糖尿病大鼠肝脏β-defensins表达及血清炎症因子的变化

目的:观察糖尿病大鼠病程中肝脏β-defensins的表达和血清炎症因子的变化,探讨在糖尿病状态时两者之间可能存在的影响.方法:实验大鼠分为普通饲料组、高糖饲料组、糖尿病4wk组和12wk组,采集血液检测血清炎症因子TNF-α,IL-1β及IL-10和血糖、胰岛素、三酰甘油等相关生化指标;收集肝脏标本,RT-PCR及Western Blot方法检测β-defensins的表达.结果:与对照组相比,糖尿病大鼠整个病程中都存在致炎因子TNF-α,IL-1β激活及抗炎因子IL-10的削弱.肝脏中表达β-defensins1(rBD-1)和β-defensins2(rBD-2).rBD-1呈少量表达,糖尿病病程中rBD-2表达呈减少趋势.高血糖或糖基化反应可能干扰rBD-2的组成性表达及炎症因子产生的诱导性表达.结论:糖尿病大鼠病程中存在慢性炎症,高血糖或糖基化反应干扰了炎症刺激重要的小分子抗菌肽β-defensins在肝脏表达的信号通路,使其表达下调.

中国南方汉族人β-defensin-1基因多态性与慢性阻塞性肺疾病易感性的关系

目的 探讨中国南方汉族人 β- defensin- 1基因多态性与慢性阻塞性肺疾病 (COPD)易感性的关系。方法 采用限制性片段长度多态性 (RFLP)技术检测 β defensin -1外显子 2基因型在 12 0例COPD吸烟患者和10 8例非COPD吸烟者中的频率。结果 COPD组同源野生型 (G/G)、杂合型 (G/A)和同源突变型 (A/A)频率分别为 82 .5 0 %、10 .83%和 6 .6 7% ,非COPD组的频率分别为 95 .37%、3.70 %和 0 .93% ,两组基因频率分布的差异有显著性 (P <0 .0 1)。两组等位基因的分布差异也有显著性 (等位基因G频率分别为 87.92 %∶97.2 2 % ;等位基因A频率分别为 12 .0 8%∶2 .78% ;P <0 .0 1) ;G→A的突变率随COPD病情的严重度增加而上升。结论 β defensin 1基因外显子 2的 16 5 4位多态性与中国南方汉族人群COPD易感性可能有关 ,而且可能还影响COPD的进展。

Human β-defensin 2 enhances IL-1β production and pyroptosis through P2X7-mediated NLRP3 expression in macrophages

Periodontal disease is the leading cause of tooth loss,which is also a high-risk factor for other diseases including oral cancer and cardiovascular disease.Periodontitis is one of the most common type of periodontal diseases.Interleukin-1β(IL-1β)plays a key role in the pathogenesis of periodontitis.However,the mechanism how IL-1βis produced during periodontitis is still unclear.In the present study,we found that humanβ-defensin 2(hBD2)enhances IL-1βproduction through an LPS-primed human acute monocytic leukemia(THP-1)macrophage model.Inhibition of P2X purinoceptor 7(P2X7)reduced hBD2-enhanced IL-1βproduction.Incubation of LPS-primed THP-1 macrophages with potassium chloride also suppressed hBD2-enhanced IL-1βproduction.Silence of inflammasome adaptor Nod-like receptor family pyrin domain containing 3(NLRP3)led to reduced hBD2-enhanced IL-1βproduction.Likewise,inhibition of caspase-1 also resulted in the decrease of IL-1β.Moreover,an ethidium bromide uptake test indicated that hBD2-activated caspase-1 mediated pyroptotic pore formation.Subsequent lactate dehydrogenase detection and flow cytometric analysis indicated that hBD2 also induced pyroptosis.In brief,these findings illustrated not only the mechanism of hBD2 in enhancing the inflammatory response,but also provided novel therapeutic targets for periodontitis.

