猪β防御素研究进展

β防御素是猪体内分泌的一类抗菌肽，广泛分布于各个组织中，在抵抗病原入侵和免疫调节中发挥着重要作用。猪β防御素对细菌、真菌和病毒都有很强的杀灭作用，并且具有分子量小、热稳定性好、无残留等优点，是理想的抗生素替代品。研究表明，猪β防御素的表达与个体抗病力正相关，增加内源性防御素表达或直接摄入外源性防御素均能增强机体免疫力并促进生长。脂肪酸、氨基酸、维生素、益生菌、病原微生物等外源刺激皆可影响β防御素的表达。随着防御素相关研究的深入，将有望解决养猪生产中长期使用抗生素而导致的耐药性、药物残留及环境污染等问题。文章介绍了猪β防御素的结构特征、表达特异性及生物学功能，主要综述了外源刺激对β防御素表达的影响及其作用机制，初步说明外源β防御素作为饲料添加剂在仔猪生产中的效果，旨在为猪β防御素的深入研究及其在生猪健康养殖中的应用提供参考。     

Effect of eleutheroside B/E on proliferation and expression of PPARγ and TGF-β1 in HBZY-1 cells cultured with high glucose

AIM: To explore the effects and mechanism of eleutheroside (ETS) B or E on the proliferation of HBZY-1 cells treated with high glucose. METHODS: The HBZY-1 cells were cultured under high glucose condition. The 4th generation of HBZY-1 cells was used for determining the optimal cell density, which was consistent with the growth regulation curve of the cells. The cells were divided into 6 groups: low glucose (LG) group, high glucose (HG) group, high glucose plus ETS-B/E (low dose, medium dose and high dose) groups, and high glucose plus losartan (LTG) group. After all cells were treated with the corresponding drugs at 24 h, 48 h and 72 h, the inhibitory rate of the proliferation was measured, and the expression of TGF-β1 and PPARγ was detected by immunocytochemistry and Western blotting. RESULTS: The best cell density was 2 000 cells/well, which was complied with the basic rules of the cell growth, and high glucose significantly promoted the HBZY-1 cell proliferation. At each time point, the inhibitory effects of ETS-B/E were significantly different between HG group and LTG group on the proliferation of the HBZY-1 cells (P<0.05). The expression of TGF-β1 was significantly inhibited, and the expression of PPARγ was significantly promoted by ETS-B/E (P<0.05). ETS-E showed stronger effect than ETS-B (P<0.05) in a concentration- and time-dependent manner. CONCLUSION: ETS-B/E significantly inhibits the proliferation of HBZY-1 cells under high glucose condition by decreasing TGF-β1 expression and promoting PPARγ expression.     

甲状腺素对猪小肠上皮细胞miR-let-7a基因表达及细胞增殖的影响

miR-let-7a在动物细胞的分化、增殖与凋亡等方面发挥越来越重要的作用。甲状腺激素(TH)作用非常广泛,机体的每个细胞几乎都是TH作用的靶细胞,其可以促进组织分化、生长和成熟。本实验用甲状腺素(T4)浓度分别为(0、0.02、0.03、0.05、0.075、0.1、0.2μmol/L)在体外培养猪的小肠上皮细胞。结果表明:T4处理组的细胞体积形态相对于空白对照组没有明显变化;当T4添加浓度为0.03μmol/L时,细胞的增殖率显著低于其他组(P0.05);当T4浓度为0~0.03μmol/L时,let-7a的表达随着添加剂量的增加而升高,浓度从0.03~0.2μmol/L变化时,let-7a的表达呈现降低趋势,浓度为0.03μmol/L时表达量极显著高于其他组(P0.01)。let-7a的表达量与细胞增殖呈负相关。     

Use of Porcine Meal in Plant‐based Practical Diets for Pacific White Shrimp,Litopenaeus vannamei

Two growth trials were conducted to evaluate and confirm the efficacy of a porcine meal (PM) with high protein content (>90%) as an alternative feed ingredient in commercial‐type feed formulation for Pacific white shrimp, Litopenaeus vannamei. Six experimental diets were formulated for the two growth trials. The first five diets contained increasing levels (0, 1, 2, 4, and 6%) of PM as a replacement for soybean meal in a plant‐based diet with low inclusion level (6%) of fish meal (FM). The last experimental diet was produced utilizing 4.2% PM to completely replace FM. In Trial 1, shrimp (1.5 g initial mean weight, 20 shrimp/tank, n = 4) were offered test diets for 6 wk in a semirecirculation system. At the end of Trial 1, shrimp fed with the diet containing 6% PM exhibited significantly enhanced weight gain (WG), feed conversion ratio (FCR), and survival compared to those fed with the diet devoid of FM. As survival was poor across all treatments and different densities could mask growth results the trial was repeated. In Trial 2, shrimp (0.85 g, 15 shrimp/tank, n = 4) were offered diets for 6 wk. Dietary supplementation of PM at 6% significantly improved WG, FCR, and apparent net protein retention in contrast with the treatment devoid of FM, confirming the same trends in Trial 1. No significant difference was detected in protein, lipid, moisture, and mineral profiles of whole‐body shrimp as well as survival across all the treatments. Results of this study indicate that PM is a good high protein source in shrimp feeds, which can be included up to 6% in the low FM‐based diet without compromising the growth of shrimp.     

