Percoll密度梯度离心法分离大鼠肺Ⅱ型上皮细胞

目的建立大鼠肺泡Ⅱ型上皮细胞（AT-Ⅱ）的改良体外原代培养法。方法采用循环冲洗、支气管灌洗、胰蛋白酶消化、滤网过滤并用轻比重及重比重PercolL密度梯度离心,分离AT-Ⅱ。原代培养后通过碱性磷酸酶（alkaline phosphatase,AKP）法和电镜鉴定AT-Ⅱ。结果经AKP染色和电镜鉴定所得的AT-Ⅱ纯度为（80．2±6．3）％,每只大鼠可分离出1×10^7AT-Ⅱ。结论采用Percoll密度梯度离心法能分离出较高纯度的AT-Ⅱ。     

AnnexinⅡ和AnnexinⅣ在大肠癌中的表达及其临床意义

目的：探讨AnnexinⅡ和AnnexinⅣ在大肠癌中的表达及其临床意义。方法：用免疫组化SP法分别检测AnnexinⅡ和AnnexinⅣ在60例大肠癌组织中的表达。结果：（1）AnnexinⅡ和AnnexinⅣ在大肠癌组织中的阳性表达率分别为65％（39／60）和60％（36／60）;（2）AnnexinⅡ和AnnexinⅣ阳性和阴性病例的平均生存期、五年生存率和中位生存期比较,差异具有显著性（P〈0．05）：（3）大肠癌组织中AnnexinⅡ和AnnexinⅣ的表达一致率为68．33％（41／60）,存在显著相关性（P〈0．01）;（4）大肠癌不同临床时期（Ⅱ、Ⅲ和Ⅳ）AnnexinⅡ和AnnexinⅣ阳性表达比较,差异具有显著性（P〈0．01）。结论：AnrlexinⅡ和AnnexinⅣ与大肠癌的临床分期和预后有关。     

ROCKⅠ／Ⅱ基因下调对血管平滑肌细胞迁移及增殖的影响

目的：探讨ROCK亚型ROCKⅠ和ROCKⅡ对血管平滑肌细胞（A7r5）迁移及增殖的影响。方法：利用Western blot技术检测ROCKⅠ和ROCKⅡ蛋白在A7r5细胞中的表达水平;利用siRNA技术使ROCKⅠ和ROCKⅡ基因表达分别下调,并检测基因下调后蛋白表达水平;利用Boyden小室法,观察ROCKⅠ和ROCKⅡ基因下调后及ROCK特异抑制剂Y-27632对PDGF诱导的A7r5细胞迁移的影响;使用MTT法检测ROCKⅠ和ROCKⅡ基因下调后对A7r5细胞生长曲线的影响。结果：ROCKⅠ和ROCKⅡ在A7r5细胞中的蛋白表达水平不同,ROCKⅡ较ROCKⅠ的表达水平高4倍;通过对A7r5细胞进行ROCKⅠ和ROCKⅡsiRNA转染,使二者蛋白表达水平分别下调83.4％和94.7％;基因表达下调后,ROCKⅠ明显抑制了PDGF诱导的A7r5细胞的迁移,而ROCKⅡ无明显影响,Y-27632也抑制了A7r5细胞的迁移;ROCKⅠ和ROCKⅡ基因下调后对A7r5细胞生长曲线的影响无明显差别。结论：ROCKⅠ在血管平滑肌细胞迁移过程中起主导作用,ROCKⅠ和ROCKⅡ对血管平滑肌细胞的增殖作用无明显差异。     

应用APACHEⅡ在百草枯中毒评估的研究

目的应用急性生理学及慢性健康状况评分Ⅱ(acute physiology and chronic health evaluationⅡ,APACHEⅡ)评估百草枯农药中毒患者的病情危重程度并判断其预后。方法收集2005~2010年入住我院急诊重症监护室(EICU)百草枯农药中毒资料完整患者28例,追踪随访患者将其分为生存组和死亡组,分别计算各自APACHEⅡ评分及死亡风险系数,并进行统计分析。结果 28例患者APACHEⅡ评分为4~32分,平均(15.26±5.77)分。生存组8例,APACHEⅡ评分为(8.86±4.14)分,死亡风险系数为0.15±0.05;死亡组20例,APACHEⅡ评分为(17.5±4.44)分,死亡风险系数为0.37±0.18。两组APACHEⅡ评分以及死亡风险系数的差异有统计学意义(前者t=4.5,P0.01;后者t=4.7,P0.01)。结论 APACHEⅡ评分系统可应用于百草枯农药中毒患者危重程度及预后的评估。     

应用逆转录聚合酶链反应法扩增类胰岛素生长因子Ⅱ基因

利用酸性异硫氰酸胍-酚-氯仿一步法从人胚胎组织中提取总RNA,经Oligo(dT)纤维柱分离纯化出mRNA。用逆转录与聚合酶链反应相结合的RT-PCR法,扩增出人类胰岛素生长因子Ⅱ(IGFⅡ)的cDNA片段,在限制性内切酶Sma Ⅰ存在的连接体系中,将扩增出的cDNA片段克隆进PUC12的Sma Ⅰ位点处。经限制性内切酶EcoR Ⅰ、Sal Ⅰ、Eco47Ⅲ酶切鉴定其方向。以重组质粒的双链DNA为模板,用末端终止法测定其全部核苷酸顺序,证实其核苷酸编码的IGFⅡ在氨基酸顺序上与文献报道的相同。     

