利用偏光影像技术定量观察大鼠胸主动脉中纤维结构

目的：本实验选取大鼠胸主动脉为研究对象,探讨定量偏光影像技术对双折光性物质的成像特点与适用研究领域。方法：采用Abrio液晶偏光影像系统在未经染色条件下对青年和衰老大鼠胸主动脉纤维结构进行观察,然后对主动脉切片进行苦味酸天狼猩红染色,对比Abrio和传统偏振光技术成像的差别。结果：较传统偏振光显微镜不同,Abfio可以对纤维双折射的方位角和光程差进行定量分析。在未经染色的条件下,Abrio可对大鼠胸主动脉中具有双折光性的纤维结构清晰成像,而传统偏光显微镜在未经染色条件下不能给出有意义的信息。经苦味酸天狼猩红染色后,对比传统偏振光技术,Abrio使成像不受纤维排列方向和人为因素的影响,较为完整地反映主动脉中纤维的结构及分布情况。结论：Abrio液晶偏光影像技术能够实现对样本双折光的强弱及方向的定性和定量分析,在未经染色的条件下给出微观结构信息,适合于心血管疾病的检测与评估,可拓展应用面宽,将在未来生物医药领域中体现更大的价值。     

龈下菌斑涂片两种染色分类方法的比较

目的:用革兰染色和刚果红负染两种不同的染色方法对同一龈下菌斑样本进行分类计数,比较两种分类方法的优缺点,提出应用中的问题以及解决方法.方法:随机抽样采集40例牙周病患者的80个位点龈下菌斑,同一标本进行生理盐水涂片革兰染色和刚果红负染.光学显微镜油镜(15×100)镜检共计数200个细菌,包括5种不同形状细菌.采用SPSS 10.01统计软件,对数据进行分析.结果:两种染色分类方法计数统计结果为螺旋体、弯曲菌、梭形菌3种菌数值P＞0.05无统计学意义,而球菌、杆菌2种菌数值经统计P＜0.01有高度统计学意义.结论:刚果红负染简便易行,但龈下菌斑球菌、杆菌进行百分计数时,不适宜用刚果红负染法,应根据实验目的选择恰当的方法.     

小鼠眼神经注入Dil后三叉神经节的形态学研究

目的：经眼神经注入DiI研究小鼠三叉神经节的形态学结构。方法：小鼠10只,体重25—30克,雌雄不拘,进行灌注固定后,在外科显微镜下开颅并确认三叉神经节和眼神经,分别于双侧眼神经植入DiI染色晶体。37℃恒温箱放置3个月,待DiI染色晶体扩散后,取出植入DiI染色晶体的眼神经和三叉神经节,再根据神经走向切片,通过荧光显微镜观察DiI染色晶体在三又神经节内的分布。结果：眼神经离三叉神经节约1cm处植入DiI染色晶体后,应用荧光显微镜明视野观察,均可见到高密度标记的眼神经纤维,行向后内,穿经眶上裂入颅。逐步靠近三叉神经节外上方,并进入三叉神经节内,眼神经标记的神经元位于三叉神经节的前内侧。在三叉神经节内可见到DiI标记的神经节细胞及神经纤维。神经纤维平行致密排列,并被神经节细胞神经纤维分隔成群或簇。神经节细胞呈圆形和卵圆形,大小不一,部分节细胞呈蜂窝状排列。亦可见神经元的突起,有的呈螺旋状连于胞体,有的呈线状连于胞体,并可见到双极神经元。结论：小鼠经眼神经注入DiI后,三叉神经节细胞和神经纤维的排列循序跟其他动物基本一致。     

带培养室的倒置显微镜下人雪旺细胞形态和运动的观察

为了显示体外培养条件下三维结构上人雪旺细胞的形态和运动方式,将引产人胎儿坐骨神经的雪旺细胞培养在聚酯纤维上,用带培养室的显微镜观察。结果显示人雪旺细胞呈梭形,绕聚酯纤维作螺旋式迁移;有的细胞同时抓住两根纤维;在相对静止期,细胞虽无位置的迁移,但有频繁的形态变化;分裂后的子细胞有形态和运动时相的不同。     

