Restricted localization of claudin-16 at the tight junction in the thick ascending limb of Henle's loop together with claudins 3, 4, and 10 in bovine nephrons

Claudin-16 is one of the tight junction protein claudins and has been shown to contribute to reabsorption of divalent cations in the human kidney. In cattle, total deficiency of claudin-16 causes severe renal tubular dysplasia without aberrant metabolic changes of divalent cations, suggesting that bovine claudin-16 has some roles in renal tubule formation and paracellular transport that are somewhat different from those expected from the pathology of human disease. As the first step to clarify these roles, we examined the expression and distribution of claudin-16 and several other major claudin subtypes, claudins 1-4 and 10, in bovine renal tubular segments by immunofluorescence microscopy. Claudin-16 was exclusively distributed to the tight junction in the tubular segment positive for Tamm-Horsfall glycoprotein, the thick ascending limb (TAL) of Henle's loop, and was found colocalized with claudins 3, 4, and 10. This study also demonstrates that bovine kidneys possess segment-specific expression patterns for claudins 2-4 and 10 that are different from those reported for mice. Particularly, distribution of claudin-4 in the TAL and distal convoluted tubules was characteristic of bovine nephrons as were differences in the expression patterns of claudins 2 and 3. These findings demonstrate that the total lack of claudin-16 in the TAL segment is the sole cause of renal tubular dysplasia in cattle and suggest that the tight junctions in distinct tubular segments including the TAL have barrier functions in paracellular permeability that are different among animal species.     

Tight junction proteins in the canine epidermis: A pilot study on their distribution in normal and in high IgE-producing canines

Epidermal tight junctions (TJ) have been well-described in human medicine and are involved in many skin diseases such as atopic dermatitis (AD). In dogs, there are no data regarding the implication of TJ in skin diseases including canine AD.The aim of this study was to compare the expression and the distribution of ZO-1, occludin and claudin-1 in the epidermis of healthy and atopic dogs.Skin biopsies from 6 high IgE-producing beagles sensitized to house dust mite (atopic group) were used. Skin specimens from nine healthy dogs without skin issues were sampled (healthy group).Immunoperoxydase staining was used to study the staining pattern of zonula occludens-1 (ZO-1), occludin and claudin-1 in the epidermis of healthy and atopic dogs. Positive controls were healthy human skin samples.Labeling patterns were assessed by 2 examiners blinded to the identities of the specimens. Comparisons between groups were performed using an exact Wilcoxon-Mann-Whitney test.The mean total expression score of claudin-1 was lower in atopic dogs as compared to healthy subjects. Occludin and ZO-1 expression remained unchanged within each group.These results suggest a defect in claudin-1 expression in the nonlesional epidermis of atopic dogs.     

宫内发育迟缓仔猪小肠形态和屏障功能相关基因的表达特征

为了研究宫内发育迟缓(IUGR)仔猪小肠形态和屏障功能相关基因的表达特征,选取24窝"长白×大白"杂交新生仔猪,每窝选取1头IUGR仔猪和1头正常出生体重(NBW)仔猪,分别于7、21和28日龄屠宰8头IUGR仔猪和8头NBW仔猪,采集小肠样品进行分析.结果表明:1)与NBW仔猪相比,IUGR仔猪7日龄时空肠绒毛高度、...     

Hepatocyte nuclear factor 4 alpha is associated with mesenchymal-epithelial transition in developing kidneys of C57BL/6 mice

