Study of transactivating effect of pre-S2 protein of hepatitis B virus and cloning of genes transactivated by pre-S2 protein with suppression subtractive hybridization

AIM： To investigate the transactivating effect of pre-S2 protein of hepatitis B virus （HBV） and construct a subtractive cDNA library of genes transactivated by pre-S2 protein with suppression subtractive hybridization （SSH） technique, and to pave the way for elucidating the pathogenesis of HBV infection. METHODS： pcDNA3.1（-）-pre-S2 containing pre-S2 region of HBV genome was constructed by routine molecular methods. HepG2 cells were cotransfected with pcDNA3.1 （-）-pre-S21pSV-lacZ and empty pcDNA3.1（-）/pSV-lacZ. After 48 h, cells were collected and detected for the expression of β-galactosidase （β-gal）. SSH and bioinformatics techniques were used, the mRNA of HepG2 cells transfected with pcDNA3.1（-）-pre-S2 and pcDNA3.1（-） empty vector was isolated, respectively, cDNA was synthesized. After digestion with restriction enzyme RsaI, cDNA fragments were obtained. Tester cDNA was then divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5α The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. RESULTS： The pre-S2 mRNA could be detected in HepG2 cells transfected with pcDNA3.1（-）-pre-S2 plasmid. The activity of β-gal in HepG2 cells transfected with pcDNA3.1 （-）-pre-S2/pSV-lacZ was 7.0 times higher than that of control plasmid （P〈0.01）. The subtractive library of genes transactivated by HBV pre-S2 protein was constructed successfully. The amplified library contains 96 positiveclones. Colony PCR showed that 86 clones contained 200-1 000 bp inserts. Sequence analysis was performed in 50 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 25 coding sequences were obtained, these cDNA sequences might be the target genes transactivated by pre-S2 protein. CONCLUSION： The pre-S2 protein of HBV has transactivating effect on SV40 early promoter. The obtained sequences may be target genes transactivated by pre-S2 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity, signal transcluction and cell apoptosis.This finding brings some new clues for studying the biological functions of pre-S2 protein and further understanding of HBV hepatocarcinogesis.

Low-density lipoprotein receptor-related protein 2(LRP2)is required for lipid export in the midgut of the migratory locust,Locusta migratoria

Low-density lipoprotein receptor-related protein 2(LRP2)is a multifunctional endocytic receptor expressed in epithelial cells.In mammals,it acts as an endocytic receptor that mediates the cellular uptake of cholesterol-containing apolipoproteins to maintain lipid homeostasis.However,little is known about the role of LRP2 in lipid homeostasis in insects.In the present study,we investigated the function of LRP2 in the migratory locust Locusta migratoria(LmLRP2).The mRNA of LmLRP2 is widely distributed in various tissues,including integument,wing pads,foregut,midgut,hindgut,Malpighian tubules and fat body,and the amounts of LmLRP2 transcripts decreased gradually in the early stages and then increased in the late stages before ecdysis during the nymphal developmental stage.Fluorescence immunohistochemistry revealed that the LmLRP2 protein is mainly located in cellular membranes of the midgut and hindgut.Using RNAi to silence LmLRP2 caused molting defects in nymphs(more than 60%),and the neutral lipid was found to accumulate in the midgut and surface of the integument,but not in the fat body,of dsLmLRP2-treated nymphs.The results of a lipidomics analysis showed that the main components of lipids(diglyceride and triglyceride)were significantly increased in the midgut,but decreased in the fat body and hemolymph.Furthermore,the content of total triglyceride was significantly increased in the midgut,but markedly decreased in the fat body and hemolymph in dsLmLRP2-injected nymphs.Our results indicate that LmLRP2 is located in the cellular membranes of midgut cells,and is required for lipid export from the midgut to the hemolymphand fat body in locusts.

