Intraocular pressure before and after capsulorhexis using two viscoelastic substances and two surgical approaches in enucleated porcine eyes

AIM:To investigate the influence of ophthalmic viscoelastic devices(OVDs)and different surgical approaches on the intraocular pressure(IOP)before and after creation of the curvilinear circular capsulorhexis(CCC)as a measure for anterior chamber stability during this maneuver.METHODS:Prospective experimental WetLab study carried out on enucleated porcine eyes.IOP was measured before and after CCC with the iCare Rebound tonometer(iCare ic200;iCare Finland Oy,Vantaa,Finland).The OVDs used were a cohesive one[Z-Hyalin,Carl Zeiss Meditec AG,Germany;hyaluronic acid(HA)]and a dispersive[Z-Celcoat,Carl Zeiss Meditec AG,Germany;hydroxy propylmethylcellulosis(HPMC)].The CCC was created using Utrata forceps or 23 g microforceps in different combinations with the OVDs.RESULTS:Using the Utrata forceps the IOP dropped from 63.65±6.44 to 11.25±3.63 mm Hg during the CCC.The use of different OVDs made no difference.Using the 23 g microforceps the IOP dropped from 65.35±8.15 to 36.55±6.09 mm Hg.The difference between IOP drop using either Utrata forceps or 23 g microforceps was highly significant regardless of the OVD used.CONCLUSION:Using the sideport for the creation of the capsulorhexis leads to a lesser drop in IOP during this maneuver compared to the main incision in enucleated porcine eyes.The use of different OVD has no significant influence on IOP drop.

Small-diameter acellular porcine corneal stroma for peripheral corneal ulceration treatment

AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 eyes)with PCU between April 2018 and December 2020.All patients had PCU and underwent lamellar keratoplasty with SAPS.Observation indicators included preoperative and postoperative best-corrected visual acuity(BCVA)and transparency of SAPS.The infection control rate in the surgical eye-lesion area was also calculated.RESULTS:Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU.None of the patients experienced rejection after 6mo(18/18)and 12mo(16/16)of follow-up.The BCVA(0.47±0.30)at the 6mo followup after operation was significantly improved compared with the baseline(0.99±0.80),and the difference was statistically significant(Z=-3.415,P<0.05).The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo(Z=0,P=1).With time,the SAPS graft gradually became transparent.At the 6mo(18/18)and 12mo(16/16)follow-up,none of the patients had recurrent corneal infection.CONCLUSION:SAPS is clinically effective in the treatment of PCU,improving the patient’s BCVA and reducing the incidence of rejection after keratoplasty.

Porcine enteric alphacoronavirus infection increases lipid droplet accumulation to facilitate the virus replication

Coronaviruses are widely transmissible between humans and animals, causing diseases of varying severity. Porcine enteric alphacoronavirus(PEAV) is a newly-discovered pathogenic porcine enteric coronavirus in recent years, which causes watery diarrhea in newborn piglets. The host inflammatory responses to PEAV and its metabolic regulation mechanisms remain unclear, and no antiviral studies have been reported. Therefore, we investigated the pathogenic mechanism and antiviral drugs of PEAV. The transcriptomic analysis of PEAV-infected host cells revealed that PEAV could upregulate lipid metabolism pathways. In lipid metabolism, steady-state energy processes, which can be mediated by lipid droplets(LDs), are the main functions of organelles. LDs are also important in viral infection and inflammation. In infected cells, PEAV increased LD accumulation, upregulated NF-κB signaling, promoted the production of the inflammatory cytokines IL-1β and IL-8, and induced cell death. Inhibiting LD accumulation with a DGAT-1 inhibitor significantly inhibited PEAV replication, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. The NF-κB signaling pathway inhibitor BAY11-7082 significantly inhibited LD accumulation and PEAV replication. Metformin hydrochloride also exerted anti-PEAV effects and significantly inhibited LD accumulation, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. LD accumulation in the lipid metabolism pathway therefore plays an important role in the replication and pathogenesis of PEAV, and metformin hydrochloride inhibits LD accumulation and the inflammatory response to exert anti-PEAV activity and reducing pathological injury. These findings contribute new targets for developing treatments for PEAV infections.