During kidney development, the metanephric mesenchyme (MM) develops into the nephron through mesenchymal-epithelial transition (MET). We have previously reported that knock-down of the expression of hepatocyte nuclear factor 4 alpha (Hnf4a) gene induces failure of cellular organization in the condensed mesenchyme (CM) of cultured embryonic kidneys. To elucidate the details of MET during nephrogenesis, embryonic mouse kidneys were analyzed by electron microscopy, immunohistochemistry, and molecular biology. The findings showed that the intercellular junction, but not the basal lamina, was present in the CM. Additionally, immediately after Hnf4a gene expression, the expression of epithelial genes (Krt8, Tjp1, and Cdh1) increased, and those of mesenchymal genes (Acta1 and Vim) decreased, in the CM compared to the MM. To clarify the relationship between MET and Hnf4α, the fibroblast cell line with forced expression of Hnf4α protein were analyzed. In this model, it was noted that Hnf4α induced increasing epithelial and decreasing mesenchymal gene expression. In these, up-regulation of Pvrl1, -2, and Mllt4 genes which mediate the formation of apico-basal polarity, were found. These results, and those of previous findings, indicate that Hnf4α protein is associated with the initiation of MET in early nephrogenesis.     

腹水综合征肉鸡空肠黏膜组织结构及相关基因表达分析

试验旨在研究肉鸡腹水综合征发生发展过程中空肠核因子κB （NF-κB）及紧密连接蛋白（闭合蛋白（occludin）、紧密连接蛋白-1（claudin-1）和胞质紧密黏连蛋白1（zonula occluden 1,ZO-1）） mRNA相对表达量,以及绒毛长度、隐窝深度和绒隐比的变化,为肉鸡腹水综合征的防治提供理论依据。选取40羽1日龄罗斯肉鸡常规饲养7 d后,随机分为对照组和模型组（饲料添加3%猪油和4%鱼粉,饮水添加0.12% NaCl,9~11℃低温饲养）,35日龄检测体重,并取心脏组织测腹水心脏指数,取空肠组织以HE染色切片测量绒毛长度和隐窝深度,实时荧光定量PCR检测空肠NF-κB和紧密连接蛋白的基因表达量。结果表明,与对照组相比,模型组肉鸡体重极显著下降（P<0.01）,腹水心脏指数极显著升高（P<0.01）,空肠绒毛长度、绒隐比极显著下降（P<0.01）,隐窝深度极显著升高（P<0.01）,NF-κB mRNA相对表达量极显著升高（P<0.01）,occludin、claudin-1和ZO-1 mRNA相对表达量极显著下降（P<0.01）。表明肠道黏膜结构与功能异常及肠通透性增高促进了肉鸡腹水综合征的发生发展。     

Developmental changes in intercellular junctions and Kv channels in the intestine of piglets during the suckling and post-weaning periods

Background: The intestinal epithelium is an important barrier that depends on a complex mixture of proteins and these proteins comprise different intercellular junctions. The purpose of this study was to investigate the postnatal and developmental changes in morphology, intercellular junctions and voltage-gated potassium(Kv) channels in the intestine of piglets during the suckling and post-weaning periods.Results: Samples of the small intestine were obtained from 1-, 7-, 14-, and 21-d-old suckling piglets and piglets on d 1, 3, 5, and 7 after weaning at 14 d of age. The results showed that the percentage of proliferating cell nuclear antigen(PCNA)-positive cells and alkaline phosphatase(AKP) activity, as well as the abundances of E-cadherin,occludin, and Kv1.5 m RNA and claudin-1, claudin-3, and occludin protein in the jejunum were increased from d 1to d 21 during the suckling period(P 0.05). Weaning induced decreases in the percentage of PCNA-positive cells,AKP activity and the abundances of E-cadherin, occludin and zonula occludens(ZO)-1 m RNA or protein in the jejunum on d 1, 3 and 5 post-weaning(P 0.05). There were lower abundances of E-cadherin, occludin and ZO-1m RNA as well as claudin-1, claudin-3 and ZO-1 protein in the jejunum of weanling piglets than in 21-d-old suckling piglets(P 0.05). The abundances of E-cadherin, occludin, ZO-1 and integrin m RNA were positively related to the percentage of PCNA-positive cells.Conclusion: Weaning at 14 d of age induced damage to the intestinal morphology and barrier. While there was an adaptive restoration on d 7 post-weaning, the measured values did not return to the pre-weaning levels, which reflected the impairment of intercellular junctions and Kv channels.     

