Embryonic tissue extracts modulate cell-fate determination in human epidermal stem cells

AIM: To investigate the phenotypic changes and cell-fate determination of keratinocyte stem cells in vitro. METHODS: Improved collagen Ⅳ-coated adhesion method was used to isolate and culture the keratinocyte stem cells after neutron-protease selectively digested the dermo-epidermal junctions. Morphological features and identification of these immature cells were studied by immunochemistry and confocal microscopy analysis. After treated with extracts from 14-day-old mouse fetuses at the concentration of 10% (W/V), phenotypic changes and the cell-fate determination of keratinocyte stem cells (KSCs) were detected by flow FACS analysis and RT-PCR assays. RESULTS: Immunocytochemical analysis proved that KSCs and their subunits were positive for α₆ and β₁ integrin, keratin 19 and 14, p63, nestin and PCNA, yet negative for keratin 10 expressions. Double staining immunofluorescence demonstrated that markers such as α₆ integrin and CD71 was co-expressed in KSCs, and there were important regional differences in the distribution of α6 integrin and CD71 in KSCs and transit amplifying cells (TA cells). Meanwhile, two-color flow cytometric analysis of α₆ integrin and CD71 consistently revealed that after treated with an extract from mouse fetuses, the percent of α+6 CD71+ and CD71+ fraction increased and reached to 68.43% and 4.51% of the total isolated cells, respectively. However, the percent of α+6 CD71- fraction reduced from 22.49% to 10.92% determined by flow cytometry. Moreover, the expression levels of β₁ integrin and some putative biological markers in human epidermal cells were analyses by RT-PCR after fetus extract treatment. The relative gene expressions of β₁ integrin, K19 and K10 increased in KSCs, whereas the expression of K14 in keratinocyte stem cells was observed to be significantly suppressed when compared to the appropriate controls. CONCLUSION: The keratinocyte stem cells isolated by collagen Ⅳ-coated adhesion method have some characteristics of adult stem cells and perhaps the potentiality to differentiate and cross-differentiate. The regional differences in the distribution of α6 integrin and CD71 are one of the transition markers to discriminate the KSCs and TA cells. Furthermore, fetus extracts contribute to not only the proliferation and self-renewal of KSCs but also the dedifferentiation of TA cell subunits.     

不同剂量氟苯尼考对猪血液生化指标及猪瘟抗体的影响

40头70日龄健康猪随机分为高、中、低剂量组和对照组,每组10头,各试验组注射猪瘟弱毒疫苗(2头份/猪),同时高、中、低剂量组分别按每千克体质量于饲料中加入120、60、30 mg氟苯尼考,连续给药7 d,停药后1、8、15、22、29 d,对相关的血液生化指标以及猪瘟抗体水平进行检测分析.结果表明,停药后1、8 d,所有氟苯尼考添加组的猪瘟抗体滴度下降,仅120 mg/kg组差异显著(P<0.05),停药15 d后抗体水平恢复至正常水平;60和120mg/kg给药组TP含量在停药后1、8 d均显著下降(P<0.05);所有给药组的ALT活性在所有检测时间内均显著上升(P<0.05或P<0.01);所有给药组的γGT活性在停药后1 d均显著上升(P<0.05),而60和120 mg/kg组γGT在第8天仍显著上升(P<0.05);所有给药组的BUN含量在停药后1 d均显著升高(P<0.05或P<0.01);AST、LDH、CHE活性和ALB、CRE含量与对照组相比无显著差异(P>0.05);表明每千克体质量60 mg以上氟苯尼考在饲料中饲喂7 d后,对部分生化指标有一定的影响,应引起重视.     

虹鳟IL-17多克隆抗体的制备及初步鉴定

本研究根据NCBI已发表序列设计引物,提取经植物血凝素刺激后的虹鳟Oncorhynchus mykiss头肾细胞总RNA,采用RT-PCR方法扩增IL-17成熟肽基因,测序结果表明所获得的序列与发表序列相一致。将该基因重组至原核表达载体pET32a中,并转化大肠杆菌Escherichia coli Rosetta,进行诱导表达。SDS-PAGE电泳结果表明:目的蛋白以包涵体形式表达,大小约为32k Da,重组蛋白经Ni-NTA系统纯化、复性后纯度达90%以上,以其免疫小鼠制备虹鳟IL-17多克隆抗体。ELISA结果显示其效价为1∶25 600,而间接免疫荧光结果显示所制备的多克隆抗体能够特异性地识别真核细胞中瞬时表达的虹鳟IL-17。本研究成功制备虹鳟IL-17多克隆抗体,为下一步IL-17在虹鳟黏膜免疫中的作用及其佐剂效应研究奠定基础。     