Exploration of cyclooxygenase-2 inhibitory peptides from walnut dreg proteins based on in silico and in vitro analysis

Walnut dreg protein hydrolysates(WDPHs)exhibit a variety of biological activities,however,the cyclooxygenase-2(COX-2)inhibitory peptide of WDPHs remain unclear.The aim of this study was to rapidly screen for such peptides in WDPHs through a combination of in silico and in vitro analysis.In total,1262 peptide sequences were observed by nano liquid chromatography/tandem mass spectrometry(nano LC-MS/MS)and 4 novel COX-2 inhibitory peptides(AGFP,FPGA,LFPD,and VGFP)were identified.Enzyme kinetic data indicated that AGFP,FPGA,and LFPD displayed mixed-type COX-2 inhibition,whereas VGFP was a non-competitive inhibitor.This is mainly because the peptides form hydrogen bonds and hydrophobic interactions with residues in the COX-2 active site.These results demonstrate that computer analysis combined with in vitro evaluation allows for rapid screening of COX-2 inhibitory peptides in walnut protein dregs.

GATA binding protein 2 mediated ankyrin repeat domain containing 26 high expression in myeloid-derived cell lines

BACKGROUND Thrombocytopenia 2,an autosomal dominant inherited disease characterized by moderate thrombocytopenia,predisposition to myeloid malignancies and normal platelet size and function,can be caused by 5’-untranslated region(UTR)point mutations in ankyrin repeat domain containing 26(ANKRD26).Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1)have been identified as negative regulators of ANKRD26.However,the positive regulators of ANKRD26 are still unknown.AIM To prove the positive regulatory effect of GATA binding protein 2(GATA2)on ANKRD26 transcription.METHODS Human induced pluripotent stem cells derived from bone marrow(hiPSC-BM)INTRODUCTION Ankyrin repeat domain containing protein 26(ANKRD26)acts as a regulator of adipogenesis and is involved in the regulation of feeding behavior[1-3].The ANKRD26 gene is located on chromosome 10 and shares regions of homology with the primate-specific gene family POTE.According to the Human Protein Atlas database,the ANKRD26 protein is localized to the Golgi apparatus and vesicles,and its expression can be detected in nearly all human tissues[4].Moreover,UniProt annotation revealed that ANKRD26 is localized in the centrosome and contains coiled-coil domains formed by spectrin helices and ankyrin repeats[5,6].The most common disease related to ANKRD26 is thrombocytopenia 2(THC2),which is a rare autosomal dominant inherited disease characterized by lifelong mild-to-moderate thrombocytopenia and mild bleeding[7-9].Caused by the variants in the 5’-untranslated region(UTR)of ANKRD26,THC2 is defined by a decrease in the number of platelets in circulating blood and results in increased bleeding and decreased clotting ability[8,10].Due to the point mutations that occur in the 5’-UTR of ANKRD26,its negative transcription factors(TFs),Runt related transcription factor 1(RUNX1)and friend leukemia integration 1(FLI1),lose their repression effect[11].The persistent expression of ANKRD26 increases the activity of the mitogen activated protein kinase and extracellular signal regulated kinase 1/2 signaling pathways,which are potentially involved in the regulation of thrombopoietin-dependent signaling and further impair proplatelet formation by megakaryocytes(MKs)[11].However,the positive regulators of ANKRD26,which might be associated with THC2 pathology,are still unknown.

Neural Wiskott-Aldrich syndrome protein(N-WASP)promotes distant metastasis in pancreatic ductal adenocarcinoma via activation of LOXL2

Pancreatic ductal adenocarcinoma(PDAC)is one of the most aggressive solid malignancies.A specific mechanism of its metastasis has not been established.In this study,we investigated whether Neural Wiskott-Aldrich syndrome protein(N-WASP)plays a role in distant metastasis of PDAC.We found that N-WASP is markedly expressed in clinical patients with PDAC.Clinical analysis showed a notably more distant metastatic pattern in the N-WASP-high group compared to the N-WASP-low group.N-WASP was noted to be a novel mediator of epithelialmesenchymal transition(EMT)via gene expression profile studies.Knockdown of N-WASP in pancreatic cancer cells significantly inhibited cell invasion,migration,and EMT.We also observed positive association of lysyl oxidase-like 2(LOXL2)and focal adhesion kinase(FAK)with the N-WASP-mediated response,wherein EMT and invadopodia function were modulated.Both N-WASP and LOXL2 depletion significantly reduced the incidence of liver and lung metastatic lesions in orthotopic mouse models of pancreatic cancer.These results elucidate a novel role for N-WASP signaling associated with LOXL2 in EMT and invadopodia function,with respect to regulation of intercellular communication in tumor cells for promoting pancreatic cancer metastasis.These findings may aid in the development of therapeutic strategies against pancreatic cancer.

Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis

BACKGROUND Recently,type 2 diabetic osteoporosis(T2DOP)has become a research hotspot for the complications of diabetes,but the specific mechanism of its occurrence and development remains unknown.Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP.Polycytosine RNA-binding protein 1(PCBP1),an iron ion chaperone,is considered a protector of ferroptosis.AIM To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes.METHODS A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose(HG)and/or ferroptosis inhibitors at different concentrations and times.Transmission electron microscopy was used to examine the morpho-logical changes in the mitochondria of osteoblasts under HG,and western blotting was used to detect the expression levels of PCBP1,ferritin,and the ferroptosis-related protein glutathione peroxidase 4(GPX4).A lentivirus silenced and overex-pressed PCBP1.Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin(OPG)and osteocalcin(OCN),whereas flow cytometry was used to detect changes in reactive oxygen species(ROS)levels in each group.RESULTS Under HG,the viability of osteoblasts was considerably decreased,the number of mitochondria undergoing atrophy was considerably increased,PCBP1 and ferritin expression levels were increased,and GPX4 expression was decreased.Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1,increased the expression levels of ferritin,GPX4,OPG,and OCN,compared with the HG group.Flow cytometry results showed a reduction in ROS,and an opposite result was obtained after silencing PCBP1.CONCLUSION PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment.Moreover,PCBP1 may be a potential therapeutic target for T2DOP.

Glucokinase regulatory protein rs780094 polymorphism is associated with type 2 diabetes mellitus, dyslipidemia, non-alcoholic fatty liver disease, and nephropathy

In this editorial,we comment on the article by Liu et al published in the recent issue of the World Journal of Diabetes(Relationship between GCKR gene rs780094 polymorphism and type 2 diabetes with albuminuria).Type 2 diabetes mellitus(T2DM)is a chronic disorder characterized by dysregulated glucose homeostasis.The persistent elevated blood glucose level in T2DM significantly increases the risk of developing severe complications,including cardiovascular disease,re-tinopathy,neuropathy,and nephropathy.T2DM arises from a complex interplay between genetic,epigenetic,and environmental factors.Global genomic studies have identified numerous genetic variations associated with an increased risk of T2DM.Specifically,variations within the glucokinase regulatory protein(GCKR)gene have been linked to heightened susceptibility to T2DM and its associated complications.The clinical trial by Liu et al further elucidates the role of the GCKR rs780094 polymorphism in T2DM and nephropathy development.Their findings demonstrate that individuals carrying the CT or TT genotype at the GCKR rs780094 locus are at a higher risk of developing T2DM with albuminuria compared to those with the CC genotype.These findings highlight the importance of genetic testing and risk assessment in T2DM to develop effective preventive strategies and personalized treatment plans.

Targeting neuronal PAS domain protein 2 and KN motif/ankyrin repeat domains 1:Advances in type 2 diabetes therapy

This editorial summarizes the latest literature on the roles of neuronal PAS domain protein 2 and KN motif/ankyrin repeat domain 1 in type 2 diabetes(T2D).We highlight their involvement inβ-cell dysfunction,explore their potential as therapeutic targets,and discuss the implications for new treatment strategies.We offer valuable insights into relevant gene regulation and cellular mechanisms relevant for the targeted management of T2D.