前列腺基质细胞的原代培养方法

目的:建立一种操作简单、成功率高、重复性好的前列腺增生组织原代基质细胞(PSC)培养方法。方法:采用胶原酶消化法、组织块贴壁法和胰酶消化组织块贴壁法,从70岁及以上男性的良性前列腺增生组织中分离培养PSC,通过显微镜观察比较PSC的数量、形态、培养周期,用免疫荧光染色法鉴定PSC的纯度。结果:胶原酶消化法得到的贴壁细胞少,细胞体积较小且形态无法铺展,增殖能力较弱;组织块贴壁法培养72h后细胞会从组织边缘缓慢爬出,生长周期长;胰酶消化组织块贴壁法,细胞培养7d后基本融合,折光性强,细胞多呈长梭形,通过免疫荧光染色鉴定,基质细胞纯度在95%以上。结论:利用胰酶消化组织块贴壁法建立了一种易行、高效且重复性好的前列腺增生组织基质细胞培养方法。     

大鼠半月板纤维软骨细胞的体外培养和生物学特征鉴定

目的:体外分离、培养和鉴定大鼠半月板纤维软骨细胞,为研究大鼠半月板纤维软骨细胞的损伤修复提供简单、可行的细胞培养方法。方法:机械分离大鼠膝关节内外侧半月板,0.1%Ⅱ型胶原酶消化配合机械吹打,10%FBS的培养基行原代和传代培养,倒置显微镜动态观察不同时间点半月板纤维软骨细胞的形态及生长情况,鉴定采用甲苯胺蓝染色法和Ⅱ型胶原免疫细胞化学染色法。CCK-8法检测大鼠纤维软骨细胞增殖。结果:在不同时间点,细胞表现出不同的生理形态,由多角形或短梭形变为梭形,最终呈现出三角形或椭圆形,并随着时间延长,细胞增殖能力逐渐增加。细胞培养48 h和72 h的OD值分别明显高于培养24 h的OD值(P0.05),差异具有统计学意义。细胞培养48 h和72 h的OD值相比较,72 h的OD值虽有增加,但并无统计学差异(P0.05)。经甲苯胺蓝染色和Ⅱ型胶原免疫荧光染色为阳性。结论:该方法培养的细胞形态稳定,增殖能力强,具有体内纤维软骨细胞的生物学基本特性,可为后续实验提供简单、可靠的原代大鼠半月板纤维软骨细胞培养方法。     

大鼠脊髓匀浆上清诱导骨髓间充质干细胞分化为神经元样细胞的实验研究

目的:观察大鼠脊髓匀浆上清诱导骨髓间充质干细胞(mesenchymal stem cells,MSCs)形成的神经元样细胞形态特征.方法:通过贴壁法培养分离大鼠骨髓MSCs,体外扩增纯化后加入正常大鼠脊髓匀浆上清诱导72h,倒置显微镜下观察诱导前后细胞的形态结构.激光共聚焦显微镜观测钙离子细胞形态和荧光强度变化,免疫细胞化学方法鉴定诱导后细胞的表型特征.结果:倒置显微镜下可见MSCs呈纺锤形和多角形,核居中,有1-2个核仁,诱导后细胞呈神经元样,细胞伸出较长的轴突样和树突样突起.免疫细胞化学法显示NSE(神经元特异性烯醇化酶)、NF(神经丝蛋白)阳性,GFAP(神经胶质细胞酸性蛋白)阴性.共聚焦显微镜扫描脊髓匀浆上清诱导前细胞形态呈细长的梭形,细胞核不明显,胞体染色强,突起染色弱,荧光像素值低;诱导后,细胞呈现神经元样形态,胞体大,有多个突起,胞体及各突起染色强,荧光像素值高.结论:大鼠脊髓匀浆上清液可在体外诱导骨髓间充质干细胞分化为神经元样细胞.     

