Dermcidin在原发性肝癌诊断及病情评估中的意义

目的明确Dermcidin(DCD)作为原发性肝癌(HCC)生物标志物的可能性。方法 82例已确诊的HCC患者纳入肝癌组,其中转移组35例,未转移组47例。33例代偿期乙型肝炎肝硬化患者纳入肝硬化组,44例健康体检者纳入健康对照组。收集血清标本,通过ELISA法检测血清中DCD的水平,并采用电化学发光免疫分析法检测甲胎蛋白(AFP)水平。结果肝癌组血清中DCD、AFP水平均明显高于健康对照组及肝硬化组,差异有统计学意义(P<0.05)。转移组患者血清中DCD的水平明显高于未转移组,差异有统计学意义(P=0.001)。而AFP水平在转移组与未转移组间比较,差异无统计学意义(P=0.697)。DCD及AFP在不同性别和不同年龄的受试者间的表达差异无统计学意义(P>0.05)。结论 DCD有望成为HCC新的血清标志物,可判断肝癌是否发生转移,DCD在不同性别和不同年龄的受试者上的表达没有明显差异,具有较广的适用性。

急性冠脉综合征早期抗栓干预治疗后血清dermcidin蛋白片段4183Da多肽的变化趋势

目的 分析急性冠脉综合征（ACS）患者早期抗栓干预治疗后不同时间点血清标本中dermcidin蛋白（也称皮离蛋白）片段4 183Da多肽水平的变化规律.方法 118例确诊的ACS患者于就诊第一时间经静脉取血后即刻嚼服阿司匹林300 mg和氯吡格雷300 mg,并根据病情,在征得患者及家属知情同意后分别采取急诊经皮冠状动脉（冠脉）介入治疗（PCI）或溶栓治疗、内科保守治疗.于抗栓治疗后2、4、6、8、10、12、16、20、24、32、40、48、60、72 h经静脉取血,分离血清,采用基质辅助激光解析离子化飞行时间质谱（MALDI-TOF-MS）技术检测血清dermcidin蛋白片段4 183Da多肽,并检测肌红蛋白（Myo）、肌钙蛋白I（cTnI）、肌酸激酶同工酶（CK-MB）水平.结果 118例ACS患者入院就诊时血清dermcidin蛋白片段4 183Da多肽相对强度的自然对数值为2.75±1.02,抗栓治疗后2 h即明显下降为1.84±1.19（P=0.005）;4 h为1.74±1.12（P=0.000）,4 h后略有回升.4 183Da多肽升高先于心肌损伤标志物.结论 阿司匹林可以显著降低血清dermcidin蛋白片段4 183Da多肽水平,此片段可以作为观察早期抗栓治疗效果的指标之一.

蛇葡萄素通过Dermcidin蛋白调节肝癌细胞株HepG2侵袭活力及侵袭基因表达的实验

目的研究蛇葡萄素(AMP)对肝癌细胞株HepG2侵袭活力及侵袭基因表达的调节作用及分子机制。方法培养肝癌细胞株HepG2后用不同剂量的AMP处理(20、40、60、80μmol/L)、转染Dermcidin的siRNA,测定细胞的迁移能力、侵袭能力以及Dermcidin、侵袭基因mRNA表达量。结果不同剂量AMP处理后细胞的迁移、侵袭数目及细胞中Dermcidin、基质金属蛋白酶(MMP)2、MMP9、MMP10mRNA水平均低于对照组,且AMP剂量越大,细胞的迁移、侵袭数目及细胞中Dermcidin、MMP2、MMP9、MMP10mRNA水平越低;80μmol/L AMP联合dermcidinsiRNA后,HepG2细胞的迁移、侵袭数目以及细胞内MMP2、MMP9、MMP10mRNA表达水平均增多。结论AMP通过下调Dermcidin蛋白来抑制肝癌细胞株HepG2的侵袭活力、降低侵袭基因的表达水平。

