细胞周期蛋白激酶抑制调控蛋白p21在HHV-8病毒裂解复制周期的作用

目的研究细胞周期蛋白激酶抑制调控蛋白p21对人类疱疹病毒8型(human herpesvirus 8,HHV-8)病毒裂解复制周期的影响。方法组蛋白去乙酰化酶(histone deacetylase,HDAC)抑制剂SAHA预处理后,倒置荧光显微镜观察红色荧光蛋白(RFP)阳性的iSLK.219细胞数,实时定量PCR检测TREx-K-Rta BCBL-1细胞中HHV-8病毒相关基因的mRNA水平。脂质体转染p21-siRNA后,免疫印迹法检测iSLK.219和TREx-K-Rta BCBL-1细胞中p21蛋白表达,计算RFP阳性iSLK.219细胞百分率,检测TREx-K-Rta BCBL-1细胞中ORF50和PAN的mRNA水平,CCK-8法和台盼蓝染色观察细胞存活情况。结果SAHA显著增强iSLK.219细胞RFP阳性率、TREx-K-Rta BCBL-1细胞中HHV-8裂解复制周期相关基因ORF50、PAN及K8.1的mRNA水平和p21蛋白表达,差异有统计学意义(P<0.05)。siRNA沉默p21后,iSLK.219细胞RFP阳性率、TREx-K-Rta BCBL-1细胞中HHV-8裂解复制周期相关基因ORF50和PAN mRNA水平显著下降,差异有统计学意义(P<0.05),且保护SAHA介导的TREx-K-Rta BCBL-1细胞死亡。结论抑制HDAC活性通过调控p21促进HHV-8病毒裂解复制。

PD-L1和CD8在HER2阳性乳腺癌患者中的表达及其与病理参数相关性分析

目的通过评估程序性死亡受体配体1(programmed cell death-ligand 1,PD-L1)和分化群8(cluster of differentiation 8,CD8)在肿瘤微环境中的表达情况及其与患者临床病理特征的相关性,明确PD-L1和CD8在判断人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)阳性乳腺癌患者预后方面的作用。方法选取2016年10月至2018年10月山东省滕州市中心人民医院收治的HER2阳性乳腺癌患者65例,所有患者均为女性。癌组织切片经免疫组织化学方法标记PD-L1和CD8;收集患者的临床病理资料,包括年龄、组织学分级、P53、KI-67、雌激素受体、孕激索受体、肿瘤淋巴结转移分类(tumor node metastasis classification,TNM)分期在内的7项与预后相关的临床病理参数。分别分析PD-L1和CD8的表达是否和上述临床病理参数相关。结果PD-L1阳性表达率为21.5%,PD-L1阳性表达与组织学分级和TNM分期相关,差异有统计学意义(χ^(2)=6.250,P=0.044;χ^(2)=13.730,P=0.001);CD8阳性表达率为21.5%,CD8表达与组织学分级和TNM分期相关,差异有统计学意义(χ^(2)=8.023,P=0.018;χ^(2)=6.117,P=0.047)。结论PD-L1和CD8表达与乳腺癌组织学分级和TNM分期相关,可能是HER2阳性乳腺癌患者重要的预后标志物。

