猪伪狂犬病毒感染PK15细胞对细胞凋亡及相关凋亡因子的影响

为研究PRV感染PK-15细胞对其细胞凋亡及相关凋亡因子的影响,本试验采用体外细胞培养技术在不同时间点收集细胞、应用噻唑蓝(MTT)法检测细胞的增殖活性、流式细胞仪测定细胞凋亡率、实时荧光定量PCR法检测凋亡相关基因caspase-3,Bcl-2,Bax和Bcl-xl的表达情况。结果显示,PRV感染24 h后可明显抑制细胞的增殖,这种抑制与时间密切相关,与感染剂量无显著差异。流式细胞仪检测也显示PRV能明显提高其凋亡率,PT-PCR显示PRV感染后caspase-3,Bax表达量明显上调,Bcl-xl,Bcl-2表达量明显下调。结果表明,PRV在感染过程中对细胞的生长的影响与时间紧密联系,能够诱发细胞凋亡,Bax,Bcl-2,Bcl-xl,caspase-3,起着重要的调控作用。     

伪狂犬病病毒Ra株对不同细胞增殖特性及致病性研究

本研究以伪狂犬病病毒(pseudorabies virus,PRV)Ra株体外感染不同的细胞系为模型,研究PRV Ra株最适细胞系。试验采用ST细胞、PK-15细胞、Vero细胞、F81细胞、DF1细胞、MDCK细胞和CEF细胞7种细胞作为该毒株的接种对象,观察毒株在这几种细胞上病变特点、细胞病变时间及病毒增殖规律等并进行比较。观察结果发现,受试的各种细胞在PRV感染期间均能表现明显的细胞病变,但不同细胞在病变时间上存在较大差异,其中PK-15和ST细胞在感染后24 h内出现明显细胞病变,时间最早。TCID₅₀测定病毒增殖力结果表明,ST细胞在病毒增殖传代中毒力保持最好,与原毒株毒力相当,为10^6.9 TCID₅₀/0.1 mL。本试验结果表明,ST细胞是较适合于PRV Ra株体外研究的细胞系。     

沙冬青生物碱对MDCC-MSB1细胞增殖及细胞线粒体膜电位的影响

采用体外细胞培养技术、噻唑蓝(MTT)还原法、流式细胞技术检测梯度浓度沙冬青总生物碱对体外培养鸡马立克氏病肿瘤HDCC-MSB1细胞株的增殖抑制及对线粒体膜电位的影响。结果显示,沙冬青生物碱可显著抑制HDCC-MSB1细胞增殖,且这种抑制具有剂量和时间效应关系。经沙冬青生物碱作用的HDCC-MSB1细胞培养样本中有明显的DNA低含量颗粒(亚G1期峰),细胞周期各时相分布发生改变,细胞增殖在G1被阻滞。细胞线粒体膜电位(△Ψm)明显下降,也呈现出剂量和时间效应关系。结果表明,沙冬青生物碱对HDCC-MSB1细胞增殖有显著抑制效应,且抑制依赖于诱导HDCC-MSB1细胞线粒体膜电位(△Ψm)的下降,并进一步诱导了该细胞的凋亡。     

伪狂犬病病毒Ra株体外感染ST细胞超微结构的研究

本研究以伪狂犬病病毒(pseudorabies virus, PRV)Ra株体外感染ST细胞为生物模型,通过透射电镜对PRV的增殖规律和致细胞病变的显微结构进行观察。结果显示,PRV能诱导ST细胞发生明显病变,细胞的病变程度与PRV感染时间密切相关。PRV Ra株感染ST细胞,病毒吸附于ST细胞表面,以膜融合内陷的方式进入细胞和细胞核内,在细胞核内复制,出现包涵体结构,以出芽方式离开细胞核,在高尔基体等细胞内膜结构处完成病毒粒子的囊膜化过程。感染前期,病毒通过膜融合方式被释放到细胞外,完成细胞间病毒的传播;感染后期,细胞溶解,大量释放病毒粒子。感染细胞超微结构的变化主要体现为:线粒体肿胀、数目减少,嵴面积减少,核内出现包涵体,细胞融合,细胞内空泡化严重,溶细胞现象。     