Molecular Docking Studies of Botanical Beverage Mix Berries (LIFEGREENTM) against Breast Cancer Cells from Targeted Protein 1QQG, 7B5Q & 7B5O & Uterine Fibroid from Targeted Protein 2AYR, 6T41 & 3GRF

Fibroids, also called leiomyomas or myomas, are communal tumors of the muscle or uterine wall that affect about 20% of females who are of reproductive age. They can look as if singly or in clusters, and they often cease to grow after menopause. Fibroids can be classified as intramural, sub serosal, pedunculated, or submucosal based on where they are positioned in the uterus. Although fibroids are benign, they can grow quickly and cause a range of symptoms, such as pelvic pressure, heavy menstrual flow, and infertility. As a result, fibroids are a main reason behind hysterectomy surgeries. The majority of cases of breast cancer are ductal and lobular cancers, making it the second utmost common cancer in women international. Gene mutations like those in BRCA1 or BRCA2 knowingly raise the risk of breast and other cancers, typically with an earlier cancer onset. Cancer risk is influenced by a complex interplay of genetic abnormalities, environmental factors, and lifestyle selections. Further research into these relations is domineering. Although they are common in uterine leiomyomas, especially multiple leiomyomas, MED12 mutations do not significantly correlate with tumor size. These mutations have also been noticed in smooth muscle tumors and leiomyosarcomas, two other types of uterine cancer. The identification of MED12 mutations as the sole genetic abnormality originates in leiomyomas raises the opportunity of a role in the genesis of cancer. 10% - 15% of women who are of reproductive age have endometriosis, which grants serious difficulties because of its chronic nature and range of clinical symptoms. Even after effective surgeries, issues reoccur often, adding to the enormous financial burden. The effects of MED12 mutations have been experiential in recent studies examining the molecular causes of endometriosis-associated infertility, which have shown anomalies in cellular connections and signaling cascades. Computational techniques were used in this study to investigate LifeGreenTM’s potential to prevent uterine fibroids and breast cancer. The efficacy of LifeGreenTM as a preventive measure or a treatment for common gynecological matters was examined and modeled. We investigated the mechanisms underlying LifeGreenTM’s benefits in the treatment of uterine fibroids and breast cancer using computational techniques. Our research contributes to our understanding of its potential therapeutic benefits for women’s health.

Pre-S1Ag、Pre-S2Ag及HBV-LP在乙肝病毒检测中的性能

目的对三种HBV表面蛋白(Pre-S1 Ag、Pre-S2 Ag和HBV-LP)在HBV检测中的性能进行比较。方法通过对乙肝患者(包括HBsAg携带者和HBV DNA阳性患者)及健康人群Pre-S1 Ag、Pre-S2 Ag和HBV-LP的检测,对比分析其灵敏度、特异性和检出限。结果在进行HBV筛查时,三种表面蛋白特异性保持在99%~100%,反观Pre-S2 Ag和HBV-LP在检测HBsAg携带者、HBV-DNA阳性患者时,其敏感性表现要比Pre-S1 Ag更高(P<0.001)。三种表面蛋白吸光度与HBV-DNA水平呈正相关(P<0.001),Pre-S2 Ag和HBV-LP在HBV对应的检出限是2~3 Log10IU/mL,反观Pre-S1 Ag对HBV的检出限高于5 Log10IU/mL。结论通过Pre-S2 Ag、HBV-LP、Pre-S1 Ag对比来看,前面两者的敏感性较高,检出限较低,故可以用于HBV筛查、识别病毒的血清标志物。

Pre-sarcopenia and Mac-2 binding protein glycosylation isomer as predictors of recurrence and prognosis of early-stage hepatocellular carcinoma

BACKGROUND The Mac-2 binding protein glycosylation isomer(M2BPGi),a fibrosis marker in various liver diseases,is reportedly a prognostic marker in patients with hepatocellular carcinoma(HCC)who underwent hepatectomy.AIM To evaluate whether the M2BPGi value,M2BP,and pre-sarcopenia before radiofrequency ablation(RFA)could be useful recurrence and prognostic markers in patients with early-stage HCC.METHODS In total,160 patients with early-stage primary HCC treated with RFA were separately analyzed as hepatitis C virus(HCV)-positive and HCV-negative.Factors contributing to recurrence and liver-related death,including M2BP,M2BPGi,and skeletal muscle mass index,were statistically analyzed.Eighty-three patients were HCV-positive and 77 were HCV-negative.RESULTS In HCV-positive patients,only des-γ-carboxy-prothrombin≥23 mAU/mL was a significant poor prognostic factor affecting survival after RFA.In HCV-negative patients,M2BPGi≥1.86 cutoff index was significantly associated with tumor recurrence,while M2BP was not.M2BPGi≥1.86 cutoff index(hazard ratio,4.89;95%confidence interval:1.97-12.18;P<0.001)and pre-sarcopenia(hazard ratio,3.34,95%confidence interval:1.19-9.37;P=0.022)were independent significant poor prognostic factors in HCV-negative patients.CONCLUSION In HCV-negative patients with primary HCC treated with RFA,lower M2BPGi contributed to a lower tumor recurrence rate and longer survival period.Pre-sarcopenia contributed to the poor prognosis independently in HCV-negative patients.These factors might be useful recurrence and prognostic markers for early-stage primary HCC.

