术中冰冻切片快速免疫组化HE染色技术诊断硬化性肺细胞瘤和肺腺癌的应用对比

目的 探讨术中冰冻切片快速免疫组化诊断硬化性肺细胞瘤（PSP）和肺腺癌的可行性及意义。方法 收集安徽医科大学第一附属医院2018年1月至2019年7月PSP术中标本12例设为PSP组以及肺腺癌术中标本26例设为肺腺癌组,使用术中冰冻切片HE染色和术中冰冻切片快速免疫组化检测甲状腺转录因子-1（TTF-1）、雌激素受体（ER）和细胞增殖核抗原（Ki-67）在两种疾病标本中的表达,以石蜡切片免疫组化结果为金标准,对比术中冰冻切片快速免疫组化和HE染色两种方法诊断PSP和肺腺癌的正确率。结果 术中冰冻切片快速免疫组化和术中冰冻切片HE染色诊断PSP正确率分别为100.00%,58.33%,差异有统计学意义（P=0.037）。术中冰冻切片快速免疫组化结果显示,PSP组TTF-1阳性、ER阳性和Ki-67阴性表达率分别为100.00%、91.67%和83.33%,均高于肺腺癌组,差异有统计学意义（P < 0.001）。结论 术中冰冻切片快速免疫组化诊断PSP和肺腺癌显著优于术中冰冻切片HE染色,TTF-1、ER和Ki-67联合可用于术中冰冻切片快速免疫组化指标鉴别诊断PSP和肺腺癌。     

不同固定液和时间对大鼠睾丸组织病理学制片质量的影响

目的 探讨不同固定液和固定时间对大鼠睾丸组织病理学制片质量的影响。方法 选用雄性SD大鼠12只,取两侧睾丸组织,共24例标本。每例标本分别用不同的固定液(10%中性福尔马林溶液、30%Davidson固定液及FAA固定液)固定不同的时间(24、48 h)后,进行脱水、包埋、切片、HE染色。用LEICA DM5000B普通光学显微镜观察。结果 3种固定液固定24 h后,可见30% Davidsons固定液组睾丸组织的异常变化程度低于10%中性福尔马林组和FAA组;固定时间对睾丸组织有一定程度的影响:固定24 h的异常变化程度低于固定48 h。结论 30% Davidson固定液组SD大鼠睾丸组织病理学变化程度最小;固定时间不宜超过24 h。     

临床前毒理学研究Beagle犬中枢神经系统一致性制片方法

目的 建立临床前毒理学研究中Beagle犬中枢神经系统一致性制片方法。方法 以Beagle犬脑组织肉眼可见的解剖部位，如额极、视交叉、动眼神经、脑桥、前庭耳蜗神经为标志，对Beagle犬脑组织（腹侧面向上）进行冠状水平单侧取材。并且对Beagle犬脊髓的颅颈段（C1~C2）、胸中段（T6~T8）和腰膨大处（L4~L5）进行横切面及倾斜横向面取材，并经常规脱水、包埋、切片、HE染色处理。结果 通过这种方法制作的Beagle犬中枢神经系统组织学切片一致性高、质量好，包括了Beagle犬中枢神经系统主要结构，例如尾状核、基底核、大脑皮质（额叶、顶叶、枕叶、颞叶）、脉络丛、海马、下丘脑、延髓、中脑、脑桥、丘脑、小脑和脊髓。结论 该制片方法简单易行且一致性高，可用于临床前毒理学研究中Beagle犬中枢神经系统组织病理学检查的一致性制片。     

6种固定液短时间固定对冰冻切片制片效果影响

目的通过探讨6种不同固定液短时间固定对冰冻切片制片效果的影响,以便在快速冰冻切片制片中选择较好的固定液。方法收集新鲜活体组织,取材冷冻包埋后,经恒温式冰冻切片机切片,以6种不同固定液固定6s,进行HE染色。结果经乙醚酒精固定后冰冻切片质量较好,染色鲜艳,镜下组织结构细胞形态清晰,细胞核浆对比分明。结论乙醚酒精固定是一种较为理想的冰冻切片快速固定液。     