钼对TM3小鼠睾丸间质细胞周期和凋亡的影响

以体外培养的小鼠睾丸间质细胞系TM3 为材料,加入不同质量浓度的钼酸钠溶液(0,10,20,40,80,160mg/L)染毒培养,分别在干预4,8,12,24,48h采用MTT法检测细胞的增殖。干预48h后,采用流式细胞术检测细胞周期和凋亡,单细胞凝胶电泳检测DNA损伤的变化。结果表明:与对照组相比,不同剂量钼酸钠作用24h后,睾丸间质细胞的增殖活性均受到抑制;不同质量浓度的钼酸钠作用48h后,细胞周期阻滞于G0/G1期,20mg/L及其以上剂量组G0/G1期细胞百分率与对照组相比显著升高(P<0.05或P<0.01);与对照组相比,各剂量组TM3 小鼠睾丸间质细胞凋亡率显著升高,差异极显著(P<0.01);细胞尾部DNA含量及细胞尾长随着钼剂量的增加呈不同程度的增加,且存在着剂量—效应关系。结论说明钼能够引起睾丸间质细胞周期的紊乱,并诱导睾丸间质细胞发生DNA损伤和凋亡。     

亮氨酸对猪胎盘滋养层细胞增殖和周期的影响

为了研究亮氨酸(Leu)对猪胎盘滋养层细胞(p Tr)增殖数目和增殖周期的影响,本试验分别利用MTT法和流式细胞仪检测不同Leu浓度(0、0.01、0.05、0.1、0.25、0.5、1、5、10 mmol/L)处理p Tr不同时间(24 h、48 h、72 h)后,其增殖活力和细胞周期分布。结果显示,Leu处理p Tr细胞24 h后,0.01 mmol/L组和0.05 mmol/L组细胞数目极显著降低(P0.01);Leu处理p Tr细胞48 h后,细胞数目极显著降低(P0.01);1~10 mmol/L Leu处理p Tr细胞72 h后,细胞数目极显著降低(P0.01)。不同浓度Leu处理p Tr 24 h和48 h后,各试验组G0~G1期细胞数目逐渐增多,S期细胞数目逐渐减少,并有时间和剂量依赖性。高浓度的Leu会阻碍正常细胞周期,从而抑制p Tr的增殖活力。     

苦杏仁精油对HaCaT细胞增殖和凋亡的影响

为了研究苦杏仁精油(bitter almond essential oil,BAEO)对人永生化角质形成细胞(HaCaT)体外增殖抑制和凋亡诱导的作用,并探讨其可能存在的抗银屑病作用机理。采用气相色谱-质谱联用仪(gas chromatography-mass spectrometer,GC-MS)分析BAEO化学成分;磺酰罗丹明B(sulforhodamine-B,SRB)法检测BAEO对HaCaT细胞增殖的影响;用流式细胞术碘化丙啶(propidium iodide,PI)单染法检测BAEO对细胞周期的影响;Annexin V-FITC/PI双染法检测细胞凋亡;Western blot测定凋亡相关蛋白Caspase-3和PARP表达水平。结果显示,BAEO主要化学成分是苯甲醛(57.36mL/L)、苯甲酸(12.73mL/L)和扁桃腈(9.54mL/L);BAEO对HaCaT增殖抑制率随药物浓度和时间的增加而显著增加(P0.01);流式细胞仪检测经BAEO(0.1、0.25、0.5mL/L)作用于HaCaT细胞24h后,细胞周期停滞在G0/G1期,早期凋亡率显著升高(P0.05);Western blot结果表明,0.1、0.25、0.5mL/L的BAEO作用于HaCaT细胞24h后Caspase-3和PARP蛋白的表达下调。结果表明,BAEO能显著地抑制HaCaT细胞增殖,以及通过G0/G1期细胞周期停滞、细胞凋亡率升高、增加Caspase-3和PARP蛋白的剪切来诱导HaCaT细胞凋亡。     