PCR检测HBV DNA与HBV血清标志物常见模式和Pre-S2相互关系研究

目的 探讨乙型肝炎病毒 (HBV)DNA与六种常见HBV血清免疫学标志物模式、前S2 蛋白抗原 (Pre -S2 )之间的相互关系及其临床检测意义。方法 采用PCR法对HBVDNA、ELISA法对HBV血清免疫学标志物、Pre -S2 同时进行检测。结果 1484例六种常见模式HBVDNA阳性率依次为 89 8% >5 5 6 % >2 1 8% >7 5 % >7 1% >6 8% ,即模式 (1) >(3) >(2 ) >(5 ) >(6 )>(4) ;而HBV血清免疫学标志阳性例数依次为 (2 ) >(1) >(3) >(5 ) >(4) >(6 )。 (2 )、(3)种模式HBVDNA与Pre S2阳性率比较P <0 0 1,其余 (1)、(4)～ (6 )种模式HBVDNA与Pre-S2 阳性检出率无显著性差异 (P >0 0 5 )。结论 HBV血清免疫学标志物、Pre -S2、HBVDNA的检测 ,三者缺一不可 ,ELISA法检测HBV血清免疫学标志物、Pre -S2 ,只是HBV的表型指标 ,提供HBV感染的间接证据。而PCR -HBVDNA的检测是HBV感染与否的直接证据。因此它们各自有其独特的临床检测意义。

Pre-S2蛋白抗体结合部位的确定

本文研究了Pre-S2蛋白体液免疫反应的精细特异性。结果表明小鼠对Pre-S2蛋白的抗体反应部位主要局限于14—24共11个连续的氨基酸残基内,与我们理论预测的B细胞表位相吻合;人的抗Pre-S2阳性血清除识别与小鼠抗血清及其Pre-S2特异性单抗同一肽段外,还识别1—13肽段。这些结果对于设计新一代合成疫苗及诊断试剂有意义。

乙型肝炎患者Pre-S1、Pre-S2抗原与HBV-DNA的相关性分析

目的探讨乙型肝炎病毒(HBV)Pre-S1、Pre-S2抗原与HBV-DNA的相关性。方法对180例标本采用ELISA方法检测前S1抗原、前S2抗原,采用荧光定量PCR方法检测HBVDNA。结果在150例HBVDNA阳性的乙肝患者中,Pre-S1、Pre-S2的阳性率分别为89.33%和86.00%;30例HBVDNA阴性的乙肝患者中,Pre-S1、Pre-S2的阳性率分别为36.67%和26.67%。HBVDNA阳性和阴性患者的Pre-S1、Pre-S2的阳性率差异有统计学意义(P<0.05)。结论随着HBVDNA载量的增加,HBV乙肝患者的Pre-S1、Pre-S2的阳性率逐渐升高。Pre-S1、Pre-S2抗原与HBVDNA载量具有高度相关性,可作为HBVDNA检测的有效补充。

