一种简单实用纯化腹水McAb方法--辛酸/硫酸铵法

采用辛酸－硫酸铵法（ＣＡ－ＡＳ）、硫酸铵沉淀法（ＣＡ）和亲合层析法,分别提取了小鼠多株腹水单抗。结果显示：ＣＡ－ＡＳ法提取的ＩｇＧ 纯度为８７％ ～９１％ ,高于ＣＡ 法（６５％ ～７０％ ）,低于亲合层析法（１００％ ）;ＣＡ－ＡＳ法提取的ＩｇＧ得率为４．８～５．５ｇ／Ｌ腹水,低于ＣＡ 法（７．０～７．９ｇ／Ｌ腹水）,远高于亲合层析法（０．６ｇ／Ｌ腹水）;ＣＡ－ＡＳ法提取ＩｇＧ回收率为３９．０％ ～４４．４％ ,低于ＣＡ法（５６．０％ ～６５．０％ ）,远高于亲合层析法（７．４％ ）。４Ｅ３、Ｈ６ 腹水抗体经ＣＡ－ＡＳ法提取前后酶联效价不变,表明ＣＡ－ＡＳ法对ＭｃＡｂ 活性无影响。作者认为,ＣＡ－ＡＳ法是一种操作简便,耗时短,效果佳,成本低的提取腹水ＭｃＡｂ 的新方法。     

一种简易高效的建立小鼠胰岛移植模型的方法

目的 探索一种简便高效的建立小鼠胰岛移植模型的方法。方法 摘取四只BALB/c小鼠的胰腺,0.25mmPenfine针对胰腺反复多点注射胶原酶P后静止消化20min;采用自制的推进式离心管,以单一密度梯度Ficoll液(1.088g/ml)纯化胰腺消化物,双硫腙对胰岛进行特异性染色,利用胰岛素释放试验检测胰岛细胞的功能;纯化后的胰岛移植到实验性糖尿病C57小鼠的肾包囊下,术后观察其血糖变化。结果 纯化后共得到（789.6±26.4）个胰岛细胞当量（IEQ）,平均纯度为（77.12±3.23）％;胰岛细胞对胰岛素释放刺激反应良好,高糖时胰岛素的释放量为低糖时的2.35倍（P<0.01）。移植后可逆转糖尿病小鼠的高血糖平均达（16.3±2.9d）。结论 提供了快速获取小鼠胰岛细胞的方法,为胰岛移植的实验研究建立了一种简易高效的制作动物模型的方法。     

一种简便经济的肝星状细胞分离方法

目的　介绍一种简易、经济、高产的肝星状细胞 (HSC)分离方法 ,为肝纤维化的研究提供细胞模型。方法　参照国内外方法加以改良 ,应用D hanks液校正淋巴细胞分离液 ,使其为 1 0 53 ,采用单层一步梯度离心法分离HSC。结果　细胞获得量为 (3 5± 0 5)× 1 0 7/只 ,存活率、纯度均在 95 %以上。结论　本方法简便、实用、高产 ,不需特殊设备 ,便于推广应用     

一种简单快速分离纯化小型猪胰岛的方法

目的 探索一种简单快速分离纯化小型猪胰岛的方法。 方法 摘取小型猪胰腺,经胰管插管后注入胶原酶V,用自制的半自动消化分离装置消化,胰腺消化物经自制的推进式离心管及单一密度（1.096g/ mL）的ficoll400纯化后行双硫腙（DTZ）染色,倒置显微镜下观察计数胰岛的数量及纯度,胰岛素释放试验检测胰岛细胞的功能。结果 纯化后平均每克小型猪的胰腺可以获得（2 645 ±234）个胰岛细胞当量（IEQ）,平均纯度为（74.2 ±5.1）%。纯化后的胰岛细胞对胰岛素释放刺激反应良好,高糖时的胰岛素释放量为低糖时的3.14倍(P<0.01)。 结论 为小型猪胰岛细胞的研究建立了一种简单快速分离纯化胰岛的方法,纯化后的胰岛细胞功能良好。     

