Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×104 were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 105, and 3D5B7 reached 107. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.

Long-term exposure to microgravity is considered to cause liver lipid accumulation, thereby increasing the risk of non-alcoholic fatty liver disease (NAFLD) among astronauts. However, the reasons for this persistence of symptoms remain insufficiently investigated. In this study, we used tandem mass tag (TMT)-based quantitative proteomics techniques, as well as non-targeted metabolomics techniques based on liquid chromatography–tandem mass spectrometry (LC–MS/MS), to comprehensively analyse the relative expression levels of proteins and the abundance of metabolites associated with lipid accumulation in rat liver tissues under simulated microgravity conditions. The differential analysis revealed 63 proteins and 150 metabolites between the simulated microgravity group and the control group. By integrating differentially expressed proteins and metabolites and performing pathway enrichment analysis, we revealed the dysregulation of major metabolic pathways under simulated microgravity conditions, including the biosynthesis of unsaturated fatty acids, linoleic acid metabolism, steroid hormone biosynthesis and butanoate metabolism, indicating disrupted liver metabolism in rats due to weightlessness. Finally, we examined differentially expressed proteins associated with lipid metabolism in the liver of rats exposed to stimulated microgravity. These findings contribute to identifying the key molecules affected by microgravity and could guide the design of rational nutritional or pharmacological countermeasures for astronauts.

Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression plasmid, pET28a-ZIKV-Eecto of ZIKV Eecto, was constructed, transformed into Escherichia coli BL21 and induced by isopropyl β-D-thiogalactoside (IPTG). The recombinant Eecto protein was expressed in the form of inclusion bodies, and purified proteins were obtained through denaturation, renaturation and ultrafiltration. After three rounds of immunization with the Eecto protein, the serum of BALB/c mice was obtained and the titer of polyclonal antibodies in serum was determined. The reactivity of polyclonal antibodies was analyzed with Western blotting and immunofluorescence assay in HEK293T cells expressing the ZIKV prME. Spleen cells from mice with higher antibody titers were prepared and fused with SP2/0 myeloma cells. The hybridoma cells secreting antibodies were screened through the limited dilution method, and the ascites containing antibody were harvested for titer measurement and subclass analysis. The Eecto from the envelope proteins of Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Dengue virus (DENV1-4), and Tick borne encephalitis virus (TBEV) were coated and used to analyze the cross-reactivity of ZIKV monoclonal antibodies by ELISA. Further specificity analysis was conducted on antibodies with high titers and strong specificity. Results The plasmid pET28a-ZIKV-Eecto was successfully constructed. The purified Eecto protein was obtained with good immunogenicity. Four monoclonal antibodies were prepared and screened, namely 1D6, 4F11, 4H7, and 4F8. Among them, 1D6, 4H7, and 4F8 are IgG (K) type antibodies, and 4F11 is an IgM (K) antibody. The ascitic fluid titer of 1D6 was higher than 1:108. Antibodies 1D6 and 4H7 are ZIKV-specific and showed no cross-reactivity with other Flaviviruses. Conclusion The mice monoclonal antibodies against ZIKV-Eecto are produced successfully, which will provide experimental materials for the establishment of ZIKV detection methods and the study of its pathogenesis.

开放性骨折、关节炎(OA)和骨质疏松(OP)等疾病所导致的骨缺损修复术后抗感染已成为临床治疗上的一个难题.骨科围手术期大量应用抗生素易导致细菌耐药并加重患者的经济负担,因此开发非抗生素途径的抗菌骨修复材料是国内外众多科学家的追求与愿景.目前,非负载抗生素的骨修复材料主要有复合无机抗菌剂、复合有机抗菌剂和仿生纳米结构表面抗菌剂三类.现从抗菌作用、生物相容性和各自优缺点对上述三类骨修复材料进行综述,以期为开发新型具有抗菌和成骨作用的多功能骨修复材料提供新思路.

