马铃薯class Ⅰ patatin基因在试管块茎形成中的功能

将正反义classⅠpatatin基因导入马铃薯品种甘农薯2号中,有2个转正义基因株系试管块茎的可溶性蛋白含量和LAH活性与对照相比有不同程度的增加,有3个转反义基因株系的可溶性蛋白质含量下降,并且所有转反义基因植株的LAH活性都不同程度地降低。试管块茎的诱导结果表明,有1个转正义基因株系的结薯株率和单株结薯数比其对照明显增加,有2个转反义基因株系的结薯株率和单株结薯数比对照明显减少,说明该classⅠpatatin基因参与了马铃薯试管块茎的形成及其调控。

中国美利奴羊MHC class Ⅰ区段基因组成预测分析

为了准确筛选中国美利奴羊MHC classⅠ区段,试验采用GenScan软件对覆盖绵羊MHC classⅠ区段的11个BAC克隆测序结果进行预测,在GenBank数据库中BlastX比对分析预测的基因,获得的信息在KEGG数据库中进行基因注释。结果表明:覆盖绵羊MHC classⅠ区段的11个BAC克隆共预测出基因数123个,其中编码抗体基因19个,占预测基因总数的15%;其他基因65个,占53%;未知基因39个,占32%;并明确了预测基因在BAC克隆上的分布情况及其编码的多肽可能具有的生物学功能。

朝鲜鹌鹑MHC class Ⅰ基因第2、3外显子多态性与NDV抗性的关联分析

【目的】研究朝鲜鹌鹑MHC classⅠ基因第1,2和3外显子的多态性,并与新城疫病毒抗性进行关联分析。【方法】采用胸肌注射的方法对14日龄朝鲜鹌鹑进行新城疫La Sota弱毒株攻毒,每周注射1次,连续攻毒3周后,心脏采血,采用β-微量法测定血清中新城疫抗体水平。PCR扩增朝鲜鹌鹑MHC classⅠ基因,测序后采用DNA Star、SPSS等分析软件研究朝鲜鹌鹑MHC classⅠ基因第1,2,3外显子的多态性,并分析不同基因型新城疫抗体水平的差异。【结果】朝鲜鹌鹑的新城疫抗体效价为26～210。PCR扩增获得了1 290bp的MHC classⅠ基因,其编码区序列为780bp,编码259个氨基酸。通过与已知序列日本鹌鹑RNA(AB005528)比对,得到长度分别为76,272和274bp的第1,2和3外显子。在第1外显子上未检测到突变为点,在第2和3外显子上共检测到11个突变位点(第2外显子上4个突变位点:257bp(A/C)、267bp(A/T)、366bp(A/G)和500bp(T/G);第3外显子上7个突变位点:795bp(T/C)、798bp(T/C)、810bp(T/G)、830bp(A/G)、873bp(A/T)、927bp(A/G)和932bp(C/G)),均为中度多态基因座,11个突变位点中,267bp(A/T)、830bp(A/G)、873bp(A/T)、932bp(C/G)突变导致编码氨基酸改变,500bp(T/G)、873bp(A/T)、932bp(C/G)突变位点基因型之间新城疫抗体水平存在显著差异(P<0.05)。【结论】朝鲜鹌鹑MHC classⅠ基因第2、3外显子具有丰富的多态性,并且与新城疫病毒抗性相关。

Effects of small interfering RNA inhibit Class Ⅰ phosphoinositide 3-kinase on human gastric cancer cells