吸入类药物临床前研究中鼻组织脱钙技术优化：用于大鼠鼻黏膜组织病理学检查

目的 研究不同脱钙液对大鼠鼻组织的脱钙作用及其病理切片染色效果。方法 选择10%乙二胺四乙酸（ethylene diamine tetraacetic acid，EDTA）、10%甲酸、5%硝酸脱钙液3种不同脱钙液，在室温静置及微波条件下，对大鼠鼻组织的脱钙时间和脱钙效果进行比较分析，综合评估骨组织经不同脱钙方法后制成病理切片质量。结果 常温条件下鼻组织经过EDTA脱钙液所需脱钙时间最长，微波条件下鼻组织经硝酸脱钙液所需脱钙时间最短，经EDTA脱钙液的鼻组织切片质量、HE染色、MASSON染色和免疫组化染色中质量最佳，硝酸脱钙液的鼻组织的切片质量最差，甲酸脱钙液的鼻组织HE染色效果较佳，MASSON和免疫组化染色质量略差。结论 EDTA脱钙液配合微波进行的鼻组织脱钙，脱钙效率明显提升，且切片和染色效果俱佳。     

皮肤组织冰冻切片在直接免疫荧光法中包埋剂的改良应用

目的用普通胶水代替OCT包埋皮肤组织来制作冰冻切片,不仅降低了成本,而且所制切片与用OCT包埋剂包埋所制切片在制片质量与染色效果上无异。方法行冰冻切片时,用普通胶水代替OCT对皮肤组织起支托固定作用。结果用普通胶水包埋制作的冰冻切片,在制片质量及染色效果上均与用OCT包埋剂包埋制片无异。结论冰冻切片行直接免疫荧光法要求冰冻切片质量优良,且在制片过程中快捷、力求保持抗原的完整性。质量优良的包埋剂在制片过程中可以达到以上要求,但其价格昂贵;用普通胶水代替质量优良的包埋剂,不仅在制片质量及染色效果上与前者无异,且价格便宜,值得推广使用。     

冰冻切片制作技术的改进及临床应用

目的：探讨冰冻切片制作过程中的影响因素及改进措施,提高冰冻切片的临床应用价值。方法：选取新鲜活体组织,取材后直接冻结,采用恒温式冰冻切片机切片,冰冻固定液固定,HE染色。结果：质量较好,染色鲜艳,显微镜下组织结构、细胞形态清晰,细胞核浆对比分明。结论：制作高质量的冰冻切片需要在取材、冷冻、切片、固定等多个环节上精益求精,不断改进,以提高病理诊断率,提高其临床应用价值。     

不同时期家兔动脉粥样硬化模型的比较

目的 从动脉粥样硬化（AS）发生进展的不同时期,对家兔模型进行比较和研究。方法 新西兰白兔30只,随机分为5组,包括对照组和模型组,模型组又分为AS 4周组、AS 8周组、AS 12周组和AS 16周组,每组6只。对照组给予普通饲料喂养,模型组给予高脂饲料喂养。分别于实验0、4、8、12、16周给各组动物称质量,耳缘静脉取血,测定血清胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白（LDL）和高密度脂蛋白（HGL）含量;通过石蜡切片HE染色及冰冻切片油红O染色对胸主动脉进行病理形态学观察。结果 实验4周时,模型组家兔体质量增加值明显高于对照组,而在实验8、12、16周时,二者比较没有明显差异。血脂测定显示,模型组TC和LDL水平增加幅度较大,TG和HDL水平增加幅度较小,但是与对照组比较差异有显著性（P<0.05、0.01）。组织病理学检查结果显示,实验4周时,模型组家兔在主动脉内皮下可见少量脂质沉积,油红O染色呈红色;实验8周和12周时,处于动脉粥样硬化脂纹期的不同阶段,病灶处内皮下可见大量的泡沫细胞聚集;实验16周时,处于动脉粥样硬化纤维斑块期,病灶表层可见覆盖以纤维帽,纤维帽下有数量不等的泡沫细胞,平滑肌细胞、炎细胞等。结论 家兔单纯给予高脂饲料喂饲4～16周,可造成不同病理分期的动脉粥样硬化模型。     

