phyla (Firmicutes, Actinobacteria, Proteobacteria, Halobacterota, Chloroflexia) observed under each treatment remained consistent regardless of the nature of the phosphate amendment applied. However, changes in the abundance of the bacterial phyla populations were observed as a function of the nature of the phosphate amendment or chemical fertilizer. It appears that the addition of excessive natural phosphate rock does not alter the number and the diversity of soil microorganisms population despite successive cultivation cycles. However, the addition of excessive chemical fertilizer reduces soil microorganisms density and structure after the 2nd cultivation cycle.

致病性猪沙门菌四环素耐药基因tetB的PCR检测

目的检测临床分离猪源致病性沙门菌四环素的耐药基因,确定沙门菌四环素耐药基因类型。方法用KB法测定分离株对四环素等21种抗生素的耐药情况,用PCR方法检测四环素耐药基因tetA、tetB、tetC、tetD、tetE、tetG及tetK,并对扩增产物进行测序。结果临床分离株全部对四环素耐药,33株沙门菌中有29株扩增出tetB特异性片段,基因序列与参考菌的同源性为99%,tetB检测与药敏试验阳性符合率为87.9%。结论tetB的PCR检测对四环素耐药性具有较高的特异性,tetB基因是决定本试验中临床分离株四环素耐药的主要基因,为四环素耐药性的分子流行病学监测提供了依据。

A型产气荚膜梭菌tetB(P)耐药基因核苷酸序列与蛋白结构分析

为了对产气荚膜梭菌tetB(P)耐药基因进行分析,通过生物软件设计引物,扩增A型产气荚膜梭菌tetB(P)耐药基因,测序后对tetB(P)基因核苷酸序列与蛋白结构进行分析.结果表明:分离株tetB(P)基因长度为1 959bp,编码652个氨基酸.与参考菌株CW92、EHE-NE18的核苷酸序列同源性依次为99.7%、99.9%,氨基酸序列同源性依次为99.4%、99.8%.分离株tetB(P)蛋白亲水性氨基酸较多,无跨膜区,含有4个N-糖基化位点,1个cAMP和cGMP依赖性蛋白激酶磷酸化位点,9个蛋白激酶C磷酸化位点,12个酪蛋白激酶Ⅱ磷酸化位点,7个N-豆寇酰化位点,1个ATP/GTP结合位点基序,1个翻译型鸟苷酸结合域,三级结构主要是由α-螺旋、β折叠和无规则卷曲形成.

Cloning and Bioinformatics Analysis of P23 Gene from Theileria sergenti

[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.

P53-inducible Gene 3(PIG-3)在弥漫性大B细胞淋巴瘤中的表达及意义

本研究旨在探究P53-inducible gene 3(PIG-3)在弥漫性大B细胞淋巴瘤(DLBCL)中的表达情况及其与淋巴瘤发病机制的相关性。应用免疫印迹(Western blot)和RT-PCR等方法,检测弥漫性大B细胞淋巴瘤患者和健康成年人PIG-3蛋白的表达情况,并判断其与淋巴瘤发病机制的相关性。结果表明,Western blot检测弥漫性大B细胞淋巴瘤细胞中PIG-3蛋白表达明显低于对照组,化疗后6个月PIG-3蛋白表达较化疗前升高。RT-PCR结果显示,扩增产物大小为1285 bp,与理论值吻合。结论:PIG-3表达下调可能与弥漫性大B细胞淋巴瘤发生密切相关,故PIG-3有可能作为弥漫性大B细胞淋巴瘤治疗及预后检测的一个重要指标。

The Promoter Hypermethylation of DAPK Gene and pl6 Gene in Sera from Chinese Non-small Cell Lung Cancer Patients

Objective： To evaluate the clinical significance of the aberrant methylation of DAPK gene and p16 gene in sera from 65 NSCLC patients from Nanjing General Hospital of Nanjing Command, China. Methods： A methylation-specific PCR （MSP） was performed for the detection of promoter hypermethylation of DAPK gene and p16 gene in blood DNA from 65 cases of NSCLC, and to analyze the relation of the aberrant methylation of DAPK gene and p16 gene and the clinicopathological data. Results： 30.8% （20/65） of the sera from 65 cases of NSCLC showed hypermethylation for DAPK promoter and 43.1% （28/65） the same for p16 promoter, whereas no methylated DAPK gene promoter and p16 gene promoter were found in sera from the patients with lung benign diseases and normal controls. Methylated DAPK gene promoter and p16 gene promoter in sera were not closely correlated with the pathological classification, stage, metastasis and differentiation in NSCLC. Conclusion： Detection of the aberrant methylation of DAPK gene and p16 gene in blood DNA from NSCLC patients might offer an effective means for the earlier auxiliary diagnosis of the malignancy.

Cloning and Prokaryotic Expression of P23 Major Surface Protein Gene from Theileria sergenti

[Objective] The aim was to study cloning and prokaryotic expression of P23 major surface protein gene of Theileria sergenti. [Method] A pair of specific primers was designed according to the sequence of P23 major surface protein of T. sergenti (D84447).The P23 gene was amplified by PCR from genomic DNA of T. sergenti and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-P23. Positive clones were identified by PCR screening and restriction digestion. A recombinant expression plasmid pGEX-4T-P23 was constructed by subcloning the cloned P23 gene into the linearized pGEX-4T-1 vector and transformed into E. coli BL21. After introduction by IPTG,the expressed fusion protein was identified by SDS-PAGE and Western-blotting. [Result] The cloned gene has a total length of 507 bp. Sequencing result showed that the nucleotide sequence of the cloned P23 gene shared 99.4% identity with that of P23 published in GenBank (D84447). The expressed fusion protein was 46 ku in molecular mass. Induction opportunity of zhours after culture inoculation was the best,the induction time of 6 h was the best,and induction temperature of 34 ℃ was the best as well,IPTG of 1 mmol/L had little effect on the expression. Western-blotting indicated that recombinant protein was recognized by specific antibody. [Conclusion] This study would lay a foundation for further research on the prevention and diagnose of T. sergenti.

Construction of DNA Vaccine for FMDV P1 Gene and Immunization Experiment

[Objective] This study aimed to construct DNA vaccine of foot-and-mouth disease （FMD）.[Method] Plasmid carriers plESZP1 and pUTK3CP1 with PRV were constructed for FMDV P1 gene expression.Mice were immunized,and their antibody level was detected.The two eukaryotic expression plasmids constructed were transfected into Vero cells.PCR,IFA and Westem-blot were carried out to detect the transcription and expression of the objective gene.Balb/C mice were intramuscularly inoculated with the DNA plasmid which expressed the target gene correctly,and the antibody level in mice was detected by the means of ELISA and serum neutralization （SN）.[Result] DNA plasmid carrying P1 gene which encodes FMDV capsid protein caused specific body fluid immunoreaction in mice,and the antibody level of anti-FMDV had no difference in the mice induced by the two recombinant plasmids.[Conclusion] This study lays a foundation for evaluating the genetically modified vaccine by immunizing animals with recombinant PRV containing the FMDV P1 gene and recombinant virus.