肝癌微环境中肝星状细胞活化及Stefin B的表达

探讨肝癌微环境中肝星状细胞(hepatic stellate cell,HSC)的活化与Stefin B表达情况。采用不同浓度TGF-β_1(5、10和20 ng/m L)、不同浓度肝癌细胞条件培养液(25%、50%和75%)以及TGF-β_1与肝癌细胞条件培养液混合的方法分别处理HSC,免疫细胞化学染色法和Western blotting法检测α-SMA和Stefin B蛋白表达,CCK-8法检测细胞增殖。结果:TGF-β_1与肝癌细胞条件培养液分别单独作用或共同作用时,HSC的α-SMA表达均升高且细胞出现活化,细胞增殖能力均增强;Stefin B在TGF-β_1单独作用时表达下调,而在肝癌细胞条件培养液单独作用及TGF-β_1与肝癌细胞条件培养液共同作用时显著升高。结果表明:肝癌微环境能够促进HSC活化及Stefin B表达。     

索拉菲尼通过下调ELK-3抑制肝纤维化的进展

目的:探讨索拉菲尼与ELK-3和上皮-间质转化以及肝纤维化的关系及其作用机制。方法:Western blot检测索拉菲尼对TGF-β1诱导的肝细胞EMT细胞模型中EMT标志蛋白、ELK-3蛋白表达以及p38 MAPK蛋白的表达的影响。Masson′s trichrome染色检测索拉菲尼对肝纤维化动物组织中胶原沉积水平以及对EMT标志蛋白、α-SMA和ELK-3蛋白表达的影响。结果:索拉菲尼组处理的EMT组与EMT组相比,E-cadherin的表达水平显著升高(P0.05),Vimentin、ELK-3蛋白表达和p38磷酸化水平均显著降低(P0.05)。索拉菲尼灌胃的肝纤维化组与肝纤维化组相比,E-cadherin的表达水平显著升高(P0.05),Vimentin、α-SMA和ELK-3蛋白表达水平均显著降低(P0.05)。结论:索拉菲尼通过下调p38MAPK/ELK-3抑制肝细胞的上皮-间质转化进而抑制肝纤维化的进展。     

两种不同给药计算方法对体外药效实验结果的影响

目的:研究按浓度与按单个细胞药物含量两种不同计算方法对甲基阿魏酸(methyl ferulic acid,MFA)抑制TGF-β1刺激的体外培养人肝星状细胞(HSC-LX2)α-SMA表达的影响,评价两种给药计算方法在细胞实验中的应用效果。方法:体外培养HSC-LX2,MTT法测定MFA抑制HSC-LX2增殖的作用,由此确定MFA 1.25、2.5、5mg/L(其相应的单个细胞MFA含量为1.13×10-7、2.25×10-7和4.5×10-7 mg)3个浓度进行后续的实验;以两种不同的细胞数量接种到培养皿,分别以上述浓度和单个细胞MFA含量干预72h,采用RT-PCR检测细胞α-SMA mRNA的表达和Western blotting检测细胞内α-SMA蛋白含量。结果:在两种不同细胞数量情况下,以单个细胞MFA含量计算方法干预后,相同MFA含量组α-SMA mRNA和蛋白的表达趋势一致,且与MTT结果相吻合;而以浓度计算方法干预后,相同MFA浓度组α-SMA mRNA和蛋白表达差异较大。结论:以单个细胞药物含量计算方法在MFA对HSC-LX2细胞体外药效实验中的应用效果更稳定,更科学,实验可重复性较好。     

TGF-β1、Caspase-3和WT1在肾活检肾小球肾炎组织中的表达及临床病理意义

目的：探讨转化生长因子β1（TGF-β1）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）及肾母细胞瘤抑癌基因蛋白（WT1）在肾活检肾小球肾炎组织中的表达及其意义。方法：采用Envision法对40例肾小球肾炎组织及7例对照组肾组织进行TGF-β1、Caspase-3和WT1免疫组织化学染色标记、分析。结果：TGF-β1、Caspase-3、WT1在各种肾炎组织中均有表达,各实验组与对照组差异有统计学意义（P〈0.05）。TGF-β1在系膜增生性肾小球肾炎组的表达明显低于膜性肾病组（t=-4.131,P=0.002）;Caspase-3在各组别中其表达差异无统计学意义;WT1在大量高蛋白尿组表达低于非大量蛋白尿组（t=-2.130,P=0.046）;其余组别差异无统计学意义。WT1的低表达与TGF-β1、Caspase-3的高表达相关。结论：TGF-β1、Caspase-3表达的上调及WT1表达的下调与肾病理改变密切相关,蛋白尿和足细胞减少可能是肾脏病进展的预测因子及肾小球硬化的原因。     

