甲胎蛋白异质体在原发性肝癌患者血清中的表达

为了揭示原发性肝癌患者血清中甲胎蛋白异质体(AFP-L3)的表达及意义,本研究选取2014年2月至2016年3月在本院治疗的原发性肝癌患者80例,同时选取肝硬化患者60例,慢性乙肝患者60例及健康志愿者60例,采用亲和吸附离心管法检测不同受试者血清中AFP-L3水平。研究显示,原发性肝癌患者的血清AFP-L3 (122.13 ng/mL)水平显著低于肝硬化患者,并且显著高于慢性乙肝患者和健康者,差异具有统计学意义(p0.05);原发性肝癌患者中,血清AFP-L3水平在肿瘤大小≤3 cm (171.14 ng/mL)和TNM分期为Ⅰ～Ⅱ期(147.53 ng/mL)患者中显著高于肿瘤大小3 cm和TNM分期为Ⅲ～Ⅳ期患者(p0.05);血清AFP-L3高表达(≥122.13 ng/mL)的肝癌患者中位生存时间(22.76月)与血清AFP-L3低表达的肝癌患者(122.13 ng/mL)(22.04月)差异不显著(p0.05)。本项研究表明原发性肝癌患者血清中AFP-L3水平明显上升,并且与患者的肿瘤大小和TNM分期具有相关性。     

甲胎蛋白异质体在肝细胞癌中的诊断价值分析

目的:探讨甲胎蛋白异质体(AFP-L3)的检测对肝细胞肝癌(HCC)的诊断价值。方法:选取2009年2月至2012年11月在本院治疗的90例HCC患者为研究组,另选择同期接受治疗的慢性乙型肝炎患者30例和肝炎肝硬化患者30例作为对照组。观察三组患者的AFP、AFP-L3阳性率。结果:肝癌患者AFP和AFP-L3阳性率显著高于乙肝和肝硬化患者,差异具有统计学意义(P0.05)。乙肝和肝硬化两组患者的AFP及AFP-L3阳性率无显著差异(P0.05)。AFP400 ng/m L:肝癌组AFP-L3阳性率显著高于乙肝组和肝硬化组,差异具有统计学意义(P0.05)。结论:AFP-L3在HCC中的诊断价值较甲胎蛋白大,更适合在临床诊断中应用。     

AFP-L3蛋白芯片的建立与应用

目的:建立一种可同时检测血清中甲胎蛋白(AFP)及甲胎蛋白异质体(AFP-L3)的蛋白芯片方法,为AFP-L3检测提供经济、便捷、省时、有效的新途径。方法:将鼠源AFP单克隆抗体和小扁豆凝集素点样固定在醛基玻片上,制备出AFP抗体和小扁豆凝集素蛋白芯片。利用抗原抗体特异性结合以及盐藻糖与小扁豆凝集素特异性结合的原理,用蛋白芯片方法检测血清样本中的AFP和AFP-L3。结果:肝癌血清39份,其中37份检测到AFP;26份肝癌血清中同时检测到AFP和AFP-L3,2份肝癌血清均未检测到AFP和AFP-L3。肝细胞癌组血清样本中AFP和AFP-L3水平明显高于健康对照组(P0.001),健康对照组与空白对照组统计学上无差异(P0.05)。结论:本研究成功地建立AFP和AFP-L3同时检测的蛋白芯片方法,与ELISA法和凝集素微量离心柱法相比,是一种切实可行,经济、便捷、省时、有效的方法。     

MRI扫描联合血清4项肿瘤标志物诊断原发性肝癌的临床价值研究

目的:探究磁共振成像(MRI)扫描联合血清4项肿瘤标志物诊断原发性肝癌(PHC)的临床价值。方法:将我院从2017年9月~2019年8月收治的65例PHC患者(肝癌组)纳入研究,另选取同期我院收治的60例良性肝病患者作为对照组。比较两组MRI图像特征。此外,检测并比较两组血清甲胎蛋白(AFP)和甲胎蛋白异质体(AFP-L3)、高尔基体蛋白73(GP73)、磷脂酰肌醇蛋白聚糖-3(GPC-3)水平。以病理诊断为金标准,分析不同诊断方式应用于PHC患者诊断中的效能。结果:PHC患者的MRI图像表现为边界清晰,呈类圆形或不规则分叶状,肝癌组T1WI呈低或稍低信号人数占比高于对照组,且T2WI、DWI呈高信号强度人数占比高于对照组(均P<0.05)。肝癌组血清AFP、AFP-L3、GP73、GPC-3水平均高于对照组(均P<0.05)。以病理诊断为金标准,MRI联合血清AFP、AFP-L3、GP73、GPC-3诊断PHC的灵敏度、特异度以及准确度分别为95.24%(40/42)、95.65%(22/23)、95.38%(62/65),均高于MRI诊断的73.81%(31/42)、69.57%(16/23)、72.31%(47/65)(P<0.05),以及血清4项肿瘤标志物联合诊断的80.95%(34/42)、73.91%(17/23)、78.46%(51/65)(P<0.05)。结论:PHC患者血清AFP、AFP-L3、GP73、GPC-3水平升高,MRI扫描联合血清AFP、AFP-L3、GP73、GPC-3检测可作为诊断PHC的有效手段。     

