清开灵注射液对登革病毒Ⅱ型的抗病毒作用研究

目的 检测清开灵注射液在细胞模型上抗登革病毒Ⅱ型(DENV-Ⅱ型)作用.方法 本实验以白纹伊蚊C6/36细胞为宿主细胞,阿昔洛韦(ACV)为阳性对照药物,通过观察细胞病变效应(CPE)和改良MTT法检测细胞存活率来测定药物的细胞毒性、药物对DENV-Ⅱ的直接灭活作用、以及药物抗DENV-Ⅱ对细胞的吸附和对DENV-Ⅱ在细胞内复制的抑制作用.结果 该药在体外对DENV-Ⅱ无直接灭活作用,也不能阻止其对细胞的吸附,但对病毒在细胞内的增殖有明显的抑制作用,且呈一定的剂量效应依赖性.结论 该药在体外有一定的抗DENV-Ⅱ感染作用.     

N400 as an index of uncontrolled categorization processing in brand extension

Chrysotoxine is a naturally occurring bibenzyl compound found in medicinal Dendrobium species. We previously reported that chrysotoxine structure-specifically suppressed 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death. Whether chrysotoxine and other structurally similar bibenzyl compounds could also inhibit the neurotoxicity of 1-methyl-4-phenyl pyridinium (MPP(+)) and rotenone has not been investigated. We showed herein that chrysotoxine inhibited MPP(+), but not rotenone, induced dopaminergic cell death in SH-SY5Y cells. The overproduction of reactive oxygen species (ROS), mitochondrial dysfunction as indexed by the decrease in membrane potential, increase in calcium concentration and NF-κB activation triggered by MPP(+) were blocked by chrysotoxine pretreatment. The imbalance between the pro-apoptotic signals (Bax, caspase-3, ERK and p38 MAPK) and the pro-survival signals (Akt/PI3K/GSK-3β) induced by MPP(+) was partially or totally rectified by chrysotoxine. The results indicated that ROS inhibition, mitochondria protection, NF-κB modulation and regulation of multiple signals determining cell survival and cell death were involved in the protective effects of chrysotoxine against MPP(+) toxicity in SH-SY5Y cells. Given the different toxic profiles of 6-OHDA and MPP(+) as compared to rotenone, our results also indicated that DAT inhibition may partially account for the neuroprotective effects of chrysotoxine.     

羧基荧光素乙酰乙酸与四甲基偶氮唑盐法检测人脐血间充质干细胞刺激脾淋巴细胞增殖作用的比较

目的 比较羧基荧光素乙酰乙酸(CFSE)与四甲基偶氮唑盐(MTT)比色法检测人脐血间充质干细胞(CB-MSCs)和神经分化的CB-MSCs对大鼠脾淋巴细胞(LCs)刺激作用的敏感程度. 方法 分别制备刺激细胞和LCs,将刺激细胞分为4组:1. CB-MSCs组;2. Dif-CB-HSCs组:脐血间充质干细胞培养7d后维甲酸(RA)处理4d诱导其神经分化细胞;3. SH-SY5Y组:人源SH-SY5Y细胞(人神经母细胞瘤细胞系)作为阳性对照组;4. Auto-LC组:自体大鼠LCs作为阴性对照组.分别将上述各组刺激细胞与LCs进行混合培养,分别通过酶标仪(MTT法),流式细胞术(CFSE法)检测LCs增殖情况( n =3). 结果 CFSE法和MTT法检测结果 均显示,CB-MSCs组和Dif-CB-MSCs组刺激LCs增殖明显弱于阳性对照组(SH-SY5Y细胞组),但CFSE法检测结果 显示,CB-MSCs组和Dif-CB-MSCs组细胞刺激LCs增殖百分率高于阴性对照Auto-LC组,MTT法检测结果 显示,CB-MSCs组和Dif-CB-MSCs组的吸光度( A )值稍低于阴性对照Auto-LC组. 结论 CFSE法比MTT法能更加客观地反映淋巴细胞增殖情况,在实际应用中更具有优势.     