Bacterial infection caused by medical material is a common problem in the field of medicine. In this work, we aim to develop a novel antimicrobial bioplastic with potential medical value. A novel composite material composed of polyamino acid (PAA) and titanium dioxide/silver nanoparticles (TiO2/Ag NPs) was synthesized by in situ melting polycondensation with different TiO2/Ag loadings. The morphological, antibacterial, mechanical, thermal, and cytotoxicity properties of the PAA/TiO2/Ag composites were investigated. The results showed that uniform nanocomposites with amide bonds were synthesized, and the nanocomposites with 10% TiO2/Ag incorporation exhibited the best thermal and mechanical properties. Cytotoxicity assay showed that the composites significantly promoted the proliferation of MG-63 cells. The composites showed obvious inhibitory effect on both Escherichia coli and Staphylococcus aureus, and this effect was increased as the TiO2/Ag NPs doping was increased. These PAA/TiO2/Ag composites are suitable for antibacterial and high-performance biomaterials in the medical field.

Morphine is the most widely used analgesic for pain management worldwide. Abstinence of morphine could lead to neuropsychiatric symptoms, including depression. Gut microbiota is believed to contribute to the development of depression. However, the characteristics and potential role of gut microbiota in morphine abstinence-induced depression remain unclear. In the present study, we first established morphine abstinence-induced depressive behavior in mice. After dividing the mice into depressive and non-depressive groups, the gut microbiota of the mice was detected by 16S rRNA gene sequencing. The difference in the diversities and abundance of the gut microbiota were analyzed between groups. Then, the representative microbial markers that could distinguish each group were identified. In addition, gene function prediction of the operational taxonomic units (OTUs) with differential abundance between the depressive and non-depressive groups after morphine abstinence was conducted. Our results suggested that four weeks of abstinence from morphine did not change the richness of the gut microbiota. However, morphine abstinence influenced the gut microbial composition. Several specific genera of gut microbiota were identified as markers for each group. Interestingly, gene function prediction found that the fatty acid metabolism pathway was enriched in the OUTs in the depressive group compared with the non-depressive group after morphine abstinence. Our data suggested that gut microbiota dysbiosis was associated with morphine abstinence-induced depressive behavior, possibly by implicating the fatty acid metabolism pathway.

Our study aims to assess whether asiaticoside promotes the recovery of SINOFH by inhibiting bone marrow stem cells (BMSCs) differentiation into osteoclasts (OC). BMMs were induced to form OC system by dexamethasone in vitro and ELISA detected the expression of OC-related genes formation by asiaticoside. BMSCs were cultured followed by analysis of BMSCs morphology under microscope, gene expression by qRT-PCR. TRACP and c-Src level by western blot, RANKL, OPG and TRACP5b level by ELISA. Asiaticoside inhibited the expression of OC formation in SIONFH. The expression of OC-related genes increased with the induction days. With the increasing of induction days, asiaticoside level in culture fluid was decreased. While after asiaticoside interference, OCrelated genes and proteins levels were significantly down-regulated. Aasiaticoside can significantly increase the RANKL signaling protein expression. In conclusion, asiaticoside promotes the recovery of SINOFH by inhibiting BMSCs differentiation into OC.

目的 研究含三元基序家族蛋白35(tripartite motif-containing protein 35,TRIM35)在骨肉瘤组织中的表达、对人骨肉瘤细胞系143B、Saos-2侵袭和迁移的作用及机制.方法 收集30例正常人骨组织与64例骨肉瘤患者的肿瘤组织,应用免疫组化技术检测TRIM35的表达情况;通过转染TRIM35的siRNA与过表达质粒载体,结合qRT-PCR与Western blot技术检测转染效率;在划痕与Transwell实验中观察TRIM35对骨肉瘤细胞侵袭与迁移能力的影响;在骨肉瘤细胞中沉默或过表达TRIM35后,利用Western blot技术检测上皮-间质转化(Epithelial-mesenchymal transtion,EMT)进程标志蛋白.结果 免疫组化实验中,TRIM35在骨肉瘤组织中高表达,正常组织低表达.划痕实验结果表明,沉默TRIM35能够抑制143B的迁移能力,过表达后可加快Saos-2细胞的迁移;Tran-swell 实验显示过表达TRIM35能够促进Saos-2细胞的侵袭,沉默TRIM35能够抑制143B细胞的侵袭;Western blot结果显示沉默或过表达TRIM35能够引起EMT进程的标志性蛋白的变化.结论 TRIM35在骨肉瘤组织中高表达,通过介导EMT促进骨肉瘤细胞的侵袭及迁移.