胰高血糖素样肽-2对断奶仔猪肠上皮紧密连接蛋白相关基因表达的影响

本文旨在研究胰高血糖素样肽-2(GLP-2)对离体培养的断奶仔猪空肠上皮紧密连接蛋白相关基因表达的影响,探讨GLP-2调节肠道黏膜屏障的可能机制。将断奶仔猪空肠组织块置于含0、1×10-8、1×10-7mol/L的GLP-2的细胞培养液中进行培养,考察不同GLP-2添加水平对断奶仔猪空肠上皮丝裂原活化蛋白激酶(MAPK)信号通路细胞外调节蛋白激酶丝裂原活化蛋白激酶激酶1/2(MEK1/2)、p90核糖体S6蛋白激酶(p90RSK)以及紧密连接蛋白ZO-1、Oc-cludin、Claudin-1基因表达的影响,待确定出GLP-2能有效促进紧密连接蛋白相关基因表达的剂量后,再向培养液中添加MEK1/2的特异性阻断剂U0126,考察阻断MAPK经典通路后紧密连接蛋白ZO-1、Occludin、Claudin-1的基因表达的变化。结果表明,与对照组相比,将空肠组织块置于含1×10-7和1×10-8mol/L GLP-2的培养液中培养72 h均能显著促进MEK1/2、p90RSK以及紧密连接蛋白ZO-1、Occludin、Claudin-1的基因mRNA相对表达量(P<0.05),除ZO-1的基因之外,上述各蛋白的基因mRNA相对表达量还随着GLP-2浓度的增高而升高(P<0.05);向1×10-7mol/L的GLP-2组添加U0126阻断p90RSK、ERK1/2的基因表达后,紧密连接蛋白ZO-1、Occludin、Claudin-1的基因mRNA相对表达量也显著下降(P<0.05)。由此可知,GLP-2能够促进断奶仔猪空肠上皮紧密连接蛋白ZO-1、Occludin、Claudin-1的基因表达,而MAPK通路可能是GLP-2调控肠道紧密连接蛋白基因表达的重要信号通路之一。     

Effects of dietary supplementation with l-arginine on the intestinal barrier function in finishing pigs with heat stress

Previous studies showed heat stress reduces body weight gain and feed intake associated with damaged intestinal barrier function, and l -arginine (L-Arg) enhanced intestinal barrier function in young animals under stress. The aim of this study was to evaluate effects of L-Arg on serum hormones, intestinal morphology, nutrients absorption and epithelial barrier functions in finishing pigs with heat stress. Forty-eight finishing pigs (Landrace) were balanced for sex and then randomly assigned to six groups: TN group, thermal neutral (22°C, ~80% humidity) with a basal diet; HS group, heat stress (cyclical 35°C for 12 hr and 22°C for 12 hr, ~80% humidity) with a basal diet; PF group, thermal neutral (22°C, ~80% humidity) and pair-fed with the HS; the TNA, HSA and PFA groups were the basal diet of TN group, HS group and PF group supplemented with 1% L-Arg. Results showed that HS decreased (p < .05) the thyroxine concentrations and increased (p < .05) the insulin concentrations in serum compared with the TN group, but 1% L-Arg had no significant effects on them. Both HS and PF significantly increased (p < .05) the mRNA expression of cationic amino acid transporters (CAT1 and CAT2) and decreased the mRNA expression of solute carrier family 5 member 10 (SGLT1) in the jejunum compared with the TN group. Compared with the TN group, HS reduced the expression of tight junction (TJ) protein zonula occluden-1 (ZO-1) and occludin, but PF only decreased ZO-1 expression in the jejunum. Results exhibited that dietary supplementation with 1% L-Arg improved the intestinal villous height, the ratio of villous height to crypt depth, and the expression of occludin and porcine beta-defensin 2 (pBD2) in the jejunum of intermittent heat-treated finishing pigs. In conclusion, dietary supplementation with 1% L-Arg could partly attenuate the intermittent heat-induced damages of intestinal morphology and epithelial barrier functions in finishing pigs.     

