大肠杆菌不耐热毒素纯化的改良法

本文介绍采用单克隆抗体亲和层析柱一步纯化大肠杆菌不耐热毒素(LT)的方法,此法提取的LT,活性较好,纯度较高。纯化后的LT经免疫家兔已获得LT的特异抗血清。     

乙型肝炎病毒基因调控的小鼠肝细胞质膜蛋白质变化

目的 通过对转HBV全基因小鼠肝细胞质膜蛋白质组的分析,研究病毒基因对宿主肝细胞质膜蛋白质表达的影响,探讨HBV感染机制. 方法 以HBV转基因C57小鼠和正常鼠肝为材料,通过蔗糖密度梯度离心结合抗体亲和法纯化肝细胞质膜.用Western blot法验证质膜的纯度和转基因鼠肝细胞质膜中HBV外膜蛋白质的表达.对纯化的质膜组分进行差异蛋白质组学分析. 结果 亲和纯化法较密度梯度离心法能进一步富集质膜近3倍,线粒体的污染减少近2倍;HBV外膜蛋白质只在转基因鼠肝细胞质膜中表达.蛋白质双向电泳分离得到500多个蛋白质点,分析得到26个具有2倍以上差异的蛋白质点,准确鉴定出7个蛋白质.结论 获得了好的质膜纯化方法;HBV基因调节了小鼠肝细胞质膜蛋白质的表达,鉴定得到了与HBV病毒感染相关蛋白质,为进一步探讨HBV感染机制打下了基础.     

免疫斑点技术检测LT和CT的研究

本文使用免疫斑点技术对大肠杆菌LT和霍乱弧菌CT进行了检测研究。并与ELISA双抗体夹心法作比较。其敏感性和特异性基本一致。对124份大肠杆菌,55份霍乱弧菌毒素提取液与65份家兔肠袢积液用两种方法同时检测,阳性率均无差异,在检测LT时两者符合率达94.3％。IDT操作简便,15小时即可获得明确结果,试剂用量少,肉眼观测结果,无需特殊仪器,故对LT和CT的检测具有较大的实用价值。     

纯化HBV促进人肝星状细胞系LX-2细胞Smad3表达

目的体外实验观察纯化HBV对人肝星状细胞系LX-2细胞表达Smad3的影响。方法采用蔗糖密度梯度离心法从HBV感染者的高滴度血清中进行病毒纯化浓缩,然后应用MTT法观察纯化HBV对LX-2细胞增殖率的影响;再用纯化HBV培养LX-2细胞24h和48h后,应用实时荧光定量PCR方法检测细胞Smad3mRNA表达的变化,以Western blot方法检测细胞Smad3蛋白表达的变化。结果经蔗糖密度梯度离心可得到高纯度的HBV颗粒,10^3～10^7copies／ml的HBV可逐渐增加LX-2细胞的增殖率,纯化HBV培养LX-2细胞24h和48h后,Smad3mRNA表达分别为阴性对照的1．7倍和4．3倍。纯化HBV培养LX-2细胞24h后阴性对照组和纯化HBV组HSmad3／β-actin吸光度比值分别为0．62、0．91;培养48h后阴性对照组和纯化HBV组Smad3／-actin吸光度比值分别为0．33、1．02。结论纯化HBV可促进人肝星状细胞系LX-2细胞Smad3表达。     

载氧血红蛋白纯化工艺研究

目的改善心脑血管缺氧载氧药物是一种创新药物。由于它半径比红细胞小400~1 000倍,易于通过毛细血管,给缺血组织及时供氧,迅速缓解或纠正缺氧状态,达到治疗抢救目的。血红蛋白的纯化工艺是载氧药物研制的重要工艺步骤。方法本研究建立了一套通过热敏法分离纯化人脐带血血红蛋白的工艺以及较为完善的纯化血红蛋白质量检测指标。结果与现有的纯化方式相比,热敏法操作简便,仪器设备造价低廉,纯化与病毒灭活同时进行,得到的纯化产品损失少,纯度高,各项理化指标达到国际水平。结论本工艺适用于规模制备纯化血红蛋白,为进一步研制治疗心脑血管缺氧载氧药物创造了有利条件。     