Enhancement of porcine in vitro embryonic development through luteolin‑mediated activation of the Nrf2/Keap1 signaling pathway

Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.

Interlayer repair with porcine small intestinal submucosa versus internal repair with tragus cartilage in endoscopic tympanoplasty

Objective Endoscopic tympanoplasty includes various surgical methods,such as internal repair,interlayer repair,and external overlay.This technique requires autologous materials,allografts,and xenografts,which are used to repair tympanic membrane(TM)perforation.To obtain good results,appropriate surgical methods and repair materials should be selected.This study aims to assess the efficacy of repairing refractory TM perforations in the porcine small intestinal submucosa(SIS)during transcanal endoscopic type I tympanoplasty.Method A retrospective chart review was performed on patients who underwent TM perforation repair with porcine SIS and tragus cartilage between January 2022 and September 2022 at Sir Run Run Shaw Hospital,Zhejiang University School of Medicine.Perforation size,tympanic status,pre-and postoperative symptoms,follow-up data,wound healing rates,and hearing improvement were analysed.Results Of the 115 patients included in the study,56 underwent interlayer repair with porcine SIS of the TM,and 59 patients underwent internal repair with tragus cartilage.No significant difference was found between the two groups at baseline in terms of age,sex,disease course,perforation side,tympanic status,underlying disease,or preoperative infection.The total postoperative effective rate of interlayer implantation with porcine SIS was 91.07%(51 patients),and that of internal implantation with tragus cartilage was 88.14%(52 patients).No significant difference was found in terms of the graft success rate between the two surgical methods(p=0.887).Postoperative pure tone auditory(PTA)and air-bone gap(ABG)density significantly increased in both groups compared with before surgery(p<0.05).However,the postoperative PTA and ABG density were not significantly different 3 months post-surgery between the two groups(p>0.05).Compared to those in the internal implantation group,the patients in the interlayer group had a shorter operation duration(51.36±6.76 min vs.59.71±7.45 min,t=6.298,p<0.001)and less blood loss(11.91±2.61 mL vs.15.27±2.57 mL,t=7.019,p<0.001).Conclusions Our study suggests that the porcine SIS,as well as the tragus cartilage,has a high success rate in repairing irreversible TM perforation.Endoscopic tympanoplasty via interlayer implantation with porcine SIS offers distinct advantages,including the absence of donor-site incision and scar formation,and ease of graft modification and manipulation.

IFN-β通过STAT1诱导SARI表达抑制AML细胞增殖并促进凋亡

目的:探讨IFN-β诱导SARI表达对急性粒细胞性白血病(AML)细胞增殖、凋亡的作用,并筛选其潜在的调控分子。方法:qPCR、Western blot筛选SARI低表达的AML细胞作为实验细胞株;不同浓度IFN-β干预AML细胞,于不同时间采用qPCR、Western blot检测SARI表达,选取IFN-β作用的适当浓度和时间;采用RNA-Seq转录组测序及KEGG富集分析初步筛选IFN-β诱导AML细胞SARI表达的潜在调控分子;通过相应分子抑制剂联合IFN-β处理AML细胞,MTS法检测细胞增殖,流式细胞术检测细胞凋亡;明确该分子参与IFN-β诱导SARI表达对AML细胞增殖及凋亡的作用。结果:HL60和NB4细胞SARI表达相对较低,选为实验细胞株;1 ng/ml IFN-β作用12 h后AML细胞SARI表达升高且细胞增殖被抑制,凋亡增多;筛选STAT1为IFN-β诱导SARI表达的潜在调控分子;抑制STAT1后,IFN-β对AML细胞SARI表达、增殖抑制、凋亡促进的作用被明显逆转。结论:IFN-β可通过STAT1诱导AML细胞SARI表达,抑制细胞增殖,促进细胞凋亡。