血清TNFAIP8、HE4及FSH在卵巢癌中的意义及相关性研究

目的探究血清肿瘤坏死因子-α诱导蛋白8(Tumor Necrosis Factor-αInducing Protein 8,TNFAIP 8)、人附睾蛋白-4(Human Epididymal Protein-4,HE4)、卵泡刺激素(Follicle Stimulating Hormone,FSH)在卵巢癌中的表达意义,并分析上述指标相关性及在卵巢癌诊断中的意义。方法选择2019年8月—2022年10月于聊城市传染病医院确诊为卵巢癌的88例患者为研究组,另取同期于聊城市传染病医院确诊为良性卵巢病变的50例患者为对照组,对比两组患者血清TNFAIP8、HE4及FSH水平差异,按照病理检测结果将研究组患者进行FIGO分期,并对比不同分期卵巢癌患者血清TNFAIP 8、HE4及FSH水平差异,采用Pearson相关性分析探究卵巢癌患者血清TNFAIP8、HE4及FSH相关性,并通过绘制ROC曲线的方式,计算血清TNFAIP 8、HE4及FSH对卵巢癌的诊断效能。结果研究组患者血清TNFAIP 8、HE4及FSH水平均明显高于对照组,差异有统计学意义(P<0.05);按照病理检测结果将研究组患者分为Ⅰ+Ⅱ期亚组和Ⅲ+Ⅳ期亚组,对比显示Ⅲ+Ⅳ期亚组患者血清TNFAIP8、HE4及FSH水平均显著高于Ⅰ+Ⅱ期亚组,差异有统计学意义(P<0.05);Pearson相关性分析显示,卵巢癌患者血清TNFAIP8水平与其HE4、FSH水平呈现正相关关系(r=0.221,P<0.05;r=0.594,P<0.05),HE4水平与其FSH水平呈现正相关关系(r=0.163,P<0.05);分别绘制血清TNFAIP 8、HE4及FSH水平鉴别卵巢癌ROC曲线,计算其AUC分别为0.924(95%CI:0.969~0.979,P<0.05)、0.909(95%CI:0.850~0.967,P<0.05)、0.757(95%CI:0.659~0.856,P<0.05)。结论卵巢癌患者血清TNFAIP8、HE4及FSH水平会出现异常升高表现,且与患者的病理分期可能存在相关性,可以考虑将上述因子应用于卵巢癌的鉴别诊断中。

基于CRISPR/Cas9技术的HCT-8细胞基因编辑系统构建

【目的】构建人回盲肠癌(human ileocecal adenocarcinoma,HCT-8)细胞系的CRISPR/Cas9基因编辑系统,实现对目的基因的高效编辑。【方法】采用慢病毒感染的方式,将慢病毒载体质粒LentiCas9-Blast与包装质粒pSPAX2和pMD2.G共转染至人胚胎肾(human embryonic kidney 293T,HEK293T)细胞系获得高滴度的重组慢病毒液,并感染HCT-8细胞系,以未感染组为阴性对照,用杀稻瘟菌素进行抗性筛选,直至阴性对照组全部死亡;采用有限稀释法对阳性细胞进行单克隆筛选,对筛选得到的单克隆细胞系进行扩大培养,并采用PCR及Western Blot试验验证Cas9基因及其蛋白表达情况,而后对阳性单克隆细胞系进行基因编辑效率及细胞系活性验证。【结果】得到能成功表达Cas9蛋白的6株HCT-8-Cas9单克隆细胞系。其中HCT-8-Cas9_2和HCT-8-Cas9_4细胞系蛋白量表达较高;对所筛选单克隆细胞系进行基因编辑效率检测,pXPR_011慢病毒表达报告基因载体系统检测结果表明,HCT-8-Cas9_4单克隆细胞系Cas9基因编辑效率为48.82%,显著高于其他HCT-8-Cas9单克隆细胞系;单克隆细胞系活性检测结果表明,HCT-8-Cas9_2和HCT-8-Cas9_4单克隆细胞系细胞活性与HCT-8细胞系相比均无显著差异。【结论】HCT-8-Cas9_4细胞系能够稳定表达Cas9蛋白,具有良好的编辑效率,可用于后续靶标基因的高效编辑。

Genotypic analysis on the ORF-K1 gene of human herpesvirus 8 from patients with Kaposi's sarcoma in Xinjiang,China