乙肝病毒Pre-S_1、S_2抗原检测在乙型肝炎临床诊断中的价值

目的探讨乙肝病毒Pre-S1、S2抗原检测在乙型肝炎临床诊断中的作用及临床价值。方法对115例乙型肝炎患者采用ELISA法对乙肝五项、Pre-S1、Pre-S2抗原进行了检测,并对其结果进行回顾性分析。结果①115例HBV患者血清中的HBsAg、HBeAg、HBcAb患者,Pre-S1抗原阳性率为40.9%,其它均为零;Pre-S2抗原阳性率为78.0%。HBsAg、HBeAg、HBcAb组和HBsAg、HBeAb、HBcAb组Pre-S2与Pre-S1比较,经χ2检验,P<0.01。而63例HBsAg、HBeAb、HBcAb患者Pre-S2抗原阳性率为19.1%;11例HBsAg、HBcAb患者Pre-S2抗原阳性率为27.3%。②乙肝各项指标与Pre-S1、Pre-S2结果显示HBeAg最高,Pre-S1和Pre-S2分别是41.5%和78.0%;HBeAb检测Pre-S1无1例阳性,而Pre-S2结果12例阳性,阳性率为19.0%,Pre-S1与Pre-S2比较,经χ2检验,P<0.01,结果有显著性意义。结论PreSl、PreS2抗原与HBV呈不同程度相关性,是病毒感染、复制的指标较HBeAg敏感,是HBV存在和复制较为直接的标志,对HBV检测起重要的补充作用,对临床上判断HBV复制和疾病预后有重要的参考价值。其Pre-S2诊断乙肝患者对HBV的病毒感染、病毒复制、传染性及对不同临床类型肝炎等均优于其他检测指标。

Pre-S2蛋白抗原表位及二级结构的预测与比较

本文采用Chou和Fasman方法预测adw,adr,ayw三种亚型的乙肝病毒Pre-S2蛋白的二级结构,并用双亲性方案和亲水性方案分析了抗原表位。结果显示三者均可能含有较多的β-片层和β-转折;N-末端30个残基所形成的二级结构较保守,而C-末端顺序的构象在亚型间差别较大;Th细胞识别的表位可能在39—49肽段,B细胞识别的表位可能主要在13—18残基或其附近,为分子免疫学的深入研究提供了线索。

Pre-S_2、抗Pre-S_2和HBV DNA在乙型肝炎患者中的相关性分析

目的 :探讨Pre_S2 、抗Pre_S2 二对半和HBVDNA对乙型肝炎患者血清学的诊断意义。方法 :用ELISA法和PCR法分别检测 93例乙型肝炎患者和 2 8例健康人血清中的Pre_S2 ,抗Pre_S2 ,二对半和HBVDNA水平。结果 :93例乙肝患者HBsAg、HBeAg、HBcAb阳性 32例 ,HBsAg、HBeAb、HBcAb阳性 39例 ,HBsAg、HBcAb阳性 18例 ,HBcAb阳性 4例。Pre_S2 结果阳性率 6 6 .7% (6 2 / 93) ,Pre_S2 抗体结果阳性率 1.1% (1/ 93) ,HBVDNA结果阳性率40 .9% (38/ 93)。 2 8例健康人二对半 ,五项全阴 16例 ,HBsAb阳性 12例 ,Pre_S2 阳性 1例 ,HBVDNA阳性 1例 ,Pre_S2 抗体阳性 5例占HBsAb阳性 41.7% (5 / 12 ) ,Pre_S2 和HBVDNA相比有明显差异 (P <0 .0 1) ,非HBeAg阳性组中HBVDNA阳性率约低于Pre_S2 。结论 :Pre_S2 ,抗Pre_S2 ,HBVDNA和二对半在乙型肝炎血清学诊断中显示 ,Pre_S2 优于HBVDNA和二对半。Pre_S2 抗体出现早于抗HBs和抗HBe ,抗Pre_S2 阳性检出率较低 ,可能同乙型肝炎疫苗中抗Pre_S2 含量低有关。