A simple method for purification of genomic DNA from whole blood using Fe3O4/Au composite particles as a carrier

Objective： To establish a method of genomic DNA extraction from whole blood using Fe3O4/Au composite particles as a carrier. Methods： Two crucial conditions （sodium chloride concentration and amount of the magnetic particles） were optimized and 8 different human whole blood samples were used to purify genomic DNA under the optimal condition. Then agarose gel electrophoresis and polymerase cbain reaction （PCR） were performed. Results： The optimal binding condition was 1.5 mol/L NaC1/10% PEG, and the optimal amount of Fe3O4/Au composite particles was 600μg. The yields of the genomic DNA from 100μl of different whole blood samples were 2-5 μg, and the ratio of A260/A280 was in the range of 1.70-1.90. The size of genomic DNA was about 23 kb and the PCR was valid. Conclusion： The purification system using Fe3O4/Au composite microparticles has advantages in high yield, high purity, ease of operating, time saving and avoiding centrifugation. The purified sample was found to function satisfactorily in PCR amplification.     

LC-MS/MS method for the quantitation of serum tocilizumab in rheumatoid arthritis patients using rapid tryptic digestion without IgG purification

The quantitation of serum tocilizumab using liquid chromatography tandem-mass spectrometry (LC-MS/MS) method has not been widely applied in clinical settings because of its time-consuming and costly sample pretreatments. The present study aimed to develop a validated LC-MS/MS method for detecting serum tocilizumab by utilizing immobilized trypsin without an immunoglobulin G purification step and evaluate its applicability in the treatment of rheumatoid arthritis (RA) patients administered intravenously or subcutaneously with tocilizumab. The tocilizumab-derived signature peptide was deciphered using a nano-LC system coupled to a hybrid quadrupole-orbitrap mass spectrometer. The serum tocilizumab was rapidly digested by immobilized trypsin for 30 min. The chromatographic peak of the signature peptide and that of the internal standard were separated from the serum digests for a total run time of 15 min. The calibration curve of serum tocilizumab concentration was linear with a range of 2–200 μg/mL. The intra- and inter-day accuracy and relative standard deviation (RSD) were 90.7%–109.4% and <10%, respectively. The serum tocilizumab concentrations in the RA patients receiving intravenous and subcutaneous injections were 5.8–28.9 and 2.4–63.5 μg/mL, respectively. The serum tocilizumab concentrations using the current method positively correlated with those using the enzyme-linked immunosorbent assay, although a systematic error was observed between these methods. In conclusion, a validated LC-MS/MS method with minimal sample pretreatments for monitoring serum tocilizumab concentrations in RA patients was developed.     

A simple and efficient method of DNA extraction from formalin-fixed and paraffin-embedded tissues for screening EGFR gene mutation by gene sequencing

Epidermal growth factor receptor （EGFR） is frequently overexpressed in non-small-cell lung cancer （NSCLC） and plays a key role in tumorigenesis.1 Small molecule tyrosine kinase inhibitors （TKIs）,such as gefitinib,erlotinib,and icotinib,which can inhibit receptor tyrosine kinase activity of EGFR become clinically available for the treatment of non-small-cell lung cancer （NSCLC）.2 NSCLC patients with EGFR mutation have experienced a marked response to EGFR-TKIs therapy.3 Detection of mutations of the EGFR gene is critical for predicting the response to therapy with TKIs.4     

血清蛋白质组学研究中白蛋白和IgG去除方法的建立与评价

目的 建立和评价血清蛋白质组学研究中白蛋白和IgG的去除方法。方法 应用Micro Bio-Spin column过滤纯化血清蛋白,去除白蛋白和IgG,利用双向凝胶电泳(2-DE)分析评价血清蛋白过滤效果。结果 建立了血清白蛋白和IgG去除方法;过滤后大部分血清白蛋白和IgG能够去除,2-DE图谱中低丰度蛋白质分辨率增加。结论 本研究建立的血清蛋白质纯化技术,可有效应用于疾病的血清蛋白质组学研究。     