High concentrations of human β-defensin 2 in gastric juice of patients with Helicobacter pylori infection

AIM: Human β-defensin (HBD)-1 and HBD-2 are endogenous antimicrobial peptides. Unlike HBD-1, the HBD-2 expression is augmented by Helicobacter pylori (H pylori). We sought to determine HBD-1 and HBD-2 concentrations in gastric juice duringH pylori infection.METHODS: HBD-1 and HBD-2 concentrations were measured by radioimmunoassay in plasma and gastric juice of 49 H pylori-infected and 33 uninfected subjects and before and after anti-H pyloritreatment in 13 patients with H pylori-associated gastritis. Interleukin (IL)-1β and IL-8 concentrations in gastric juice were measured by enzyme-linked immunosorbent assay (ELISA). Histological grades of gastritis were determined using two biopsy specimens taken from the antrum and corpus. Reverse phase high performance liquid chromatography (RP-HPLC)was used to identify HBD-2.RESULTS: HBD-2 concentrations in gastric juice, but not in plasma, were significantly higher in H pylori-positive than -negative subjects, albeit the post-treatment levels were unchanged. Immunoreactivity for HBD-2 was exclusively identified in H pylori-infected mucosa by RPHPLC. HBD-2 concentrations in gastric juice correlated with histological degree of neutrophil and mononuclear cell infiltration in the corpus. IL-1β levels correlated with those of IL-8, but not HBD-2. Plasma and gastric juice HBD-1concentrations were similar in H pylori-infected and uninfected subjects.CONCLUSION: Our results place the β-defensins, especially HBD-2, in the front line of innate immune defence.Moreover, HBD-2 may be involved in the pathogenesis of H pylori-associated gastritis, possibly through its function as immune and inflammatory mediator.

Human β-defensin-3 induction in H pylori-infected gastric mucosal tissues

AIM: To examine human β-defensin-3 (hBD-3) expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H pylori infection for better understanding the innate immune response to H pylori. METHODS: We used reverse transcription-polymerase chain reactions and immunohistochemistry to examine hBD-3 expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H pylori. Effects of hBD-3 against H pylori were also evaluated. RESULTS: The mean mRNA expression of hBD-3 in H pylori -positive specimens was significantly higher than that in H pylori-negative specimens (P = 0.0002, Mann-Whitney). In addition, unlike uninfected samples, 8 of 15 (53.33%) infected mucosal samples expressed hBD-3 protein. H pylori dose-dependently induced mRNA expression of hBD-3 in MKN45 cells, an effect inhibited by adding anti-toll-like receptor (TLR)-4 antibody. HBD-3 protein completely inhibited H pylori growth. CONCLUSION: Our results suggest that like hBD-2, hBD-3 may be involved in the pathophysiology of H pylori-induced gastritis.

Concentrations of α-and β-defensins in gastric juice of patients with various gastroduodenal diseases

AIM: To determine the concentration of α- and β-defensins in gastric juice of patients with various gastroduodenal diseases.METHODS: Concentrations of human neutrophil peptides (HNPs) 1-3, the major forms of α-defensins, and human β-defensin (HBD)-1 and HBD-2 were measured by radioimmunoassay in plasma and gastric juice of 84 subjects,consisting of 54 Helicobacter pylori-infected and 30 uninfected subjects. They included 33 patients with chronic gastritis (CG),12 with gastric ulcer (GU), 11 with duodenal ulcer (DU), 11with benign gastric polyp (BGP) and 16 with normal mucosa (N group) on upper endoscopy. Plasma pepsinogen Ⅰ and Ⅱ levels, biomarkers for gastric mucosal inflammation and atrophy, were also measured.RESULTS: Gastric juice HNPs 1-3 levels in patients with CG, GU and BGP were significantly higher than those in patients with DU and N. Gastric juice HBD-2 concentrations in patients with CG and GU were significantly higher than those in the N group, but were significantly lower in DU patients than in GU patients. Gastric juice HBD-1 levels and plasma levels of these peptides were similar in the patient groups.Concentrations of gastric juice HNPs 1-3 and HBD-2 of in Hpylori-infected patients were significantly different from those in uninfected subjects. HNPs 1-3 concentrations in gastric juice correlated negatively with plasma pepsinogen I levels and Ⅰ/Ⅱ ratios. HBD-2 levels in gastric juice correlated positively and negatively with plasma pepsinogen Ⅱ concentrations and Ⅰ/Ⅱ ratios, respectively.CONCLUSION: HNPs 1-3 and HBD-2 levels in gastric juice are diverse among various gastrointestinal diseases, reflecting the inflammatory and atrophic events of the background gastric mucosa affected by H pylorri.