Pre-S1Ag和Pre-S2Ag及HBV-LP在乙肝病毒筛查中的价值

目的:探讨乙肝病毒(HBV)前S1抗原(Pre-S1 Ag)、前S2抗原(Pre-S2 Ag)及乙肝大蛋白(HBV-LP)在乙肝病毒(HBV)筛查中的价值。方法:258例乙肝患者分为乙肝表面抗原(HBs Ag)携带组(n=182)及HBV DNA阳性组(n=76),另选100例健康体检者作为对照组;采用双抗体夹心(ELISA)法检测受检者血清Pre-S1Ag、Pre-S2 Ag及HBV-LP水平,比较Pre-S1 Ag、Pre-S2 Ag及HBV-LP筛查HBV的灵敏度和特异度,分析Pre-S1Ag、Pre-S2 Ag及HBV-LP对HBs Ag携带组3种血清学模式患者的HBV检出能力;采用线性回归法分析Pre-S1Ag、Pre-S2 Ag、HBV-LP与HBV DNA的相关性,推算3个指标的ABV病毒检出限。结果:Pre-S1 Ag、Pre-S2 Ag及HBV-LP应用于HBV筛查的特异度为99%～100%,但Pre-S2 Ag和HBV-LP筛查HBV的灵敏度显著高于Pre-S1 Ag(P <0. 01); HBe Ag阴性人群中(HBs Ag-HBeAb-HBcAb阳性和HBs Ag-HBcAb阳性模式)的Pre-S2 Ag、HBV-LP检出率均显著高于Pre-S1 Ag (P <0. 01); Pre-S1 Ag、Pre-S2 Ag及HBV-LP与HBV DNA正相关(r=0. 82、0. 76、0. 70,P <0. 01),Pre-S2 Ag、HBV-LP对HBV的检出限接近106IU/L,Pre-S1 Ag对HBV的检出限≥108IU/L。结论:Pre-S2 Ag、HBV-LP相较于Pre-S1 Ag具有更高的灵敏度和更低的检出限,更适合作为HBV筛查的血清标志物。

Pre-S1Ag、Pre-S2Ag和HBeAg联合测定用于判断乙肝病毒传染性的研究

我国乙肝病毒感染率较高,目前判断乙肝病毒感染的最主要血清标志物为乙型肝炎“两对半”,其中乙肝表面抗原（HBsAg）是乙肝病毒感染检测中最为重要的一种标志物,但HBsAg阳性只能说明感染乙肝病毒,不能反映病毒是否复 制及传染性强弱。判断乙肝病毒是否复制就要进行HBV—DNA的检测。HBV—DNA检测步骤繁琐．条件要求较高。且成本高昂，一般基层医院无法开展。我们采用EUSA方法检测乙肝病毒前S1抗原（Pre-S1Ag）和前S2抗原（Pre-S2Ag），结合乙肝病毒e抗原（HBeAg）检测结果来判断乙肝病毒传染性强弱．取得了较好的效果。

下调HMGB2表达对肝癌LM3细胞上皮-间质转化的抑制作用及其AKT/mTOR信号通路机制

目的:探讨下调肝癌细胞中高迁移率族框蛋白2 (HMGB2)表达对肝癌细胞生物学行为及上皮-间质转化(EMT)进程的影响,并阐明其作用机制。方法:对数生长期的人肝癌LM3细胞分为阴性对照组和HMGB2 RNA干扰组(HMGB2 siRNA组),分别以Lipofectamin 2000为载体转染无关序列的RNA寡核苷酸(RNA oligo)和敲除HMGB2序列的RNA oligo。采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测2组细胞中HMGB2 mRNA和蛋白表达水平,分别采用细胞划痕实验和Transwell小室实验检测2组细胞的迁移和侵袭能力,采用Western blotting法检测2组细胞中E-钙黏蛋白(E-cadherin)、 N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达水平。结果:与阴性对照组比较,HMGB2 siRNA组细胞中HMGB2 mRNA和蛋白表达水平均明显降低(P<0.05),HMGB2 siRNA组细胞划痕愈合率明显降低(P<0.01),侵袭细胞数明显减少(P<0.01),细胞中E-cadherin蛋白表达水平明显升高(P<0.01),N-cadherin、Vimentin、mTOR、AKT和磷酸化AKT (p-AKT)蛋白表达水平明显降低(P<0.05或P<0.01)。结论:下调HMGB2的表达可降低肝癌LM3细胞迁移和侵袭能力并抑制EMT,其作用机制可能与参与调节AKT/mTOR通路相关蛋白表达有关。