AIM: To investigate the effects of small interfering RNA (siRNA)-mediated inhibition of Class?I?phosphoinositide 3-kinase (Class?I?PI3K) signal transduction on the proliferation, apoptosis, and autophagy of gastric cancer SGC7901 and MGC803 cells.METHODS: We constructed the recombinant replication adenovirus PI3K(I)-RNA interference (RNAi)-green fluorescent protein (GFP) and control adenovirus NC-RNAi-GFP, and infected it into human gastric cancer cells. MTT assay was used to determine the growth rate of the gastric cancer cells. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment. Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with MDC, LC3 staining, and transmission electron microscopy. Western blotting was used to detect p53, Beclin-1, Bcl-2, and LC3 protein expression in the culture supernatant.RESULTS: The viability of gastric cancer cells was inhibited after siRNA targeting to the Class?I?PI3K blocked Class?I?PI3K signal pathway. MTT assays revealed that, after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 27.48% ± 2.71% at 24 h, 41.92% ± 2.02% at 48 h, and 50.85% ± 0.91% at 72 h. After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 24.39% ± 0.93% at 24 h, 47.00% ± 0.87% at 48 h, and 70.30% ± 0.86% at 72 h (P < 0.05 compared to control group). It was determined that when 50 MOI, the transfection efficiency was 95% ± 2.4%. Adenovirus PI3K(I)-RNAi-GFP (50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells, and the results described here prove that RNAi of Class?I?PI3K induced apoptosis in SGC7901 cells. The results showed that adenovirus PI3K(I)-RNAi-GFP transfection induced punctate distribution of LC3 immunoreactivity, indicating increased formation of autophagosomes. The results showed that the basal level of Beclin-1 and LC3 protein in SGC7901 cells was low. After incubating with adenovirus PI3K(I)-RNAi-GFP (50 MOI), Beclin-1, LC3, and p53 protein expression was significantly increased from 24 to 72 h. We also found that Bcl-2 protein expression down-regulated with the treatment of adenovirus PI3K(I)-RNAi-GFP (50 MOI). A number of isolated membranes, possibly derived from ribosome-free endoplasmic reticulum, were seen. These isolated membranes were elongated and curved to engulf a cytoplasmic fraction and organelles. We used transmission electron microscopy to identify ultrastructural changes in SGC7901 cells after adenovirus PI3K(I)-RNAi-GFP (50 MOI) treatment. Control cells showed a round shape and contained normal-looking organelles, nucleus, and chromatin, while adenovirus PI3K(I)-RNAi-GFP (50 MOI)-treated cells exhibited the typical signs of autophagy.CONCLUSION: After the Class?I?PI3K signaling pathway has been blocked by siRNA, the proliferation of cells was inhibited and the apoptosis of gastric cancer cells was enhanced.

Impact of preformed donor-specific antibodies against HLA class Ⅰ on kidney graft outcomes:Comparative analysis of exclusively anti-Cw vs anti-A and/or-B antibodies

AIM To analyze the clinical impact of preformed antiH LA-Cw vs antiH LA-A and/or-B donor-specific antibodies(DSA) in kidney transplantation.METHODS Retrospective study, comparing 12 patients transplanted with DSA exclusively antiH LA-Cw with 23 patients with preformed DSA antiH LA-A and/or B.RESULTS One year after transplantation there were no differencesin terms of acute rejection between the two groups(3 and 6 cases, respectively in the DSA-Cw and the DSA-A-B groups; P = 1). At one year, eG FR was not significantly different between groups(median 59 mL /min in DSA-Cw group, compared to median 51 mL /min in DSA-A-B group, P = 0.192). Moreover, kidney graft survival was similar between groups at 5-years(100% in DSA-Cw group vs 91% in DSA-A-B group, P = 0.528). The sole independent predictor of antibody mediated rejection(AMR) incidence was DSA strength(HR = 1.07 per 1000 increase in MFI, P = 0.034). AMR was associated with shortened graft survival at 5-years, with 75% and 100% grafts surviving in patients with or without AMR, respectively(Log-rank P = 0.005).CONCLUSION Our data indicate that DSA-Cw are associated with an identical risk of AMR and impact on graft function in comparison with "classical" class I DSA.