雷帕霉素对小鼠百草枯中毒导致肺纤维化的治疗作用(英文)

目的:评估雷帕霉素(RAPA)对小鼠百草枯(PQ)中毒导致肺纤维化的治疗作用,并初步探讨其作用机制。创新点:首次在小鼠PQ中毒的模型中证明RAPA可明显抑制肺纤维化,且此作用与转化生长因子-β1(TGF-β1)的抑制相关。方法:将C57BL/6J雄鼠分为对照组(腹腔注射生理盐水)和实验组(腹腔注射10 mg/kg PQ)。实验组根据采用的治疗不同,可分为以下四组:PQ组、PQ+RAPA组、PQ+MP(甲强龙)组和PQ+MP+RAPA组。用苏木精-伊红染色法(H&E)和马松(Masson)三色染色法观察肺组织病理结构变化,用酶联免疫吸附测定(ELISA)试剂盒检测肺组织中的羟脯氨酸(HYP)含量,用免疫组化和免疫印迹的方法检测TGF-β1和α-平滑肌肌动蛋白(α-SMA)的表达水平。结论:本实验中小鼠的肺组织病理染色结果显示,RAPA治疗能缓解肺纤维化导致的病理改变(图3)。ELISA实验结果显示,RAPA治疗28天后可显著降低肺组织中HYP的含量(图4)。免疫组化和免疫印迹实验结果显示,RAPA治疗能明显下调肺组织中TGF-β1和α-SMA的高表达(图5和6)。综上所述,RAPA在治疗PQ中毒导致的肺纤维化中有重要价值。     

NE对THP-1细胞IL-6 HMGB1和CD137表达的影响

目的:探讨去甲肾上腺素对人THP-1细胞CD137、IL-6及HMGB1表达的影响.方法:加入101μmol/L的去甲肾上腺素处理THP-1细胞1小时.运用逆转录多聚酶链反应检测其CD137、IL-6及HMGB1mRNA的表达.结果:10μmol/L的去甲肾上腺素能上调THP-1细胞CD137及IL-6mRNA的表达,与对照组比较差异有显著性(P<0.01);但对HMGB1mRNA的表达没有影响.结论:去甲肾上腺素上调THP-1细胞CD137及IL-6mRNA水平.     

ELABELA衍生肽ELA13通过抑制Smad和ERK信号通路减轻肾纤维化（英文）

肾脏纤维化是各种慢性肾脏疾病发展为终末期肾病的关键过程。目前尚无针对肾纤维化的特异性治疗方法。ELA13（氨基酸序列：RRCMPLHSRVPFP）是ELABELA在所有脊椎动物中的保守片段,目前对其生物学活性的研究却很少。本研究评估了ELA13对转化生长因子β1(TGF-β1)处理的NRK-52E细胞和单侧输尿管闭塞（UUO）小鼠的作用效果。首先,体外实验表明在TGF-β1诱导的NRK-52E细胞中,ELA13可以降低纤维化标志物I型胶原（CollagenI）、纤连蛋白（fibronectin）和α-平滑肌肌动蛋白（α-SMA）的表达水平。随后,在UUO诱导的小鼠肾纤维化模型中,我们发现ELA13可以通过降低血清中肌酐和尿素氮的含量来改善肾功能,并通过Masson染色、免疫组织化学、实时定量聚合酶链式反应（RT-PCR）和蛋白质印迹（westernblot）的结果证实纤维化标志物和炎症标志物的表达降低了。进一步机制研究发现,ELA13处理可抑制Smad和细胞外调节蛋白激酶（ERK）信号通路。综上所述,ELA13通过抑制Smad和ERK信号通路发挥抗肾纤维化的作用,有望成为抗肾纤维化治疗...     