利巴韦林对肠道病毒体外抑制作用的研究

目的研究抗病毒药物利巴韦林体外抗肠道病毒（EV）的效果。方法采用细胞病变效应（CPE）法和MTT分析法,观察利巴韦林对肠道病毒（EV71、CAV16、CBV3、ECH011、EV84）的抑制作用。结果利巴韦林对Vero细胞的半数毒性浓度（TC50）为2．09mg／mL,0．2mg／mL浓度的利巴韦林对5种肠道病毒均有抑制作用,对CAV16、EV71、ECHO11、EV84、CBV3的抑制率分别为13％、27％、36％、23％、58％。对EV84、EV71病毒,利巴韦林浓度为0．1mg／mL时其抑制率为16．5％、29．5％;对CBV3病毒,利巴韦林抗病毒作用与其剂量呈正相关,半数有效浓度（IC50）为0．125mg／mL,治疗指数（TI）为16．72。结论利巴韦林在体外对肠道病毒具有抑制作用,对不同的肠道病毒其抑制效率不同,对CVB3的抑制率较高。     

Human apolipoprotein A-I Gly26Arg stimulation of inflammatory responses via NF-kB activation: Potential roles in amyloidosis?

The cascade of molecular events leading to Human apolipoprotein A–I (apoA–I) amyloidosis is not completely understood, not even the pathways that determine clinical manifestations associated to systemic protein deposition in organs such as liver, kidney and heart. About twenty natural variants of apoA–I were described as inducing amyloidosis, but the mechanisms driving their aggregation and deposition are still unclear. We previously identified that the mutant Gly26Arg but not Lys107-0 induced the release of cytokines and reactive oxygen species from cultured RAW 264.7 murine macrophages, suggesting that part of the pathogenic pathway could elicit of an inflammatory signal. In this work we gained deep insight into this mechanism and determined that Gly26Arg induced a specific pro-inflammatory cascade involving activation of NF-κB and its translocation into the nucleus. These findings suggest that some but not all apoA–I natural variants might promote a pro-oxidant microenvironment which could in turn result in oxidative processing of the variants into a misfolded conformation.     

Study on In Vitro Anti-Tumor Activity of Bidens Bipinnata L. Extract

We studied the in vitro anti-tumor activity of Bidens Bipinnata L. extract. MTT assay was used to investigate the inhibitory effect of different concentrations of the extracts on human hepatocellular carcinoma (HepG2) cell lines and human cervical carcinoma (Hela) cell lines, and the IC₅₀ values were calculated. The Bidens Bipinnata L. extract had different degrees of inhibitory effects on these two cells, and when exposure time was 48 h, the inhibition rate reached its peak, with IC₅₀ values of 14.80 µg/mL and 13.50 µg/mL respectively. The Bidens Bipinnata L. extract had a good inhibitory effect on human HepG2 cell lines and Hela cell lines, and thus has certain development prospects.     

A Study on the Inhibitory Effect of Matrine on Gastric Cancer SGC-7901 Cells

The objective of this paper was to investigate the antitumour mechanism of action of matrine by studying its inhibitory effect on gastric cancer SGC-7901 cells. SGC-7901 cells were chosen, and cell-killing capacity of matrine on gastric cancer SGC-7901 cells was determined using MTT assay and single PI staining assay. The results showed that matrine had an inhibitory effect on gastric cancer SGC-7901 cells, which was somewhat dose-dependent. The study concluded that matrine has a significant in-vitro inhibitory effect on SGC-7901 tumour cells, influences cell cycle of SGC-7901 cells, and induces their apoptosis.     

