Effects of growth factors (EGF,PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing

Objective The physiologic mechanisms involving growth factors, including PDGF‐BB, EGF, and TGF‐β1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types. Procedures Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF‐BB, EGF, TGF‐β1, keratocytes (1 × 10⁵/well) and epithelial cells (2 × 10⁵/well) were each cultured in 12 well plates and exposed separately to the growth factors. The cells were exposed to concentrations of EGF between 0 and 50 ng/mL; PDGF‐BB between 0 and 75 ng/mL; and TGF‐β1 between 0 and 10 ng/mL. Cell proliferation was measured using ³H‐thymidine assay and differences in growth determined using anova and Tukey's HSD test (P < 0.05). Results Epithelial cell and keratocyte cultures were successfully established. EGF maximally stimulated keratocyte and epithelial cells at 25 ng/mL and 5 ng/mL, respectively. PDGF‐BB maximally stimulated keratocytes and epithelial cells at 50 ng/mL and 5 ng/mL, respectively. TGF‐β1 inhibited keratocytes at 5 ng/mL and 10 ng/mL, and epithelial cells at 1 ng/mL and 2 ng/mL. Conclusions Methods were established to maintain epithelial cells and keratocytes in vitro. PDGF‐BB and EGF stimulate, while TGF‐β1 inhibits the proliferation of epithelial cells and keratocytes. These growth factors may play a role in maintenance and repair of the equine cornea.     

一例细菌对MDCK细胞污染的检测

MDCK细胞培养液（含双抗）表面有一层薄的油状膜，培养液不混浊。细菌学检测为一种球菌污染。对污染菌株进行药敏试验。使用MEM（含双抗）多次清洗细胞培养瓶及细胞表面，细胞培养液中改加菌株敏感的恩诺沙星。细菌学检测结果表明，加入恩诺沙星有效地控制和清除了细胞中污染的该株细菌。     

Resection cystoplasty of a squamous cell carcinoma in a mare

A 22‐year‐old Arabian mare was examined for evaluation of haematuria of 2 months' duration. Complete blood cell count and chemistry revealed anaemia of chronic disease (i.e. normocytic, normochromic) and hypoproteinaemia. Cystoscopy revealed a haemorrhagic mass protruding into the lumen of the urinary bladder in the apical region. The mass was surgically removed by partial cystectomy performed through a caudal ventral midline laparotomy. Histological examination of the mass confirmed the diagnosis of squamous cell carcinoma. Tumours of the equine urinary bladder are rare, and when a partial cystectomy is indicated, a caudal ventral midline laparotomy may provide adequate surgical exposure even in adult horses.     

以超滤技术浓缩猪细小病毒含毒细胞液的试验

在研制猪细小病毒灭活疫苗时,应用了超滤浓缩技术,提高了合毒细胞培养液中的抗原含量。为了保证浓缩的流量,减少对滤膜的污染,对含毒细胞培养液作了预处理一离心及微滤,以便除去细胞培养液中的细胞碎片、蛋白质等大分子物质及固形微粒。超滤时选用50000Da滤膜,在20psig压力下,经过5倍浓缩的PPV细胞培养液,血凝滴度提高2个滴度以上。病毒含量测定表明,TCID50／0．2mL由107左右升至109以上,以其配制疫苗,能显著地提高疫苗的免疫原性。超滤技术具有易于操作、高效、分离精度高、没有二次污染等优点,可以根据需要选择不同的浓缩浓度,对保证疫苗的质量具有重要作用。     

