THE DETECTION AND CLINICAL SIGNIFICANCE OF hEGF AND β＿2－MG IN BALF OF PATIENTS WITH LUNG CANCER

Human epidermal growth factor (hEGF) and β2-Microglohulin(β2-MG) in bronchoalveolar lavage fluid (BALF) and serum were detected in 74 patients with lung cancer and 76 patients with benign lung disease. The results showed that the positive rates or both hEGF (58.1%) and β2-MG (62. 2%) in BALF of the affected side of lung cancer were higher than those of either the healthy side of lung cancer or benign lung disease (P<0.05), and also higher than that in serum.The level of BALF-hEGF was highest in squamous cell carcinoma and adenocarcinoma, and it was higher in stages Ⅲ, Ⅳ than in stages Ⅰ, Ⅱ. The combinative detection or hEGF and β2-MG in BALF and serum will he valuable to early diagnosis of lung cancer.

镁合金浸提液对肠上皮细胞Bcl-2及Caspase-3的影响

目的研究不同浓度Mg-Ca合金浸提液对人结肠黏膜上皮细胞NCM460凋亡调控因子Bcl-2及Caspase-3表达的影响。方法将Mg-Ca合金制成不同浓度(100%、50%和10%)的浸提液,体外培养人结肠黏膜上皮细胞NCM460,以含10%胎牛血清的高糖DMEM培养基作为阴性对照。采用逆转录聚合酶链反应法及Western-bloting法检测不同浓度合金浸提液对NCM460细胞Bcl-2及Caspase-3 RNA及其表达的影响。结果在NCM460细胞分别经100%、50%、10%浸提液处理5 d后,Bcl-2基因在50%或100%浸提液的表达显著低于在对照组细胞的表达。而Caspase-3基因在100%、50%和10%浸提液处理过的细胞中的表达显著高于在对照组的表达。结论 Mg-Ca合金浸提液在一定范围内浓度较高时易诱导NCM460细胞Caspase-3的表达,抑制Bcl-2的表达,这可能与镁离子及钙离子浓度有关。

Differential effects of Ca^(2+) and Mg^(2+) on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonuclease activity in non apoptotic HL 60 promyelocytic leukemia cell nuclei cleaved the chromatin of the autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL 60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca 2+ , but not by exogenous Mg 2+ . In Ca 2+ /Mg 2+ free nuclei digestion buffer, addition of Ca 2+ （1\10 mmol/L） induced endonuclease activity in the isolated nuclei, while addition of Mg 2+ had no effect. In the presence of Ca 2+ （0.1 mmol/L）, endonuclease activity was enhanced by exogenous Mg 2+ （0.1\10 mmol/L）. These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL 60 cells during apoptosis is activated by Ca 2+ and further modulated by Mg 2+ in the presence of Ca 2+ .

色素调节剂对体外培养的人表皮黑素细胞的影响

目的观察几种色素调节剂对体外培养的人表皮黑素细胞形态及功能的影响。方法体外培养正常表皮黑素细胞,分别加入合适浓度的表没食子儿茶素没食子酸酯(EGCG)、氢醌(HQ)、烟酰胺(NAA)、α-黑素细胞刺激素(α-MSH)和维生素C磷酸酯镁(AP-Mg)干预细胞72h,测定酪氨酸酶活性和黑素生成量。用黑素体相关蛋白单克隆抗体(NKI-beteb)染色鉴定黑素细胞,观察并摄片。结果3mmol/LEGCG,0.5mmol/L HQ,6mmol/L AP-Mg与黑素细胞共同孵育72h后,其细胞酪氨酸酶活性及黑素生成受到抑制,但只有EGCG组细胞形态发生变化;50nmol/Lα-MSH干预后,细胞酪氨酸酶活性及黑素生成显著增加,形态改变较显著。lmg/mL NAA组细胞酪氨酸酶活性无改变,而黑素生成减少,细胞异形化。结论EGCG,HQ,NAA,AP-Mg和α-MSH对黑素细胞都有显著的影响,虽然作用于黑素细胞的机制有所不同,但也有一些相似的地方,其具体机理有待于进一步研究。

Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes

AIM： To establish a simplified method for generating peptide-major histocompatibility complex （MHC） class I tetramers.METHODS： cDNAs encoding the extracellular domain of human lymphocyte antigen （HLA）-A＊0201 heavy chain （A2） and β2-microglobulin （132m） from total RNA extracted from leukocytes of HLA-A2＋ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and 132m proteins were expressed in ~/a oo/i^uain BL21（DE3） and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain 132m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme （BirA） and purified by low pressure anion exchange chromatography on a Q-Sepharose （fast flow） column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4：1. Flow cytometry was used to confirm the expected tetramer staining of CD8^＋ T cells.RESULTS： Recombinant genes for HLA-A＊0201 heavy chain （A2） fused to a BirA substrate peptide （A2-BSP） and mature β2m from HLA-A2＋ donor leukocytes were successfully doned and highly expressed in E. coli, Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of 132m and peptides loaded with either human cytomegalovirus pp65495-503 peptide （NLVPMVATV,NLV; designated as A2-NLV） or influenza virus matrix protein Mp58-66 peptide （GILGFVFTL, GIL; designated as A2-GIL）. Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplicationas revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8^＋ T cells from a HLA-A2^＋ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION： The procedure is simple and efficient for generating peptide-MHC tetramers.

人β2微球蛋白在不同原核表达载体中的表达及活性比较

目的在不同原核表达载体中表达人β2微球蛋白(β2-microglobulin,β2MG),并比较两种蛋白的活性。方法将人β2MG基因分别插入原核表达载体pET-28a(+)和pET-32a(+)中,构建重组表达质粒pET-28a(+)-β2MG和pET-32a(+)-β2MG,转化E. coli BL21(DE3),IPTG诱导表达。获得的两种β2MG融合蛋白菌液经离心、超声破碎处理后,分别取上清和沉淀进行SDS-PAGE分析,利用Ni柱亲和层析法纯化目的蛋白,间接ELISA法检测两种融合蛋白的免疫学活性。结果不同表达载体所表达的β2MG溶解性不同,pET-28a(+)-β2MG于37℃诱导表达的His-β2MG以包涵体形式表达,pET-32a(+)-β2MG于25℃诱导表达的Trx-His-β2MG以可溶性蛋白形式表达。纯化的两种融合蛋白纯度均可达95%以上,Trx-His-β2MG的免疫学活性显著高于His-β2MG,且优于对标抗原。结论人β2MG在不同原核表达载体中的表达位置和活性均不同,可溶性表达的Trx-His-β2MG具有较高的免疫学活性,应用价值较大,为免疫诊断试剂的开发奠定了基础。

姜黄素对人成骨肉瘤MG-63细胞增殖抑制和凋亡相关基因表达的影响

目的:探讨姜黄素对人成骨肉瘤MG-63细胞增殖抑制及凋亡相关基因表达的影响。方法:用姜黄素处理MG-63细胞,细胞计数法检测细胞增殖抑制的效果;荧光染色观察细胞的凋亡;流式细胞仪(FCM)进行细胞周期时相分析;免疫细胞化学法和Western blotting免疫法检测细胞凋亡相关基因的表达水平。结果:随着姜黄素浓度的增加及其作用时间延长,对细胞的增殖抑制作用增强,最高抑制率可达89.07%;光镜下可观察到细胞发生染色质浓缩,细胞核凝聚和碎裂等典型的凋亡形态学改变;FCM检测结果显示细胞经姜黄素处理后出现明显的凋亡峰;免疫反应结果显示,凋亡相关基因bcl-2和P53表达水平降低,而bax和Fas表达水平升高。结论:姜黄素能显著抑制MG-63细胞增殖并可有效诱导其凋亡,其作用机制可能与bcl-2和bax二者的比值发生变化从而接受了凋亡刺激信号有关。