Human herpesvirus 8 （HHV-8） is thought to be essential for the development of all forms of Kaposi＇s sarcoma （KS）. HHV-8 DNA is present virtually in all KS tumor biopsy samples. Genes at both ends of the HHV-8 genome have been shown to vary considerably. Seven major molecular subtypes of HHV-8 were defined based on the amino acid sequence of the open reading frame K1 （ORF-K1）, generally known as A, B, C, D, E, F, and Z. Most strains collected worldwide were clustered into two subtypes （A and C）. Here, the K1/VRI region of HHV-8 was amplified by nested PCR in 22 （81.48%） of 27 cases from Xinjiang Uygur Autonomous Region, a province in northwestern China. Phylogenetic analysis on the basis of the K1/VR1 amino acid sequence indicated that the majority of these KS patients were infected by subtype C HHV-8 （n = 18, including 15 belonging to the C2 group）, and several by subtype A （n = 4, including 3 being the A1 group）. This is the first report of subtype A HHV-8 in China. Furthermore, the correlations between different forms and lesions of KS and different subtypes of HHV-8 were analyzed. The findings showed that subtype A HHV-8 resulted in significantly more frequent mucosal KS lesions than subtype C. However, there was no obvious correlation between different forms of KS and different subtypes of HHV-8.

8q24.13q24.23微重复病儿1例的临床及遗传学分析

目的明确1例8q24.13q24.23微重复胎儿的遗传学病因及其来源,并对该胎儿做产前诊断。方法采集2020年3月厦门大学附属第一医院行无创产前筛查的1对夫妻外周血及胎儿羊水细胞行染色体G显带分析;孕妇外周血行无创产前筛查;胎儿羊水细胞行染色体微阵列分析。结果父亲染色体核型正常,母亲和胎儿染色体核型46,XX,dup(8)(8q24.13q24.23);孕妇无创产前筛查:8号染色体长臂8q24.13q24.23存在11.3 Mb的重复(chr8:g.126215101-139315100dup)。染色体微阵列分析:arr[GRCh37]8q24.13q24.23(126646442-137947833)x3。结论8q24.13q24.23微重复为偏良性的拷贝数变异(CNV),建议孕妇继续妊娠。

Human herpesvirus-8 positive iatrogenic Kaposi's sarcoma in the setting of refractory ulcerative colitis

Although Kaposi sarcoma(KS) has been more traditionally considered an AIDS-defining illness,it may also be seen in individuals on immunosuppresive therapy.We report a case of a patient who presented to the hospital in the setting of increasingly refractory ulcerative colitis.Computed tomography scan of the abdomen was consistent with sigmoid diverticulititis and blood cultures were positive for Klebsiella.After a course of antibiotics with resolution of infection,a colonoscopy was performed to evaluate his diverticulitis and incidentally revealed a new rectal tumor.Immunohistochemistry showed the tumor was consistent with KS,with cells staining strongly positive for human herpesvirus-8.This case not only illustrates a rare case of KS found in an HIV-negative individual,but it also highlights the importance of considering an alternative diagnosis in a patient refractory to medical treatment.We discuss the management and care of an ulcerative colitis patient diagnosed with KS on immunosuppressive therapy.

Human leukocyte antigen DQ2/8 prevalence in non-celiac patients with gastrointestinal diseases