Expression of Occludin in Canine Testis and Epididymis

Tight junctions (TJ) in inter-Sertoli junctional areas and epididymal epithelia are important for the formation of blood-testis barrier (BTB) and blood-epididymal barrier (BEB). In this study, the expression of occludin, an integral member of the TJ, was verified in canine testis and epididymis. Both low molecular weight (MW) (25-28 kDa) forms as well as high MW (68-72 kDa) forms of occludin were detected in the testis and epididymis using Western blot. The relative amount of the high MW forms of occludin vs low MW forms was higher in the testis than in the epididymis. Some difference in the composition of different MW forms of occludin was found along the segments of epididymis, suggesting the possible correlation between cellular composition of occludin proteins and paracellular permeability of epithelia along the epididymal tubule. In the testis, intense occludin immunoreactivity was found in the basally located inter-Sertoli junctional area. Diffused immunoreactivity of occludin was also found in the cytoplasm of Sertoli cells. A similar pattern of zonula occludens-1 immunoreactivity was found in the cytoplasm of Sertoli cells, suggesting that occludin was not confined to the inter-Sertoli junctional areas and that subcellular localization of occludin in the Sertoli cells was dynamically regulated during spermatogenesis in canine testis. In the epididymis weak immunoreactivity was found in the apical sides and cytoplasm of epithelial cells.     

自噬调节因子Atg5和Beclin1在不同来源小鼠胚胎早期发育过程中的表达分析

旨在探究自噬调节因子Atg5和Beclin1在胚胎早期发育过程中的表达模式及胚胎的不同生产方式对两种因子表达的影响。本研究将6~8周龄雌性小鼠进行超数排卵,分为2组,一组收集小鼠卵母细胞,孤雌激活处理后进行体外培养;另一组超排小鼠与公鼠1：1合笼,第2天收集小鼠受精卵进行体外培养;分别在2细胞期、4~8细胞期、桑葚胚期和囊胚期收集不同阶段小鼠孤雌激活胚胎和自然受精胚胎。提取RNA和蛋白,通过实时荧光定量PCR、Western blot等方法检测自噬关键因子Atg5和Beclin1的表达,通过间接免疫荧光法检测Atg5和Beclin1在小鼠囊胚中的表达定位。结果显示,小鼠自然受精和孤雌激活胚胎在发育各时期均可表达Atg5和Beclin1,表达量在胚胎发育的早期呈现出较高的水平,其中二者的表达在小鼠自然受精胚胎中从2细胞期起逐渐降低,而在孤雌激活胚胎的4~8细胞阶段表达量最高,与同期自然受精胚胎差异极显著（P<0.01）;从4细胞期开始,各时期孤雌激活胚胎中Atg5和Beclin1蛋白表达水平均高于自然受精胚胎,差异极显著（P<0.01）;在囊胚中,滋养层细胞和内细胞团中均可检测到Atg5和Beclin1蛋白的荧光,但内细胞团中的荧光强度高于滋养层细胞,且Beclin1蛋白在孤雌激活胚胎囊胚内细胞团中荧光强度高于自然受精胚胎。自噬关键因子Atg5和Beclin1在不同来源小鼠胚胎早期发育各时期均有不同程度的表达,提示自噬对早期胚胎发育的调控作用与胚胎的生产方式存在一定关联,研究结果为进一步探索细胞自噬参与哺乳动物胚胎发育的生理调控提供理论依据。     

Expression Analysis of Autophagy Regulators Atg5 and Beclin1 during Early Embryonic Development of Mouse (Mus musculus) from Different Sources