Establishment of a Real-time Fluorescent Quantitative RTPCR Method for Detecting NP Gene of Class Ⅰ Newcastle Disease Virus(NDV)

Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( class Ⅰ,and class Ⅱ) according to their genetic relationship.To develop a method for rapid quantitative detection of class Ⅰ NDV,a pair of primers and a TaqM an probe were designed and synthesized according to the conservative sequence of NP gene of class Ⅰ NDV.The positive recombinant plasmid harboring NP gene of JS-18-05 isolate was used as a positive template to establish the standard curve.A real-time fluorescent quantitative RT-PCR method was established for rapid detection of class Ⅰ NDV with strong specificity,high sensitivity and good repeatability.The established method exhibited a good linear relationship within the concentration of 102 to 108 copies of NDV,by which 1 μl of 10 copy of NDV nucleic acid could be detected in the initial template.Compared with conventional virus isolation methods,the established method had similar sensitivity and led to the same results in detecting33 class Ⅰ,class Ⅱ NDV isolates.The study provided the basis for rapid quantitative detection of class Ⅰ NDVs and further clarification of their pathogenicity and pathogenic mechanism in poultry.

肯氏Ⅰ、Ⅱ类牙列缺损数字化印模及模型在可摘局部义齿中的应用

目的评估肯氏Ⅰ、Ⅱ类牙列缺损数字化印模及树脂模型技术在可摘局部义齿(RPD)中的应用效果。方法选择肯氏Ⅰ、Ⅱ类牙列缺损患者,按照义齿制作流程分组:数字化印模/树脂模型/钴铬合金铸造支架组(A组)、数字化印模/树脂模型/激光打印钛支架组(B组)、藻酸盐印模/石膏模型/钴铬合金铸造支架组(C组)、藻酸盐印模/石膏模型/激光打印钛支架组(D组),每组40例。对最终完成的RPD在口内就位情况进行检查,评估指标包括卡环固位力、连接体和基托在口内的密合度、咬合准确度,各项指标评估分值使用Kruskal-Wallis秩和检验进行分析。结果4组RPD各项指标的评分值差异无统计学意义。结论利用数字化印模及树脂模型完成的铸造钴铬合金和激光打印钛支架式RPD能够满足肯氏Ⅰ、Ⅱ类牙列缺损患者的临床修复要求。

Ⅰ类切口围术期抗菌药物预防使用合理性分析

目的分析濮阳油田总医院Ⅰ类切口围术期抗菌药物预防使用合理性情况,为后续临床抗菌药物的管理提供参考性依据。方法将濮阳油田总医院2023年1月至2023年6月开展的635例行Ⅰ类切口手术患者作为本次研究对象,回顾性分析接受Ⅰ类切口手术患者在围术期接受抗菌药物的治疗效果。结果本次研究中收入的635例Ⅰ类切口手术患者中,共有214例患者使用抗菌药物,针对每个科室Ⅰ类切口患者围术期抗菌药物预防性使用情况进行分析,脊柱外科患者使用抗菌药物占66.04%,神经外科占64.00%,手足与创伤骨科占50.90%;甲状腺血管外科、乳腺外科、疝与腹壁外科与泌尿外科中未使用任何抗菌药物,差异有统计学意义(P<0.05)。Ⅰ类切口围术期使用抗菌药物的种类主要为第一、二代头孢菌素类,以静脉给药为主。共使用抗菌药物7种,其中头孢唑林占59.35%,头孢呋辛占26.17%,差异有统计学意义(P<0.05);抗菌药物合理使用率为86.45%,不合理使用率为13.55%,主要以无指征预防用药、超疗程用药为主,差异有统计学意义(P<0.05)。结论本院接受Ⅰ类切口手术的患者中在围术期采取预防使用抗菌药物情况大部分合理,但仍存在少部分不合理情况,故应加强干预,保证抗菌药物的合理使用,提高抗菌药物合理率,保证患者用药安全。