SV40LT基因重组腺病毒载体的包装、鉴定及用于转染日本血吸虫童虫细胞的研究

目的构建携带SV40LT基因的重组腺病毒表达载体,制备具有感染力的重组腺病毒,观察其转染日本血吸虫（Schistosoma japonicum,Sj）童虫细胞后的表达情况。方法采用体外连接法构建好的携带SV40LT基因的重组腺病毒质粒（AdHu5-SV40LT）转化Stbl2感受态菌,获得重组腺病毒质粒后,经Pac I酶切线性化后转染293A细胞,获得出重组腺病毒（AdHu5-SV40LT）。将重组腺病毒感染Sj童虫细胞,采用RT-PCR和免疫组织化学检测SV40LT基因的表达情况。结果重组腺病毒载体质粒可转染293A细胞并可在293A细胞内进行有效的复制;提取病毒DNA进行PCR检测证实含有SV40LT目的基因。以重组腺病毒能感染Sj童虫细胞,经RT-PCR和免疫组化检测有SV40LT基因在细胞中的表达。结论成功包装了具有感染能力的含SV40LT基因的重组腺病毒,感染Sj童虫细胞后目的基因有表达,为进一步探索日本血吸虫细胞永生化提供了实验依据。     

IL-4、IFN-γ在淋巴细胞性甲状腺炎合并乳头状癌中的表达及意义

目的探讨IL-4、IFN-γ在淋巴细胞性甲状腺炎（LT）合并甲状腺乳头状癌（PTC）发病中的作用。方法采用免疫组化SABC法检测32例LT、23例PTC及39例LT合并PTC患者IL-4、IFN-γ的表达。结果淋巴细胞中IL4在单纯LT表达强于LT合并PTC（P〈0．05）,IL-4、IFN-γ在LT合并PTC中的表达强于单纯PTC（P〈0．05）。IL-4、IFN-γ在LT、LT合并PTC及PTC中均有表达,病变周围相对正常滤泡上皮细胞无表达。在LT成分的滤泡上皮细胞中,单纯LTIL-4、IFN-1,表达强于LT合并PTC（P〈0,05）。LT合并PTC中IL-4、IFN-γ在癌变滤泡上皮细胞的表达强于LT成分中的滤泡上皮细胞（P〈0．05）。IL4在LT合并PTC有淋巴结转移中的表达高于无淋巴结转移（P〈0．05）。结论IL4、IFN-γ在LT合并PTC发病中起重要作用,癌变后的滤泡上皮分泌IL-4、IFN-γ增强;IL-4阳性预示淋巴结转移可能性大。     

纯化及非纯化法培养肝癌细胞对化疗药物敏感性的测定

采用纯化法及非纯化法分别培养肝癌细胞,并进行7种化疗药物的敏感性测定。结果显示,7种化疗药物对纯化法培养肝癌细胞的平均抑制率均高于非纯化法培养的肝癌细胞,但同一种化疗药物对非纯化和纯化法培养的肝癌细胞抑制率相比,P均〉0.05,两者呈正相关（r=0.715,P〈0.01）。认为非纯化与纯化法所培养的肝癌细胞对化疗药物的敏感性测定结果都能为肝癌患者实施个体化化疗提供实验依据。     

东方田鼠血清IgG抗体的快速纯化法

目的　探索快速、高效纯化东方田鼠血清IgG抗体的方法。　方法　采用G蛋白或A蛋白亲和层析法,对3种东方田鼠血清IgG抗体进行纯化,比较抗体纯度和回收率。　结果　获得的IgG抗体纯度和回收率均以G蛋白亲和层析法为高。纯化的抗体活性高,与酶标二抗的吸附力分别为非IgG洗脱物的8.5倍和未纯化血清IgG的3.1倍。　结论　G蛋白亲和层析法纯化东方田鼠血清IgG快速、活性高,具有实用价值。     