概述近年来没食子酸对人宫颈癌HeL:a细胞凋亡机制的研究进展.没食子酸主要是通过调控P53信号通路、调节Caspase-3水平变化、促进ROS水平升高及调控ADAM17、EGFR、AKT/p-AKT和ERK/p-erk信号通路,实现对宫颈癌HeLa细胞凋亡的调控.

白芷是中医临床的常用药,可用于治疗风寒感冒、风疹湿痒、鼻渊等.研究表明,白芷有效成分主要为香豆素类和挥发油,对多种肿瘤细胞的抑制有显著作用.本文对近年来国内外相关文献进行归纳总结得出,白芷中有效成分主要通过抑制肿瘤细胞增殖、促进凋亡、细胞周期阻滞、促进自噬、抑制肿瘤细胞转移能力、提高耐药肿瘤细胞对化疗药物的敏感性等发挥抗肿瘤作用,此外,提出当前研究存在的不足之处,希望为将来的实验、临床研究提供参考.

目的:采用文献计量学的方法分析国内中药外用治疗阴道炎的研究现状,为后续中药相关研究提供理论参考.方法:在中国知网数据库中以"阴道炎""中药""带下病""中药熏洗""中医药疗法""Vaginitis"为关键词进行中英文扩展检索.时间:2006—2016年.结果:经筛选有476篇文献符合要求,平均发表文献47.6篇/年,其中核心期刊144篇,占总文献量的30.25％;基金资助率为7.98％.结论:国内对于中药外用治疗阴道炎的研究关注度在逐步增加,多倾向于临床应用研究,而且基金支持率较低,中医药治疗阴道炎的研究尚具有较大挖掘潜力.

Cancer-associated fibroblasts (CAFs) exhibit tumor-stimulating properties and are associated with poor survival in several types of cancer, making them potential therapeutic targets. The present study aimed to determine whether CAFs were associated with cell migration and invasion in lung squamous cell carcinoma (LUSC), as well as their association with microRNA-369 (miR-369) in these processes. Firstly, the changes of the malignant biological behavior were observed by treating the LUSC cells with the CAFs-derived extracellular vesicles (CAFs-EVs). Subsequently, the differentially expressed miRNAs in the cells treated with CAFs-EVs were analyzed by microarray analysis. Following inhibition of miR-369 expression in CAFs-EVs, LUSC cells were co-cultured, and the malignant biological behavior of the cells was re-examined. Then, through bioinformatics analysis and verification, the mRNA targets of miR-369 and the corresponding downstream signaling pathway were screened out. Finally, the effects of CAFs-EVs on the growth and metastasis of LUSC were demonstrated byin vivotumor formation and metastasis experiments. It was identified that miR-369 was expressed at a relatively high level in the CAFs-EVs. Neurofibromin-1 (NF1) was hypothesized as a direct target of miR-369 in LUSC. Also, the overexpression of miR-369 activated the mitogen-activated protein kinase signaling pathway by interacting with NF1, consequently potentiating LUSC cell growth. The present study provided novel insights into the action of miR-369 in CAFs-EVs in controlling LUSC cell migration, invasion and tumorigenesis, and identified miR-369 in CAFs-EVs as an important prognostic marker and therapeutic target.

目的 探究酸柏栀油软胶囊对糖尿病肾病(DN)大鼠的作用.方法 60只4周龄雄性SD大鼠随机分为正常对照组和模型组,运用高糖高脂饲料联合小剂量链脲佐菌素(STZ)制备DN模型.成模后大鼠随机分为模型对照组、阳性对照组、酸柏栀油软胶囊高、中、低剂量组.正常对照组和模型对照组给予等量生理盐水,阳性对照组给予盐酸二甲双胍、酸柏栀油软胶囊高、中、低剂量组分别给予不同剂量酸柏栀油软胶囊灌胃.干预8w末,检测大鼠血糖、血脂、血肌酐(SCr)、尿素氮(BUN)水平.结果 在干预末,各给药组体重均显著高于模型对照组(P＜0.05),酸柏栀油软胶囊高剂量组体重、血糖、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、总胆固醇(TC)、BUN与阳性对照组相比差异无统计学意义(P＞0.05),而与模型组相比差异有统计学意义(P＜0.05).结论 酸柏栀油软胶囊具有改善DN大鼠糖代谢、脂代谢紊乱的作用,能促进糖尿病大鼠肾功能的恢复.