SD大鼠脑梗死急性期血清HIF-1α、VEGF因子的变化特点及相关性研究

目的：研究低氧诱导因子（HIF-1α）、血管内皮细胞生长因子（VEGF）表达水平在SD大鼠脑梗死急性期不同时间点的动态变化;探讨两个因子之间的变化是否具有相关性;为脑梗死的治疗和相关的药物研制提供新的靶点。方法：将雌雄各半的36只SD大鼠（月龄2-3月,体重250-320g）随机分为三个组,手术组（简称甲组,16只）、假手术组（简称乙组,16只）、空白组（4只）。甲、乙两组又分为ABCD四个小组。每个小组有4只大鼠,雌雄鼠各两只。参照改良线栓法闭塞右侧大脑中动脉,制作大鼠局灶性脑梗死模型。取血后用ELISA法测定血液中HIF-1α与VEGF的浓度;通过方差分析统计HIF-1α与VEGF两个因子在手术组、假手术组、空白组不同时间点的变化特点;通过线性相关分析的统计方法分析HIF-1α与VEGF因子之间表达的相关性。结果：（1）HIF-1α因子的表达在1/2h出现上调,2h、3h、6h持续少量增高,12h达到高峰。随后表达有所下降,但在168h（7d）时表达又有所增高。（2）VEGF因子的表达也在1/2h出现上调,2h、3h、6h、12h持续少量增高,并在24h达到高峰。随后表达有所下降,在168h（7d）时表达又有所增加。（3）HIF-1α因子和VEGF因子的表达在12小时内表现明显的相关性。结论：（1）HIF-1α自1/2h开始变化,在脑梗死超早期就开始出现表达增加,12h达高峰,对脑梗死后一系列缺氧反应起到重要作用;（2）VEGF自1/2h开始变化,24h达高峰,也参与了脑梗死后的应急表达;（3）HIF-1α因子和VEGF因子在12h内的表达具有明显的相关性。     

安石榴苷对脂多糖诱导奶牛子宫内膜上皮细胞炎症损伤的保护作用（英文）

目的:评估安石榴苷对脂多糖诱导奶牛子宫内膜上皮细胞炎症损伤的保护作用,并初步探讨其作用机制。创新点:首次证明安石榴苷对脂多糖诱导奶牛子宫内膜上皮细胞炎症损伤具有保护作用,且此作用与核转录因子κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)信号通路的抑制相关。方法:用不同浓度的脂多糖(1、10、30、50和100μg/ml)刺激奶牛子宫内膜上皮细胞3、6、9、12和18 h,筛选出建立炎症损伤的最佳作用浓度和时间。安石榴苷预处理细胞2 h后用脂多糖刺激12 h,用逆转录聚合酶链式反应(RT-PCR)检测炎症因子白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、白细胞介素8(IL-8)及肿瘤坏死因子α(TNF-α)的表达。用蛋白质免疫印迹试验(Western blotting)的方法检测核因子κB抑制蛋白α(IκBα)、磷酸化的p65、p38、c-Jun氨基末端激酶(JNK)和细胞外调节蛋白激酶(ERK)的表达水平。结论:MTT结果显示,30μg/ml脂多糖刺激奶牛子宫内膜上皮细胞12 h能够造成细胞活力下降和形态改变(图2和3)。RT-PCR结果显示,安石榴苷预处理后炎症因子显著降低(图4)。Western blotting结果显示,安石榴苷预处理能显著抑制IκBα降解以及p65、p38、JNK和ERK的磷酸化表达水平(图5和6)。综上所述,安石榴苷对脂多糖诱导奶牛子宫内膜上皮细胞炎症损伤具有保护作用,在治疗奶牛子宫内膜炎中具有重要价值。     

蛋白激酶Cα-EGFP及其突变体真核表达载体的构建和在C2C12细胞中的表达

目的研究PKCα结构与转位的关系.方法用PCR的方法构建了4个PKCα突变体,在DNA连接酶的作用下与pEGFP-N1结合后转移至质粒中,构建PKCα突变体-EGFP真核表达载体.观察它们在C2C12细胞中的表达及转位情况.结果pPKCα-EGFP-N1转染C2C12肌细胞24h后,细胞内表达的PKCα-GFP融合蛋白主要定位于细胞浆,细胞核中未见分布.pPKCα-βI-EGFP-N1、pPKCβI-α-EGFP转染C2C12细胞24h的表达产物集中在胞浆.pPKCα(-)-EGFP-N1、pPKCα(m)-EGFP-N1转染C2C12细胞24h的定位与野生型明显不同.结论PKCα的调节区(C1V1C2)尤其是RACK结合区(C2)、PKCα的催化区(C3V4C4)尤其是ATP结合位点与PKCα的转位密切相关.     