钼对TM3小鼠睾丸间质细胞周期和凋亡的影响

以体外培养的小鼠睾丸间质细胞系TM3 为材料,加入不同质量浓度的钼酸钠溶液（0,10,20,40,80,160mg/L）染毒培养,分别在干预4,8,12,24,48h采用MTT法检测细胞的增殖。干预48h后,采用流式细胞术检测细胞周期和凋亡,单细胞凝胶电泳检测DNA损伤的变化。结果表明：与对照组相比,不同剂量钼酸钠作用24h后,睾丸间质细胞的增殖活性均受到抑制;不同质量浓度的钼酸钠作用48h后,细胞周期阻滞于G0/G1期,20mg/L及其以上剂量组G0/G1期细胞百分率与对照组相比显著升高（P〈0.05或P〈0.01）;与对照组相比,各剂量组TM3 小鼠睾丸间质细胞凋亡率显著升高,差异极显著（P〈0.01）;细胞尾部DNA含量及细胞尾长随着钼剂量的增加呈不同程度的增加,且存在着剂量—效应关系。结论说明钼能够引起睾丸间质细胞周期的紊乱,并诱导睾丸间质细胞发生DNA损伤和凋亡。     

PRV基因缺失株在体外细胞中增殖的超微结构

用伪狂犬病痛毒(Pseudorabies virus,PRV)Fa株和本实验室构建的系列PRV基因缺失株(PRV-TK-、PRV-gI\gE-、PRV-TK\gI\gE-株)感染不同的细胞系(IBRS-2、Marc145、ST、Vero、MDBK细胞),对感染细胞的形态学观察发现,受试各PRV毒株在以上细胞均能增殖并引起细胞病变,但对不同的细胞却表现差异,其中在ST细胞上增殖滴度最低,在MDBK上最高;同PRV Fa株相比,PRV-gI\gE-和PRV-TK＼gI\gE-株的增殖力有所降低,而PRV-TK-则变化不明显;对细胞感染后的超微结构分析发现,基因缺失对该毒株在细胞上的吸附和穿入过程没有影响,但与Fa株相比,主要表现为增殖速度的减缓和病毒粒子数量的减少.     

Production of stem cell factor in canine mast cell tumors

Mast cell tumor (MCT) is the most common cutaneous tumor in dogs. We recently revealed that production of stem cell factor (SCF) contributes to the proliferation of neoplastic mast cells in an autocrine/paracrine manner. The aim of the present study was to determine the contribution of the mechanism in clinical MCTs. In consequence, high SCF expression (>10 times compared to HRMC cells) was observed in 5 of 7 MCT samples used in the study regardless of KIT mutation, which was confirmed in immunohistochemical analysis. In addition, production of SCF was observed in Ki-67-positive cells in the MCT xenograft. These results indicate the broad contribution of SCF autocrine/paracrine mechanism on clinical MCTs, providing the rationale for the clinical use of KIT inhibitors regardless of KIT mutation.     

Mast cell and plasma cell collision tumor in the spleen of a dog

A 9‐year‐old spayed female English Mastiff was referred for outpatient ultrasound due to a 3‐week history of weight loss, vomiting, and decreased appetite. Abdominal ultrasound showed multiple splenic masses of varying sizes and serum chemistry panel showed hyperglobulinemia. Cytologic examination of fine‐needle aspirates of the splenic masses indicated a mast cell and plasma cell collision tumor. Results of serum and urine protein electrophoresis and immunofixation indicated the plasma cell neoplasia was producing IgA immunoglobulins.     