GENETIC POLYMORPHISM IN HUMAN β-DEFENSIN-1 AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE IN HAN POPULATION IN SOUTH OF CHINA

Objective To investigate the correlation between human β-defensin-1 (HBD-1) exon 2 variations and chronic obstructive pulmonary disease susceptibility in Han population in south of China. Methods The frequency of polymorphic genotypes of HBD-1 exon 2 (1654G/A) was examined in 120 COPD patients (COPD group) and 108 smokers without COPD (control group) by restriction fragment length polymorphism. Results The frequencies of polymorphic genotypes in HBD-1 exon 2 in COPD group were G/G 82.50%, G/A 10.83%, and A/A 6.67%. The frequencies of polymorphic genotypes in control group were G/G 95.37%, G/A 3.70%, and A/A 0.93%. It showed significant difference between two groups(P<0.01). The differences in allele frequencies were also significant between two groups (G allele frequency: 87.92% vs 97.22%; A allele frequency: 12.08% vs 2.78%; P<0.01). The G→A mutation rised along with the severity of the COPD. Conclusion The genetic polymorphism in HBD-1 exon 2 gene might be associated with the susceptibility to COPD in Han population of South China.

血清HBD-3、DCR3对复杂性肾结石患者经皮肾镜碎石术后尿路感染的预测价值

目的分析血清β-防御素-3(HBD-3)、诱骗受体3(DCR3)对复杂性肾结石患者经皮肾镜碎石术后尿路感染的预测价值。方法选取2020年1月—2022年12月河北省沧州中西医结合医院收治的112例复杂性肾结石患者为研究对象进行回顾性研究,所有患者均行经皮肾镜碎石术(PCNL),根据术后感染情况将患者分为非尿路感染组52例、尿路感染组60例。比较两组一般资料及HBD-3、DCR3水平。受试者工作特征(ROC)曲线分析C反应蛋白(CRP)、降钙素原(PCT)、HBD-3、DCR3水平对术后尿路感染的预测价值。结果与非尿路感染组比较,尿路感染组的HBD-3[(0.77±0.08)ng/mL vs.(1.36±0.25)ng/mL,P=0.001]、DCR3[(4.68±0.53)ng/mL vs.(13.21±0.28)ng/mL,P=0.001]水平较高。多因素logistics回归分析显示,泌尿道手术史、术前尿路感染、手术时间、导尿管留置时间、结石负荷、抗菌药物种类、合并肾功能障碍、术中通道类型CRP、PCT、HBD-3、DCR3为患者术后尿路感染的影响因素(P<0.05)。ROC曲线显示,CRP、PCT、CRP联合PCT准确度分别为70.54%、72.32%、78.57%;HBD-3、DCR3、HBD-3联合DCR3准确度分别为69.64%、75.89%、86.61%。结论复杂性肾结石患者术后尿路感染与多种因素相关,且术后尿路感染患者HBD-3、DCR3表达水平较高,联合检测对术后尿路感染具有较高的预测价值。

Human β-defensin2(hBD-2)在幽门螺杆菌相关性胃炎中的表达

目的检测humanβdefensin2(hBD2)在幽门螺杆菌(Hp)相关性胃炎胃黏膜上皮中的表达,评价其在Hp感染的胃黏膜表面的抗菌作用。方法随机选择患者18例,取胃窦黏膜组织,以快速尿素酶及改良Giemsa染色两种方法判断Hp的存在与否。两者同时阳性诊断为Hp阳性,同时阴性为Hp阴性。18例患者中,Hp阳性10例,阴性8例。另以RTPCR法检测标本中hBD2的表达。结果10例Hp阳性标本中8例呈现hBD2mRNA的高表达,1例呈现低表达,总表达率90%;8例Hp阴性标本中6例未检测出hBD2mRNA,2例呈现hBD2mRNA的表达,表达率25%。结论Hp感染可诱导hBD2在胃黏膜上皮中的表达。体外试验表明hBD2抑制Hp的生长,推测hBD2在Hp相关性胃炎中起到一定的抗菌作用。

