Effects of two commercial diets and technical feed treatment on stomach lesions and immune system of fattening pigs

The impact of technical feed treatment and diet on stomach lesions and traits of the local and systemic immune system were investigated in fattening pigs. Feeding groups differed in technical feed treatment (standard ground meal vs. finely ground and pelleted feed) and diet (soya bean meal vs. rapeseed meal/DDGS/soya beans). Pigs were fattened approximately 10 weeks by ad libitum feeding and slaughtered subsequently. Gastric alterations were assessed by a macroscopic scoring system [macroscopic stomach score (MSC) 0 =  normal to 4 =  severe lesions]. For immunological investigations, lymphocytes from blood and jejunal tissues were isolated. T‐cell phenotyping was carried out by staining intestinal lymphocytes with monoclonal antibodies for CD4 and CD8 and flow cytometric measurements. MSC was higher in animals fed finely ground and pelleted feed compared with their counterparts. Significant interactions between diet and feed treatment considering the MSC were observed (p = 0.027). There was no effect of diet or technical feed treatment on T cells of blood, Lymphonodi gastrici or lamina propria (LP) and intraepithelial cells. However, technical feed treatment significantly affected subsets of CD4⁺, CD8⁺, CD8^low, CD4/CD8 double‐positive T cells, the mean fluorescence intensity of CD4⁺ T cells and the ratio of CD8^low/CD8^high T cells in Peyer's patches (PP). All named parameters were reduced in PP of animals fed finely ground and pelleted feed compared with animals fed standard ground meal. Furthermore, significant differences between T cells of lymph nodes and LP were observed between animals with middle MSC (MSC = 1–2.5) and animals with high MSC (MSC = 3–4). Significant alterations in T cells of PP were observed between animals of low (MSC = 0–0.5) and high MSC. The observed effects provide the evidence that the impact of technical feed treatment is not limited on the stomach lesions. Possible stimuli and consequences of the immune system should be studied in more detail.     

百里香酚对山羊子宫内膜上皮细胞的体外毒活性研究

为了探究百里香酚对山羊子宫内膜上皮细胞(goat endometrial epithelial cells,g EECs)的毒性作用,采用不同浓度的百里香酚作用于g EECs后,观测其对细胞生长曲线,细胞形态学变化,以及细胞膜结构完整性的影响。结果表明,百里香酚对EECs的毒性作用与其浓度呈剂量依赖效应(y=-0.008x+1.2202,R~2=0.9767),即随着药物浓度的增加细胞的增殖抑制率增大,药物对细胞的半数增殖抑制率(IC50)为100μg/m L。当50、100μg/m L的百里香酚作用于EECs时,g EECs的细胞核、细胞浆的形态均未产生明显变化,但当150μg/m L的百里香酚作用于g EECs时,细胞膜破裂,细胞死亡;而且药物浓度≥100μg/m L时细胞LDH的释放量显著高于正常细胞组,g EECs细胞膜的完整性受到破坏。百里香酚作用于山羊子宫内膜上皮细胞后呈现一定的毒性作用,且毒性作用和药物的浓度有关。百里香酚作用于g EECs的安全范围在0~100μg/m L,研究结果为百里香酚治疗奶牛等经济动物的炎症性疾病的临床用药浓度提供理论依据。     

Isolation,proliferation and characterization of endometrial canine stem cells

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT‐PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue‐derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue‐derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the “Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy” for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue‐derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow‐derived MSC in dog.     

印楝素对min6细胞增殖及胰岛素分泌的影响

【目的】研究印楝素对min6细胞增殖、葡萄糖摄取及胰岛素分泌的影响,并探讨其作用机制。【方法】不同浓度印楝素处理min6细胞48 h,检测细胞增殖量。在5.5和25.0 mmol·L~(-1)葡萄糖条件下,检测印楝素处理细胞的葡萄糖摄取水平,胰岛素ELISA检测试剂盒检测细胞胰岛素的分泌。【结果】与对照组相比,在5.5和25.0mmol·L~(-1)葡萄糖条件下,印楝素均表现促进细胞增殖活性。印楝素处理下,与25.0 mmol·L~(-1)葡萄糖条件相比,5.5 mmol·L~(-1)葡萄糖表现出更显著地促进葡萄糖摄取和胰岛素分泌的作用。【结论】印楝素可能通过调控葡萄糖水平影响胰岛素的分泌。     