Establishment of cell line and in vivo mouse model of canine Langerhans cell histiocytosis

A cell line named FB‐LCH01, derived from a dog diagnosed with Langerhans cell histiocytosis (LCH), was established and characterized. FB‐LCH01 had C‐shaped nucleoli, characterized by modal chromosome aberrations. The original tumour cells as well as established FB‐LCH01 cells were immunopositive for human leukocyte antigen‐DR, Iba‐1 and E‐cadherin, and immunonegative for CD163 and CD204, suggesting Langerhans cell origin. Furthermore, the characteristics of FB‐LCH01 were compared with those of two canine histiocytic sarcoma cell lines (PWC‐HS01 and FCR‐HS02) established previously. Expression of E‐cadherin was detected only in FB‐LCH01, but not in PWC‐HS01 and FCR‐HS02. All (n = 9) the severe combined immunodeficiency mice inoculated with the FB‐LCH01 cells developed subcutaneous tumour masses after 3 weeks. Eight of nine mice also developed metastatic lesions in the lymph nodes (8/8; 100%), lung (5/8; 62.5%), stomach (5/8; 62.5%), heart (4/8; 50%), pancreas (4/8; 50%), kidney (3/8; 37.5%), skin (3/8; 37.5%) and bone marrow (1/8; 12.5%). Tumour cells were pleomorphic and round‐ to polygonal‐shaped with prominent anisocytosis and anisokaryosis. The xenotransplanted tumour cells maintained the immunohistochemical features of the original tumour with persistent E‐cadherin expression at injection site and some visceral organs. In conclusion, the established cell line as well as the mice xenotransplant model in this study reflect the nature of canine LCH and may serve as promising models for investigating the patho‐tumorigenesis and therapy of the disease.     

Differentiation of human umbilical cord mesenchymal stem cell into germ-like cell under effect of co-culture with testicular cell tissue

Supplements produced by mouse testicular cells (mTCs) and the interaction between cells can increase the differentiation rate of human umbilical cord mesenchymal stem cells (hUCMSCs) into the germ-like cells. We studied the differentiation rate of hUCMSCs into the germ-like cells under effect of mTCs co-culturing. Isolated hUCMSCs from postpartum human umbilical cords were cultured. Then, the expression of mesenchymal (CD73, CD90 and CD105) and haematopoietic (CD34 and CD45) markers of hUCMSCs were confirmed by flow cytometry. Then, the hUCMSCs were cultured in four distinct groups: (a) control, (b) co-culture until D0, (c) co-culture until D5 and (d) co-culture until D10, in order to differentiate into the germ-like cells. After 10 days, the expression of OCT4, VASA, Fragilis and SYCP3 genes were examined by Real-Time qPCR. The flow cytometry indicated a high expression of mesenchymal markers and a low expression of haematopoietic markers (CD73:98.6%, CD90: 99.1%, CD105: 99.5%, CD34: 4.22% and CD45: 2.54%). The expression of OCT4 decreased during the time while the expression of VASA, Fragilis and SYCP3 markers increased in the co-culture with testicular cells (p value <.05). Co-culture with mTCs may be used as an effective method to differentiate hUCMSCs into germ-like cells.     

胰腺干细胞的研究进展

概述了胰腺干细胞的分布、胰腺的发生、胰腺干细胞的相关标记物以及胰腺干细胞的体外分离培养、增殖、分化等领域的研究进展，并对胰腺干细胞的生物学特征、体外分离培养时遇到的困难等进行了探讨，针对所遇到的困难提出了一些改进意见。     

Reliability in somatic cell count measurement of clinical mastitis milk using DeLaval cell counter