冰冻切片技术在神经病理诊断中的应用

目的探讨改进术中冰冻切片质量的技巧和方法 ,以利于提高冰冻切片的染色效果,提高冰冻切片病理诊断的准确率。方法对2011年1月至2014年8月首都医科大学附属北京天坛医院神经外科手术切除的3 000例新鲜脑肿瘤标本进行取材、冰冻、切片、染色,制作冰冻切片。其中神经上皮组织肿瘤2 103例,神经肿瘤102例,脑膜肿瘤388例,淋巴造血系统肿瘤23例,生殖细胞肿瘤79例,鞍区肿瘤129例,转移性肿瘤79例,其他肿瘤97例,对上述肿瘤的冰冻切片进行回顾性分析,改进染色方法,观察染色效果,分析诊断准确率。结果 3 000例脑肿瘤标本的冰冻切片染色效果良好,组织结构完整,细胞形态清晰,一般制片过程约需10 min。术后与石蜡切片诊断对照,其中神经上皮组织肿瘤2 078例,神经肿瘤101例,脑膜肿瘤378例,淋巴造血系统肿瘤22例,生殖细胞肿瘤75例,鞍区肿瘤127例,转移性肿瘤76例,其他肿瘤86例,诊断符合率为98.1%,未发现良、恶性质相反的诊断。结论改进冰冻切片的染色方法,可提高冰冻切片的染色质量,提高病理诊断的准确率。     

红豆杉提取物对慢性阻塞性肺疾病小鼠的药效学评价

目的 观察红豆杉提取物灌胃给药对吸烟致慢性阻塞性肺病（COPD）模型小鼠的影响,对其药效进行评价,并对其作用机制进行初步探讨。方法 C57BL/6小鼠烟熏3个月后,收集肺泡灌洗液（BALF）和肺组织,对BALF进行白细胞分类计数,取肺组织做组织切片进行HE染色和AB/PAS染色,观察炎症细胞浸润情况,并对超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、髓过氧化物酶（MPO）活性和TNF-a、KC、MIP-2表达量进行测定。结果 红豆杉提取物具有降低白细胞总数的作用,可显著增加SOD、GSH含量,抑制MPO活性,且具有显著抑制TNF-a、KC炎症因子的作用。结论 红豆杉提取物对COPD动物模型有一定的治疗效果,其作用机制可能与抑制中性粒细胞迁移、抑制炎症细胞因子的产生、抑制黏液分泌有一定关系。     

CLOCK基因在甲状腺乳头状癌组织中的表达及临床意义

目的 探讨生物钟基因CLOCK在甲状腺乳头状癌组织中的表达,并分析其与甲状腺癌的关系,为甲状腺癌的早期诊断提供科学依据。方法 采用免疫组化方法检测CLOCK基因在74例甲状腺乳头状癌及癌旁组织中的表达水平,并分析其表达与临床病理特征的关系。结果 在甲状腺乳头状癌组织与其配对的癌旁正常组织中,CLOCK基因阳性表达分别为68.9%(51/74)和40.5%(30/74)。两类组织中阳性率比较,差异有统计学意义(P<0.05)。CLOCK基因表达与甲状腺乳头状癌淋巴结转移及TNM分期有关(P<0.05)。结论 CLOCK基因可能与甲状腺乳头状癌的发生有重要关系,但其与肿瘤的侵袭和转移的关系需更深入的研究。     

Characterization of Murine Monoclonal Antibody to Tumor Necrosis Factor (TNF-MAb) Formulation for Freeze-Drying Cycle Development

Purpose. This study was designed to characterize the formulation of protein pharmaceuticals for freeze-drying cycle development. Thermal properties of a protein formulation in a freezing temperature range are important in the development of freezing and primary drying phases. Moisture sorption properties and the relationship between moisture and stability are the bases for the design of the secondary drying phase. Methods. We have characterized the formulation of TNF-MAb for the purpose of freeze-drying cycle development. The methods include: DTA with ER probes, freeze-drying microscopy, isothermal water adsorption, and moisture optimization.Results. The DTA/ER work demonstrated the tendency to noneutectic freezing for the TNF-MAb formulation at cooling rates of –1 to –3°C/min. The probability of glycine crystallization during freezing was quite low. A special treatment, either a high subzero temperature holding or annealing could promote the maximum crystallization of glycine, which could dramatically increase the T_g' of the remaining solution. The freeze-drying microscopy further indicated that, after the product was annealed, the cake structure was fully maintained at a T_p below –25°C during primary drying. The moisture optimization study demonstrated that a drier TNF-MAb product had better stability. Conclusions. An annealing treatment should be implemented in the freezing phase in order for TNF-MAb to be dried at a higher product temperature during primary drying. A secondary drying phase at an elevated temperature was necessary in order to achieve optimum moisture content in the final product.     