团头鲂外周血细胞的显微结构及细胞化学特征

采用常规瑞氏染色和细胞化学染色方法对团头鲂(Megalobrama amblycephala)外周血细胞的显微结构及细胞化学特征进行了观察。在团头鲂外周血细胞中可区分出六类细胞: 红细胞、淋巴细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞和血栓细胞。其中淋巴细胞是除红细胞外含量最多的细胞, 其次分别为血栓细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞。成熟红细胞多为卵圆形, 表面光滑, 胞核呈椭圆形或圆形, 染色质较为致密; 淋巴细胞多呈圆形, 胞质较少, 胞核常偏位; 单核细胞多为圆形, 胞核呈圆形或椭圆形, 胞质内可见空泡状结构; 嗜中性粒细胞近似圆形, 胞核常偏于细胞一侧, 呈分叶状、肾形或椭圆形, 核质界限清晰; 嗜酸性粒细胞一般为圆形, 胞核为肾形或椭圆形, 胞质中充满紫红色颗粒; 血栓细胞形态多样, 主要有椭圆形、纺锤形、长杆状和泪滴形, 核质比较大。淋巴细胞呈α-醋酸萘酚酯酶(ANAE)阳性, 呈过碘酸-雪夫(PAS)、氯乙酸AS-D萘酚酯酶(AS-DCE)弱阳性, 呈苏丹黑B(SBB)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)及过氧化物酶(POX)阴性; 单核细胞呈POX、ACP强阳性, PAS、SBB、AS-DCE和ANAE为阳性, 呈AKP阴性; 嗜中性粒细胞除PAS和ANAE为弱阳性外, 其他染色结果和单核细胞相同; 嗜酸性粒细胞呈POX、ANAE强阳性, SBB、ACP阳性, PAS及AS-DCE则为弱阳性, 呈AKP阴性; 血栓细胞呈PAS、AS-DCE及ANAE弱阳性, 呈SBB、ACP、AKP及POX阴性。团头鲂外周血细胞的显微结构及细胞化学特征与其他鱼类具有相似之处, 但亦有其明显的物种特异性。该研究结果可作为监测团头鲂健康状态的依据, 为其养殖及病理诊断提供基础资料。     

肾上腺皮质细胞的单层培养方法

目的: 体外单层培养大鼠肾上腺皮质细胞,以便为肾上腺皮质细胞的有关实验提供必要条件.方法: 采用胶原酶Ⅱ、透明质酸酶消化等步骤分离Wistar大鼠的肾上腺皮质细胞;分离的细胞于含15% NBS的DMEM培养基、37℃、5% CO2的环境中培养,3β-羟基类固醇脱氢酶特异染色鉴定.结果: 培养的肾上腺皮质细胞呈梭形,胞体较大、有少量突起,3β-羟基类固醇脱氢酶特异染色后胞体显著着蓝色.肾上腺皮质细胞不呈典型的"S"形增殖趋势,适于作原代培养.上皮样细胞和成纤维样细胞同时存在.结论: 通过细胞形态观察及特异酶染色鉴定,本文培养的细胞为大鼠肾上腺皮质细胞.     

我国首次发现暗孢节菱孢所致皮肤真菌病——一株新的条件致病性真菌

本文报告暗孢节菱孢(又名球形阜孢霉)所致皮肤真菌病。此菌是我国首次发现的条件致病真菌。患者头部发生多处皮下结节及萎缩性秃发。皮损活检可见毛囊和皮脂腺严重破坏。肉芽肿内有褐色孢子和分隔菌丝,尤以皮下脂肪浸润明显。多次从皮损外皮屑内检出圆形的厚壁孢子。在沙氏琼脂培养基上,37℃培养生长良好。菌落初呈淡黄色,渐变为橙色。小培养镜检时(放大400倍),可见菌丝分枝有隔(1—2μm)。单细胞性小分生孢子呈球形,晶体形等,光滑发亮呈褐色。有的孢子中央有一黑色纵条带。扫描电镜下发现球形或卵形分生孢子,将暗孢节菱孢制成的菌液接种于实验动物皮肤,2周后出现斑块、小结节、小脓肿和脱毛等症状,与患者临床症状相同。对照组动物正常。动物皮损活检所见病理损害与患者皮损病理损害相同,证实了此菌的致病性。     