This study aimed to explore the expression patterns of autophagy regulators Atg5 and Beclin1 in the early embryonic development and the effects of different embryonic production methods on the expression of the two factors. Female mice aged 6-8 weeks were subjected to superovulation and divided into 2 groups. The mouse oocytes of one group were collected, and cultured in vitro after parthenogenetic activation. The other group of female mice were caged with male mice (1:1), and the next day, the mouse fertilized eggs were collected for in vitro culture. Parthenogenetic activated embryos and naturally fertilized embryos were collected at 2 cell stage, 4-8 cell stage, mulberry embryo stage and blastocyst stage, respectively. RNA and protein were extracted, real-time fluorescence quantitative PCR, Western blot and other methods were used to detect the expression of key autophagy factors Atg5 and Beclin1. And indirect immunofluorescence was used to detect the expression and location of Atg5 and Beclin1 in mouse blastocysts. The results showed that Atg5 and Beclin1 were expressed in all development stages of naturally fertilized and parthenogenetic activated embryos in mice, and showed a high level in the early stage of embryonic development. The expression of Atg5 and Beclin1 were gradually reduced from the 2 cell stage in mouse naturally fertilized embryos. The expression levels of Atg5 and Beclin1 in parthenogenetic activated embryos were the highest in the 4-8 cell stage, which was extremely significantly different from the naturally fertilized embryos of the same period (P<0.01). From the 4 cell stage, the expression levels of Atg5 and Beclin1 in parthenogenetic activated embryos were higher than naturally fertilized embryos at all subsequent stages, the difference was extremely significantly different (P<0.01). In mouse blastocysts, the fluorescence of Atg5 and Beclin1 protein could be detected in the trophoblast cells and the inner cell mass, but the fluorescence intensity in the inner cell mass was higher than that in the trophoblast cells. In addition, the fluorescence intensity of Beclin1 protein in the inner cell mass of parthenogenetic activated embryos was higher than that in naturally fertilized embryos. Atg5 and Beclin1, the key autophagy factors, are expressed at different levels in the early development of mouse embryos from different sources. It is suggested that the regulation of autophagy on early embryonic development is related to embryo production modes. The results will provide a theoretical basis for further exploring the role of autophagy in the physiological regulation of mammalian embryo development.     

蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响

本试验旨在研究中国蜂胶乙醇提取物（ethanol extract of Chinese propolis，EECP）对细菌脂多糖（lipopolysaccharide，LPS）刺激下体外培养奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接渗透性的影响。EECP中总酚酸和总黄酮含量测定采用福林酚法和硝酸铝法，并建立LPS诱导奶牛乳腺上皮细胞（bovine mammary epithelial cells，MAC-T）炎症模型，采用CCK-8法测定EECP对MAC-T相对增殖率的影响，利用实时荧光定量PCR（RT-qPCR）评估EECP对LPS诱导的MAC-T细胞炎症相关因子（IL-6、IL-8、TNF-α和IL-1β）相对mRNA转录水平；以及对紧密连接蛋白（occludin、ZO-1）相对mRNA转录水平进行检测，并进一步利用免疫荧光技术对紧密连接膜蛋白进行定位，确定EECP对LPS诱导MAC-T细胞炎症紧密连接渗透性的影响。结果显示：EECP中总酚酸含量为106.35 mg没食子酸当量（GAE）·g^-1、总黄酮含量为320.85 mg芦丁当量（RE）·g^-1；CCK-8结果显示EECP的安全浓度为0~15 μg·mL^-1，并可有效提高LPS刺激下MAC-T的活力；LPS刺激显著增加了细胞炎症相关因子IL-6、IL-8、TNF-α和IL-1β mRNA的转录量（P<0.001）；但2.5~15.0 μg·mL^-1 EECP预处理显著降低了IL-6、IL-8、TNF-α和IL-1β mRNA的转录量；与此类似，LPS刺激显著抑制了紧密连接蛋白基因（occludin、ZO-1）mRNA的转录量（P<0.01），而EECP预处理后紧密连接蛋白基因（occludin和ZO-1）mRNA的转录量显著增加（P<0.05）；免疫荧光染色试验也证实EECP能通过上调紧密连接蛋白（occludin、ZO-1）的表达，缓解LPS诱导的乳腺上皮细胞屏障功能紊乱。该结果证实，EECP对细菌脂多糖诱导奶牛乳腺上皮细胞炎症具有良好的保护作用，这为利用中国蜂胶预防奶牛乳腺炎提供了试验基础。     