人源性多肽Dermcidin的特性与功能

人源性多肽dermcidin(DCD)是最近从人汗液中分离的一种天然活性多肽。它在汗腺中组成性表达并分泌到体表的汗液中,作为抗菌肽参与调节皮肤菌群结构。以前在人类神经细胞中发现的一种存活促进肽以及人体内与一种鼠恶病质因子同源的多肽均被证实为DCD衍生肽。最新研究发现dermcidin基因在多种类型肿瘤及人胎盘组织中表达,表明DCD可能作为多功能的活性多肽在肿瘤发生及人体天然免疫系统、神经系统疾病、妊娠相关疾病的病理生理过程中发挥作用。

A study on the relationship between changes in serum hs-CRP levels and Chinese ischemic stroke subclassification

Objective: To explore the effects of combined application of culture supernatant of human umbilical cord mesenchymal stem cells (hUCMSCs) and ciprofloxacin on Staphylococcus aureus (SA) in vitro. Methods: hUCMSCs were isolated from umbilical cord tissues of full-term healthy fetuses after cesarean section and then cultured. Cells in the third passage were chosen for the use of experiment after identification. SA strains isolated from wounds of burn patients in our burn wards were used in the following experiment. Cells were divided into 0, 10, 100 and 1,000 ng/ml lipopolysaccharide (LPS) groups by use of the random number table (similarly hereinafter). Cells were cultured with culture medium containing mesenchymal stem cells (MSCs) after being treated with medium containing corresponding mass concen-trations of LPS for 12 h. At post culture hour (PCH) 6, 12 and 24, 6 wells of culture supernatant of cells in each group were obtained to measure the content of LL-37 with enzyme-linked immunosorbent assay (ELISA). Ninety blood agar culture plates were divided into ciprofloxacin control group (CC), ciprofloxacin + supernatant group (CS), and ciprofloxacin + supernatant+ LL-37 antibody group (CSL), with 30 blood agar culture plates in each group. Blood agar culture plates in group CC were coated with 1.5 × 108 colony forming unit (CFU)/ml bacteria solution prepared with normal saline. Blood agar culture plates in group CS were coated with 1.5 × 108 CFU/ml bacteria solution prepared with normal saline and hUCMSC culture supernatant (cultured by MSC culture medium, the same below) in double volume of normal saline. Blood agar culture plates in group CSL were coated with 1.5 × 108 CFU/ml bacteria solution prepared with normal saline, hUCMSC culture supernatant in double volume of normal saline, and 2.6 μL of LL-37 antibodies at the concentration of 2 μg/ml. At PCH 12, 24 and 48, 10 blood agar culture plates were taken out from each group to observe the distribution of SA colonies on blood agar culture plates and to measure diameters of zones of inhibition of ciprofloxacin. The minimum inhibitory concentration (MIC) of ciprofloxacin against SA in each group was recorded. Fractional inhibitory concentration (FIC) indexes of ciprofloxacin in group CS and group CSL at PCH 12, 24 and 48 were calculated, with the synergistic effect evaluated. Data were processed with factorial design ANOVA, one way ANOVA, LSD-t test, Kruskal-Wallis test and Mann-Whitney U test. Results: (1) At each PCH, the content of LL-37 in cell culture supernatant in 10 ng/ml LPS group, 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 0 ng/ml LPS group (with t values ranging from 11.22 to 33.36, p values all below .01);the content of LL-37 in cell culture supernatant in either 100 ng/ml LPS group or 1,000 ng/ml LPS group was higher than that in 10 ng/ml LPS group (with t values ranging from 2.24 to 18.73, p < .05 or p < .01);the content of LL-37 in cell culture supernatant in 1,000 ng/ml LPS group was higher than that in 100 ng/ml LPS group (with t values ranging from 12.46 to 14.70, p values all below .01). (2) At PCH 12, 24 and 48, the bacterial colonies in groups CC, CS and CSL became integrated over time. In CC group, diameters of zones of inhibition of ciprofloxacin at PCH 12, 24 and 48 were 26 mm, 24 mm and 23 mm respectively, with no obvious changes. At PCH 12, 24 and 48, diameters of zones of inhibition of ciprofloxacin in groups CS and CSL were 82 mm, 71 mm, 68 mm and 74 mm, 59 mm, 56 mm respectively, which were significantly larger than those in group CC. (3) At each PCH, MIC of ciprofloxacin against SA in group CC was significantly higher than that in groups CS and CSL respectively (with Z values ranging from 6.22 to 6.71, p values all below .01);MIC of ciprofloxacin against SA in group CSL was significantly higher than that in group CS (with Z values all equal to 6.72, p values all below .01). (4) FIC indexes of ciprofloxacin in groups CS and CSL at PCH 12, 24 and 48 were 0.011, 0.032, 0.032 and 0.122, 0.350, 0.350, respectively. The results indicated that hUCMSC culture supernatant had a synergistically antibacterial effect when combined with ciprofloxacin. Conclusions: hUCMSCs can secrete LL-37, and the secretion level is improved with increase of LPS concentration. The com-bination of hUCMSC culture supernatant with ciprofloxacin can decrease the dosage of ciprofloxacin in resisting SA effectively. Once LL-37 is neutralized, the synergistically antibacterial effect of hUCMSC culture supernatant is decreased thereby.