DNA聚合酶γ的提取、纯化和鉴定

目的: 提取并纯化人宫颈癌细胞(HeLa)的线粒体DNA聚合酶gamma(DNA polymerase gamma, Pol gamma), 鉴定其纯度和活性. 方法: 运用离子交换层析等方法提取纯化HeLa细胞的线粒体Pol gamma, 并用Bradford法检测蛋白浓度. 经SDS-PAGE检测蛋白纯度和相对分子量, Western blotting验证蛋白. 用alpha-(32)P-dTTP掺入法, 液体闪烁计数器进行放射性测量以确定Pol gamma的活性. 结果: 成功提取并纯化HeLa细胞的线粒体Pol gamma, 经SDS-PAGE鉴定, 有一个大约140 kDa的亚基单位, Western blotting证实为Pol gamma. 对其进行150倍纯化, 收得率为6%, 酶的总活力为4.81 U, 比活力为36.17 U/mg. 结论: HeLa细胞的线粒体Pol gamma通过离子交换层析法提取和纯化后活性较高, 可用于体外药物线粒体毒性的检测.     

Effects of either LT4 monotherapy or LT4/LT3 combined therapy in patients totally thyroidectomized for thyroid cancer.

After total thyroidectomy all thyroid cancer patients require lifelong treatment with thyroid hormones; the treatment of choice is synthetic levothyroxine (LT4). The question of whether these patients might benefit from the combined LT4 and liothyronine (LT3) treatment has been addressed with conflicting conclusions. The aim of the present study was to compare the effects of combined low LT4/LT3 molar ratio therapy versus LT4 monotherapy on various target organs and tissues in patients thyroidectomized for thyroid cancer. Urine collection (24 hour), a fasting blood sample for laboratory examinations, thyroid function clinical score, and cardiovascular, neurological, and neuropsychological evaluations were obtained. Clinical parameters and peripheral markers of thyroid function were measured during the two different treatment regimens in 20 patients. Mean serum aspartate aminotransferase, alanine aminotransferase, sex hormone binding globulin, and osteocalcin values were significantly higher during the combined treatment. No significant differences in the clinical score, the systolic and diastolic performance, and the neurological and neuropsychological evaluations were observed between the two treatment regimens. Moreover, no alteration due to subclinical hyperthyroidism or to the fluctuations in serum T3 concentrations during the combined therapy was observed. In conclusion, we found no evidence that combined therapy with a low LT4/LT3 molar ratio resulted in improved well-being and cognitive function or in increased thyroid hormone action on peripheral tissues in respect to LT4 monotherapy. Until future large, blind, randomized, and controlled trials prove otherwise, LT4 should remain the standard treatment for thyroid cancer patients.     

The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages

The heat-labile toxin (LT) is one of the major virulence factors of enterotoxigenic Escherichia coli (ETEC). We recently described that 20 polymorphic LT variants are present in ETEC strains isolated globally. Two of the variants, LT1 and LT2, are particularly common and we found that they were associated with clonal ETEC lineages that express the colonization factors (CFs), CFA/I, CS1+CS3, CS2+CS3, and CS5+CS6. ETEC expressing these CFs are frequently found among ETEC strains isolated from cases with diarrhea. ETEC expressing the colonization factors CS1+CS3, and CS2+CS3 are found in 2 discrete clonal lineages and express the LT1 variant and heat stable toxin (STh). Although they clearly are virulent they neither produce, nor secrete, high amounts of LT toxin. On the other hand ETEC strains expressing LT, STh, CFA/I and LT, STh, CS5+CS6, carry the LT2 variant and produce and secrete significantly more LT toxin. Despite differences in toxin production, LT1 and LT2 are found in ETEC lineages that have managed to spread globally confirming that these variants are important for ETEC virulence.     

Benefit of combined triiodothyronine (LT(3)) and thyroxine (LT(4)) treatment in athyreotic patients unresponsive to LT(4) alone