In-Situ Near-Infrared Spectroscopy Monitoring of the Lyophilization Process

Purpose. The purpose of this work was to demonstrate the feasibility of using near-infrared spectroscopy (NIRS) to monitor the freeze-drying process in-situ. Methods. The experiment was performed in a pilot-scale freeze-dryer, in which the NIRS probe was interfaced using a lead-through to the lyophilizer. Special equipment for the sample presentation was developed. NIRS measurements were made using a FT (Fourier transform)-NIR spectrometer fitted with a single fiber reflectance probe. Results. The physical changes, that is, freezing, sublimation, and desorption, generated significant spectral changes. There was good agreement between NIRS monitoring and product temperature monitoring about the freezing process and the transition from frozen solution to ice-free material. The NIRS monitoring also provided new information about the process that was not possible to detect with product temperature monitoring, such as the rate of the desorption process and the steady-state where the drying was complete. The NIRS monitoring yields significantly more information about the actual process and essentially explains the observed changes of the product temperature during the lyophilization process. Conclusions. NIRS monitoring is a viable tool for in-situ monitoring, both qualitatively and quantitatively. It can facilitate investigations of the drying process within a sample. The small volume monitored makes sample presentation very important.     

Effect of Freezing Rate on the Stability of Liposomes During Freeze-Drying and Rehydration

Purpose. In the present study we examined the effect of the freezing protocol on carboxyfluorescein (CF) retention in liposomes after freeze-drying and rehydration. Methods. Liposomes were frozen slowly at 0.5°C/min, or quickly by submerging the samples in boiling nitrogen before freeze-drying. The thermal behaviour of the frozen dispersions was analysed by Modulated Temperature Differential Scanning Calorimetry (MTDSC). The dried cakes were analysed by SEM, MTDSC and FTIR. The % encapsulated CF of the (re)hydrated liposomes was determined by fluorimetry after GPC, their vesicle size was measured by the Dynamic Light scattering Technique and their bilayer transition was studied by DSC. Results. Slow freezing resulted in a markedly higher CF retention after freeze-drying and rehydration as compared to quick freezing. The effect of the freezing rate depended on the lipid composition and was most pronounced for rigid liposomes. The damage caused by quick freezing did not occur after a freezing/thawing cycle. The freezing protocol did not influence the interaction between the phospholipids and the lyoprotectants (sucrose, trehalose or glucose) in the freeze-dried state. However, analysis by DSC of dipalmitoylphosphatidylcholine (DPPC): dipalmitoylphosphatidylglycerol (DPPG) =10:1 and DPPC liposome dispersions showed that the freezing protocol affected the bilayer melting characteristics of these liposomes after freeze-drying and rehydration. Conclusions. A proper design of the freezing protocol is essential to achieve optimal stability of rigid liposomes during a freeze-drying and rehydration cycle.     

Thermophysical Properties of Trehalose and Its Concentrated Aqueous Solutions

Purpose. To address the lack of fundamental thermophysical data for trehalose and its aqueous systems by measuring equilibrium and non-equilibrium properties of such systems. Methods/Results. Differential scanning calorimetry (DSC) and dynamic mechanical analysis were used to measure glass transition temperatures of trehalose and its solutions. X-ray diffractometry was used to verify the structure of amorphous trehalose. Controlled-stress rheometry was used to measure viscosity of several aqueous trehalose systems at ambient and sub-ambient temperatures. Over this temperature range, the density of these solutions was also measured with a vibrating tube densimeter. The equilibrium phase diagram of aqueous trehalose was determined by measuring the solubility and freezing point depression. Conclusions. Our solubility measurements, which have allowed long times for attainment of chemical equilibrium, are substantially different from those reported earlier that used different techniques. Our measurements of the glass transition temperature of trehalose are higher than reported values. A simple model for the glass transition is presented to describe our experimental observations.     