生物碱是自然界中广泛存在的一类含氮碱性有机物,具有抗肿瘤、抗癌、降糖等多种生理活性.目前具有减轻或延缓实验动物糖尿病肾病肾脏的病变过程作用的生物碱主要有以下几大类:吡啶衍生物类、吡咯啶衍生物类、莨菪烷衍生物类、异喹啉衍生物类、吡嗪衍生物类、有机胺类和萜类生物碱等.常见的生物碱有小檗碱、川芎嗪、山莨菪碱、甜菜碱、苦参碱、秋水仙碱等.生物碱的肾脏保护作用机制多种多样,包括调节氧化应激、改善胰岛素抵抗、减少炎症因子的浸润、以及下调或增加相关因子的表达等.

目的 探讨坤泰合洗剂对小鼠白色念珠菌性阴道炎的治疗疗效及作用机制,为其临床治疗及应用提供参考依据.方法 在小鼠注射苯甲酸雌二醇油剂的基础上给予白色念珠菌混悬液,建立白色念珠菌阴道炎模型.将成模小鼠随机分为模型对照组、坤泰合洗剂高、中、低剂量组及氟康唑组,另取正常小鼠作为空白对照组.模型和空白对照组给予生理盐水,观察组给予不同剂量坤泰合洗剂和氟康唑治疗,连续治疗14 d.治疗结束后,取阴道分泌物进行真菌培养,摘眼球取血,分离血清,ELISA法测定血清中白细胞介素-10 (IL-10)和白细胞介素-8(IL-8)的水平,评价各组疗效.结果 阴道分泌物培养出的白色念珠菌计数比较显示,模型组与空白组比较明显升高(P＜0.05),坤泰合洗剂组菌落数均低于模型组,差异有统计学意义(P＜0.05).坤泰合洗剂高剂量组与氟康唑组对比差异无统计学意义(P＞0.05),坤泰合洗剂中剂量和低剂量组菌落数均高于氟康唑组,差异有统计学意义(均P＜0.05).IL-10水平比较显示:坤泰合洗剂高剂量组与氟康唑组对比差异无统计学意义(P＞0.05),而中低剂量组均高于氟康唑组,差异有统计学意义(均P＜0.05),坤泰合洗剂组均低于模型对照组,差异有统计学意义(均P＜0.05);IL-8水平比较显示:坤泰合洗剂组与氟康唑组相比均差异无统计学意义(均P＞0.05),而坤泰合洗剂高剂量组要低于模型对照组,差异有统计学意义(P＜0.05),且坤泰合洗剂高剂量组与空白对照组对比均差异无统计学意义(均P＞0.05).结论 坤泰合洗剂对白色念珠菌所致的小鼠阴道炎模型真菌感染具有抑制作用,可能是通过抑制炎症因子的产生来发挥作用,以高剂量作用最为明显.

目的 系统评价枣仁安神胶囊治疗失眠症的临床效果.方法 计算机检索PubMed、The Cochrane Library、EMbase、CNKI、WanFang Data、维普数据库中有关枣仁安神胶囊治疗失眠症的文献,以“枣仁安神胶囊、失眠症、不寐、Zaoren Anshen Capsule、Insomnia”为检索关键词,检索时限均从建库至2019年2月.根据设定的纳入与排除标准选择文献并提取相关资料,采用RevMan 5.3软件进行Meta分析.结果 共纳入研究16篇,包括792例治疗组和674例对照组人群.枣仁安神胶囊组的治疗总有效率高于对照组[OR=1.66,95％CI(1.27,2.16),P=0.0002],匹兹堡睡眠质量指数总分与对照组比较差异无统计学意义[WMD=-0.88,95％CI(-1.88,0.12),P=0.09],汉密尔顿焦虑量表总分低于对照组[WMD=-1.93,95％CI(-2.61,-1.25),P＜0.000 01].结论 枣仁安神胶囊治疗失眠症的效果良好,但需要开展更高质量的研究,以进一步佐证枣仁安神胶囊治疗失眠症的临床效果.