PAS染色在骨骼肌糖原贮积症诊断中的应用

目的探讨PAS染色在骨骼肌糖原贮积症诊断中的作用。方法用组织化学方法高碘酸-schiff(periodic acid schiff,PAS)染色方法显示糖原贮积症肌纤维胞浆内糖原的贮积。结果糖原贮积症患者肌纤维胞浆内聚集的红至红紫色颗粒为糖原。结论 PAS染色对于判断细胞内糖原贮积的糖原病和多糖体贮积性疾病的诊断是必要的。     

Fine needle aspiration of tophi for crystal identification in problematic cases of gout. A report of two cases

BACKGROUND: The definitive diagnosis of gout is best established by demonstration of monosodium urate (MSU) crystals in the synovial fluid or biopsy. Fine needle aspiration cytology (FNAC) of tophi can play a crucial role in diagnosis. CASES: A 36-year-old chronic alcoholic male developed subcutaneous nodules on both malleoli without a history of arthropathy and with normal serum uric acid levels. FNAC of the nodules demonstrated stacks and sheaves of needle-shaped crystals of MSU. A 50-year-old diabetic male developed multiple nodules on the feet. He gave a past history of painful athropathy. A roentgenogram of the feet was suspicious for gout; however, joint aspiration failed, and the serum uric acid levels were normal. At this juncture FNAC of the feet tophi clinched the diagnosis of gout. In both cases, polarization of needle washings (wet mount) and the fixed, Papanicolaoustained smears showed negatively birefringent, needle-shaped crystals of MSU, thus confirming the diagnosis of gout. CONCLUSION: FNAC of gouty tophi is an easy alternative to synovial biopsy and joint fluid analysis. It is simpler, easier and less painful. As crystals are preserved in stained smears, they can be employed for polarization and confirmation of gout.     

Diagnostic value of fine needle aspiration cytology in gouty tophi: a report of 7 cases

BACKGROUND The diagnosis of gout can be problematic when the presentation is atypical and serum uric acid is borderline elevated. Demonstration of monosodium urate (MSU) crystals in fine needle aspiration (FNA) smears from nodular masses clinically suspected to be tophi establishes the diagnosis unequivocally. CASES: Of the 7 cases in this study, 4 were suspected clinically to have gouty tophi. Giant cell tumor of tendon sheath, giant cell tumor of bone and metastatic tumor with multicentric involvement of bone were the clinical diagnoses in 1 case each. Serum uric acid levels high enough to be in the diagnostic range for gout were reported in 3 cases, within normal limits in 3 cases and low in 1 chronic alcoholic patient. Bright field microscopy of FNA smears revealed singly scattered or stacks of MSU crystals with variable number of inflammatory cells, with or without foreign body giant cells in 6 cases. In 1 patient, FNA showed stacks of MSU crystals only. Characteristic birefringence of MSU crystals was observed on polarizing microscopy. CONCLUSION: FNA demonstration of MSU crystals on polarizing microscopy can easily establish the nature of the nodules in and around the joints and in soft tissue as gouty tophi and is thus an investigation differentiating this lesion from other masses clinically simulating it.     