Despite some reports, the usefulness of levothyroxine (LT(4)) and levotriiodothyronine (LT(3)) combination therapy in hypothyroidism remains controversial. The objective of this paper is to study a benefit of additional LT(3) in athyreotic patients who failed to normalize TSH on LT(4) alone even with hyperthyroid serum T(4) values. In a survey of 200 athyreotic patients treated between 2006 and 2009, about 7% failed to normalize serum TSH levels following treatment with LT(4), though serum T(4) values in the hyperthyroid range were achieved. These patients (characterized by serum T(4)≥160?nmol/L and TSH≥5.0?mIU/L), were additionally treated with 10?μg b.?i.?d LT(3). LT(3) and LT(4) combination therapy resulted in decreased serum TSH levels into the normal range (12.8 vs. 1.22?mIU/L; p<0.01) and reduced LT(4) dose (153.3 vs. 117.5?μg; p<0.01) required for normalization of serum T(4) values (170.6 vs. 123.3?nmol/L; p<0.01). Serum T(3) values were higher (1.3 vs. 2.26?nmol/L; p<0.01) than those during monotherapy with LT(4). Our results indicate a subpopulation of athyreotic patients that could significantly benefit from combined LT(4)?+?LT(3) therapy in restoring normal TSH and thyroid hormone patterns. Further research should be undertaken to provide a genetic basis for these findings.     

LT4-monopreparation versus LT4-LT3-compound preparation in the treatment of diffuse endemic goitre

Fifty-four patients with diffuse non-toxic goitre were observed before and under therapy either with 125 micrograms LT4 (group A) or 75 micrograms LT4 + 15 micrograms LT3 (group B) in a prospective double blind study, using 1-, 3- and 6-months controls. Changes in goitre size have been estimated by ultrasonic scanning. Both treatment forms provided a significant reduction of goitre size, even after 1 month of therapy: a 20%-decrease in group A and 16% in group B. The 6-month reduction was about 30% in group A and 27% in group B. Between the 2 groups there were no differences in the reduction of volume. The suppression of the TSH-response to TRH was identical in both groups, too. In group A there was a predominant increase of the TT4- and FT4-serum levels, both reaching the hyperthyroid range. In group B there was a predominant increase of the TT3- and FT3-serum levels and a slight increase of the FT4-levels. The TT3- and FT3-serum levels also exceeded the upper normal range. As the blood samples were drawn about 2 h after medication, acute hormone resorption influenced these data. In the 3-month controls only there was a significant correlation between the reduction of the thyroid volume and the suppression of TSH-release. In the 6-month controls we found a weak correlation of the reduction of volume and the decrease of the pertechnetate uptake value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid identification of LT+ E. coli by means of PCR and its test comparisons

AIM To select a test method for specifical, sensitive and rapid identification of LT+ E. coli.METHODS Stool samples inoculated into LB solution were cultured for 4 hours at 35℃. 10 μ boiled culturesolution was taken to template. Two oligonucleotide primers were used in a polymerase chain reaction (PCR)procedure to amplify a highly conserved DNA sequence of the A subunit of the heat-labile enterotoxin.Detection of the 110 bp amplified product can be done by agarose gel electrophoresis. Thirty strains ofknown bacteria (LT+ E. coli (EC-129), ST+ E. coli (EC-130)and LT+ ST+ E. coli (EC-142), Salmonellatyphimurium , Salmonella typhi , Salmonella paratyphi A, Salmonella group C, Shigella sonnei , Enterobacteraerogenes, Alcaligenes sp, Providencia rettgeri, Proteus mirabilis, Morganella morganii, Pseudomouasaeruginosa, Aeromonas hydrophila, Klebsiella pneumoniae, Citrobacter diversus, Enterobacter cloacae, 12strains of E. coli isolated from bile samples ) and 108 diarrhea samples were detected. A total of 108 diarrheasamples were compared with LT probe hybridization, modified Eleck (M-Eleck) and ELISA simultaneously.RESULTS By PCR, of the 30 strains of bacteria, only LT+ E. coli and LT+ ST+ E. coli were positive; in40 of the 108 diarrhea samples, 20 were positive and in the other 68 samples from infants, only five werefound to be positive. Of the 25 positive samples by PCR, 23 were also found to be positive in the other 3tests; 1 was found to be positive by M-Eleck and ELISA. Of the 83 negative samples by PCR, the samenegative results were found by M-EIeck and ELISA, but 2 were found to be positive by LT probehybridization. The overall coincidence rate was about 95%. Analysis of correlation showed a significantdifference between PCR and other three tests (P<0.01) and analysis of difference showed no significantdifference (P>0.05) between them. In the detection of LT+ E. coli by means of PCR, the minimumnumber of target bacteria required was 50 CFU. The whole test was finished in 7 hours.CONCLUSION Detection of LT+ E. coli by PCR showed that the method is specific, sensitive and rapid.     