大孔树脂分离纯化辣椒叶总黄酮的工艺研究

目的:筛选适合分离纯化辣椒叶总黄酮的一种大孔树脂,同时用响应面法进行优化得到最佳纯化工艺。方法:采用热回流法提取辣椒叶总黄酮,以吸附率和解吸率为考察指标,考察6种不同型号的大孔树脂(HPD100、HPD450、HPD600、HPD826、D101、AB-8)对辣椒叶总黄酮的吸附能力与解吸能力,确定最佳树脂。通过动态吸附解吸实验考察此树脂对辣椒叶总黄酮的最佳分离纯化工艺。结果:通过对辣椒叶总黄酮吸附分离性能的分析显示HPD600为最佳树脂,最优工艺为:上样浓度为10 mg/mL,上样量为10 mL,洗脱体积为4 BV,洗脱液流速为4 mL/min,洗脱液pH为7,依次用水、10%、30%乙醇冲洗树脂柱,50%乙醇为洗脱液。纯化后的黄酮纯度435.4 mg/g。结论:该方法简便,操作简单,对辣椒叶总黄酮的纯化效果较好。     

复方苦参注射液联合TACE对中晚期肝癌患者预后、生存质量和血清AFP、AFP-L3水平的影响

摘要 目的：观察肝动脉化疗栓塞术（TACE）联合复方苦参注射液对中晚期肝癌患者预后、生存质量和血清甲胎蛋白（AFP）、甲胎蛋白异质体（AFP-L3）水平的影响。方法：选择我院于2016年3月~2019年10月期间收治的98例中晚期肝癌患者,经随机数字表法分为对照组（49例,TACE治疗）和研究组（49例,复方苦参注射液联合TACE治疗）。对比两组疗效、生存率、生存质量改善率、血清AFP、AFP-L3水平及不良反应发生率。结果：与对照组比较,研究组的临床总有效率明显升高（P<0.05）。研究组的1年生存率高于对照组（P<0.05）。与对照组比较,研究组的生存质量改善率明显升高（P<0.05）。两组治疗后血清AFP、AFP-L3水平较治疗前下降,且研究组低于对照组（P<0.05）。两组不良反应总发生率组间对比无统计学差异（P>0.05）。结论：TACE基础上联合复方苦参注射液治疗中晚期肝癌患者,可改善其短期预后及生存质量,降低其血清AFP、AFP-L3水平。     

黑果枸杞色素的提取和精制工艺研究

本文采用正交实验法对黑果枸杞色素的提取和精制工艺进行了研究。结果表明,黑果枸杞色素的最佳提取条件为:以pH 3.0的80%乙醇作浸提剂,提取温度50℃,提取时间3 h,固液配比1:40;用X-5大孔吸附树脂对色素进行精制,以树脂柱径高比1:15、流速3 mL/minp、H 3.0、色素液浓度1 g/L为最佳吸附条件,色素的吸附量可达0.03715 g/mL湿树脂体积;而以95%乙醇做洗脱液,在pH 2.0、流速5 mL/min、3倍于柱床体积的洗脱液条件下解吸附效果最佳,色素回收率达到97.78%;制取的色素产品外观呈紫红色,色价为36.7。     

丹参酮和丹参酚酸的同步提取分离纯化工艺研究

本文研究丹参中丹参酮和丹参酚酸同步提取分离纯化工艺条件。以丹参酮ⅡA和丹酚酸B的含量为综合评价指标,应用正交试验设计,考察乙醇浓度、渗漉液体积和渗漉速度对提取效果的影响;采用单因素实验对大孔树脂型号、上样液p H值、乙醇浓度、洗脱剂用量等进行考察,最终确定了提取分离纯化工艺为:丹参粉碎过10目筛,用80%乙醇以4 m L/(min·kg)的速度渗漉,收集10倍量的渗漉液,回收乙醇,加水稀释至0.5 g/m L生药,用盐酸调节p H值3.0,过滤,即得丹参酮提取物。滤液上HPD100大孔吸附树脂柱,2 BV水洗,50%乙醇3 BV洗脱,收集洗脱液,即得丹参酚酸提取物。结果表明优化后提取分离效果好,提取物中丹参酮ⅡA和丹酚酸B的含量及提取率高,该工艺操作简便、易行、稳定性好,适合在生产中推广应用。     