结核分枝杆菌特异性IFN-γ、IL-2联合检测在肺结核与细菌性肺炎鉴别诊断中的应用

目的评价结核分枝杆菌特异性细胞因子干扰素-γ(IFN-γ)、白细胞介素-2(IL-2)双因子联合检测在肺结核与细菌性肺炎鉴别诊断中的应用价值。方法选择阜阳市人民医院2022年1月-2023年10月呼吸科住院患者91例,明确诊断为肺结核患者45例(肺结核组)和细菌性肺炎患者46例(肺炎组),均进行双因子联合检测,比对分析双因子联合检测与C反应蛋白(CRP)对肺结核和细菌性肺炎鉴别诊断的效果。结果使用双因子联合检测对肺结核与细菌性肺炎进行鉴别诊断,灵敏度为86.7%、特异度为84.8%,受试者工作特征曲线下面积(AUC)值为0.928(95%CI:0.870~0.986),与CRP的鉴别诊断效果相比差异有统计学意义(P<0.05)。结论结核分枝杆菌特异性细胞因子IFN-γ、IL-2联合检测在鉴别肺结核与细菌性肺炎时具有较高的应用价值,能为临床肺结核和细菌性肺炎的鉴别诊断提供依据。

藏獒IFN-γ基因在毕赤酵母中的表达与抗病毒活性分析

采用毕赤酵母表达系统表达藏獒γ-干扰素(Tibetan mastiff interferon gamma,TmIFN-γ)并分析其生物活性,为抗病毒生物制品的研发与临床应用奠定基础。将构建的TmIFN-γ的真核表达质粒pPIC9k-TmIFN-γ转化至毕赤酵母GS115细胞,用不同浓度的G418筛选得到多拷贝重组菌株;利用甲醇进行诱导表达,以镍层析柱纯化目的蛋白,对表达产物进行SDS-PAGE和Western blotting鉴定;通过CCK-8试验检测重组TmIFN-γ蛋白的细胞毒性作用,利用VSV-MDBK系统检测其抗病毒生物活性。结果显示,成功筛选得到高拷贝pPIC9k-TmIFN-γ重组转化毕赤酵母菌株,利用甲醇诱导可获得TmIFN-γ重组蛋白,该蛋白对细胞无毒性,具有显著抗病毒活性,其抗病毒生物效价为1.727×10~5IU/mL。本研究获得了具有抗病毒活性的TmIFN-γ,有助于进一步探索TmIFN-γ的功能和开发新型基因工程抗病毒药物。

表达IFN-α的重组猪繁殖与呼吸综合病毒的构建及生物学特性分析

猪繁殖与呼吸综合征(PRRS)是危害我国生猪业的重要疫病之一,其病原是猪繁殖与呼吸综合征病毒(PRRSV),是一种免疫抑制性病毒。干扰素(IFN)是一类具有免疫调节功能和抗病毒作用的细胞因子。IFN-α除更直接的抗病毒作用外,还可以调节宿主的先天性和适应性免疫。论文构建表达IFN-α的重组PRRSV,分析重组病毒的生物学特性以及IFN-α的生物学活性。通过反向遗传操作方法将IFN-α插入到PRRSV ORF1b和ORF2a之间,重组质粒转染细胞后可以拯救出重组病毒(rGXAM-P-IFN-α)。插入到PRRSV基因组中的IFN-α可遗传稳定9代。重组病毒生长特性分析可发现rGXAM-P-IFN-α复制能力显著低于亲本病毒。rGXAM-P-IFN-α感染猪肺泡巨噬细胞(PAM)可显著上调抗病毒基因(PKR,ISG15和ISG54)mRNA表达水平,为进一步研发新型PRRSV疫苗提供参考。

Isorhamnetin protects porcine oocytes from zearalenone-induced reproductive toxicity through the PI3K/Akt signaling pathway

Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.

Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome(PRRS)is recognized as one of the most infectious viral diseases of swine.Although Cluster of differentiation 163(CD163)is identified as an essential receptor for mediating PRRS virus(PRRSV)infection,the important residues involved in infection on CD163 are still unclear.Therefore,it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs.In this study,we first generated immortalized porcine alveolar macrophage(IPAM)cell lines harboring 40-residues(residues 523-562,including R561(arginine(R)at position 561))deletion of CD163.PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection.We then generated cloned pigs carrying CD163-R561A(an arginine(R)to alanine(A)substitution at position 561 of CD163).PRRSV challenge experiments in porcine alveolar macrophages(PAMs)isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs.Through this study,we show that CD163523-562 contains essential residues for mediating PRRSV infection,and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection.These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection.Furthermore,CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus,African swine fever virus,and atypical porcine pestivirus

With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.