昆明鼠卵母细胞的孤雌发育及对其干扰核移植试验的预防措施

本文着重研究了在昆明鼠细胞核移植过程中，卵母细胞孤雌发育的激活原因、形成过程，并提出了一些防止其干扰细胞核移植的措施。     

鹌鹑源火鸡隐孢子虫在小白鼠和雏鸡体内内生发育阶段的超微结构比较

用鹌鹑源火鸡隐孢子虫（Cryptos poridium meleagridis）卵囊分别感染昆明系小白鼠和固始雏鸡，用透射电镜和扫描电镜观察比较了C．meleagridis在两种试验动物体内的内生发育虫体超微结构和致病性的差异。利用扫描电镜观察发现，鹌鹑源火鸡隐孢子虫（C.meleagridis）在小白鼠和雏鸡体内的寄生部位有较大差异，在小白鼠体内寄生于十二指肠，但在雏鸡体内主要寄生于回肠；C．meleagridis深嵌于小白鼠肠微绒毛丛内，微绒毛较为完整；但在雏鸡回肠，C．meleagridis似黏附在肠黏膜表面，微绒毛脱落明显，对雏鸡致病性明显比对小白鼠的致病性强。利用透射电镜在两种试验动物的样品中均观察到不同发育阶段的滋养体、裂殖体和大配子体以及正在孢子化的卵囊。滋养体和裂殖体在发育过程中可明显见到粗面内质网结构，裂殖生殖中期阶段粗面内质网尤其发达；小白鼠体内的C．meleagridis虫体与肠黏膜接触处形成一凹陷，寄生部位较深，而在雏鸡体内无此现象。此外，利用透射和扫描电镜均观察到虫体寄生部位周围微绒毛密度高而且也比其它部位长。形成这些差异的原因有待于进一步探讨。     

CD20片段抗体在红花悬浮细胞中的表达研究

【目的】研究用红花愈伤组织建立红花悬浮细胞培养体系的条件,并利用红花悬浮细胞通过农杆菌介导转化法表达CD20复合片段抗体。【方法】以红花子叶为外植体,研究不同种类激素组合对愈伤诱导率的影响,筛选优质愈伤组织进行悬浮培养,探索不同初始接种量、蔗糖质量分数、水解酪蛋白质量浓度对红花悬浮细胞生长的影响。将CD20复合片段抗体基因两端引入XmaⅠ/EcoRⅠ酶切位点,并将其与pEASY-T1和pBasta载体相连,构建pEASY-T1-CD20克隆载体和pBasta-CD20表达载体,再将构建的pBasta-CD20表达载体转入根瘤农杆菌,利用根瘤农杆菌介导红花悬浮细胞转化法表达CD20复合片段抗体。【结果】红花子叶愈伤诱导最佳条件为MS+NAA 2mg/L+6-BA 0.5mg/L、温度(25±0.1)℃、光照70μmol/(m2·s)连续光照,继代培养条件为MS+NAA 1mg/L+6-BA 0.5mg/L、30μmol/(m2·s)连续光照、温度(25±0.1)℃。悬浮细胞体系培养最佳条件为不加琼脂粉的MS+NAA 1mg/L+6-BA 0.5mg/L、接种量2.5g(50mL培养基)、蔗糖质量分数2%、水解酪蛋白800mg/L。pBastaCD20表达载体构建成功,且目的蛋白在红花悬浮细胞中成功表达。【结论】成功构建了红花悬浮细胞培养体系,并用该体系成功表达了CD20复合片段抗体。     

合肥野生动物园5种雉鸡接种新城疫疫苗后抗体水平测定

选择成年白冠长尾雉5只,白腹锦鸡5只,白鹇3只,红腹锦鸡3只和孔雀11只,同时用L系弱毒冻干苗和新城疫油乳剂灭活疫苗进行免疫接种,通过血凝抑制试验(HI)测定其抗体滴度.结果表明,在免疫接种后26 d,白冠长尾雉抗体效价为9.8log2,白腹锦鸡为8.0log2,白鹇为8.0log2,红腹锦鸡为10.7log2;孔雀在免疫后接种0、8、15、20、25 d,抗体水平分别为3.8log2、5.0log2、6.2log2、7.5log2及8.2log2.     

监测口蹄疫抗体的液相阻断ELISA法的改进和体会

液相阻断ELISA（LBE）法由于具有高度的敏感性、特异性和重复性,目前被广泛应用于动物免疫抗体效果监测实践工作中,特别是为防控牛、羊亚洲I型口蹄疫（FMD）做出了积极的贡献。但该方法操作繁琐,稍有不慎就会导致实验结果发生偏差或试验失败,笔者就该方法应用于亚I型口蹄疫抗体检测中的改进和体会作一总结。     

粮食污染物残留免疫分析技术研究与示范

随着人们对粮食安全问题的重视,政府部门不断加强对粮食安全的监测工作。针对粮食生产、加工、储运过程中的化学污染物安全性问题,研究粮食污染物快速监测技术,研究污染物免疫分析方法通过利用抗原、抗体的特异性反应来检测农药含量,设计并筛选吡虫啉、多溴联苯醚、功夫菊酯、氰戊菊酯4种半抗原,制备相关高特异性的抗体,分析吡虫啉、多溴联苯醚、功夫菊酯与气相色谱法(GC)相比,稻谷、大米、玉米样品通过相关性分析均高度相关,操作简便、快速。