Somatic cell counts (SCC) measurements are typically performed using quantitative methods, such as the Breed method (Breed) and the Fossomatic method (FSCC). The DeLaval cell counter (DCC) developed recently is a quantitative somatic cell counter with a low initial cost and superior portability. However, since the DCC was specifically developed for measuring SCC of ≤ 4 × 10⁶ cells/mL milk from bulk tanks or individual cows, its reliability for estimating SCC that exceed this concentration has not yet been clarified. This study therefore examined whether it is possible to accurately measure SCC by diluting milk samples with initial SCC of 4 × 10⁶ cells/mL, as seen in clinical mastitis milk. We collected milk samples from 99 quarters of 99 Holstein cows with clinical mastitis. These milk samples were diluted 10‐fold with saline and thoroughly mixed before performing SCC measurement with the DCC. The correlation coefficients of SCC measured by the FSCC, Breed and DCC methods indicated strong correlations between each pair of methods. The findings showed that DCC can be used to identify bovine clinical mastitis milk and is useful as a quantitative SCC measurement device on farm sites.     

从原始生殖细胞分离和克隆兔的胚胎生殖细胞

研究采用 1 4～ 1 8d兔胎儿的生殖嵴及周围组织与其同源成纤维细胞共培养 ,低糖DMEM +1 0 %NBS +1 0 %FCS +1 0ng/mLLIF +1 0ng/mLSCF+0 1mol/Lβ 巯基乙醇 +1 0 0U/mL青霉素 +80U/mL链霉素作培养基 ,分离出兔原始生殖细胞 (PGC) ,克隆并多次传代。从原始生殖细胞 (PGC)中获得胚胎生殖细胞 (EG)细胞集落 ,1 4d胎儿原代观察到类EG细胞集落 ,传至 4代后丢失。 1 6d胎儿的类EG只传 2代 ,1 8d胎儿没有得到EG细胞集落。EG细胞具有干细胞的诸多特征 ,呈典型的团块状聚集生长 ,碱性磷酸酶 (AKP)染色呈阳性 ,在衰老饲养层的培养基中生长形成类胚体、上皮细胞、神经细胞和成纤维细胞等     

Photoreceptor cell dysplasia in two Tippler pigeons

Two 12-week-old Tippler pigeons were evaluated for ocular abnormalities associated with congenital blindness. The pigeons were emaciated and blind. Biomicroscopy and direct and indirect ophthalmoscopy findings of the Tippler pigeons were normal with the exception of partially dilated pupils at rest. Scotopic (blue stimuli) and photopic monocular electroretinograms were extinguished in the blind Tippler pigeons. Histological and electron microscopy studies revealed reduced numbers of rods and cones, and an absence of the double cone complex. The photoreceptor cells' outer segments were absent, and the inner segments were short and broad. The number of cell nuclei in the outer and inner nuclear layers was decreased, and the internal and external plexiform layers were reduced in width. Photoreceptor cell endfeet with developing synaptic ribbons were present in the external plexiform layer. Inflammatory cell and subretinal debris was not seen. The electroretinographic, histopathological, and ultrastructural findings of the blind Tippler pigeons support the diagnosis of a photoreceptor cell dysplasia.     

成纤维上皮细胞炎症反应模型的建立

利用培养金黄色葡萄球菌培养基过滤液建立成纤维上皮细胞炎症反应模型，用于观察和研究炎症反应过程中细胞形态的变化。将培养金黄色葡萄球菌培养基除菌过滤冻干，用细胞培养基稀释，按照一定蛋白含量加到上皮细胞的培养基中，制作细胞炎症反应模型。结果显示，成纤维上皮细胞发生炎症反应，高浓度细菌滤过液的细胞形态发生变化，细胞边缘模糊不清，细胞出现空泡，随着培养时间的延长低浓度的大量大型细胞开始增加；在添加金黄色葡萄球菌培养基滤过液培养24h后利用^3H—TdR方法测定细胞增殖率，细胞增殖差异是显著的。结果表明，建立细胞炎症反应模型是可行的，并为以后炎症反应机理研究和抗炎性反应药物研究奠定了基础。