医嘱点评干预对医院Ⅰ类切口手术患者围术期抗菌药物使用的影响

目的:分析医嘱点评干预对医院Ⅰ类切口手术患者围术期抗菌药物使用的影响,为临床Ⅰ类切口手术患者围术期抗菌药物的合理使用提供参考。方法:选取2020年1月—2021年6月(点评干预前,164例)和2021年7月—2022年12月(点评干预后,164例)抚州市东乡区人民医院收治的328例Ⅰ类切口手术患者作为研究对象,比较和分析点评干预前后不同手术患者抗菌药物的预防使用率和不合理用药类型。结果:医嘱点评干预后Ⅰ类切口手术患者抗菌药物的预防使用率明显低于医嘱点评干预前(28.66%vs 79.27%,P<0.05),并且在腹股沟疝手术、乳腺手术、骨科手术、白内障手术、甲状腺疾病手术等术式上的抗菌药物预防使用率均低于医嘱点评干预前(P<0.05);而医嘱点评结果显示,医嘱点评干预后患者围术期抗菌药物在给药时机、药品选用、用药疗程、用法用量、联合用药等方面的不合理率均明显低于医嘱点评干预前(P<0.05)。结论:医嘱点评干预确实可以有效改善医院Ⅰ类切口手术患者围术期抗菌药物的不合理使用情况,从而提升临床的合理用药水平,进而保障患者的用药安全。

五龙鹅MHC ClassⅠ基因克隆及同源建模研究

主要组织相容性复合体（MHC）与动物机体对外源性抗原的免疫应答之间存在关联。从GenBank/DDBJ/EMBL基因库中读取鸡、其他鸟类、爬行类和哺乳类的MHC ClassⅠ基因进行序列分析设计引物，使用LA—PCR法从五龙鹅的基因组中克隆了MHC ClassⅠ基因序列（DNA序列和mRNA序列GenBank登录号分别为：AM114925和AM114924），并分析其基因组结构。运用生物信息学技术对测序结果进行分析显示：基因组DNA由8个外显子和7个内含子组成，与鸡基因序列同源率为60．8％～64．1％，与人的同源率为42．9％。分子进化树进一步揭示了五龙鹅与鸡、其他鸟类、爬行类、哺乳类以及人类的进化关系，同源建模分析发现该基因由氨基末端结构域和羧基末端结构域构成。

新城疫病毒ClassⅠ强毒株HN单克隆抗体的研制及免疫学反应

目的:制备抗新城疫病毒(NDV)ClassⅠ强毒分离株9a5b的单克隆抗体。方法:以9a5b尿囊液为免疫原,接种6周龄BALB/c小鼠,以血凝抑制(HI)和间接免疫荧光(IFA)检测所获得的mAb。结果:成功制备获得NDV血凝素(HN)特异性mAb4株。免疫特性鉴定表明:在这4株mAb中,3H7和3H9株仅具有IFA和HI效价,且呈现ClassI毒株特异性。结论:该抗体材料为NDVClassⅠ和Ⅱ毒株的血清学鉴定提供极大的便利工具,并为NDVHN功能及受体学研究奠定了基础。

ClassⅠ新城疫病毒概述

新城疫病毒是影响养禽业健康发展的主要病原之一。新城疫病毒只有一个血清型,但根据亲源关系可以划分为ClassⅠ和ClassⅡ两大类。ClassⅠ新城疫病毒于2006年被证实存在,当前可被细分为9个基因型(1～9)。ClassⅠ所有自然分离株均为无毒株或弱毒株,但有经人工传代返强的报道,由于其分布广泛,数量庞大,对养禽业有巨大的潜在威胁。为了更好的了解ClassⅠ新城疫毒株的生物学特性,为研究ClassⅠ新城疫病毒提供参考,现将其基因组结构、分子流行病学及检测方法等综述如下。