Dynamic compressive response of porcine muscle measured using a split Hopkinson bar system with a pair of PVDF force transducers

The classic metallic Split Hopkinson Pressure Bar(SHPB)cannot capture the transmitted signal accurately when measuring soft biological tissue,because of the very low wave impedance and strength of this material.So the dynamic compressive response of porcine muscle has been investigated by using a modified SHPB.The forces on both ends of the sample measured using Polyvinylidene fluor(PVDF)transducers were applied to calculate the stress in the specimen instead of the strain gauge signal on the transmitted bar.Moreover,a circular cardboard disk pulse shaper was applied for generating a suitable incident pulse to achieve stress equilibrium and constant strain rates in the specimens.Then,the dynamic mechanical properties of porcine muscle parallel and perpendicular to the fiber directions were measured,and the stress equilibrium process during loading was analyzed,as well as the inertia-induced extra stress being corrected.Furthermore,quasi-static tests were conducted at two different strain rates to investigate the strain rate dependence using a universal material testing machine.The results show that the stress-strain curves are sensitive to strain rate in the two different loading directions.The compressive stress perpendicular to the fiber direction is stiffer than that parallel to the fiber direction.In addition,a strain rate-dependent constitutive model was developed based on the mechanical response of the muscle at different strain rates and fitted to the experimental data.The results show that the overall fit is good,and the constitutive model could describe the muscle's dynamic mechanical properties.

Anethole improves the developmental competence of porcine embryos by reducing oxidative stress via the sonic hedgehog signaling pathway

Background Anethole(AN)is an organic antioxidant compound with a benzene ring and is expected to have a positive impact on early embryogenesis in mammals.However,no study has examined the effect of AN on porcine embryonic development.Therefore,we investigated the effect of AN on the development of porcine embryos and the underlying mechanism.Results We cultured porcine in vitro-fertilized embryos in medium with AN(0,0.3,0.5,and 1 mg/mL)for 6 d.AN at 0.5 mg/mL significantly increased the blastocyst formation rate,trophectoderm cell number,and cellular survival rate compared to the control.AN-supplemented embryos exhibited significantly lower reactive oxygen species levels and higher glutathione levels than the control.Moreover,AN significantly improved the quantity of mitochondria and mitochondrial membrane potential,and increased the lipid droplet,fatty acid,and ATP levels.Interestingly,the levels of proteins and genes related to the sonic hedgehog(SHH)signaling pathway were significantly increased by AN.Conclusions These results revealed that AN improved the developmental competence of porcine preimplantation embryos by activating SHH signaling against oxidative stress and could be used for large-scale production of high-quality porcine embryos.

血清RAGE、HMGB1水平与重症肺炎急性呼吸窘迫综合征发病及IFN-γ/IL-4变化的关系

目的探究血清晚期糖基化终产物受体(RAGE)、高迁移率族蛋白B1(high mobility group protein B1,HMGB1)水平与重症肺炎(SP)急性呼吸窘迫综合征(ARDS)发病及γ-干扰素(IFN-γ)/白细胞介素4(IL-4)变化的关系。方法前瞻性选取2020年3月至2022年2月我院收治的100例SP患儿为研究对象,根据患儿是否发生继发性ARDS将患儿分为ARDS组(n=56)和对照组(n=44),收集患儿一般资料,采集外周血以酶联免疫吸附法进行RAGE、HMGB1、IFN-γ和IL-4表达水平检测,采用多因素logistic回归分析SP患儿继发ARDS的影响因素,采用Pearson相关性分析其与IFN-γ/IL-4的相关性,并采用受试者工作曲线(ROC)分析RAGE、HMGB1表达对SP患儿继发ARDS的预测价值。结果两组SP患儿性别、年龄、体温以及发病季节之间无显著差异,ARDS组致病菌种类多于对照组,PaO_(2)/FiO_(2)和APS评分、血清RAGE、HMGB1、IFN-γ和IL-4表达水平以及IFN-γ/IL-4比值均高于对照组(P<0.05)。经多因素logistic回归分析可知,致病菌种类、PaO_(2)/FiO_(2)、RAGE、HMGB1表达、IFN-γ、IL-4和IFN-γ/IL-4均为SP患儿继发ARDS的影响因素。经Pearson相关检验,SP患儿血清RAGE、HMGB1表达水平与IFN-γ、IL-4和IFN-γ/IL-4均呈正相关(P<0.05)。经ROC曲线分析可得,血清RAGE、HMGB1水平预测SP患儿发生ARDS的AUC分别为0.707和0.750,灵敏度分别为73.2%、64.3%,特异度分别为68.2%、77.3%,两者联合预测的AUC为0.848,灵敏度和特异度分别为80.4%和81.8%。结论SP继发ARDS患儿血清中RAGE、HMGB1表达水平较高,与IFN-γ/IL-4呈正相关,监测患儿血清RAGE、HMGB1表达对SP患儿继发ARDS的风险有一定的预测价值。