AIM: To investigate the prevalence of human leukocyte antigen (HLA) DQ2/8 alleles in Southern Italians with liver and gastrointestinal (GI) diseases outside of celiac disease. METHODS: HLA DQ2/8 status was assessed in 443 patients from three ambulatory gastroenterology clinics in Southern Italy (University of Federico Ⅱ, Naples, Loreto Crispi Hospital, Ruggi D'Aragona Hospital, Salerno). Patients were grouped based on disease status [pre-post transplant liver disease, esophageal/gastric organic and functional diseases, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD)] and DQ2/8 alleles, which correspond to a celiac disease genetic risk gradient. Subject allele frequencies were compared to healthy Italian controls. RESULTS: One hundred and ninety-six out of four hundred and forty-three (44.2%) subjects, median age 56 years and 42.6% female, were DQ2/8 positive. When stratifying by disease we found that 86/188 (45.7%) patients with liver disease were HLA DQ2/8 positive, 39/73 (53.4%) with functional upper GI diseases and 19/41 (46.3%) with organic upper GI diseases were positive. Furthermore, 38/105 (36.2%) patients with IBS and 14/36 (38.9%) with IBD were HLA DQ2/8 positive (P = 0.21). Compared to healthy controls those with functional upper GI diseases disorders had a 1.8 times higher odds of DQ2/8 positivity. Those with liver disease had 1.3 times the odds, albeit not statistically significant, ofDQ2/8 positivity. Both those with IBS and IBD had a lower odds of DQ2/8 positivity compared to healthy controls. CONCLUSION: The proportion of individuals HLA DQ2/8 positive is higher in those with liver/upper functional GI disease and lower in IBS/IBD as compared to general population estimates.

Reactivation of Latent Infection of Human Herpesvirus 8 in BC-3 Cells from Primary Effusion Lymphoma by Recombinant CytokinesSimilar to that Produced by HIV-1-infected T Cells

Objective: To study and confirm that recombinant cytokines similar to those produced by HIV-1 infected T cells induced lytic cycle replication of human herpesvirus 8 (HHV-8) in BC-3 cells, another cell line from primary effusion lymphama(PEL). Methods: The persistent stimulation of BC-3 was conducted by several cytokines known to be produced by HIV-1-infected T cells and important in growth and proliferation of Kaposi's sarcoma(KS)cells in vitro, such as the interferon-γ (IFN-γ) , tlie hepatocyte growth factor/scatter factor (HGF / SF) , the Oncostain M(OSM) , and the tumor necrosis factor-α (TNF-α)which is not produced by HIV-1-infected T cells. Treated and untreated BC-3 cells were collected at the 3rd and 7th day of persistent stimulation, respectively. Immuno-histochemical (IHC) staining, Northern blot, quantitative PCR (real- time PCR ) and electron microscopy (EM) were carried out to detect the expression of immunogenic protein ORF59, messenger RNA (mRNA) of minor capsid protein ORF26, and the presence of viral particles of HHV-8 from treated and untreated BC-3 cells. Results: It showed that IFN-γ, HGF/SF, OSM, and TNF-α were found to induce an increase in mRNA expression of ORF26 when added individually to BC-3 cells. Particularly, ORF26 expression stimulated with IFN-γ and TNF-α respectively, increased 6. 1 and 2. 5-fold(from real-time PCR results)at the 7th day when compared with untreated BC-3 cells. Meanwhile, about 20% of IFN-γ stimulated BC-3 cells expressed ORF59 at the 7th day as compared with 1. 5% of untreated BC-3 cells when IHC staining was employed. In addition, viral particles of HHV-8 were readily identified in BC-3 cells stimulated with IFN-γ at the 7th day with EM analysis. Conclusion;TNF-α and recombinant cytokines being similar to those produced by HIV- 1 infected T Cells could really induce HHV- 8 lytic cycle replication in BC-3 cells, another cell line of PEL.

Human immune suppression is inducible by trichosanthin via CD8 cell-mediated pathway

Thichosanthin(Tk), a polypeptide with 249 amino acid residues isolated and purified from a Chinese medicinal herb, showed the capability of inducing abortion and was able to inhibit tumor growth and HIV replication. Owing to sequence homology of the peptide with a ribosomeinactivating protein, the downward activity of Tk was suggested to be related to its cytotoxic property. We report here, however, that Tk could exert potent inhibitory effects on human lymphoproliferative responses in vitro to allogeneic, mitogenic and soluble antigens with 50% inhibition doses ranged between 0.05 and 0.5 μg/ml. The lowresponsiveness caused by Tk was not due to toxic cytolysis. Rather, evidences suggested that, in the dose range adopted, the Tk-induced inhibition was attributable, at least in part, to immune suppression, in view of (1) Tk was more effective in the early stage of alloreactivity; (2)Suppression also occurred if responder cells were pulsetreated with Tk rather than cocultured; (3) Irradiated Tk-pulsed cells were capable of inducing suppression in a Tk-free culture; (4) Suppression could also be transferred by the supernatants of Tk-pulsed cultured cells; (5) Tkinduced immune suppression was diminished by depletion of CD8+ cells from the culture, and, finally; (6) Adding CD8+ cells back to the culture could restore the suppres Trichosanthin-induced humall immune suppression sion. Thus the possibility that Tk might function as a down-regulator by immunological mechanisms in human immune responses is discussed.