黄芪甲甙对吸烟大鼠气道-defensin 2表达的影响

目的了解熏烟对大鼠肺组织β-防御素2(β-defensin2)表达的影响,黄芪甲甙对β-defensin2表达的调控作用及其可能的机制。方法大鼠接受染毒柜被动吸烟三周,建立熏烟模型。分别给于黄芪甲甙1.2mg/kg·d,2.4mg/kg·d,3.6mg/kg·d灌胃。提取肺组织和支气管肺泡灌洗液,采用免疫组化、RT-PCR等方法检测β-defensin 2的表达水平。结果吸烟可以使气道上皮β-defensin 2表达升高,使BALF中炎细胞总数,中性粒细胞,巨噬细胞,淋巴细胞数增高。黄芪甲甙可以降低吸烟诱导的β-defensin 2的高表达,减少支气管肺泡灌洗液白细胞及中性粒细胞,淋巴细胞的数量。结论黄芪甲甙下调吸烟诱导的气道上皮β-defensin 2的高表达,减轻气道炎症。

Relevance of α-defensins(HNP1-3) and defensin β-1 in diabetes

AIM: To investigate the genetic background of human defensin expression in type 1 and 2 diabetes.METHODS: Associations between DEFA1/DEFA3 gene copy number polymorphism and diabetes as well as between the promoter polymorphisms of DEFB1 and diabetes were studied. The copy number variation of the DEFA1/DEFA3 genes was determined in 257 diabetic patients(117 patients with type 1 and 140 with type 2 diabetes). The control group consisted of 221 age- and gender-matched healthy blood donors. The cumulative copy numbers of the DEFA1/DEFA3 genes were detected by using quantitative PCR analysis. To evaluate the HNP 1-3(human neutrophil peptide 1-3 or α-defensin) levels in the circulation, plasma HNP 1-3 concentrations were measured by ELISA. The expression of DEFA1/A3 in peripheral leukocytes of the diabetic patients was measured by quantitative RT PCR analysis. Three SNPs of the human DEFB1(human defensin β-1) gene: DEFB1 G-20A(rs11362), DEFB1 C-44G(rs1800972) and DEFB1 G-52A(rs1799946) were genotyped by Custom TaqMan? Real Time PCR assay.RESULTS: Significant differences were observed in HNP1-3 levels between the healthy subjects and both groups of diabetic patients. The mean ± SE was 28.78 ± 4.2 ng/mL in type 1 diabetes, and 29.82 ± 5.36 ng/mL in type 2 diabetes, vs 11.94 ± 2.96 ng/mL in controls; P < 0.01 respectively. There was no significant difference between patients with type 1 and type 2 diabetes in the high plasma concentrations of HNP1-3. The highest concentrations of α-defensin were found in diabetic patients with nephropathy(49.4 ± 4.8 ng/mL), neuropathy(38.7 ± 4.8 ng/mL) or cardiovascular complications(45.6 ± 1.45 ng/L). There was no significant difference in the cumulative copy numbers of DEFA1/DEFA3 genes between controls and patients, or between patients with the two types of diabetes. Comparisons of HNP 1-3 plasma level and DEFA1/A3 copy number of the same patient did not reveal significant relationship between defensin-α levels and the gene copy numbers(r2 = 0.01). Similarly, no positive correlation was observed between the copy numbers and the mRNA expression levels of DEFA1/A3. Regarding the C-44G polymorphism of DEFB1, the GG "protective" genotype was much less frequent(1%-2%) among both groups of patients than among controls(9%).CONCLUSION: Elevated HNP1-3 levels in diabetes are independent of DEFA1/DEFA3 copy numbers, but GG genotype of C-44G SNP in DEFB1 gene may result in decreased defensin β-1 production.