Glycine Crystallization During Freezing: The Effects of Salt Form,pH, and Ionic Strength

Purpose. The purpose of the study is to characterize glycine crystallization during freezing of aqueous solutions as a function of the glycine salt form (i.e., neutral glycine, glycine hydrochloride, and sodium glycinate), pH, and ionic strength. Methods. Crystallization was studied by thermal analysis, microscopy, x-ray diffraction, and pulsed Fourier transform nmr spectroscopy. Results. A solution of neutral glycine with no additives undergoes rapid secondary crystallization during freezing, forming the polymorph, with a eutectic melting temperature of –3.4°C. Glycine hydrochloride solutions undergo secondary crystallization relatively slowly, and the eutectic melting temperature is –28°C. Sodium glycinate crystallizes from frozen solution at an intermediate rate, forming a eutectic mixture with a melting temperature of –17.8°C. Where secondary crystallization does not occur rapidly, a complex glass transition is observed in the –70° to – 85°C temperature range in the DSC thermograms of all systems studied. Rates of secondary crystallization and the type of crystal formed are influenced by solution pH relative the the pKs of glycine, and also by the change in ionic strength caused by adjustment of pH. Increased ionic strength significantly slows the crystallization of neutral glycine and promotes formation of the polymorph. Thermal treatment or extended holding times during the freezing process may be necessary in order to promote secondary crystallization and prevent collapse during freeze drying. Conclusions. The results underscore the importance of recognizing that seemingly minor changes in formulation conditions can have profound effects on the physical chemistry of freezing and freeze drying.     

Investigations into the Stabilization of Drugs by Sugar Glasses: III. The Influence of Various High-pH Buffers

Purpose. To study the effect of the high-pH buffers ammediol, borax, CHES, TRIS, and Tricine on the glass transition temperature of the freeze concentrated fraction (Tg) of trehalose/buffer and inulin/buffer solutions at pH 6.0 and pH 9.8. Also, the glass transition temperature (Tg) of sugar glasses obtained after freeze drying of these solutions was elucidated. Additionally, the effect occurring during the freezing process on the pH of the various buffers was investigated. Furthermore, the stability of alkaline phosphatase (AP) incorporated in these sugar glasses prepared from solutions at pH 9.8 was evaluated. Methods. The Tg and Tg were measured using differential scanning calorimetry (DSC), and the change of pH during freezing was estimated by using an indicator solution added to the respective solutions. The enzymatic activity of AP after freeze drying and storage at 60°C was evaluated by an enzymatic activity assay. Results. It was found that the Tg and Tg of the samples investigated are strongly influenced by the presence of the buffer. On freezing, only minor changes of the pH were observed. The samples with the lowest Tg and the samples containing buffers that formed complexes with the sugars showed the poorest stability of the AP. Conclusions. The stabilizing capacities of sugars that are currently recognized as excellent stabilizers for proteins during drying and storage can be completely lost if certain high-pH buffers such as ammediol, borax, and TRIS are used at high concentrations. Loss of stabilizing capacities can be ascribed to strong depression of the Tg and Tg or to complex formation.     

昆明种乳鼠肝细胞原代培养方法的比较研究

目的 通过比较3种昆明鼠乳鼠肝细胞原代培养的方法,探索一种更有效、快捷的原代培养方法。方法 取30只1~3日龄昆明种乳鼠,随机分为3组,分别采用组织块培养法、胰酶消化法、组织块联合胰酶消化法进行肝细胞原代培养,用倒置显微镜观察实验过程中肝细胞的形态及生长情况。结果 3种方法均能进行乳鼠肝细胞的原代培养,其中组织块联合胰酶消化法培养的肝细胞形态学和生物学特性优于其他两种方法培养的细胞。结论 组织块联合胰酶消化法是3种方法中最快速和高效的一种方法,获得的乳鼠肝细胞形态及生物学特性好,为体外建立乳鼠肝细胞实验模型奠定了实验基础。