Super-resolution immunohistochemistry study on CD4 and CD8 cells and the relation to macrophages in human cochlea

Recently, the human cochlea has been shown to contain numerous resident macrophages under steady-state. The macrophages accumulate in the stria vascularis, among the auditory nerves, and are also spotted in the human organ of Corti. These macrophages may process antigens reaching the cochlea by invasion of pathogens and insertion of CI electrode. Thus, macrophages execute an innate, and possibly an adaptive immunity. Here, we describe the molecular markers CD4 and CD8 of T cells, macrophage markers MHCⅡ and CD11b, as well as the microglial markers TEME119 and P2Y12, in the human cochlea. Immunohistochemistry and the advantageous super-resolution structured illumination microscopy(SR-SIM) were used in the study. CD4^+ and CD8^+ cells were found in the human cochleae. They were seen in the modiolus in a substantial number adjacent to the vessels, in the peripheral region of the Rosenthal's canal, and occasionally in the spiral ligament. While there are a surprisingly large number of macrophages in the stria vascularis as well as between the auditory neurons,CD4^+ and CD8^+ cells are hardly seen in these areas, and neither are seen in the organ of Corti. In the modiolus,macrophages, CD4^+ and CD8^+ cells appeared often in clusters. Interaction between these different cells was easily observed with SR-SIM, showing closely placed cell bodies, and the processes from macrophages reaching out and touching the lymphocytes. Otherwise the CD4^+ and CD8^+ cells in human cochlear tissue are discretely scattered. The possible roles of these immune cells are speculated.

Functional characterization of the human-specific （type II） form of kallikrein 8, a gene involved in learning and memory

Kallikrein 8 （KLK8） is a serine protease functioning in the central nervous system, and essential in many aspects of neuronal activities. Sequence comparison and gene expression analysis among diverse primate species identified a human-specific splice form of KLK8 （type II） with preferential expression in the human brain, which may contribute to the origin of human cognition. To gain insights into the physiological and biochemical role of this novel form, we conducted functional analyses of human type II KLK8. Our results show that type II KLK8 is abundantly expressed in human embryonic stem cells and in embryo brain samples, suggesting a potential role in embryogenesis. There are dramatic expression variations in different individuals and brain regions, which is a reflection of its dynamic role in neural activities. Furthermore, the transcription start site （TSS） of KLK8 is tissue-specific, with a brain-specific TSS found in humans indicating functional specialization. Our in vitro biochemical assay shows that there is a type II-specific intermediate protein form, although the processed end-point enzymes are the same for both type 1 and type II KLK8, suggesting that the emergence of type II KLK8 in the human brain likciy leads to functional modifications of KLK8.

Up-regulation interleukin-6 and interleukin-8 by activated protein C in lipopolysaccharide-treated human umbilical vein endothelial cells

Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.