TNF and LT binding capacities in the plasma of arthritis patients: effect of etanercept treatment in juvenile idiopathic arthritis

BACKGROUND: Etanercept (Enbrel) induces a rapid and sustained decline in disease activity in the majority of patients with refractory juvenile idiopathic arthritis (JIA). For unknown reasons, however, a number of JIA patients fail to respond to this therapy. During this treatment neutralisation of tumour necrosis factor (TNF, previously termed TNF alpha) and lymphotoxin (LT, previously termed TNF beta) may be mediated by etanercept itself as well as by naturally occurring soluble TNF receptors. In light of this, it was of interest to study the total TNF neutralizing capacity in plasma before and during treatment with etanercept. RESULTS: In initial experiments plasma samples from healthy individuals were incubated with etanercept, and spiked with TNF or LT to a final concentration of 1000 pg/mL. Detection of TNF and LT by ELISA was found to be reduced by approximately 50% and 80% respectively, at a concentration of etanercept of 5-500 ng/mL, which is close to the pharmacological plasma concentrations. Plasma samples (n = 80) were then collected from 12 JIA patients (5 with pauciarticular, 5 with polyarticular and 2 with the systemic onset type) during treatment with etanercept (0.4 mg/kg twice weekly) for a period of 20.8 (15.6-23.9) months (median, range). The plasma samples were spiked with LT, and the inhibition of LT detection in ELISA was measured. In samples obtained 3 months after the start of etanercept, the inhibition of LT detection was augmented [72% (60-85)] compared with pre-treatment samples [16% (0.32)] (p = 0.0039). These findings were confirmed in binding assays using radiolabelled TNF. Among patients who responded insufficiently to therapy, reduced LT binding capacity, coinciding with flares of disease activity, was observed. CONCLUSION: We have developed an assay by which LT binding capacity, reflecting the level of free, pharmacologically active etanercept, may be monitored in the blood of patients treated with etanercept. This assay may prove to be useful in guiding dose adjustments in patients with an incomplete response to etanercept.     

端粒酶催化亚单位和猿猴病毒40大T抗原致人脐静脉内皮细胞永生化

目的用端粒酶催化亚单位(hTERT)和猿猴病毒40大T抗原(SV40大T)异位表达建立永生化人脐静脉内皮细胞系。方法构建含有hTERT和SV40大TcDNA片段的逆转录病毒载体,转染人原代脐静脉内皮细胞,连续传代培养。通过细胞免疫组织化学观察、Western blot分析hTERT和SV40大T抗原表达、PCR—ELISA检测端粒酶活性、ELISA分析内皮特异性E-选择素和RT—PCR测定内皮细胞脂肪酶进行细胞形态学和功能学鉴定。结果转染的人脐静脉内皮细胞为扁平多角形,呈单层铺路石状镶嵌排列。有Ⅷ因子、SV40大T、hTERT表达、E-选择素和内皮细胞脂肪酶表达阳性。转染细胞中端粒酶活性经测定在第12代时为0．36,在第50代时为0．38,而对照的原代细胞在第1代时为1．12,第3代时仅为0．06。结论导入外源性端粒酶催化亚单位和SV40大T抗原能使人脐静脉内皮细胞永生化,保持内皮细胞性状不改变。     

Ultrastructure of ovarian teratomas in LT mice.

Stem cells of these tumors formed nests consisting of undifferentiated embryonic cells in the center with more differentiated cells towards the periphery. A type viral particles were seen in the cisternae of rough endoplasmic reticulum. Ultrastructurally the stem cells of ovarian teratomas did not differ from stem cells of testicular or embryo-derived teratomas. They were however distinct from the cleavage stage embryonic cells and/or the unfertilized ovum, they stemmed from. They correspond both cytologically and developmentally to ectodermal embryonic cells from the egg-cylinder, the most advanced developmental stage of parthenotes observed previously in the ovary of LT mice.