Low crude protein diets supplemented with casein hydrolysate enhance the intestinal barrier function and decrease the pro-inflammatory cytokine expression in the small intestine of pigs

To reduce nitrogen excretion and lower feeding costs, low crude protein (CP) diets are sometimes proposed, however, a great reduction of dietary CP concentration (>4% reduction vs. recommended concentration), even supplemented with essential and nonessential amino acids (AA) can detrimentally affect small intestinal barrier function and immunity, possibly due to the excessive lack of peptides. Here we hypothesize that with an extremely low CP concentration diet, protein-derived peptides, rather than AA supplementation, can improve intestinal barrier development and health. To test this hypothesis, 21 growing pigs (19.90 ± 1.00 kg body weight) were randomly assigned to 3 treatments with control diet (16% CP), or low CP diets (13% CP) supplemented with AA (LCPA) or casein hydrolysate (LCPC) for 28 days. In comparison with the control diet, the LCPA diet decreased the protein expression level of jejunal barrier factor zonula occludens-1 (ZO-1) and stem cell proliferation factor leucine-rich repeat-containing G-protein-coupled receptor-5, whereas the LCPC diet enhanced intestinal barrier function by increasing the protein expression level of jejunal occludin and ZO-1 and ileal mucin-2. The LCPA diet reduced Lactobacillus counts, whereas the LCPC diet increased Lactobacillus counts and reduced Escherichia coli counts in the ileum. The LCPA diet also increased protein expression levels of pro-inflammatory cytokine interleukin-6 (IL-6) and IL-22, whereas the LCPC diet decreased protein expression levels of pro-inflammatory IL-1β, IL-17A and tumor necrosis factor-α in the ileum. Collectively, the casein hydrolysate supplementation of low CP diets showed beneficial effects on the small intestinal barrier, bacterial community, and immunity in pigs, pointing to the important role of protein-derived peptides in small intestinal health in cases of low crude protein diets.     

敌草快和硫辛酸对育肥猪肠道结构及消化功能的影响

本文旨在研究敌草快和硫辛酸(LA)对育肥猪肠道结构及消化功能的影响,以探究LA对敌草快引起的应激是否有缓解作用。选取24头(70.64±3.61)kg的健康大白阉公猪,按照2×2因子设计,随机分为对照组、LA组、敌草快组和LA+敌草快组,每个组设6个重复,每个重复1头猪。试验期29 d。LA添加剂量为800 mg/kg饲粮,饲喂贯穿试验全程,而一次性腹腔滴注敌草快于试验第15天进行,无腹腔滴注敌草快试验猪滴注等量的生理盐水。于试验第29天对所有试验猪前腔静脉采血后屠宰取样,运用试剂盒检测血浆和肠道氧化损伤标志物的含量以及空肠食糜消化酶的活性,苏木精-伊红(HE)染色后运用图像分析软件观察肠道形态结构并测定绒毛高度、隐窝深度,并计算绒毛高度/隐窝深度(V/C),荧光定量PCR法检测闭合蛋白(occludin)和紧密结合蛋白(ZO-1)的相对表达量。结果表明:1)敌草快极显著升高了试验猪血浆和肠道丙二醛(MDA)、蛋白质羰基(PCO)和8-羟基鸟苷(8-OHd G)的含量(P0.01),极显著降低了十二指肠、空肠及回肠绒毛高度、隐窝深度(P0.01),极显著增大了十二指肠和空肠V/C(P0.01),显著增大了回肠V/C(P0.05),极显著降低了空肠occludin和ZO-1 m RNA相对表达量(P0.01),极显著降低了空肠食糜中淀粉酶、胰蛋白酶以及脂肪酶的活性(P0.01)。2)饲粮中添加LA对试验猪血浆和肠道MDA、PCO和8-OHd G含量的影响均不显著(P0.05),对十二指肠、空肠和回肠的绒毛高度、隐窝深度以及V/C的影响均不显著(P0.05),对空肠occludin和ZO-1 m RNA相对表达量的影响也不显著(P0.05),对空肠食糜中淀粉酶、胰蛋白酶以及脂肪酶活性的影响差异也均不显著(P0.05)。3)在应激状态下,饲粮中添加LA使猪血浆8-OHd G含量显著降低(P0.05),十二指肠绒毛高度、空肠绒毛高度以及回肠隐窝深度显著升高(P0.05),空肠V/C显著降低(P0.05),回肠V/C极显著降低(P0.01),空肠occludin和ZO-1 m RNA相对表达量显著升高(P0.05),空肠食糜淀粉酶和胰蛋白酶的活性显著提升(P0.05)。由此可见,敌草快引起了育肥猪强烈的氧化应激,导致了肠道结构的严重损伤并减弱了肠道的消化功能;在敌草快引起的应激状态下,饲粮中添加800 mg/kg LA能够减缓肠道结构的损伤并能一定程度地提高肠道的消化功能。     