丹参酮和丹参酚酸的同步提取分离纯化工艺研究北大核心CSCD

本文研究丹参中丹参酮和丹参酚酸同步提取分离纯化工艺条件。以丹参酮ⅡA和丹酚酸B的含量为综合评价指标,应用正交试验设计,考察乙醇浓度、渗漉液体积和渗漉速度对提取效果的影响;采用单因素实验对大孔树脂型号、上样液p H值、乙醇浓度、洗脱剂用量等进行考察,最终确定了提取分离纯化工艺为:丹参粉碎过10目筛,用80%乙醇以4 m L/(min·kg)的速度渗漉,收集10倍量的渗漉液,回收乙醇,加水稀释至0.5 g/m L生药,用盐酸调节p H值3.0,过滤,即得丹参酮提取物。滤液上HPD100大孔吸附树脂柱,2 BV水洗,50%乙醇3 BV洗脱,收集洗脱液,即得丹参酚酸提取物。结果表明优化后提取分离效果好,提取物中丹参酮ⅡA和丹酚酸B的含量及提取率高,该工艺操作简便、易行、稳定性好,适合在生产中推广应用。     

响应面法优化AB-8大孔树脂纯化玉米须总黄酮的工艺

本研究采用超声波法提取玉米须总黄酮,继而研究AB-8大孔树脂纯化玉米须总黄酮的最佳工艺。以动态吸附率为评价指标,考察上样液流速、上样液浓度、上样液pH对玉米须总黄酮动态吸附的影响,以动态洗脱率为评价指标,考察洗脱液浓度、洗脱液用量对玉米须总黄酮动态解吸附的影响。在AB-8大孔树脂单因素试验的基础上,应用响应面法优化了玉米须总黄酮的纯化工艺条件。研究表明,最佳纯化工艺条件:上样液浓度0.03 mg/mL、上样液流速0.50 mL/min、洗脱液浓度91%。在此工艺条件下,玉米须总黄酮洗脱率为87.90%,与理论值基本吻合。该工艺稳定、可行,可用于玉米须总黄酮的分离纯化。     

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis

Implementation of the on-chip immunoassay for α-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10 min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1 ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r² = 0.981 and slope = 1.03.     

Clinical evaluation of lentil lectin-reactive alpha-fetoprotein-L3 in histology-proven hepatocellular carcinoma

INTRODUCTION: Serum alpha-fetoprotein (AFP) is a useful marker of hepatocellular carcinoma (HCC), although the serum AFP concentration is also increased in patients with chronic liver diseases (CLD). The analysis of AFP glycoforms has been known to be of diagnostic value. We applied the lectin-affinity electrophoresis and antibody-affinity blotting techniques to HCC patients in Vietnam in order to better understand the role of lentil lectin-affinity AFP-L3 in the diagnosis and differential diagnosis of HCC, and its relationship with the biological characteristics of HCC. METHODS: Lens culinaris agglutinin-reactive AFP (AFP-L3) was measured in 65 patients with histologically proven HCC and 25 patients with CLD. All patients had serum AFP levels above 54 ng/mL. AFP-L3 levels were determined by lectin affinity electrophoresis coupled with antibody-affinity blotting. The diagnosis of HCC was confirmed histologically by ultrasound-guided biopsy. RESULTS: The mean value of AFP-L3 in the HCC patients was 49.6 +/- 21.6%, which was significantly higher (p<0.001) than that in the 25 CLD patients (10.7 +/- 4.3%). When the cutoff level for AFP-L3 was set at 15% (mean +/- SD), the sensitivity was 96.9%, the specificity 92.0% and the accuracy 95.5% in the 65 HCC patients. There was no clear correlation between serum AFP level and AFP-L3 percentage (r=0.16). There was no correlation between AFP-L3 and the maximum diameter of HCC nodules (r=0.05). However, the mean AFP-L3 value was higher in moderately or poorly differentiated HCC than in well differentiated tumors (p<0.001). CONCLUSIONS: AFP-L3 is potentially a clinically useful marker for the differentiation of increased AFP levels in hepatocellular carcinoma and chronic liver diseases. The AFP-L3 percentage is closely related to HCC differentiation. We consider the analysis of AFP-L3 a useful adjunct in the diagnosis of HCC.     