新方法简便高效建立小鼠原位膀胱癌模型

目的 用物理化学的方法建立小鼠原拉表浅膀胱癌模型,并对影响成瘤率的因素进行探讨,筛选出高效简便建立小鼠原位表浅膀胱痛模型的方法.方法 用改造过的静脉留置针经小鼠尿道插入膀胱腔内,实验组用酸碱腐蚀的方法对小鼠膀胱粘膜进行预处理,按观察目的 的不同分为4组:对照组不作预处理.PBS冲洗后将MB49灌注入膀胱,构建小鼠原位表浅膀胱癌模型.所有小鼠观察一般情况和肿瘤生长情况.按计划处死小鼠并解剖观察膀胱肿瘤生长情况及有无转移,取标本做病理切片.结果 病理显示膀胱粘膜已预处理的小鼠接种肿瘤细胞后可在膀胱部位成瘤(成瘤率100%);丝裂霉素能显著减缓膀胱肿瘤的生长(P<0=0.000,P<0.05);对照组接种肿瘤细胞后未见成瘤.结论 膀胱粘膜处理的时间以酸20 S,碱5 S较合适;简便的物理化学方法成功建立了可靠性、稳定性和重复性均好的小鼠原位表浅膀胱癌模型,为表浅膀胱癌术后复发的防治研究尤其是抗癌药物的筛选和免疫治疗研究提供了理想的动物实验模型.     

简便快速的血中DNA提取纯化法

目的 为了探讨血中DNA提取纯化的简便快速的方法。方法 用Tritonx-100同时反复破碎红细胞和白细胞，然后用NaClO4、SDS解聚核蛋白，用PCI抽提，Sephader G-25柱层析。结果 A260／A280可达1．78～1．80，平均产量20．3μg/ml，全过程2～3h。结论 这种方法用于临床经济、快速、得率高，诊断结论可靠。     

An improved method for directional differentiation and efficient production of neurons from embryonic stem cells in vitro

Summary To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells)in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM)in Vitro. The totipotency of ES cells was identified by observation of cells morphology and formations of teratoma in immunocompromised mice. The cells differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM. ZHOU Yufeng, male, born in 1974, Doctor in Charge     

碱提酸沉法提取银杏外种皮中银杏酸的工艺研究

目的优化银杏外种皮的有效部位总银杏酸的提取工艺。方法采用碱提酸沉法提取银杏外种皮中银杏酸,通过单因素试验和正交试验,以白果酸含量为指标,采用高效液相色谱法测其含量,考察料液比、碱液浓度、碱提时间、酸沉pH、酸沉时间对提取工艺的影响。结果银杏酸的最佳提取工艺条件为料液比1:5,碱液浓度3%,碱提时间1 h,酸沉pH 5,酸沉时间1 h。结论经优选的碱提酸沉法提取银杏酸工艺,操作简单,稳定易行,提取效果好,重复性好,适合大量提取银杏外种皮中银杏酸。     

透射电子显微镜原位细胞或组织简便快速包埋法

为了满足临床病理诊断及科研的需要，克服电镜原位细胞或组织包埋法之不足，作者研究出一咱改良的透射电子显微镜细胞或组织原位简便快速包埋法。其特点是：（１）操作简便，全部包埋步骤（固定、脱水、浸泡及包埋）均在同一载体（培养瓶 或显微镜观察的组织切片）上进行，能保存原位组织或细胞的超微结构；（２）快速，人固定到制备成超薄切片全过程仅需５小时；（３）易从载体上将包埋片分离下来，分离下来的包埋片完整，超微结构     