Previous studies have demonstrated that numerous tumor‑specific microRNAs (miRNAs) are upregulated or downregulated in hepatocellular carcinoma (HCC), and that their dysregulation is implicated in HCC occurrence and development. Therefore, investigation of crucial miRNAs involved in HCC oncogenesis and progression may provide novel insights into the therapy of patients with this malignant tumor. In the present study, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) assays were performed to detect tissue and cellular expression levels of miRNA‑663b (miR‑663b) in HCC. The effects of miR‑663b overexpression on the proliferation and invasion of HCC cells were examined using Cell Counting Kit‑8 and Transwell invasion assays, respectively. The direct target of miR‑663b in HCC cells was determined by bioinformatics analysis, luciferase reporter assay, RT‑qPCR and western blot analysis. It was observed that miR‑663b was expressed at low levels in HCC tissues and cell lines. miR‑663b upregulation suppressed the proliferative and invasive abilities of HCC cells. Additionally, Grb2‑associated binding 2 (GAB2) was regarded as a direct target gene of miR‑663b in HCC cells. Furthermore, GAB2 was overexpressed in HCC tissues, and overexpression of GAB2 was inversely correlated with levels of miR‑663b. GAB2 overexpression was able to rescue the suppressive effects of miR‑663b on HCC cells. These results demonstrated that this newly‑identified miR‑663b/GAB2 axis may be implicated in HCC occurrence and development.

肝癌是我国常见的恶性肿瘤之一,其恶性程度高、发病隐匿,缺乏特异性的早期诊断与治疗标志物.肝癌的发生发展与其代谢过程密切相关.与其他大多数肿瘤细胞类似,肝癌细胞的代谢过程较正常肝细胞发生了诸多显著改变,具体表现在糖酵解异常活跃、脂肪酸从头合成增强而氧化减弱、谷氨酰胺分解代谢加快等.这些代谢的异常改变为肝癌细胞提供了中间物质与能量,以满足其快速生长、增殖与转移所需.同时,参与肝癌细胞不同代谢过程中的多种酶与信号分子也在其中发挥着不可替代的作用,因此调控上述代谢过程中关键的代谢酶及通路被认为是肝癌诊断与治疗的重要靶点.近年来,关于肝癌细胞代谢重编程及其临床应用研究取得了令人瞩目的进展.同时,寻找新型标志物分子也一直是肝癌研究领域的重点.本文主要围绕肝癌细胞的糖、脂、氨基酸及核苷酸代谢过程中发生的异常改变及其背后的分子机理进行了综述,同时对肝癌诊断与治疗标志物分子的潜在临床应用价值进行总结与展望.

探讨坤泰洗剂体外抗阴道毛滴虫的作用.体外培养阴道毛滴虫,选择坤泰洗剂进行体外抗阴道毛滴虫实验,坤泰洗液浓度经倍比稀释分别为1:1、1:2、1:4、1:8、1:16,同时以甲硝唑、 洁尔阴洗液为阳性对照,RPMI-1640为阴性对照.实验表明坤泰洗剂在24 h时杀死阴道毛滴虫的最低稀释倍数为1:8,其杀虫效果与甲硝唑无统计学差异;但对阴道毛滴虫的抑制作用强于洁尔阴洗液,可见坤泰洗剂在体外具有显著的抑制阴道毛滴虫的作用.

目的 研究依鲁替尼对急性T淋巴细胞白血病细胞CCRF-CEM细胞株增殖抑制的作用.方法 使用不同浓度的依鲁替尼作用于CCRF-CEM(人急性T淋巴细胞白血病细胞)细胞株,分别作用24、48、72 h后,采用CCK8检测细胞抑制率,采用Annexin V-FITC/PI荧光双染色测定细胞的凋亡率,观察依鲁替尼对CCRF-CEM的凋亡影响.结果 不同浓度依鲁替尼作用CCRF-CEM细胞后,细胞增殖抑制率明显升高(P＜0.01)并且抑制率随药物浓度增高而增高.在同一浓度依鲁替尼作用细胞后,不同的作用时间,抑制率也有明显的变化(P＜0.01),呈时间依懒性,但在低浓度下,72h细胞的增殖抑制率比48h低.依鲁替尼作用细胞后,细胞凋亡率明显升高(P＜0.01),并且呈药物浓度依赖性.结论 依鲁替尼可以诱导T淋巴细胞CCRF-CEM凋亡,并且依赖药物浓度和作用时间.