苦荞凝集素对结肠癌细胞增殖的抑制作用

采用醋酸铵缓冲液抽提、硫酸铵分级沉淀、阴离子交换和分子排阻层析等方法,首次从苦荞麦中提取出一种苦荞凝集素(tatary buckwheat lectin,TBL). MTT检测发现,TBL对3种结肠癌细胞 DLD-1、HCT116及SW480的增殖有显著的抑制作用,且呈剂量依赖效应,而对正常细胞及其它类型癌细胞的增殖无明显影响. 采用兔网织红细胞裂解系统和荧光素酶检测系统检测TBL对蛋白质合成的影响,结果表明,随着TBL浓度的增大,蛋白质翻译抑制作用逐渐增强,当TBL浓度为40 μg/mL时荧光素酶活性下降至50%. 另外,TBL与兔网织红细胞裂解系统作用后,核糖体RNA出现新的片段,表明TBL具有N-糖苷酶的活性. 将HCT116细胞总RNA与TBL体外作用后,发现二者存在明显的相互作用,核糖体RNA被降解. qRT-PCR检测显示,TBL明显下调HCT116细胞中多种microRNAs (miRNAs) 表达. 综合以上实验得出,TBL是一种Ⅱ型核糖体失活蛋白(type-Ⅱ ribosome-inactivating protein, Ⅱ-RIP)类的植物凝集素,具有N-糖苷酶活性,可下调肠癌细胞中多种miRNAs的表达,抑制结肠癌细胞增殖.     

Effects of Doubled CO2 on Contents of Photosynthetic and on Kinetic Parameters of Fluorescence Induction in Different Genotypes of Soybean

Effects of doubled C02 on photosynthetic characteristics of soybean ( Glycine max L. ) Bragg (wild type) and its different monogene mutation strains--Nts 382 (supemodulation mutant) and Nod 49 (non-nodulation mutant) were studied. The experimental results showed that the contents of chlorophyll and carotenoid of Bragg, Nts 382 and Nod 49 were increased under doubled CO2, respectively, although to different extent. The determination results of fluorescence induction kinetic parameters showed that PS Ⅱ activity, the efficiency of primary conversion of light energy of PS Ⅱ and the efficiency of potential photosynthetic quantum conversion of a leaf from Bragg and its mutants were raised in doubled CO2. Fluorescence photochemical quenching coefficient and the overall photochemical quantum yield of PS Ⅱ were raised and non-photochemical quenching coefficient was reduced with CO2 enrichment; such changes were bigger in Nts 382 than those in Bragg and Ned 49. It might be that atmospheric N2 was more effectively utilized by Nts 382 than by Bragg and Ned 49.     

Effect of Doubled CO2 Concentration on Leaf Chlorophyll-protein Complexes in Several Plants

The effect of doubled CO2 on the chlorophyll-protein complexes of the leaves of soybean ( Glycine max L., Ca plants), cucumber ( Cucumis sativus L., C3 plant), millet ( Setaria italica (L.) Beauv., not a very typical C4 plant) and corn (Zea mays L. ,C4 plant) was studied. Experi- mental plants were pot-cultured in polyethylene membrane (or glass) open top cultured chambers. After sowing, C02 was kept immediately either at ambient ( (350 ± 10) x 10-6) concentration for the control or at doubled CO2 ((700 ± 10) x 10-6) concentration for the treatment chambers. The chlorophyll-protein complexes of the thylakoid membrane of the plants were resolved by disk SDS- PAGE. The results showed that after doubled CO2 treatment,either in the soybean and cucmnber,or in the millet, the quantity of polymer state of PS Ⅱ light-harvesting chlorophyll a/b-protein complex (LHC Ⅱ ) had increased as the monomer state of LHC Ⅱ decreased. But such response to doubled CO2 was not found in corn, the C4 plant. The change of the state of LHC Ⅱ in soybean etc. might be an adaptive effect of plant photosynthetic mechanism to the long term elevated CO2. Thus it could increase the efficiency of the absorption, transfer and conversion of light energy in plant photosynthesis, and support the high efficiency of photosynthetic carbon assimilation.     

Light-Induced Damage of Pheophytin a Molecule in the Isolated Photosystem Ⅱ Reaction Center D1/D2/Cyt b559 Complex

Photodamage of some pigments in the isolated photosystem Ⅱ (PS Ⅱ ) reaction center D1/D2/Cyt b559 complex from spinach has been investigated by means of high performance liquid chromatography. The light-induced damage of pheophytin a (pheo a) in the complex was observed for the first time. The content of pheo a decreased about 47 % by illumination, suggesting only one of the two pheo a molecules in the PS Ⅱ reaction center complex was damaged. No damage of β-carotene was found.