Upregulation of stromal cell-derived factor-1 alpha/CXCR4 axis-induced migration of human neural progenitors by tumor necrosis factor-alpha and interleukin-8

BACKGROUND： Studies of several animal models of central nervous system diseases have shown that neural progenitor cells （NPCs） can migrate to injured tissues. Stromal cell-derived factor 1 alpha （SDF-la）, and its primary physiological receptor CXCR4, have been shown to contribute to this process. OBJECTIVE： To investigate migration efficacy of human NPCs toward a SDF-1α gradient, and the regulatory roles of tumor necrosis factor-α （TNF-α） and interleukin-8 （IL-8） in SDF-1α/CXCR4 axis-induced migration of NPCs. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, cellular and molecular biology study was performed at the Laboratory of Department of Cell Biology, Medical College of Soochow University between October 2005 and November 2007. MATERIALS： SDF-1α and mouse anti-human CXCR4 fusion antibody were purchased from R＆D Systems, USA. TNF-αwas purchased from Biomyx Technology, USA and IL-8 was kindly provided by the Biotechnology Research Institute of Soochow University. METHODS： NPCs isolated from forebrain tissue of 9 to 10-week-old human fetuses were cultured in vitro. The cells were incubated with 0, 20, and 40 ng/mL TNF-α, or 0, 20, and 40 ng/mL IL-8, for 48 hours prior to migration assay. For antibody-blocking experiments, cells were further pretreated with 0, 20, and 40 μg/mL mouse anti-human CXCR4 fusion antibody for 2 hours. Subsequently, the transwell assay and CXCR4 blockade experiments were performed to evaluate migration of human NPCs toward a SDF-1α gradient. Serum-free culture medium without SDF-1α served as the negative control. MAIN OUTCOME MEASURES： The transwell assay was performed to evaluate migration of human NPCs toward a SDF-1α gradient, which was blocked by fusion antibody against CXCR4. In addition, CXCR4 expression in human NPCs stimulated by TNF-α and IL-8 was measured by flow cytometry. RESULTS： Results from the transwell assay demonstrated that SDF-1α was a strong chemoattractant for human NPCs （P 〈 0.01）, and 20 ng/mL produced the highest levels of migration. Anti-human CXCR4 fusion antibody significantly blocked the chemotactic effect （P 〈 0.05）. Flow cytometry results showed that treatment with TNF-α and IL-8 resulted in increased CXCR4 expression and greater chemotaxis efficiency of NPCs towards SDF-1α（P 〈 0.01）. CONCLUSION： These results demonstrated that SDF-la significantly attracted NPCs in vitro, and neutralizing anti-CXCR4 antibody could block part of this chemotactic function. TNF-α and IL-8 increased chemotaxis efficiency of NPCs towards the SDF-1αgradient by upregulating CXCR4 expression in NPCs.

Prevalence of human herpesvirus 8 infection in patients undergoing hemodialysis using nested-PCR

Objective:To study the prevalence of HHV-8 infection in patients undergoing hemodialysis.Methods:In this study,blood samples of 89 patients undergoing hemodialysis were collected.DNA was extracted from peripheral blood mononuclear cells and HHV-8 DNA was evaluated by nested-PCR.Results:Of total 89 patients,51(57.3%)were males and 38(42.7%)were females.The patients'age ranged from 24 to 90 years and the mean age was(57.5±1.4)years.HHV-8 DNA was found in 9 of 89(10.1%)peripheral blood mononuclear cell samples,8/51(15.7%)in males and 1/38(2.6%)in females(P=0.07).All patients who were positive for HHV8-DNA were more than 50 years old.Conclusions:This study shows high prevalence of HHV-8.Since hemodialysis patients are candidates for kidney transplantation and due to the possibility of HHV8-reactivation and its serious complications in immunocompromised patients,routine screening for detection of the virus should be implemented for all hemodialysis patients.