Dermcidin促进猪流行性腹泻病毒复制的研究

猪流行性腹泻病(PED)是由猪流行性腹泻病毒(PEDV)引起的一种高度传染性的猪胃肠道疾病,可导致哺乳仔猪产生急性水样腹泻,造成严重的经济损失。为调查抗菌肽Dermcidin(DCD)对PEDV复制的影响,本研究利用Western blot、荧光定量PCR(RT-qPCR)以及测定病毒半数组织细胞感染量TCID_(50)等方法,通过检测在DCD的作用下PEDV在细胞中的基因转录、蛋白表达和病毒滴度的水平,并利用Western blot检测信号通路中蛋白表达水平,探究其作用的分子机制。研究表明,DCD能够参与PEDV感染;DCD过表达促进了PEDV在Vero细胞和LLC-PK1细胞中的复制,而敲低DCD抑制了PEDV复制;DCD通过诱导PTEN表达来抑制Akt-mTOR-p70S6K通路相关蛋白位点的磷酸化,从而介导自噬促进PEDV复制。研究DCD对PEDV复制的作用机制,对今后PEDV防治工作有重要参考意义。

皮离蛋白多肽在急性冠脉综合征早期诊断及鉴别诊断中的价值探讨

目的探讨皮离蛋白（dermcidin）4183Da多肽片段是否具有早期诊断和鉴别诊断缺血性心脏病的预警价值。方法采用前瞻性对照研究方法，选择解放军第306医院收治的161例急性冠脉综合征（ACS）患者[其中不稳定型心绞痛（uA）46例，急性非sT段抬高型心肌梗死23例，急性sT段抬高型心肌梗死92例]，以111例常规体检者（其中有高血压病史45例，冠心病病史42例，糖尿病病史22例）和54例非ACS疾病患者（包括肺栓塞、主动脉夹层、心律失常、心肌炎、冠状动脉肌桥、胸膜炎、气胸、纵膈气肿、肋骨骨折、反流性食管炎、消化性溃疡、胰腺炎等）作为对照。采用基质辅助激光解析离子化飞行时间质谱（MALDI—TOF—MS）技术测定皮离蛋白4183Da多肽水平，并与髓过氧化物酶[MPO，分别采用床旁快速检测法（POCT）和酶联免疫吸附试验（ELISA）检测]、超敏C-反应蛋白（hs—CRP）、心脏型脂肪酸结合蛋白（H—FABP）、肌红蛋白（MYO）、心肌肌钙蛋白I（cTnI）、肌酸激酶同工酶（CK—MB）等多种标志物诊断和鉴别诊断缺血性心脏病的特异性及敏感性进行对比分析。结果①单因素方差分析显示，4183Da多肽水平在ACS患者中高于体检对照组（相对丰度：22．05±16．97比15．52±14．09，P=0．001），但ACS组与非ACS组之间水平相当（相对丰度：22．05±16．97与19．99±17．63，P=0．416）。②利用受试者工作特征曲线（ROC）分析4183Da多肽和MPO早期预警ACS、UA的特异性及敏感性，结果显示4183Da多肽对ACS和UA都具有一定的预警意义，ROC曲线下面积（AUC）分别为0．625和0．651（均P〈0．01），明显优于ELISA检测MPO对二者的诊断价值（AUC分别为0．440和0．336，均P〉0．05）。⑧比较4183Da多肽和多种标志物在急性心肌梗死（AMI）、UA与非ACS疾病鉴别诊断中的价值，结果显示4183Da多肽鉴别诊断AMI与非ACS疾病的特异性、敏感性不如MYO、cTnI、H—FABP（AUC分别为0．569比0．796、0．833、0．838），但与MPO（POCT／ELISA）和hs—CRP的价值相当[AUC分别为0．569比0．505（POCT）／0．477（ELISA）、0．545]；而在UA与非ACS疾病的鉴别诊断中，4183Da多肽与MYO、cTnI、H—FABP均无鉴别诊断价值（AUC分别为0．465、0．525、0．658、0．568）。结论4183Da多肽是皮离蛋白的一个片段，与缺血性心脏病发病有一定关系，对诊断早期ACS有一定的预警价值。

Dermcidin Enhances the Migration, Invasion, and Metastasis of Hepatocellular Carcinoma Cells In Vitro and In Vivo