β-defensins and the epididymis： contrasting influences of prenatal, postnatal, and adult scenarios

β-defensins are components of host defense, with antimicrobial and pleiotropic immuno-modulatory properties. Research over the last 15 years has demonstrated abundant expression of a variety of β-defensins in the postnatal epididymis of different species. A gradient of region- and cell-specific expression of these proteins is observed in the epithelium of the postnatal epididymis. Their secretion into the luminal fluid and binding to spermatozoa as they travel along the epididymis has suggested their involvement in reproduction-specific tasks. Therefore, continuous attention has been given to various β-defensins for their role in sperm function and fertility. Although β-defensins are largely dependent on androgens, the underlying mechanisms regulating their expression and function in the epididymis are not well understood. Recent investigation has pointed out to a new and interesting scenario where β-defensins emerge with a different expression pattern in the Wolffian duct, the embryonic precursor of the epididymis, as opposed to the adult epididymis, thereby redefining the concept concerning the multifunctional roles of β-defensins in the developing epididymis. In this review, we summarize some current views of β-defensins in the epididymis highlighting our most recent data and speculations on their role in the developing epididymis during the prenatal-to-postnatal transition, bringing attention to the many unanswered questions in this research area that may contribute to a better understanding of epididymal biology and male fertility.

血清高迁移率族蛋白B1、人β防御素2与腺病毒肺炎患儿闭塞性细支气管炎发生的关系

目的探讨血清高迁移率族蛋白B1(HMGB1)、人β防御素2(hBD-2)与腺病毒肺炎(AP)患儿闭塞性细支气管炎(BO)发生的关系。方法选取193例AP患儿,根据出院3个月后是否发生BO将AP患儿分为BO组58例和非BO组135例。收集两组临床资料并用酶联免疫吸附试验检测血清HMGB1、hBD-2,用单因素及多因素Logistic回归分析AP患儿发生BO的影响因素,以受试者工作特征(ROC)曲线分析血清HMGB1、hBD-2对AP患儿发生BO的预测价值。结果BO组月龄小于非BO组,热程长于非BO组,低氧血症、机械通气发生比例及血液白细胞计数、血小板计数、C反应蛋白、降钙素原、HMGB1、hBD-2高于非BO组(P均<0.05)。多因素Logistic回归分析显示,低氧血症、机械通气和HMGB1、hBD-2升高为AP患儿发生BO的独立危险因素(P均<0.05)。ROC曲线分析显示,血清HMGB1、hBD-2联合应用预测AP患儿发生BO的曲线下面积为0.818,与血清HMGB1、hBD-2单独预测的曲线下面积(0.739、0.726)比较差异有统计学意义(P均<0.05)。结论血清HMGB1、hBD-2水平升高是AP患儿发生BO的危险因素,二者联合检测对AP患儿发生BO的预测价值较高。

鱼源β-防御素的生物信息学分析

鱼源β-防御素是抗菌肽家族的重要成员,具有广谱抗菌、不损伤真核细胞、不易产生耐药性等特点,可应用于渔业养殖和临床医学等方面,但目前面临着提取工艺复杂、含量较低、提取所用蛋白酶需求量大及以生产成本高等问题。因此分析其理化性质、结构和杀菌功能,对构造便捷和低廉的衍生肽具有重要的意义。本研究结合生物信息学软件和工具预测分析其理化性质、二级结构、磷酸化和糖基化位点、疏水性、细胞内定位和信号肽。结果提示鱼源β-防御素均为疏水性阳离子型抗菌肽。β-防御素抗菌肽通过C端亲水作用与细菌膜结合,引导N端氨基酸的疏水作用插入膜磷酸二酯键中改变细胞膜的通透性使细菌生物膜破裂而死亡,其中AJA33388.1(红牙鳞鲀)破膜杀菌能力最强。经检测发现β-防御素耐热性,稳定性相对较好,尤其是ACO88907.1(鳜鱼)和QNV47918.1(条石鲷)具有极高耐热性,可将其应用于杀菌以外的多方面领域。其二级结构以α螺旋为主,具有一定的刚性结构,且均具有3~4个磷酸化位点,分布于细胞外,具有19~20个氨基酸的信号肽序列,指导其在胞外蛋白产物的分泌等。通过预测分析结构,可为β-防御素的深入研究和应用以及多领域的抗菌肽衍生物设计提供思路。