Identification and Molecular Characteristics Analysis of Porcine Epidemic Diarrhea Virus Variants

To ascertain a diarrhea case in a pig farm in Shandong province,the pathological changes of dead piglets were observed and nested RT-PCR test was carried out on 7 diarrhea samples for porcine epidemic diarrhea virus (PEDV).Pathological examination revealed that intestine was detected as enlargement,hyperemia and edema.After histological examination,the typical microscopic lesions of intestine were disappearance of epithelial cells,villus shrinkage and shortening.The result of nested RT-PCR showed that all of the 7 samples could amplify a specific target band of PEDV.Molecular characteristics of two field strains showed that they had an amino acid homology of 92.3% to 92.4% with vaccine CV777 and 96.6% to 98.6% with other previous field strains which sequences were downloaded from GenBank.Phylogenetic tree analysis further revealed that all of PEDV strains could be mainly divided into two clusters of G1 and G2. G2 consisted of the field strains of our study and other field strains from USA,China and so on,which had the same sequence characteristics of two insertions and one deletion,while G1 consisted of all vaccines of CV777 and several older field strains from China and Korea.These results indicated that our field strains were the dominant strains in recent epidemic diarrhea occurrence.Moreover,its molecular characteristics might be a characterization of differentiating the field and vaccine strains.     

超氧化物歧化酶模拟物对氧化应激IPEC-J2细胞抗氧化性能的影响

试验旨在探究在H₂O₂诱导的氧化应激状态下，超氧化物歧化酶模拟物（SODm）对仔猪空肠上皮细胞系（IPEC-J2）的保护作用。利用MTT法筛选出构建氧化应激模型H₂O₂的适宜浓度；将IPEC-J2细胞分别用0、0.05、0.5、5、50、500、2 000 U/mL SODm进行培养，利用MTT法分别在2、4、8、12 h时测定各组细胞存活率，筛选出SODm作用的适宜浓度和时间；根据构建的氧化应激模型和SODm适宜浓度和时间，将IPEC-J2细胞随机分为空白组、模型组、SODm处理组（SODm_0.5、SODm₅、SODm₅₀）和超氧化物歧化酶（SOD）处理组（SOD_0.5、SOD₅、SOD₅₀），分别测定各组细胞存活率、活性氧（ROS）含量及细胞内SOD、谷胱甘肽过氧化物酶（GSH-Px）活性及丙二醛（MDA）含量和总抗氧化能力（T-AOC）。结果表明：①H₂O₂构建细胞氧化应激模型的适宜浓度是1.0 mmol/mL。②当不同浓度SODm分别作用4、8、12 h时，0.5~50 U/mL SODm组细胞存活率显著高于空白组（P<0.05）；且8 h时存活率最高，在12 h时处于下降趋势。③除空白组外，SODm₅和SOD₅预处理的IPEC-J2存活率显著高于其他组（P<0.05）；且SODm和SOD组中细胞ROS含量显著低于模型组（P<0.05）；模型组与空白组相比，均显著降低了SOD、GSH-Px活性和T-AOC水平，提高了MDA含量（P<0.05）；与模型组相比，所有SODm和SOD处理组均显著提高了SOD、GSH-Px活性和T-AOC水平，降低了MDA含量（P<0.05），同时SODm₅₀组T-AOC水平显著低于SOD₅₀组（P<0.05）。综上，SODm对H₂O₂诱导氧化损伤状态下IPEC-J2具有保护作用。     

基于S基因序列分析新型猪流行性腹泻病毒的遗传演化

最近，笔者实验室在青藏高原地区发现两种新亚型藏猪源猪流行性腹泻病毒（PEDV），为进一步调查新型PEDV是否在四川腹泻猪群中存在或流行，对实验室2018-2019年保存的116份猪腹泻粪便或肠组织样本进行PEDV的检测及其纤突蛋白基因（spike）分子特征研究。结果表明：腹泻样本的PEDV检出率为42.2%（49/116，95% CI=33.1%~51.8%），并获得了13条完整的S基因序列，全长为4 149~4 170 bp，序列相似性为94.2%~99.9%，其中SWUN-H3-CH-SCYA-2019的S基因与藏猪源新G1亚群PEDV的序列相似性高达97.0%~98.6%。遗传演化研究结果表明13株PEDV S基因划分为G1和G2大群，其中SWUN-H3-CH-SCYA-2019位于藏猪源新G1亚群；SWUN-19-CH-SCZY-2018、SWUN-4-CH-SCXC-2018、SWUN-1-CH-SCNJ-2019和SWUN-3CH-CH-SCZG-2019位于G2亚群中一个独立的分支，且与藏猪源新G2亚群毒株有着较近的亲缘关系。为了进一步研究13株PEDV的演化过程，以贝叶斯进化分析软件包（BEAST）进行分歧时间估算，结果表明SWUN-H3-CH-SCYA-2019的分歧时间约为2012.3年，早于藏猪源新G1亚群其余毒株的最早分歧时间（2015.7年）；SWUN-4-CH-SCXC-2018、SWUN-19-CH-SCZY-2018和SWUN-3CH-CH-SCZG-2019的分歧时间约为2014.2年，早于G2亚群的藏猪源毒株2014.7年，所有藏猪源PEDV的分歧时间均晚于四川毒株。本研究在四川地区首次发现了藏猪源PEDV，并且从毒株的分歧时间推断青藏高原的藏猪源PEDV来源于四川，为新型PEDV分子遗传进化的监测提供了依据。     