SRSF10对山羊肌内前体脂肪细胞分化的影响

旨在克隆山羊SRSF10基因序列,明确其生物学特性,并通过过表达和干扰手段阐明SRSF10对山羊肌内脂肪细胞分化的影响。本研究以简州大耳羊(Capra hircus)为试验对象,利用RT-PCR技术克隆山羊SRSF10基因序列,并进行生物信息学分析; 利用实时荧光定量PCR(real-time quantitative PCR,qPCR)技术研究其在成脂诱导分化不同阶段细胞中的表达水平; 转染pcDNA3.1-SRSF10过表达载体和SRSF10-siRNA至山羊肌内前体脂肪细胞并诱导分化,通过油红O和Bodipy染色法从形态学上明确其对脂滴积聚的影响; 通过qPCR方法检测脂肪细胞分化标志基因表达的变化。结果显示,获得山羊SRSF10基因序列为1 026 bp,其中CDS区552 bp,共编码183个氨基酸; SRSF10在诱导分化96 h的肌内脂肪细胞中表达量最高; 过表达和干扰山羊SRSF10分别促进和抑制了肌内脂肪细胞中的脂滴积聚; 过表达后基因SREBP1、PPARγ和C/EBPα的相对表达量极显著上调(P < 0.01),干扰后分化标志基因SREBP1、PPARγ和C/EBPα相对表达水平极显著降低(P < 0.01)。结果表明,山羊SRSF10是肌内脂肪细胞分化的正调控作用因子,并且这种调控作用可能主要通过调节SREBP1、PPARγ和C/EBPα的表达来实现。     

高非蛋白氮利用能力酵母菌的筛选与诱变

蛋白质饲料资源短缺一直是制约着中国饲料工业健康平稳发展的瓶颈问题之一。单细胞蛋白（single cell protein,SCP）在解决世界粮食与蛋白饲料短缺问题存在较大潜质。经多年研究,单细胞蛋白在菌种选育、生产工艺、发酵原料等方面均取得了较大进展,但单细胞蛋白饲料成本仍居高不下,在饲料工业中一直未得到大量应用。提高生产单细胞蛋白生产菌株非蛋白氮（non-protein nitrogen,简称NPN）利用效率和生长速度,是降低单细胞蛋白生产成本的关键。本试验从多株酵母菌中筛选出NPN利用能力较强的菌株,并对其进行紫外照射诱变,以期获得菌体蛋白产量高的突变菌株。通过对14株酵母菌对不同碳、氮源利用的研究,确定其最适碳、氮源,并依据14株酵母的生长潜力、菌体蛋白产量、NPN利用能力等进行排名,筛选出综合性能较优的酿酒酵母菌M（Saccharomyces cerevisiae strain YI59）和N2（Saccharomyces cerevisiae isolate AA2）。将酵母菌M和N2作为出发菌,进行紫外照射诱变。以葡萄糖、硫酸铵为碳、氮源,依平板菌落大小与液体发酵菌体蛋白产量进行初筛和复筛,最终获得菌落较大的3株突变菌MU23、MU3和MU5。经液体发酵复筛发现,MU3和MU5菌体蛋白含量较原出发菌M有显著提高,分别提高12.93%和11.82%（P<0.05）,但菌体蛋白产量与出发菌相比无显著差异（P>0.05）;菌株MU23菌体蛋白含量与出发菌M无显著差异（P>0.05）,但菌体蛋白产量较原出发菌M有较大幅度提升,为0.26 g/L,提高13.04%（P>0.05）。综合上述试验结果,使用紫外照射诱变可以达到提高酵母菌NPN利用能力和菌体蛋白产量的目的。     

成年兔睾丸支持细胞的分离纯化研究

本试验以5月龄公兔为试验动物,去除睾丸被膜及血管,分离出完整的曲精细管后 用0．5％胰蛋白酶和0．1％胶原酶消化并进行低渗处理,将制成的细胞悬液植入25 cm2的培 养瓶,于38．7℃、5％CO2条件下培养,结果表明:经1:3Hanks超纯水低渗处理后,离体支持 细胞纯度和活率分别达到72．04％和84．5％,显著提高了离体支持细胞的获取量和活率。     

鹿茸干细胞体外培养技术的研究

利用消化酶消化法,用DMEM培养基对鹿茸干细胞进行了体外培养,分别从原代培养、传代、冻存、以及复苏等几个环节进行了试验。摸索鹿茸干细胞合适的体外培养条件,为鹿茸的各项相关研究打基础。结果表明,用透明质酸酶和胶原酶对组织样进行消化,然后用10%DMEM培养基进行培养细胞生长效果较好。适宜培养条件是37℃、5%CO2最大饱和湿度。