血清hBD-2、NT-proBNP、sICAM-1在急性下呼吸道感染患儿中的表达水平及对其病情预测的研究

目的 观察急性下呼吸道感染患儿血清β-防御素-2(hBD-2)、N末端脑钠肽前体(NT-proBNP)、可溶性细胞间黏附分子1(sICAM-1)表达水平,并分析3项指标对其病情检测的意义。方法 纳入2020年10月至2022年10月该院80例急性下呼吸道感染患儿为急性期组,另选取该院同期60例缓解期下呼吸道感染患儿为缓解期组,所有患儿入院时均接受血清hBD-2、NT-proBNP、sICAM-1检测,对比急性期组与缓解期组患儿血清hBD-2、NT-proBNP、sICAM-1表达水平。依据临床肺部感染评分(CPIS)将急性期组患儿分为轻症组和重症组,对比轻症组和重症组临床资料及实验室指标,分析血清hBD-2、NT-proBNP、sICAM-1表达水平与急性下呼吸道感染患儿病情的相关性,并绘制受试者工作特征(ROC)曲线分析3项指标对患儿病情的预测价值。结果 急性期组血清hBD-2、NT-proBNP、sICAM-1表达水平高于缓解期组,差异有统计学意义(P<0.05);经CPIS评分结果显示,80例急性下呼吸道感染患儿CPIS评分为(5.83±1.92)分,其中重症32例(40.00%),轻症48例(60.00%);经Pearson相关性分析,结果显示,CPIS评分与血清hBD-2、NT-proBNP、sICAM-1表达水平呈正相关(r=0.337、0.325、0.386,P=0.002、0.003、<0.001);重症组血清hBD-2、NT-proBNP、sICAM-1表达水平高于轻症组,差异有统计学意义(P<0.05);经Logistic回归分析发现,血清hBD-2、NT-proBNP、sICAM-1表达水平是急性下呼吸道感染患儿病情加重的危险因素(OR>1,P<0.05);绘制ROC曲线,血清hBD-2、NT-proBNP、sICAM-1表达水平对重症急性下呼吸道感染具有一定预测价值,曲线下面积(AUC)分别为0.728、0.769、0.786,联合检测预测价值更高(AUC=0.830)。结论 急性下呼吸道感染患儿血清hBD-2、NT-proBNP、sICAM-1表达水平升高,3项指标水平与患儿病情严重程度密切相关,可用于预测重症急性下呼吸道感染。

Protective eff ect and mechanism of nanoantimicrobial peptide ND-C14 against Streptococcus pneumoniae infection

BACKGROUND:Streptococcus pneumoniae(S.pneumoniae)is a common pathogen that causes bacterial pneumonia.However,with increasing bacterial resistance,there is an urgent need to develop new drugs to treat S.pneumoniae infections.Nanodefensin with a 14-carbon saturated fatty acid(ND-C14)is a novel nanoantimicrobial peptide designed by modifying myristic acid at the C-terminus of humanα-defensin 5(HD5)via an amide bond.However,it is unclear whether ND-C14 is effective against lung infections caused by S.pneumoniae.METHODS:In vitro,three groups were established,including the control group,and the HD5 and ND-C14 treatment groups.A virtual colony-count assay was used to evaluate the antibacterial activity of HD5 and ND-C14 against S.pneumoniae.The morphological changes of S.pneumoniae treated with HD5 or ND-C14 were observed by scanning electron microscopy.In vivo,mice were divided into sham,vehicle,and ND-C14 treatment groups.Mice in the sham group were treated with 25μL of phosphate-buffered saline(PBS).Mice in the vehicle and ND-C14 treatment groups were treated with intratracheal instillation of 25μL of bacterial suspension with 2×108 CFU/mL(total bacterial count:5×10^(6) CFU),and then the mice were given 25μL PBS or intratracheally injected with 25μL of ND-C14(including 20μg or 50μg),respectively.Survival rates were evaluated in the vehicle and ND-C14 treatment groups.Bacterial burden in the blood and bronchoalveolar lavage fluid were counted.The lung histology of the mice was assessed.A propidium iodide uptake assay was used to clarify the destructive eff ect of ND-C14 against S.pneumoniae.RESULTS:Compared with HD5,ND-C14 had a better bactericidal eff ect against S.pneumoniae because of its stronger ability to destroy the membrane structure of S.pneumoniae in vitro.In vivo,ND-C14 significantly delayed the death time and improved the survival rate of mice infected with S.pneumoniae.ND-C14 reduced bacterial burden and lung tissue injury.Moreover,ND-C14 had a membrane permeation eff ect on S.pneumoniae,and its destructive ability increased with increasing ND-C14 concentration.CONCLUSION:The ND-C14 may improve bactericidal eff ects on S.pneumoniae both in vitro and in vivo.