Evaluation of antigenicity and cell mediated immunity of Hepatitis E virus patients: Using non radioactive MTT assay

Hepatitis E virus (HEV) is an important cause of hepatitis in developing nations. Disease spans from asymptomatic infection to acute viral hepatitis (AVH) and acute liver failure (ALF). Cell-mediated immunity (CMI) is less studied. Studies document CMI in HEV patients using [³H]-thymidine incorporation (radioactive in nature). The aim of this study was to evaluate the antigenicity of recombinant HEV ORF 2 peptide (452-617 a.a) (pORF2) by non-radioactive MTT assay and detecting the proliferation indices of primary PBMC culture. A total of 27 laboratory confirmed HEV patients (16 AVH and 11 ALF) and 20 apparently healthy individuals (HC) were included. PBMCs were isolated, plated and stimulated with pORF2. After an incubation of 4 days, cells were looked for blastogenic transformation and subjected to MTT assay. PI of AVH, ALF and healthy controls were found to be 3.249 ± 0.219, 1.748 ± 0.076 and 0.226 ± 0.017, respectively. PI of AVH Vs HC, ALF Vs HC and AVH Vs ALF were found to be significantly higher (P < 0.0001). This study demonstrates MTT to be an adaptable technique to evaluate CMI in HEV patients. Recombinant pORF2 was found to be antigenic in nature and PBMCs from AVH patients were immunologically more reactive than ALF patients.     

Esculetin modulates cytotoxicity induced by oxidants in NB4 human leukemia cells

Esculetin is a polyphenolic compound with cytoprotective properties. We previously demonstrated the induction of apoptosis by esculetin in NB4 human leukemia cells, as a model, by a mechanism not well understood. To analyse the antioxidant activity of esculetin on apoptosis, we have studied the influence of co-treatments of esculetin at a concentration of 100 μM with exogenous ROS donors, namely tert-butyl-hydroperoxide and hydrogen peroxide, on NB4 cells. Esculetin (100 μM) exerts a protective effect on cell viability and death necrosis or late apoptosis caused by the oxidant t-BHP whereas it potentiates decrease of cell viability and cell death caused by H₂O₂. In the first case, the O₂^? scavenging activity of esculetin (100 μM) could be implicated. In the last one, cytotoxicity by apoptosis induction seems to be related to the increase in O₂^?, among other possible mechanisms. These results contribute to the study of the antitumor properties of esculetin by regulation of redox balance in leukemia cells.     

SUMO表达系统高效、可溶性表达重组鼠源IL-1β

目的:从LPS刺激过的小鼠胸腺细胞中克隆IL-1β基因,通过原核表达,获得具有生物活性的可溶性鼠源IL-1β蛋白,为深入研究和利用IL-1β基因奠定基础。方法:提取LPS刺激的小鼠胸腺细胞总RNA,反转录成cDNA,以此为模板,根据GenBank报道的mIL-1β序列设计引物,进行巢式PCR,得到成熟mIL-1β的编码序列基因,并插入到原核表达载体pHisSUMO ex-press中SUMO标签的下游,构建重组表达载体pHisSUMO express-mIL-1β。将该载体转化大肠杆菌Rosetta(DE3),IPTG诱导表达mIL-1β/SUMO融合蛋白,Ni-NTA Agarose纯化后,表达产物经SDS-PAGE和Western blot进行鉴定,用SUMO protease-1切去SUMO标签,经纯化并切去融合标签后获得成熟mIL-1β蛋白。用MTT方法检测目的蛋白对L929细胞的生物学活性。结果:DNA测序证明所克隆基因序列与GenBank报道的完全一致,SDS-PAGE分析表明融合蛋白的相对分子质量为37kD,切割后的成熟蛋白为17kD,与理论值相符。且蛋白表达量高,主要以可溶形式存在。Western blot证实该蛋白为mIL-1β。成熟蛋白纯化产物纯度超过95%以上,通过MTT的方法检测证明其具有使L929细胞增殖的作用,从而说明其具有生物学活性。结论:利用大肠杆菌表达系统可高效可溶性表达高纯度的具有生物活性的mIL-1β蛋白。