ClassⅠ新城疫病毒NP基因分子特征的研究

为分析2002～2007年Class Ⅰ新城疫病毒分离株NP基因的分子特征,以及其在新城疫病毒演化过程中所起的作用,采用RT-PCR方法扩增分离株的核衣壳蛋白(NP)基因,并进行了测序,应用生物信息学软件分析NDVNP基因的分子特征。根据NP基因序列的遗传发生分析表明,ClassⅠ NDV2型毒株与DE/R49-99和美国株亲缘关系较近,而3型毒株与其他ClassⅠ毒株遗传关系较远,具有地区特色,形成一个独立的分支。生物信息学软件分析表明,NP蛋白N端1～400氨基酸比较保守,而C端序列(特别是氨基酸残基400～480)高度变异。ClassⅠ病毒2型毒株与国外分离株亲缘关系较近,可能有共同的起源;而3型毒株独立于其他ClassⅠ毒株,具有独特性,这两种毒株的来源不同;不同基因型NP蛋白之间比较保守,但是P蛋白的C端发生了多个氨基酸的变异,对其功能的影响还需进一步探讨。

新城疫病毒ClassⅠ强毒株磷蛋白在细胞中的表达与定位

根据ClassI新城疫病毒分离株9a5b编码序列设计特异性引物扩增P蛋白基因，原核表达后利用纯化的蛋白免疫BALB／C小鼠制备单克隆抗体，经二次亚克隆后筛选到4株针对P蛋白的单克隆抗体，利用所得单抗进行P蛋白在细胞内定位分析，NDV9a5b株按5M．O．I感染量接种DFl单层细胞，当PI=4h时，P蛋白呈点状弥散分布于细胞浆内；PI=6～8h时，P蛋白聚集于细胞核周围；PI=12h时，P蛋白呈单极化聚集于细胞核一侧；而PI=16h后开始形成合胞体，P蛋白在单个细胞中仍呈现单极分布；当PI=48h时，细胞核已发生碎裂，荧光强度有所减弱。本研究为深入开展P蛋白在病毒增殖以及细胞周期中的作用机制研究奠定了基础。

ClassⅠ新城疫病毒核衣壳蛋白在体外细胞感染过程中表达定位的检测

为分析核衣壳蛋白(NP)在新城疫病毒(NDV)感染真核细胞过程中细胞内定位的情况,本研究以NDV Class I强毒株9a5b作为免疫原制备4株NP特异性单克隆抗体(MAb),并将病毒株9a5b按5 MOI感染量接种DF1单层细胞,当接种后1 h～3 h时,NP有极少量表达并逐量增加,主要呈点状分布于细胞浆内;当接种后4 h～20 h时,NP聚集于细胞浆中,聚集数量与感染时间成正比;感染24 h后,细胞核碎裂,NP散在分布于细胞浆中。表明NP作为立早期蛋白,始终在胞浆中进行复制;在病毒感染初期,NP介导核糖核蛋白复合物发挥转录和翻译功能。本研究为深入开展NP与病毒mRNA转录、复制和包装的相互关系机制的研究奠定了基础。

建鲤MHC classⅠ基因全长cDNA的克隆与序列分析

采用同源克隆和末端快速扩增(RACE)方法克隆了建鲤(Cyprinus carpio var.jian)MHC classⅠ基因全长cD-NA并进行了序列分析。结果显示:实验得到了1914 bp的建鲤的MHC classⅠ全长cDNA序列;建鲤MHC classⅠ基因包括1044 bp的开放阅读框(ORF),118 bp的5'非编码区(UTR)以及752 bp的3'非编码区(UTR),含有保守的半胱氨酸残基,N-糖基化位点。氨基酸序列比对结果显示,建鲤MHC classⅠ与日本鲤的MHC classⅠ相似性最高,为66.0%;与虹鳟、大西洋鲑、青鳉、红鳍东方鲀的相似性分别为54.5%、57.9%、44.3%、42.0%,与小鼠、大鼠、人的相似性分别为29.1%、28.7%、29.7%。