Human β-defensin-2 Enhances Anti-tumor Efficacy of Survivin-based Broad-spectrum DNA Vaccine in Mouse Tumor Model

Objective:Malignant tumors greatly endanger and affect the quality of human life.Among antitumor biotherapy strategies,DNA vaccines hold promise in part because of their unique advantages.In this study,an effective broad-spectrum antitumor DNA vaccine expressing human survivin T34A dominant-negative mutant fused with humanβ-defensin-2(HBD2)was investigated.Methods:The expression profiles of genes of interest were examined using western blotting,reverse transcription-polymerase chain reaction(RT-PCR),and enzyme-linked immunosorbent assay(ELISA)in vitro.The immune function of the fusion gene vaccine(FGV)was assessed in BALB/c mice,which included detection of serum antibody,spleen lymphocyte proliferation,interferon-gamma(IFN-γ)secretion,and lactate dehydrogenase(LDH)release.In vivo antitumor effects of FGV were examined in a mouse breast cancer(4T1)model,whereas in vitro effects were assessed using tumor cells derived from different origins.Caspase-3activity in tumor cells was also assessed after vaccine transfection.Results:The FGV triggered humoral as well as cellular immune responses against survivin.It exhibited more potent inhibition of tumor growth as well as prolonged the survival of immunized mice compared to mice immunized with only either survivin T34A or HBD2 vaccines.In addition,FGV displayed stronger cytotoxicity against tumor cells derived from different origins compared to the other vaccines and facilitated increased caspase-3 activity in transfected tumor cells.Conclusion:The novel DNA vaccine consisting of a fusion of the universal tumor antigen survivin T34A mutant with molecular adjuvant HBD2generates enhanced broad-spectrum antitumor efficacy against cancers derived from various origins.

枯草芽孢杆菌肽聚糖对绵羊瘤胃上皮细胞β-防御素-1(SBD-1)表达的影响

为了探究来源于枯草芽孢杆菌细胞壁的肽聚糖对绵羊瘤胃上皮细胞(ORECs)中β-防御素-1(SBD-1)表达的影响,本试验使用30μg/mL肽聚糖刺激ORECs不同时间(2、4、6、8、10和12 h),采用荧光定量PCR(qPCR)和酶联免疫吸附试验(ELISA)方法分别检测SBD-1的mRNA和蛋白表达量,确定最佳刺激时间;再使用不同浓度的肽聚糖(0、10、20、30、40和50μg/mL)刺激ORECs 2 h,采用qPCR和ELISA方法检测SBD-1的mRNA和蛋白表达量,确定最佳刺激浓度。结果显示:(1)使用30μg/mL肽聚糖刺激ORECs不同时间时,SBD-1的mRNA和蛋白表达量随着时间的增加呈先下降后升高的趋势,2 h时表达量最多并极显著高于对照组(P<0.001)。(2)使用不同浓度肽聚糖刺激ORECs 2 h时,SBD-1的mRNA和蛋白表达量随着肽聚糖浓度的升高呈现先升高后降低的趋势,在20μg/mL时表达量最多并极显著高于对照组(P<0.001)。(3)不同时间刺激试验和不同浓度刺激试验均不会对ORECs产生细胞毒性作用。结果表明,来源于枯草芽孢杆菌的肽聚糖能够诱导ORECs表达SBD-1,最佳刺激条件为20μg/mL枯草芽孢杆菌肽聚糖刺激ORECs 2 h。