Endostatin inhibits fibrosis by modulating the PDGFR/ERK signal pathway: an in vitro study

Accumulating evidence indicates that endostatin inhibits fibrosis. However, the mechanism is yet to be clarified. The aim of this study is to evaluate the effect of endostatin on platelet-derived growth factor-BB (PDGF-BB)-or transforming growth factor β1 (TGF-β1)-induced fibrosis in cultured human skin fibroblasts, and to further examine the molecular mechanisms involved. Human dermal fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and serum-starved for 48 h before treatment. Cells were grouped as follows: “PDGF-BB”, “PDGF-BB+ endostatin”, “TGF-β1”, “TGF-β1+endostatin”, “endostatin”, and “blank control”. The fibroblasts were stimulated with either TGF-β1 or PDGF-BB for 72 h in order to set up the fibrosis model in vitro. The cells were co-cultured with either TGF-β1 or PDGF-BB and endostatin and were used to check the inhibiting effect of endostatin. A blank control group and an endostatin group were used as negative control groups. The biomarkers of fibrosis, including the expression of collagen I, hydroxyproline, and α-smooth muscle actin (α-SMA), were evaluated using an enzyme-linked immunosorbent assay (ELISA) and Western blot. The expression of phosphorylated PDGF receptor β (p-PDGFRβ), PDGFRβ, phosphorylated extracellular signal-regulated kinase (p-ERK), and ERK was detected using Western blot and immunofluorescent staining was used to explore the mechanisms. Both PDGF-BB and TGF-β1 significantly up-regulated the expression of collagen I, hydroxyproline, and α-SMA. Endostatin significantly attenuated both the PDGF-BB- and TGF-β1-induced over-expression of collagen I, hydroxyproline, and α-SMA. PDGF-BB and TGF-β1 both promoted the expression of PDGFR, ERK, and p-ERK. Endostatin inhibited the expression of PDGFR and p-ERK but did not affect the expression of total ERK. Endostatin inhibited hypertrophic scar by modulating the PDGFRβ/ERK pathway. Endostatin could be a promising multi-target drug in future fibrosis therapy.     

Down-regulation of eIF5A-2 prevents epithelial-mesenchymal transition in non-small-cell lung cancer cells

Background

Epithelial-mesenchymal transition (EMT) is believed to be the critical process in malignant tumor invasion and metastases, and has a great influence on improving the survival rate in non-small-cell lung cancer (NSCLC) patients. Recent studies suggested that eukaryotic initiation factor 5A-2 (eIF5A-2) might serve as an adverse prognostic marker of survival. We detected eIF5A-2 in NSCLC A549 cells, and found that the invasive capability correlates with the eIF5A-2 expression.

Methods

Transforming growth factor (TGF)-β1 was used to induce EMT in A549 cells. Western blotting, immunofluorescence, wound healing assay, and transwell-matrigel invasion chambers were used to identify phenotype changes. Western blotting was also used to observe changes of the expression of eIF5A-2. We down-regulated the eIF5A-2 expression using an eIF5A-2 siRNA and identified the phenotype changes by western blotting and immunofluorescence. We tested the change of migration and invasion capabilities of A549 cells by the wound healing assay and transwell-matrigel invasion chambers.

Results

After stimulating with TGF-β1, almost all A549 cells changed to the mesenchymal phenotype and acquired more migration and invasion capabilities. These cells also had higher eIF5A-2 protein expression. Down-regulation of eIF5A-2 expression with eIF5A-2 siRNA transfection could change the cells from mesenchymal to epithelial phenotype and decrease tumor cell migration and invasive capabilities significantly.

Conclusions

The expression of eIF5A-2 was up-regulated following EMT phenotype changes in A549 cells, which correlated with enhanced tumor invasion and metastatic capabilities. Furthermore, in the A549 cell line, the process of EMT phenotype change could be reversed by eIF5A-2 siRNA, with a consequent weakening of both invasive and metastatic capabilities.     

白肋烟TN86晾制期叶绿体色素降解动态及呼吸强度等变化规律研究

TN86晾制期叶绿素(chl)、叶绿素a(chla)、叶绿素b(chlb)、类胡萝卜素(CTK)等的含量都随叶位升高而增加、随晾制进程而下降,其降幅是chla>chl>chlb>CTK,chla降幅最大的时段下、中、上各叶位分别在0～6 d,0—9 d,0-12 d,其余各类色素的降速则比较平缓;chla/chlb及chl/CTK的比值也随晾制进程和叶位升高而下降,使更加有利于烟叶品质的形成.随着晾制过程中水分的散失,烟叶含水率逐渐下降,生命活动也随之减弱,表现在呼吸强度和比叶重也随晾制进程及叶位下降而减小.     

Inhibition of chemotherapy-related breast tumor EMT by application of redox-sensitive siRNA delivery system CSO-ss-SA/siRNA along with doxorubicin treatment

Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) down-regulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.