新城疫病毒ClassⅠ与ClassⅡ毒株交叉血凝抑制试验快速分类

ClassⅠ新城疫病毒(NDV)分离自健康家禽泄殖腔棉拭子,其基因序列与ClassⅡNDV存在很大差异,为了建立简单快速的NDV分类方法,用SPF鸡制备ClassⅠ和ClassⅡ毒株多价抗血清分别与选取的ClassⅠ和ClassⅡ代表毒株进行交叉血凝抑制反应。结果表明所有ClassⅠ毒株比同ClassⅡ毒株的交叉血凝抑制值高8～64(3～6log2)倍;而ClassⅡ毒株与ClassⅡ抗血清的HI效价比同ClassⅠ毒株的交叉血凝抑制值高2～8(1～3log2)倍,结果与基因分型高度一致,说明可以用交叉血凝抑制试验进行初步分类。本研究结果显示,NDVClassⅠ弱毒株血凝性与广泛应用的NDV弱毒疫苗株不同,基因序列分析可以部分解释这种差异,另外可能HN蛋白的空间结构也会影响其与血凝素结合的特性。

Serum major-histocompatibility-complex class Ⅰ-related chain A antibody detection for the evaluation of graft dysfunction in renal allograft recipients

Background In addition to the well-known antibodies against human leukocyte antigens （HLA）-induced kidney-graft rejection, polymorphic major-histocompatibility-complex （MHC） class Ⅰ-related chain A （MICA） antigens can elicit antibodies and have been suggested to play a role in the antibody-mediated allograft rejection （AMR）. We carded out a prospective study of MICA antibodies in post-renal transplant patients to determine the association between MICA antibodies, C4d staining, histological features, and graft outcome.Methods We tested 52 patients who had biopsy results due to graft dysfunction. The MICA antibodies in concurrent sera were determined by Luminex. All patients were followed up for one year after renal biopsy. The influence of antibody production on the function of graft was analyzed.Results Antibodies against MICA were positive in 15 out of the 52 patients （28.9%）. The presence of MICA antibodies was associated with renal-allograft deterioration. During one-year follow-up, the estimated glomerular filtration rate （eGFR） decreased （24.0±3.4）% among recipients with anti-MICA antibodies. However, among recipients without anti-MICA antibodies, the eGFR has declined only （8.4＋3.0）% （P=0.017）. The association between C4d staining,histological features and MICA antibody production was found no significant difference.Conclusion Besides anti-HLA antibodies, the presence of post-transplant MICA antibody is associated with poor graft outcome and increases the risk of graft failure.

MHC Class Ⅰ Antigen Presentation-Recently Trimmed and Well Presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is the key to the cellular immune response.Non-self intracellular proteins are processed into short peptides and transported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by several chaperone proteins to form trimeric complex.MHC class I complex loaded with optimised peptides travels to the cell surface of antigen presentation cells to be recognised by T cells.The cells presenting non-self peptides are cleared by CD8 positive T cells.In order to ensure that T cells detect an infection or mutation within the target cells the process of peptide loading and class I expression must be carefully regulated.Many of the cellular components involved in antigen processing and class I presentation are known and their various functions are now becoming clearer.Cellular & Molecular Immunology.2004;1(1):22-30.

虎MHC ClassⅠ基因的克隆及测序

根据猫的主要组织相容性复合体Ⅰ类分子cDNA序列设计PCR引物,从基因组上成功扩增并克隆了MHCClassⅠ基因片段,命名为TLA-A。该片段全长2 820 bp,包含MHC ClassⅠ分子基因约85%的长度,包括Exon 1部分序列、intron 1、exon 2、intron 2、exon 3、intron 3、exon 4、intron 4、exon 5、intron 5、exon 6、intron 6和exon 7部分序列。与其他物种的ClassⅠ基因相比,虎与家猫、猎豹、豹猫、大熊猫、狗、马、松鼠猴、人、黄猩猩等物种的同源性依次为93.4%、91.8%、87.3%、86.4%、85.1%、85.1%、84.6%、84.3%、83.7%,种间序列差异主要表现在exon 2、exon 3两个肽结合区。