改良一步法制备人外周血白细胞线粒体DNA

目的建立从少量外周血中提取白细胞线粒体DNA(mtDNA)的快速、简便方法.方法 58份抗凝血静置后取白细胞层,采用碱变性法裂解细胞,酚/氯仿/异戊醇抽提,RNA酶消化后再抽提及沉淀,PCR扩增产物电泳鉴定.结果获得的mtDNA样品不存在蛋白质等污染,用特异性mtDNA PCR引物从58例核酸样品中均能扩增出1 528bp的mtDNA基因片段.结论改良一步法是一种省时、经济、实用、重复性好、能满足常规分子生物学和遗传学分析需要的mtDNA制备新方法.     

血清β—羟丁酸测定方法的建立及其在糖尿病酮症酸中毒中的意义

OBJECTIVE: This study was to establish enzyme-rate method for determining serum beta-hydroxybutyric acid, and to discuss its meaning in diabetes ketosis acidism. METHODS: Enzyme-rate method was used to determine serum beta-hydroxybutyric acid in 60 cases of normals, 30 cases of diabetes, and 8 cases of ketosis acidism by Hitachi 7170A autochemistry analyzer. RESULTS: The recovery rate is 108%, the linerity extent 0-4.0 mmol.L-1, the coefficient of variation within-run is 2.2% and day-to-day is 2.6%. CONCLUSION: The enzyme-rate method for determining serum beta-hydroxybutyric acid save time, gives accurate results and has wide linerity, so it can used for diagnosis of diabetes ketosis acidism.     

一种简单的分离、培养及鉴定小鼠外周血单核巨噬细胞方法的建立

目的 在L929条件培养基的作用下体外分离、培养小鼠外周血单核巨噬细胞,并进行鉴定。方法 密度梯度离心法分离小鼠外周血单核巨噬细胞,用L929条件培养基培养7 d。采用免疫荧光的方法检测F4/80的表达以及细胞的吞噬作用,用流式细胞术进一步检测细胞吞噬率。结果 用L929条件培养基诱导培养7 d后小鼠外周血单核细胞表达F4/80,加入细胞吞噬珠之后,在不同时间点用荧光显微镜可以看到红色荧光颗粒聚集在小鼠外周血单核巨噬细胞核周围,用流式细胞仪检测细胞的吞噬率为24.8%±0.79%。结论 该方法是一种简单、易操作的体外分离培养和鉴定小鼠外周血单核巨噬细胞的方法。     

正交试验比较金银花中绿原酸、异绿原酸A的提取工艺

目的优化金银花的提取工艺,建立高效液相色谱法（HPLC）快捷检测绿原酸和异绿原酸A的方法。方法以HPLC同时测定绿原酸、异绿原酸A的含量为指标,通过比较醇浓度、回流时间、超声功率、超声时间4因素对提取工艺的影响,优化超声波辅助醇法提取金银花的工艺。结果以绿原酸、异绿原酸A的综合评分为指标,最佳提取工艺是60%乙醇超声（功率500 W）45 min,再回流提取1 h。结论建立了准确灵敏的检测方法,优化的提取工艺经济、高效,能为生产提供理论依据。     

化疗药物联合细胞因子对bcr—abl转基因小鼠骨髓细胞生长及？…

应用Ｈｕ或５－Ｆｕ和鼠干细胞因子及鼠白细胞介素－３培养含金属硫蛋白启动子和增强子序列、ｂ ｒ－ａｂｌｃＤＮＡ（ｐ２１０）序列及ＳＶ４０剪切和Ｐｏｌｙ（Ａ）信号号序列的转基因慢粒白血病小鼠骨髓细胞,观察化疗药物及细胞因子单独或联合应用对ＭＴ／ｐ２１０（ｂｒ－ａｂｌ）转基因小鼠骨骨细胞生长的影响,并采用ＲＴ－ＰＣＲ