     

Impairment of liver regeneration by the histone deacetylase inhibitor valproic acid in mice

Background and objective: Liver regeneration is a complex process regulated by a group of genetic and epigenetic factors. A variety of genetic factors have been reported, whereas few investigations have focused on epigenetic regulation during liver regeneration. In the present study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was used to investigate the effect of HDAC on liver regeneration. Methods: VPA was administered via intraperitoneal injection to 2/3 partially hepatectomized mice to detect hepatocyte proliferation during liver regeneration. The mice were sacrificed, and their liver tissues were harvested at sequential time points from 0 to 168 h after treatment. DNA synthesis was detected via a BrdU assay, and cell proliferation was tested using Ki-67. The expressions of cyclin D1, cyclin E, cyclin dependent kinase 2 (CDK2), and CDK4 were detected by Western blot analysis. Chromatin immunoprecipitation (ChIP) assay was used to examine the recruitment of HDACs to the target promoter regions and the expression of the target gene was detected by Western blot. Results: Immunohistochemical analysis showed that cells positive for BrdU and Ki-67 decreased, and the peak of BrdU was delayed in the VPA-administered mice. Consistently, cyclin D1 expression was also delayed. We identified B-myc as a target gene of HDACs by complementary DNA (cDNA) microarray. The expression of B-myc increased in the VPA-administered mice after hepatectomy (PH). The ChIP assay confirmed the presence of HDACs at the B-myc promoter. Conclusions: HDAC activities are essential for liver regeneration. Inhibiting HDAC activities delays liver regeneration and induces liver cell cycle arrest, thereby causing an anti-proliferative effect on liver regeneration.     

子宫内膜异位症患者外周血T细胞亚群分析

目的:检测子宫内膜异位症患者手术前后外周血T淋巴细胞亚群变化,了解病人细胞免疫功能状况。方法:采用间接免疫荧光法检测子宫内膜异位症患者体内CD3~ 、CD4~ 、CD8~ 细胞水平,并取同龄正常妇女作为对照。结果:(1)手术前与正常妇女相比,子宫内膜异位症患者体内存在CD8~ 细胞比例升高,CD4~ 细胞比例和CD4~ /CD8~ 比值下降;(2)手术后病人CD8~ 细胞逐渐回降,而CD4~ 细胞和CD4~ /CD8~ 比值呈现回升趋势。结论:子宫内膜异位症患者存在T细胞免疫异常,这种异常可在手术治疗后得以恢复。     

小鼠Bmi1基因的克隆及其在支持细胞中的表达

目的从昆明鼠睾丸中克隆Bmi1基因,构建真核表达载体,并转染支持细胞,以便用作培养精原干细胞（SSCs）的滋养层.方法以5日龄昆明鼠为材料,提取小鼠睾丸组织中总RNA后,以RT-PCR技术克隆小鼠睾丸Bmi1基因,构建真核表达载体,并转染TM4细胞（睾丸支持细胞株）,在转染后40 h进行免疫荧光鉴定.结果成功克隆小鼠睾丸Bmi1基因的cDNA,测序正确;免疫荧光细胞染色显示,转染后的支持细胞中有Bmi1蛋白表达.结论本研究为以转染了Bmi1基因的支持细胞作饲养层培养SSCs奠定了基础.     

大鼠神经胶质瘤和嗜铬细胞瘤中P2X受体表达特征的研究

目的：采用实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）技术,研究P2X受体在大鼠神经胶质瘤和嗜铬细胞瘤及原代培养皮层神经元和星形胶质细胞上的表达差异。方法：取新生1-2 d SD大鼠大脑皮层,分离纯化神经元和星形胶质细胞,并采用实时荧光定量PCR技术,比较P2X受体在大鼠胶质瘤C6细胞、大鼠肾上腺嗜铬细胞瘤PC-12细胞、星形胶质细胞和皮层神经元上的表达差异。结果：C6细胞P2X2、P2X3和P2X5表达水平显著高于星形胶质细胞,P2X4、P2X6和P2X7表达水平显著低于星形胶质细胞,PC-12细胞P2X1、P2X2、P2X3和P2X6表达水平显著高于皮层神经元,P2X5和P2X7表达水平则显著低于皮层神经元。此外,还发现P2X2、P2X5和P2X6在C6和PC-12细胞上的表达水平存在显著差异。结论：大鼠胶质瘤和嗜铬细胞瘤细胞表达多种P2X受体,且与原代培养细胞存在表达差异,提示核苷酸介导的信号传递系统可能作为潜在的肿瘤治疗靶点。