Morphometrical approach for predicting regional lymph node micrometastatic load in canine mast cell tumours: preliminary results

Canine cutaneous mast cell tumours (MCTs) have a variable biologic behaviour, and accurate staging is necessary to dictate therapy and predict outcome. Regional lymph node (RLN) involvement is a relevant prognostic factor. While obvious lymph node (LN) metastases are relatively easy to be diagnosed, micrometastatic disease recognition is challenging. The main aim of the study was to evaluate the number of mast cells (MCs) in the LNs of clinically healthy dogs ( n = 4, group 1), dogs with inflammatory diseases ( n = 31, group 2) and dogs with cutaneous MCT ( n = 27, group 3), including animals with no RLN metastases (subgroup 3.1), those with occasional MCs in RLNs (3.2) and those with obvious RLN metastasis (3.3). MCs also were morphometrically evaluated for the following nuclear parameters: mean nuclear area (MNA), mean nuclear perimeter (MNP), largest to smallest diameter length (LS ratio), mean nuclear form factor and coefficient of variation of nuclear area. The average percentages of MCs were 0.0 and 0.01 in groups 1 and 2, respectively, and 0.07, 2.4 and 47.1 in subgroup 3.1, 3.2 and 3.3. MNA and MNP were significantly higher in subgroup 3.3 than in group 2 ( P < 0.05). MNA and MNP in subgroup 3.2 suggested the presence of neoplastic MCs; this prediction of micrometastatic load correlated with outcome. Analysis of preliminary results shows that nuclear morphometry is useful to detect micrometastatic disease in RLN of dogs bearing cutaneous MCTs.     

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。     

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。     

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。     

铝中毒对蛋鸡白细胞和红细胞免疫功能的影响

采用连续腹腔注射相同体积不同浓度梯度的三氯化铝，建立不同程度的鸡亚慢性铝中毒型，检测铝中毒雏鸡外周血白细胞数(WBC)、淋巴细胞总数、ANAE+T数及红细胞C3b受体花环率和红细胞免疫复合物花环率。结果表明，铝中毒雏鸡外周血白细胞数、淋巴细胞总数、ANAE+T数及红细胞C3b受体花环率均明显低于健康对照组雏鸡，而红细胞免疫复合物花环率明显高于健康对照组雏鸡。上述结果说明, 铝中毒对雏鸡细胞免疫和非特异性免疫功能有明显的抑制作用。     

胚胎干细胞向神经细胞定向诱导分化方法的研究进展

胚胎干细胞具有自我更新和多向分化潜能,有望成为治疗神经系统疾病重要的种子细胞来源。如何高效地诱导胚胎干细胞向特定神经细胞分化是目前研究的热点。本文就胚胎干细胞定向分化成神经细胞的3种方法:RA诱导法、谱系选择法和SDIA法及其移植研究做一综述。     

辣蓼黄酮正丁醇部分体外抗猪繁殖与呼吸综合征病毒的研究

为明确辣蓼黄酮正丁醇部分（n-butanol part of flavonoids from Polygonum hydropiper L.,FNB）体外抗猪繁殖与呼吸综合征病毒（PPRSV）的效果。本研究以Marc-145细胞和PPRSV弱毒疫苗毒株（TJM-F92）为对象,通过CCK-8法检测FNB对细胞的毒性作用,并检测先给药后接毒、先接毒后给药、药物与病毒同时作用这3种方式处理细胞后药物对病毒的抑制率。结果发现,FNB对细胞的最大安全浓度为500 μg/mL,因此,选择25~500 μg/mL浓度范围的FNB进行后续试验。各浓度的FNB处理病毒后,能不同程度的抑制PRRSV在细胞上的增殖,并呈现一定的剂量效应关系,药物的浓度越高,抗病毒效果越好。其中,先接毒后给药、药物与病毒同时作用这两种方式抗PRRSV效果显著,在25~500 μg/mL浓度范围内细胞存活率分别为21.55%~65.23%和24.85%~73.60%。而先给药后接毒,不能有效降低病毒的感染力,在药物最高剂量（500 μg/mL）时细胞存活率仅为7.00%,抗病毒效果不明显。FNB预先作用于Marc-145细胞虽未降低PRRSV感染细胞的能力,即药物对于PRRSV预防作用效果不理想,但是FNB对病毒感染细胞后呈现一定的作用,药物能够通过抑制病毒的合成、释放及直接杀灭病毒,进而能够有效抑制PRRSV在细胞上的增殖。本试验结果不仅为FNB在临床上治疗猪繁殖与呼吸综合征（PRRS）提供参考依据,而且可以为辣蓼的深度开发和利用提供理论依据。     

The use of quantitative PCR to detect Felis catus papillomavirus type 2 DNA from a high proportion of queens and their kittens

Squamous cell carcinomas are common feline skin cancers that have been associated with infection with Felis catus papillomavirus type 2 (FcaPV-2). Currently, little is known about the epidemiology of FcaPV-2 infection. The aim of this study was to develop a real-time PCR assay to quantify FcaPV-2 DNA in plucked hairs and skin swabs from 11 healthy breeding queens and their kittens. Samples were taken prior to kittening and then 2, 7 and 28 days after kittening to determine the age at which the kittens were first exposed to the virus. FcaPV-2 DNA was amplified from all of the queens and from 91% of the kittens at 2 days of age. There was a wide range in the quantity of FcaPV-2 DNA detected, from 1 to 92,520 copies per swab, and from 0.01 to 234 copies per copy of reference gene DNA in the hair plucks. The quantity of FcaPV-2 DNA detected in samples collected from the kittens was strongly correlated to that of their respective queens and the mean viral DNA load was similar for cats within a household but varied significantly between households. This is the first time that quantitative PCR has been used to detect FcaPV-2 DNA and the results suggest that the virus is ubiquitous but there is a wide variation of viral DNA loads. Kittens appear to be exposed to FcaPV-2 early in life, presumably from direct contact with their queen. These results are important when determining if FcaPV-2 infection of cats is preventable.