高脂肪饮食促进TLR2的表达促进3T3L1脂肪细胞分泌IFN-γ诱导胰岛素抵抗的发生及发展

目的:探讨高脂饮食诱导胰岛素抵抗的机制,以及了解高脂饮食诱导胰岛素抵抗的脂肪细胞的表型变化。方法:雄性C57 BL/6 J小鼠,给予正常饮食和高脂饮食。从正常饮食或高脂饮食喂养2周的小鼠中分离附睾脂肪组织。实时荧光定量RT-PCR检测γ-干扰素(Interferonγ,IFN-γ)和toll样受体2(toll-like receptor 2,TLR2)mRNA的表达。流式细胞术来检测表达TLR2或IFN-γ的脂肪细胞的数量。苏木精-伊红染色分析胰腺组织。免疫组化分析脂肪组织中TLR2和IFN-γ的表达。FFA或Zymosan A处理3T3-L1脂肪细胞,并通过实时荧光定量RT-PCR检测IFN-γ和TLR2 mRNA的表达。结果:对脂肪细胞中基因表达谱的分析表明,高脂肪摄入诱导了IFN-γ和TLR2的表达提高。流式细胞术分析显示存在共表达TLR2和IFN-γ的脂肪细胞(TLR2/IFN-γ脂肪细胞),与皮下脂肪组织相比,高脂肪摄入增加了内脏脂肪组织中TLR2/IFN-γ脂肪细胞的数量。游离脂肪酸通过TLR2信号增加3T3-L1脂肪细胞中IFN-γ的表达。结论:TLR2/IFN-γ脂肪细胞可能通过诱导内脏脂肪组织IFN-γ的表达,参与高脂诱导的胰岛素抵抗的发生。

茶花鸡2号IFN-α基因克隆及生物信息学分析

本研究旨在克隆茶花鸡2号α干扰素基因(IFN-α)CDS区序列并进行生物信息学分析,为后续研究IFN-α基因对茶花鸡2号免疫功能的影响提供参考。以茶花鸡2号为实验对象设计IFN-α引物,提取总RNA,反转录PCR扩增并克隆其编码区序列,进行生物信息学分析。结果显示茶花鸡2号IFN-α基因CDS序列全长582 bp,IFN-α蛋白等电点5.05,平均疏水指数-0.514,为酸性亲水蛋白,且存在信号肽和1个跨膜区,主要定位于细胞核。IFN-α蛋白被4个N糖基化位点、5个O糖基化位点和20个磷酸化位点修饰,主要由α-螺旋与无规卷曲构成。茶花鸡2号与固始鸡、海兰鸡、惠阳胡须鸡、罗曼鸡和乌骨鸡的氨基酸同源性分别为95.9%、97.9%、97.9%、97.9%和95.9%。IFN-α蛋白与IRF7、IFNAR1、IFNAR2和IFNK等蛋白存在互作关系。本研究结果可为进一步探讨IFN-α基因在茶花鸡2号病毒疾病防治中的作用提供理论依据。

Adjuvant Effects of Recombinant Plasmids of Porcine IL-4 and IFN-γ to Cysticercosis cellulosae Vaccines in Mice and Pigs