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。     

安淮山羊骨骼肌卫星细胞的分离培养与鉴定

为了分离得到安淮山羊骨骼肌卫星细胞,成功构建山羊骨骼肌卫星细胞系,为相关研究提供实验材料。以安淮山羊背最长肌肌肉组织为材料,采取胶原酶和胰蛋白酶结合的二步消化法,结合差速贴壁法纯化原代骨骼肌卫星细胞。对骨骼肌卫星细胞中特异性基因Pax7用免疫荧光染色法及PCR扩增加以鉴定。通过分离、纯化,得到纯度较高的山羊骨骼肌卫星细胞,其形态为圆形,折光性强。培养一段时间后密度增大,细胞呈梭型。所得骨骼肌卫星细胞生长状态良好,具有较为一致的生长方式和形态特点。经免疫荧光染色法以及PCR鉴定发现细胞系中有Pax7基因的表达。通过上述材料和方法,可获得较纯的山羊骨骼肌卫星细胞,并稳定传代。     

成年SD大鼠胃窦Cajal间质细胞分离、培养及鉴定

目的 探讨成年SD大鼠胃窦起搏细胞Cajal间质细胞（interstitial cells of cajal,ICC）的原代分离、培养及鉴定方法,为深入研究其生理功能提供条件。方法 8~9周龄SD成年大鼠,禁食24 h,乙醚吸入麻醉,颈椎脱臼处死。无菌条件下采用精细解剖方法快速取出胃窦平滑肌组织,将其剪成1~2 mm³小块后,使用Ⅱ型胶原酶消化法于37℃培养箱内消化60 min,离心后过200目筛,接种于DMEM/F12完全培养基（含5 ng/mL干细胞因子）中进行培养。用酪氨酸蛋白激酶受体c-kit特异性抗体免疫荧光染色鉴定细胞类型。结果 培养72 h后,于倒置显微镜下观察,可见细胞贴壁,呈不规则三角形、星形或梭形,有多个突起;随着培养时间的延长,各突起彼此相互连接形成网络;此原代培养细胞c-kit抗体免疫荧光染色呈阳性。结论 成功建立了一套由成年SD大鼠胃窦组织采用Ⅱ型胶原酶消化法原代培养ICC的方法,为ICC的生理功能、病理改变以及胃肠道生理与病理关系的研究奠定了基础。     

The evolution of chicken stem cell culture methods

1. The avian embryo is an excellent model for studying embryology and the production of pharmaceutical proteins in transgenic chickens. Furthermore, chicken stem cells have the potential for proliferation and differentiation and emerged as an attractive tool for various cell-based technologies.

2. The objective of these studies is the derivation and culture of these stem cells is the production of transgenic birds for recombinant biomaterials and vaccine manufacture, drug and cytotoxicity testing, as well as to gain insight into basic science, including cell tracking.

3. Despite similarities among the established chicken stem cell lines, fundamental differences have been reported between their culture conditions and applications. Recent conventional protocols used for expansion and culture of chicken stem cells mostly depend on feeder cells, serum-containing media and static culture.

4. Utilising chicken stem cells for generation of cell-based transgenic birds and a variety of vaccines requires large-scale cell production. However, scaling up the conventional adherent chicken stem cells is challenging and labour intensive. Development of a suspension cell culture process for chicken embryonic stem cells (cESCs), chicken primordial germ cells (PGCs) and chicken induced pluripotent stem cells (ciPSCs) will be an important advance for increasing the growth kinetics of these cells.

6. This review describes various approaches and suggestions to achieve optimal cell growth for defined chicken stem cells cultures and use in future manufacturing applications.     

山羊皱胃平滑肌细胞的培养及鉴定

用外科手术方法取山羊皱胃的平滑肌组织,采用组织块培养的方法,在体外进行山羊皱胃平滑肌细胞的分离培养,为反刍动物皱胃疾病的研究提供了实验材料。结果表明,本研究成功培养出山羊皱胃的平滑肌细胞。倒置显微镜下,细胞生长状态良好,呈现平滑肌细胞特征性的"峰-谷"状结构,活细胞达95%以上,免疫组化染色显示细胞浆内平滑肌肌动蛋白呈阳性表达。     

Hepa1-6细胞的最佳培养条件研究

[目的]探索Hepa1-6细胞的最佳培养条件。[方法]以小鼠Hepa1-6肝癌细胞为材料,采用正交试验设计研究RPMI1640和DMEM2种不同培养基及细胞接种密度、血清浓度、双抗浓度、D-Hanks漂洗次数、胰蛋白酶浓度和消化时间6个不同因素对Hepa1-6传代培养的影响;根据细胞在6个时间点的生长状况评分,筛选出Hepa1-6细胞的最佳培养条件。[结果]Hepa1-6细胞在含有20%血清的RPMI-1640培养基,接种密度为4×104个/cm2,100U/ml的双抗浓度,贴壁率在90%以上时,D-Hanks漂洗1次,0.5%胰蛋白酶消化2min,传代效果最好。[结论]通过正交设计筛选,为Hepa1-6细胞体外培养探索出最佳培养条件。