Background and Aims:Hepatocellular carcinoma(HCC)is a common primary liver neoplasm with high mortality.Dermcidin(DCD),an antimicrobial peptide,has been reported to participate in oncogenesis.This study assessed the effects and underlying molecular events of DCD overexpression and knockdown on the regulation of HCC progression in vitro and in vivo.Methods:The serum DCD level was detected using enzyme-linked immunosorbent assay.DCD overexpression,knockdown,and Ras-related C3 botulinum toxin substrate 1(Rac1)rescue were performed in SK-HEP-1 cells using plasmids.Immunofluorescence staining,quantitative PCR,and Western blotting were used to detect the expression of different genes and proteins.Differences in HCC cell migration and invasion were detected by Transwell migration and invasion assays.A nude mouse HCC cell orthotopic model was employed to verify the in vitro data.Results:The level of serum DCD was higher in patients with HCC and in SK-HEP-1 cells.DCD overexpression caused upregulation of DCD,fibronectin,Rac1,and cell division control protein 42 homologue(Cdc42)mRNA and proteins as well as actin-related protein 2/3(Arp2/3)protein(but reduced Arp2/3 mRNA levels)and activated Rac1 and Cdc42.Phenotypically,DCD overexpression induced HCC cell migration and invasion in vitro,whereas knockout of DCD expression had the opposite effects.A Rac1 rescue experiment in DCD-knockdown HCC cells increased HCC cell migration and invasion and increased the levels of active Rac1/total Rac1,Wiskott-Aldrich syndrome family protein(WASP),Arp2/3,and fibronectin.DCD overexpression induced HCC cell metastasis to the abdomen and liver in vivo.Conclusions:DCD promotes HCC cell migration,invasion,and metastasis through upregulation of noncatalytic region of tyrosine kinase adaptor protein 1(Nck1),Rac1,Cdc42,WASP,and Arp2/3,which induce actin cytoskeletal remodeling and fibronectin-mediated cell adhesion in HCC cells.