To investigate the adjuvant potential of porcine IL-4 and IFN-γ in mice and pigs, the genes of porcine IL-4 and IFN-γ were cloned and the recombinant mammalian expression plasmids were constructed for in vivo expression of the cytokines. Adjuvant effects of recombinant expression plasmids of IL-4 and IFN-γ （pcDNA-IL-4, pcDNA-IFN-γ） co-administrated with Cysticercus cellulosae crude antigen or TSOL18 recombinant protein antigen have been carried out in mice and pigs, respectively. We have demonstrated that recombinant plasmids of the cytokines as an adjuvant could induce stronger immune response in mice and pigs. With the C. cellulosae parasite antigen, porcine pcDNA-IL-4 induced higher specific antibody of immunized mice than pcDNA-IFN-γ. But pcDNA-IFN-γ is significantly stronger than that of no adjuvant or empty plasmids with the antigen control group. For the TSOL18 recombinant protein antigen vaccine, pcDNA-IL-4 still had a stronger ability to enhance specific antibody in swine than pcDNA-IFN-γ （P 〈 0.01）, but the immune protective rate was lower in challenged pigs （only 68.7%）. Although pcDNA-IFN-γ showed lower specific antibody, the protection rate was very high （91%） than other group （P 〈 0.01）. This study indicated that the recombinant expression plasmids of porcine IL-4 and IFN-γ display stronger adjuvant effects to C. cellulosae vaccine, further research should be carried out for understanding of the interaction mechanism.

血红素加氧酶-1通过诱导抗病毒蛋白的表达增强IFN-α抗HBV效应

目的探讨血红素加氧酶-1(HO-1)对HBV复制的作用及HO-1联合α-干扰素(IFN-α)的抗病毒效应。方法以HepG2.2.15细胞和HBV 1.3质粒转染HepG2细胞即HepG2-HBV1.3为HBV复制细胞模型;血红素(Hemin)分别处理HepG2.2.15和HepG2-HBV1.3细胞,诱导HO-1表达;CCK-8评估Hemin对HepG2、HepG2.2.15的毒性作用;化学发光法分析Hemin处理组及si-HO-1等实验组上清液中HBsAg、HBeAg;RT-qPCR分析HO-1、IFN-β、HBV-DNA;Western blot分析IRF-3、JAK/STAT信号通路中相关分子的表达;Hemin联合IFN-α处理HepG2.2.15,监测HO-1是否具有协同IFN-α抗病毒效应。结果Hemin剂量依赖性诱导HO-1,HO-1被诱导后发挥显著的抗HBV效应,同时IFN-β、IRF-3及JAK/STAT信号通路中IRF-9、MxA的表达均增加。沉默HO-1表达能逆转Hemin诱导组的抗病毒效应,同时I型干扰素IFN-β也呈现低表达,JAK/STAT信号通路中的IRF-9、MxA的表达也被抑制。Hemin联合IFN-α发挥更强的抗病毒作用。结论HO-1能够发挥抗HBV效应,这种效应可能是增加IRF-3的磷酸化诱导I型干扰素表达来激活JAK/STAT信号通路发挥抗病毒效应;HO-1可以协同IFN-α发挥抗病毒作用。

Associations between maternal vitamin D status and porcine litter characteristics throughout gestation

Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy,and the regulation of foetal growth across mammalian species.However,the temporal changes in maternal vitamin D sta-tus throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated.The abundance of 25(OH)D in maternal plasma was quantified by HPLC–MS/MS at gestational days(GD)18,30,45,60 and 90(n=5–11 gilts/GD).Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30(P<0.05).The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight,ovulation rate,mean litter weight,number of live foetuses,percentage prenatal survival,and sex ratio of the litter was assessed.Maternal 25(OH)D(P=0.059)concentrations tended to be positively associated with percentage prenatal survival on GD60.On GD90,maternal 25(OH)D(P<0.05)concentrations were inversely associated with gilt weight.Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90(P<0.05).This study has provided novel insights into temporal changes in maternal vitamin D status throughout ges-tation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight,percentage prenatal survival and percentage of male foetuses in the litter.Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species.