Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

基于COⅠ基因片段的瓜实蝇遗传多样性分析

瓜实蝇是一种重要入侵害虫,广泛分布于热带和部分亚热带国家和地区,寄主广,危害性大。本研究对中国6个主要分布省份、19个地区瓜实蝇线粒体COⅠ基因中的部分序列进行了测定,分析了种群的遗传学关系并建立系统进化树。结果表明,在获得的640 bp的DNA序列中,可变位点16个,其中简约性信息位点7个,单变异位点9个。Fst值为0.107 88,Nm值为2.07,遗传距离为0~0.001 8。单倍型多样性云南、广西最高。聚类分析发现国内各地理种群间差异不显著。中国19个种群的瓜实蝇存在一定的遗传分化,但分化程度很低。     

马兜铃酸Ⅰ对大鼠五脏的毒性及功能影响

【目的】探究马兜铃酸Ⅰ（aristolochic acid Ⅰ，AA-Ⅰ）对大鼠五脏（心脏、肝脏、脾脏、肺脏和肾脏）的毒性及功能影响，为今后马兜铃属类中药临床用药提供参考。【方法】将SD大鼠随机分为AA-Ⅰ给药组和空白对照组，给药组按20 mg/kg灌服AA-Ⅰ，连续7 d。于给药后0.25、0.5、1、2、24 h及停药后15 d，采集大鼠五脏组织。利用HPLC法检测各个时间点五脏组织中的AA-Ⅰ及马兜铃内酰胺Ⅰ（aristolactams-Ⅰ，AL-Ⅰ）含量，并检测大鼠五脏主要生理功能及病理组织学变化。【结果】给药后0.25 h，AA-Ⅰ主要分布于心脏、肝脏、脾脏和肺脏，同时在肝脏和脾脏中检测到AL-Ⅰ；给药后0.5 h，AA-Ⅰ主要分布于心脏、肝脏、脾脏、肺脏和肾脏，除心脏外，其余四脏中AA-Ⅰ含量均达到高峰值，且在肝脏、肺脏、肾脏中检测到AL-Ⅰ；给药后1 h，AA-Ⅰ主要分布于肝脏和肺脏，且在肝脏中检测到AL-Ⅰ；给药后2 h，AA-Ⅰ主要分布于心脏、肝脏、肺脏和肾脏，且在肾脏中检测到AL-Ⅰ；给药后24 h，AA-Ⅰ主要分布于心脏和肝脏，且在肝脏中检测到AL-Ⅰ；停药后15 d，五脏中均未检测到AA-Ⅰ及AL-Ⅰ。功能检测结果显示，大鼠灌服AA-Ⅰ可引起五脏发生氧化应激损伤。【结论】连续7 d给大鼠灌服20 mg/kg AA-Ⅰ，在五脏中均可检测到不同含量的AA-Ⅰ，且在肝脏、肾脏、肺脏及脾脏中检测到AL-Ⅰ。AA-Ⅰ可对五脏造成不同程度的损伤，其损伤与氧化应激有关。     

抗菌肽Tachyplesin Ⅰ的分子设计、结构与活性分析

本研究旨在抗菌肽Tachyplesin Ⅰ(TP Ⅰ)的结构活性基础上，重新设计一种全新的抗菌肽TP Ⅰ-Y4。保持母肽链长与电荷数不变，将序列中形成TP Ⅰ两个二硫键的4个半胱氨酸用酪氨酸进行替换后得到TP Ⅰ-Y4。经生物信息学软件分析，与TP Ⅰ相比，TP Ⅰ-Y4的热稳定性更好，亲水性更强，同时具有与母肽相似的结构与抗菌活性。化学合成后经圆二色谱检测发现，TP Ⅰ-Y4在水相及在模拟细胞膜疏水环境的50%三氟乙醇中都表现出β-折叠结构，疏水环境中TP Ⅰ-Y4的β-折叠含量高于水相，也高于不同环境中的TP Ⅰ。抑菌试验表明，TP Ⅰ-Y4对细菌和真菌都有较强抑菌活性，且对细菌的抑菌活性强于TP Ⅰ。TP Ⅰ-Y4对小鼠红细胞溶血性降低，并保留了很强的内毒素中和能力，浓度为40 μg·mL^-1时中和率可达50%以上。TP Ⅰ-Y4抗菌活性的提高以及溶血活性的降低，可能与β-折叠强形成者酪氨酸的替代有关，进而导致抗菌肽分子中β-折叠结构增强。     

检测猫杯状病毒RT-qPCR方法的优化与应用

为建立一种通用性良好的SYBR GreenⅠ荧光定量RT-PCR方法,根据GenBank上猫杯状病毒(feline calicivirus,FCV)ORF1保守区域序列,设计合成1对特异性引物,用于FCV的快速检测。灵敏性试验结果显示,该方法的检测下限为4.93×10;拷贝/μL,比普通RT-PCR检测下限高100倍;特异性试验结果显示,该检测方法与猫疱疹病毒、猫细小病毒、猫传染性腹膜炎病毒等无交叉反应;通用性试验结果显示,针对引物靶位点区间,该方法对所有已知变异位点的人工合成质粒和本实验室保存的不同变异位点的FCV毒株,均能够良好检出;重复性试验结果显示,组内的变异系数为0.09%~0.71%,组间变异系数为0.05%~0.82%,均小于1%。对23份临床样品进行检测,该方法的阳性检出率为60.9%,检出的14份阳性病料分别接种到CRFK细胞中,对细胞病变产物进行扩增并测序,BLAST对比后结果证实为FCV毒株。结果表明,本试验建立的荧光定量RT-PCR检测方法可用于FCV的快速诊断及流行病学的调查。