鸭源新城疫病毒强毒株感染对番鸭致病性和β-防御素基因表达的影响

为了探讨鸭源新城疫病毒(NDV)感染对番鸭β-防御素(AvBD)基因表达的影响,本实验选取80只15日龄SPF番鸭,将其随机分为两组,每组40只。其中一组以滴鼻点眼方式接种107 EID50/0.2 mL的鸭源基因Ⅶ型NDVMd/CH/LGD/1/2005毒株;另一组以相同途径接种等量灭菌的磷酸盐缓冲液,作为对照组。攻毒后持续观察临床症状25 d。并在攻毒后36 h、72 h、7 d和25 d每组分别剖杀10只番鸭,采集5个免疫器官的样品,包括骨髓、脾脏、盲肠扁桃体、哈德氏腺和法氏囊,运用real-time RTPCR方法检测其中11种防御素基因表达量的变化差异。并在攻毒后36 h、72 h、7 d以及16 d、20 d和25 d每组各采集10只番鸭的血清,用血凝抑制试验(HI)检测新城疫病毒特异性抗体水平。结果表明:番鸭感染该鸭源NDV强毒株后出现新城疫病毒感染的典型临床症状,死亡率为7.5%;感染番鸭体内组织中AvBD1、AvBD2、AvBD5、AvBD10在广泛表达,且AvBD3、AvBD5、AvBD7在法氏囊内均有不同程度表达上调,番鸭感染该鸭源NDV强毒株后72 h开始抗体开始转阳,第7 d即达到高峰,之后16 d、20 d和25 d的抗体水平趋于平稳。以上结果表明,该鸭源NDV强毒株感染可在番鸭体内诱导产生抗病毒免疫应答反应。

枯草芽孢杆菌细胞壁通过TLR-2-MyD88-NF-κB/MAPK信号通路调控绵羊瘤胃上皮细胞β-防御素-1的表达

为了探索枯草芽孢杆菌细胞壁诱导绵羊β-防御素-1(SBD-1)表达的主要信号转导途径,本试验首先使用反复冻融法与超声波破碎法相结合的方法破碎枯草芽孢杆菌并收集其细胞壁,酶联免疫吸附测定(ELISA)检测方法分析细胞壁成分及其含量,然后利用实时荧光定量PCR(RT-qPCR)和蛋白质免疫印迹(Western blot,WB)方法检测枯草芽孢杆菌细胞壁刺激绵羊瘤胃上皮细胞(ORECs)后通路因子(TLR-2、MyD88、NF-κB、p38、JNK和ERK1/2)mRNA和蛋白的表达变化,最后使用通路抑制剂分别阻断NF-κB、p38、JNK和ERK1/2信号通路,用枯草芽孢杆菌细胞壁刺激ORECs并检测刺激后SBD-1 mRNA表达变化,从而确定诱导SBD-1表达的主要信号通路。结果显示,枯草芽孢杆菌细胞壁制备成功,细胞壁主要成分为肽聚糖和磷壁酸,含量分别为(90.85±0.91)%和(8.85±0.11)%。与对照组相比,枯草芽孢杆菌细胞壁刺激ORECs后信号通路因子(TLR-2、MyD88、NF-κB、p38、JNK和ERK1/2)的mRNA和蛋白的表达水平均显著升高(P<0.05或P<0.01),使用通路抑制剂阻断各个信号通路后再刺激ORECs,SBD-1 mRNA表达水平均极显著下降(P<0.01),且添加NF-κB和JNK抑制剂组下降幅度最大。结果表明,枯草芽孢杆菌细胞壁可能通过TLR-2-MyD88-NF-κB/MAPK信号通路调控ORECs SBD-1的表达。