Prognostic Role of Pre-Treatment Serum AFP-L3% in Hepatocellular Carcinoma: Systematic Review and Meta-Analysis

Background

Serum lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3%) has been widely used for HCC diagnosis and follow-up surveillance as tumor serologic marker. However, the prognostic value of high pre-treatment serum AFP-L3% in patients with hepatocellular carcinoma (HCC) remains controversial. We therefore conduct a meta-analysis to assess the relationship between high pre-treatment serum AFP-L3% and clinical outcome of HCC.

Methods

Eligible studies were identified through systematic literature searches. A meta-analysis of fifteen studies (4,465 patients) was carried out to evaluate the association between high pre-treatment serum AFP-L3% and overall survival (OS) and disease-free survival (DFS) in HCC patients. Sensitivity and subgroup analyses were also conducted in this meta-analysis.

Results

Our analysis results showed that high pre-treatment serum AFP-L3% implied poor OS (HR: 1.65, 95%CI: 1.45–1.89 p<0.00001) and DFS (HR: 1.80, 95% CI: 1.49–2.17 p<0.00001) of HCC. Subgroup analysis revealed that there was association between pre-treatment serum AFP-L3% and endpoint (OS and DFS) in low AFP concentration HCC patients (HR: 1.96, 95% CI: 1.24–3.10, p = 0.004; HR: 2.53, 95% CI: 1.09–5.89, p = 0.03, respectively).

Conclusion

The current evidence suggests that high pre-treatment serum AFP-L3% levels indicated a poor prognosis for patients with HCC and AFP-L3% may have significant prognostic value in HCC patients with low AFP concentration.     

Primary isolation of Geotrichum klebahnii polygalacturonase by capturing with glass fiber microfilters

This study reports the use of borosilicate glass fiber microfilters (GFMs) for capturing and concentrating Geotrichum klebahnii polygalacturonase (GkPG) from the culture media through a simple coupled filtration–sorption process. GFM was characterized by scanning electron microscopy and intrusion mercury porosimetry. Adsorption of GkPG was almost instantaneous with maximum effectiveness at pH 3.0. Using the parameters of Langmuir model and a material balance, a filter bed consisting of a stack of GFM was designed for recovering GkPG from a certain volume of culture media. After adsorption and washing, the enzyme was efficiently eluted with a buffer solution at pH 5.0 containing 0.5 M NaCl. The filter bed was reused in several sorption cycles and enzyme recovery could be also done without prior cell removal with the benefits of using microfiltration for enzyme cell separation in a single step. Under the process conditions employed, GkPG recovery was about 80% with a concentration factor of about 4-fold.     

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.     

Two methods for the separation of human alpha-fetoprotein and albumin. (A) Affinity chromatography using Blue Sepharose CL-6B and (B) ampholyte displacement chromatography.

Two methods for the separation of α-fetoprotein (AFB) and albumin using (A) affinity chromatography on Blue Sepharose CL-6B and (B) ampholyte displacement chromatography are described. The heterogeneity of immunoreactive human AFP is demonstrated by ampholyte displacement chromatography; possibly seven variants of AFP can be distinguished by this method.     

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4 A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO₄ concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5–150 ng/ml concentration range [r²=0.999 for (+)- and (−)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (−)-tramadol, respectively.     

余甘原汁中多酚的大孔树脂提取分离及其抗氧化活性

为研究余甘子多酚的分离提取方法,本文以余甘原汁为原料,采用NKA-Ⅱ大孔吸附树脂,对上样量、洗脱剂、洗脱体积及洗脱速度等条件进行了考察,并对提取物的抗氧化活性进行了对比分析。通过试验确定了余甘原汁多酚的大孔树脂分离提取条件为:上样速度2BV/h,上样量0.8BV,洗脱剂为70%乙醇溶液,洗脱体积3BV,洗脱速度8BV/h,在此条件下NKA-Ⅱ大孔树脂对余甘原汁中多酚的吸附率可达到88.42%,洗脱率为90.93%,提取率为80.39%;对提取物的抗氧化活性分析显示,与分离前的余甘原汁干燥物相比,提取物的多酚和维生素C含量均提高了0.53倍,类超氧化物歧化酶活性(SODL)提高了1.4倍;铁离子还原能力(FRAP)明显高于分离前,对DPPH自由基、ABTS自由基及脂质氧化的半抑制浓度(IC50)均显著低于分离前的原汁干燥物,表明通过大孔吸附树脂分离,使余甘原汁中的多酚等抗氧化活性成分得到了有效的分离纯化。