Expression and localisation of claudin-1, -2, -3, -4, -5, -7 and -10 proteins in the normal canine mammary gland

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumour development. The aim of the present study was to analyse the expression of claudin-1, -2, -3, -4, -5, -7 and -10 in normal canine mammary glands. Samples from the inguinal mammary regions of 20 non-castrated, 1-13 years old female dogs were studied. Immunohistochemical analysis was performed on conventional specimens and tissue microarrays. The results of the immunohistochemical reactions detecting claudins in tissue sections were photodocumented. The immunoreactivity of claudins was quantitatively analysed on digital images using Leica QWin morphometry software. Intense membranous immunolabelling was found for claudin-1, -3 and -7, intense membranous with non-granular cytoplasmic immunolabelling for claudin-2, moderate membranous immunolabelling for claudin-4 and -5, and weak membranous immunolabelling for claudin-10. The occurrence of tight junctions was confirmed by ultrathin section electron microscopy. The available data suggested that claudins might be proteins preserved throughout the evolution of mammals. The results of our study support the concept that they are indeed preserved, since the same type of claudins, in identical distribution, could be detected in our canine mammary tissue samples as could be found in human mammary tissue.     

Labelling of rat endothelial cells with antibodies to vWF, RECA-1, PECAM-1, ICAM-1, OX-43 and ZO-1

Labelling with endothelium specific monoclonal antibodies, von Willebrand Factor (vWF), rat endothelial cell antigen-1 (RECA-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), OX-43 and zonula occludentes-1 (ZO-1), was investigated in cryostat sections of vessels from rats of different ages using a confocal microscope. The results showed that labelling of the vWF was positive in endothelial cells from adult, fetal and different ages of embryonic rat. Labelling with RECA-1 was weakly positive in adult rat aorta and lung endothelial cells but not in embryonic yolk sac endothelial cells. Labelling using PECAM-1, ICAM-1 and OX-43 was negative in both adult and embryonic endothelial cells. ZO-1 showed positive but very weak reactivity in embryonic yolk sac endothelial cells. The expression of vWF on vessels from adult and 19.5-day fetal tissues was strongly positive. However, the expression of vWF in embryonic endothelial cells was dependent on the gestational age. While the 11.5-day yolk sac vessels stained weakly, staining gradually increased in 13.5-, 15.5- and 17.5-day-old yolk sac vessels. The results suggest that vWF is a reliable endothelial cell marker in rat vascular endothelial cells, including both fetal and embryonic stages.     

Localization of extracellular matrix receptors in ICGN mice, a strain of mice with hereditary nephrotic syndrome.