苦参碱对奶牛乳腺上皮细胞增殖、凋亡及抗氧化能力的影响

【目的】探究苦参碱对体外培养的奶牛乳腺上皮细胞(BMECs)增殖、凋亡及抗氧化能力的影响。【方法】利用含0(A组),25(B组),50(C组),75(D组)和100μg/mL(E组)苦参碱的培养基培养奶牛乳腺上皮细胞。通过四甲基偶氮唑盐(MTT)法检测BMECs活性,采用流式细胞仪(AnnexinV/PI双染法)检测苦参碱对BMECs凋亡的影响,并检测苦参碱对BMECs抗氧化酶活性及丙二醛(MDA)含量的影响,采用real-time PCR对BMECs中Caspase-3、p53、STAT1和SOCS3基因的相对表达量进行检测。【结果】用药5d时,低质量浓度(25和50μg/mL)苦参碱对BMECs增殖具有促进作用,高质量浓度(75和100μg/mL)苦参碱对细胞增殖具有抑制作用;B~E组BMECs的凋亡率均极显著高于A组(P0.01);B~E组BMECs培养上清液中NO和乳酸脱氢酶(LDH)水平明显高于A组。B~E组BMECs的过氧化氢酶(CAT)活性均比A组高,其中C组极显著高于A组(P0.01);B~E组的谷胱甘肽过氧化物酶(GSH-Px)活性均极显著高于A组(P0.01),E组的超氧化物歧化酶(SOD)水平极显著高于A组(P0.01),各组MDA含量无显著性差异。与A组相比,苦参碱上调了B~E组BMECs中Caspase-3、p53、STAT1和SOCS3基因的相对表达量。【结论】低质量浓度苦参碱能够促进BMECs增殖,高质量浓度苦参碱则会抑制BMECs增殖;不同质量浓度苦参碱均可提高BMECs的抗氧化能力,其中50μg/mL苦参碱提高BMECs抗氧化能力的效果最明显。     

Ultrastructural impairment of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice

AIM: To investigate the ultrastructural changes of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice. METHODS: BALB/c mice were randomly divided into diabetic group and control group. The expression of insulin and platelet-endothelial cell adhesion molecule-1 (CD31) in islet microvessels was detected by immunohistochemical staining. The ultrastructural changes of islet β cells and islet microvessels were observed under transmission electron microscope. RESULTS: Compared with control group, the number of islet β cells, ratio of β cells/α cells, average number of secretory granules in β cells and insulin expression area per islet in diabetic group were significantly decreased (P<0.01). Besides, diabetic group had fewer microvessels with lower expression of CD31 (P<0.01). Mitochondria in islet microvascular endothelial cells and pericytes in diabetic group were swelling. The basement membrane of islet microvessels became thicker in diabetic group (P<0.01). CONCLUSION: Islet microvascular endothelial cells were impaired in type 1 diabetic mice.     

Vitamin C enhanced myocardial differentiation of dedifferentiated fat cells

AIM: In order to observe the myocardial differentiation capacity of the dedifferentiated fat (DFAT) cells treated with vitamin C in vitro. METHODS: DFAT cells were dedifferentiated from the mature rat adipocytes with ceiling adherent culture. The DFAT cells of passage 3 were used in the study. Vitamin C and/or neonatal rat heart tissue lysate were added into the culture medium to induce myocardial differentiation for 3 weeks. The cell morphology was observed under microscope. The myocardial-specific markers, such as cTnT, GATA-4 and NKx2.5, were examined by the methods of immunofluorescence, PCR and Western blot. RESULTS: Mature rat adipocytes dedifferentiated into fibroblast-like DFAT cells after ceiling adherent culture. The DFAT cells spontaneously differentiated into cardiomyocyte-like cells under normal culture condition with a low incidence. After treated with neonatal rat heart cell lysate, the DFAT cells became cardiomyocyte-like cells that had bigger size, longer shape and myotubule-structure. The expression of cTnT, GATA-4 and NKx2.5 was remarkably increased at both mRNA and protein levels as compared with the normal cultured DFAT cells. The expression of cTnT, GATA-4 and NKx2.5 was further increased in DFAT cells after treating with vitamin C. No spontaneous beating cell was observed. CONCLUSION: Vitamin C enhances the differentiation of DFAT cells into cardiomyocyte-like cells.