The Role of Human Herpesvirus 8 Molecular Characterization in the Management of HIV Infected Patients Diagnosed with Malignancies Associated with Its Infection

Despite the progress has been reached with Human herpesvirus 8 (HHV-8) research, there are gaps in the knowledge of viral induced oncogenesis. The aim of the present study was to identify possible associations between HHV-8 subtypes, HHV-8 loads and clinical manifestations of HIV infected patients diagnosed with different malignancies associated with HHV-8 infection. Forty six HIV-1 infected individuals diagnosed with different HHV-8 associated diseases were studied [37 epidemic Kaposi’s sarcoma (KS), 3 pleural effusion lymphoma (PEL);5 peripheral lymphadenopathies (PL);1 Hodgkin’s lymphoma (HL);1 non Hodgkin’s lymphoma (NHL)]. HHV-8 loads were determined by quantitative real time PCR (qRT-PCR) whilst HHV-8 subtypes were determined by open-reading frame (ORF)-K1 gen genotyping. HHV-8 subtypes B, A, C, A5 and E were exhibited by 31.8%, 23.4%, 19.1%, 17% and 8.5% of the studied patients, respectively. The median HHV-8 viral load did not differ between subtypes (p > 0.05) but HHV-8 viral loads were significantly higher in PEL than in epidemic KS lesion or lymph nodes (p = 0.04). Subtype B was detected in 60% of patients with B cell lymphoma (NHL, PEL and HL) whereas subtype E was only detected in patients with epidemic KS diagnosis. Our data suggest that HHV-8 DNA quantification instead of subtype identification could be used as a surrogate marker for monitoring its infection, not only in epidemic KS patients but also in HIV infected individuals with lymphoproliferative disorders.

Establishment of method for seroepidermiological detection of human herpes virus 8 infection by using the fusion protein in the prokaryotic expression system as antigen for testing

To establish a sensitive and specific method for seroepidermiological detection of human her- pes virus 8(HHV-8)infection,three potent antigenic proteins encoded by open reading frames(ORFs) K8.1,65 and 73C in genome of HHV-8 were produced as glutathione S-transferase fusion protein in the prokaryotic expression system and was used as antigen for testing.The recombinant fusion protein ex- pressed in the prokaryotic expression vector E.coli BL21 was purified by glutathione Sepharose 4B affin- ity chromatography and was quantitated with SDS-PAGE.All these 3 fusion proteins produced in the pro- karyotic expression system showed good immunogenicity as demonstrated by Western blotting and could be recognized by mixed sera of patients with Kaposi′s sarcoma(KS).The immuno-reactivities of the single or compound fusion protein were determined by means of ELISA and compared with the traditional immu- nofluorescence assay(IFA)to determine their sensitivity and specificity of the test.It was demonstrated that the sensitivity of mixed-antigen ELISA method was significantly higher than that of IFA(81.8% vs 34.4%),while the specificity of the former was demonstrated to be 97.9%.The coincidence of the de- tection rate between these two methods was considerably high,approaching up to 90.0%.These results suggest that the mixed antigen ELISA assay appears to be a sensitive and specific method for sero-epide- miological detection of human herpesvirus 8 infection.

高危型HPV阳性患者血清lncRNA SNHG8表达对宫颈癌的预测价值

目的研究高危型人乳头瘤病毒(HPV)阳性患者血清长链非编码RNA核仁小RNA宿主基因8(lncRNASNHG8)表达水平对宫颈癌的预测价值。方法纳入2019年1月至2021年12月郑州大学附属郑州中心医院收治的82例高危型HPV阳性宫颈癌患者为病例组,纳入因宫颈良性病变行手术切除的78例高危型HPV阳性患者作为对照组。检测两组血清lncRNA SNHG8表达水平及患者高危型HPV;采用多因素Logistic回归分析高危型HPV阳性患者发生宫颈癌的影响因素;绘制ROC曲线分析血清lncRNASNHG8表达水平对高危型HPV阳性患者发生宫颈癌的预测价值。结果病例组血清lncRNASNHG8表达水平及年龄≥50岁患者比例显著高于对照组,差异均有统计学意义(P<0.05)。lncRNASNHG8是高危型HPV阳性患者发生宫颈癌的独立危险因素(P<0.05)。血清lncRNASNHG8预测高危型HPV阳性患者发生宫颈癌的曲线下面积为0.835,敏感度为0.756,特异性为0.910。FIGO分期Ⅲ期+Ⅳ期、低分化、淋巴结转移的宫颈癌患者血清lncRNASNHG8表达水平显著高于FIGO分期Ⅰ期+Ⅱ期、中/高分化、无淋巴结转移的患者,差异均有统计学意义(P<0.05)。结论血清lncRNASNHG8表达水平与高危型HPV阳性患者发生宫颈癌有关。