Fibrotic degeneration was examined in the kidneys of ICR-derived glomerulonephritis (ICGN) mice, a novel inbred mouse line with a hereditary nephrotic syndrome of unknown etiology considered to be a good model of human idiopathic nephrotic syndrome. In the present study, we histochemically revealed changes in accumulation of extracellular matrix (ECM) components and in localization of integrins, cellular receptors for ECM, in the kidneys of ICGN mice with the progression of renal failure. Excessive accumulation of basement membrane (laminin and collagen IV) and interstitial (type III collagen) ECM components were demonstrated in the glomeruli and tubulointerstitum of ICGN mice. Marked deposition of type I collagen and tenascin was seen only in the glomeruli of ICGN mice but not in those of ICR mice as normal controls. Increased expression of integrin alpha1-, alpha2-, alpha5- and beta1-subunits in glomeruli with fibrotic degeneration and abnormal distribution of alpha6-subunit were noted in the kidneys of ICGN mice. Excessive laminin, a ligand of alpha6beta1-integrin, was demonstrated on the tubular basement membrane, but alpha6-subunit diffusely disappeared on the basal side of the tubular epithelial cells. We presumed that abnormal integrin expression in renal tubules causes epithelial cell detachment, and consequently tubular nephropathy, and results in disorder of ECM metabolism causing excessive accumulation of ECM components in the kidneys of ICGN mice.     

Beclin-1基因shRNA慢病毒载体的构建及其对B16F10细胞自噬及活力的影响

利用RNA干扰技术构建稳定沉默Beclin-l基因的B16F10小鼠黑色素瘤细胞系，分析稳转细胞系的自噬水平和活力，为探讨Beclin-1基因、自噬与肿瘤三者关系奠定基础。构建小鼠Beclin-l基因干扰载体，转染至293T细胞，获得慢病毒颗粒，将病毒颗粒感染B16F10细胞，通过加压筛选、有限稀释、细胞驯化得到稳定转染m-Beclin-1-shRNA1的B16F10细胞系。采用RT-PCR和免疫荧光技术检测对Beclin-l的干扰效果，Western blot和透射电镜分析稳转细胞系中自噬标志物LC3蛋白表达及自噬小体形成情况，CCK-8法检测稳转细胞系的活力。结果表明，成功构建了靶向抑制Beclin-l基因表达的shRNA，纯化的慢病毒滴度为1×10⁸ TU·mL^-1，建立的细胞系中Beclin-1 mRNA和蛋白表达都受到不同程度的抑制，mRNA的沉默效率约为75%（P<0.01），发现干预Beclin-1表达能够抑制LC3-Ⅱ蛋白的表达及自噬小体的形成数量，降低B16F10细胞活力。以上结果表明，干扰Beclin-1表达能够有效降低B16F10细胞自噬活性，促进B16F10细胞死亡，这为探讨Beclin-1基因功能及自噬在抗肿瘤中的作用奠定重要基础。     

Expression and localization of tight junction-related proteins in adult rat pituitary stem/progenitor cell niches

Pituitary endocrine cells are supplied by Sox2-expressing stem/progenitor cells in the anterior lobe of the adult pituitary gland. These SOX2-positive cells are maintained in two types of microenvironments (niches): the marginal cell layer (MCL)-niche and the parenchymal-niche. Recently, we isolated dense SOX2-positive cell clusters from the parenchymal-niche by taking advantage of their resistance to protease treatment as parenchymal stem/progenitor cell (PS)-clusters. In the present study, by analyzing these isolated PS-clusters, we attempted to identify novel structural characteristics of pituitary stem/progenitor cell niches. Quantitative real-time PCR showed that tight junction-related genes were distinctly expressed in the isolated PS-clusters. Immunocytostaining showed that the tight junction molecules, ZO-1 and occludin, were localized in the apical membrane facing the pseudo-follicle-like structure of the isolated PS-clusters regardless of the expression of S100β, which distinguishes the sub-population of SOX2-positive cells. Furthermore, immunohistochemistry of the pituitary glands of adult rats clearly demonstrated that ZO-1 and occludin were densely present in the parenchymal-niche encircling the pseudo-follicle, while they were observed in the apical membrane in the MCL-niche facing the residual lumen. Collectively, these tight junction-related proteins might be involved in the architecture and maintenance of the plasticity of pituitary stem/progenitor cell niches.