大鼠睾丸生精小管上皮精子发生周期的PAS法判定

精子发生是一个包含生殖细胞成熟分裂的连续、复杂的动态过程,不同的生精小管,或同一生精小管不同区段的生精细胞的组合、分布均不相同。本文应用PAS染色法观察了大鼠睾丸生精小管上皮中各级生精细胞在精子发生过程的形态学变化特点。参照Clermont及Russell等制定的生精上皮时相的判定标准,根据生精上皮在精子发生过程中的各级生精细胞组合分布特点,把生精上皮分为ⅩⅣ个期。通过观察精子发生过程中生精上皮细胞组合的周期性形态变化特点,对精子发生过程进行精确划分,把精子发生这一连续、复杂的动态过程静止化,具体化,可以更加准确地描述和比较不同影响因素对生精小管上皮中各级生精细胞的组织学、病理学、毒理学变化。     

小鼠生精周期判定方法的改进

目的探讨一种可以在普通HE染色或苏木精复染标本上对精子发生周期进行简单划分的方法。方法应用PAS染色法与普通HE染色法相对照,对比观察小鼠睾丸各级生精上皮在精子发生不同时期的形态学变化特点。结果参照Clermont及Russel等制定的生精上皮时相的判定标准,把小鼠生精上皮分为XⅡ个期。结论通过对比总结,可以在普通HE染色或苏木精复染标本上对精子发生周期进行简单划分,并进一步辨别各型生精细胞。     

Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serum

Objectives: Translational research using adult stem cells derived from various tissues has been highlighted in cell‐based therapy. However, there are many limitations to using conventional culture systems of adult stem cells for clinically applicability, including limited combinations of cytokines and use of nutrients derived from animals. Here, we have investigated the effects of placental extract (PE) for culture of placenta‐derived stem cells (PDSCs) as well as their potential for hepatogenic differentiation. Materials and methods: Placental extract, extracted using water‐soluble methods, was used as a supplement for culture of PDSCs. Cell viability was determined using the MTT assay, and cytokine assay was performed using Luminex assay kit. Gene expression, indocyanine green (ICG) up‐take, PAS (Periodic Acid‐Schiff) staining and urea production were also analysed. Results: The placental extract contained several types of cytokine and chemokine essential for maintenance and differentiation of stem cells. Expression of stemness markers in PDSCs cultured with PE is no different from that of PDSCs cultured with foetal bovine serum (FBS). After hepatogenic differentiation, expression patterns for hepatocyte‐specific markers in PDSCs cultured with PE were consistent and potential for hepatogenic differentiation of PDSCs cultured with PE was similar to that of PDSCs cultured with FBS, as shown by PAS staining and urea production assays. Conclusions: Our findings revealed that placental extract could be used as a new component for culture of adult stem cells, as well as for development of human‐based medium, in translational research for regenerative medicine.     

Multiple feather follicle cysts in a wild turkey

A hunter-killed wild turkey (Meleagris gallopavo silvestris) was submitted for examination because of numerous 2 to 30 mm diameter, yellowish, hard nodules in the skin. The nodules were confined to the skin and did not involve subcutaneous tissues. Nodules consisted of dilated feather follicles packed with a caseous tan to pale yellow material. Histologically, affected feather follicles were markedly dilated and filled with laminated keratin debris. The lesions were determined to be multiple feather follicle cysts of unknown etiology.     

水稻可溶性糖蛋白的纯化与鉴定研究

从水稻鲜叶中提取总蛋白,对总蛋白中的蛋白质含量进行了测定;通过硫酸铵沉淀将总蛋白提取液进行分级,从而达到了总蛋白细分和放量的目的。四级份的分级蛋白分别通过ConA-Sepharose 4B 亲和层析进行糖蛋白纯化,按吸收峰收集的各级糖蛋白混合物进行冷冻干燥,得到干粉;结合PAS法染色和考马斯亮蓝R-250染色对四级份的糖蛋白样品鉴定,在其中3个级份中均检测出糖蛋白;由于感度的差异,按考马斯亮蓝R-250染色可检测出近30种糖蛋白(包括部分糖肽),按PAS法染色可检测到7种糖蛋白;对3种含量较高的糖蛋白进行了胶上纯化,3种糖蛋白的PAS法染色均证实了3种样品为单一的糖蛋白或者糖肽,分别命名为RG1、RG2和RG3。