PEG10对人类绒毛滋养层细胞系HTR-8/Svneo细胞增殖和凋亡影响的初步研究

目的初步研究父系表达印记基因PEG10低表达对人类绒毛滋养层细胞系HTR-8/Svneo细胞增殖和凋亡的影响。方法构建4组PEG10干扰载体[si-898、si-1854、si-1951及对照(si-NC)],然后转染到HTR-8/Svneo细胞株并进体外培养,si-NC组为对照组。流式细胞术检测HTR-8/Svneo细胞的细胞周期和细胞凋亡,qRT-PCR检测Cytochrome C、Caspase3、Bid、Bak、Bax及Cyclin D1 mRNA的表达情况,MTT法检测细胞增殖活力。结果与对照组比较,转染3组siRNA片段均能显著抑制PEG10基因的表达(P<0.05),其中si-898、si-1854抑制作用更显著(P<0.01)。转染si-898或si-1854的HTR-8/Svneo细胞Cytochrome C、Caspase3、Bid、Bak、Bax mRNA的表达显著升高(P<0.01),而Cyclin D1 mRNA的表达显著降低(P<0.05),并且细胞凋亡率也显著高于对照组(P<0.05)。MTT法检测细胞增殖活力显示,与对照组相比,干扰PEG10基因后细胞在48 h和72 h的增殖活力也显著降低(P<0.05)。流式细胞术检测显示,干扰PEG10基因后细胞周期阻滞在G1期。结论干扰PEG10基因能诱导人类绒毛滋养层细胞系HTR-8/Svneo细胞凋亡,降低细胞增殖活力,阻止细胞周期从G1期到S期的转化进程,导致细胞周期阻滞。

阿司匹林对缺氧诱导人HTR-8/SVneo滋养细胞凋亡及sFlt-1、HIF-1α表达的影响

目的:探讨阿司匹林对缺氧诱导人HTR-8/SVneo滋养细胞凋亡及可溶性类fms酪氨酸激酶-1(sFLT-1)、低氧诱导因子1α(HIF-1α)表达的影响。方法:人HTR-8/SVneo滋养细胞分为正常对照组、缺氧模型组(2%O_(2)、5%CO_(2)、93%N_(2))、阿司匹林低(0.05mmol/L)、中(0.1mmol/L)、高剂量组(0.2mmol/L);培养结束后,MTT法测定细胞活力,流式细胞仪测定细胞凋亡水平及细胞周期,血清培养基基质胶培养测定细胞血管形成能力,RT-PCR法及蛋白印记法测定细胞sFlt-1、HIF-1α水平。结果:缺氧模型组OD值、存活率、血管形成能力、HIF-1α mRNA和蛋白表达均低于正常对照组及阿司匹林各剂量组,但随着阿司匹林剂量增上述各指标逐渐降低;缺氧模型组凋亡率、G1期、sFlt-1 mRNA和蛋白表达均高于正常对照组和阿司匹林各剂量组,但随着阿司匹林剂量增加凋亡率、G1期、sFlt-1 mRNA和蛋白表达逐渐升高(均P<0.05)。结论:低剂量阿司匹林可促进缺氧环境下HTR-8/SVneo细胞增殖、血管形成,抑制细胞凋亡。其机制可能与低剂量阿司匹林能抑制缺氧环境下HTR-8/SVneo细胞sFlt-1表达,促进HIF-1α表达有关。