扶正化瘀方含药血清对肝星状细胞activinA/smad信号通路活化的影响

目的　探讨扶正化瘀方含药血清对肝星状细胞（HSC）激活素A（activinA）/smad信号通路活化的影响。方法　20只SD大鼠按照随机数字表法分为对照组及扶正化瘀（Fzhy）-低、中、高剂量含药血清组，每组5只，分别以蒸馏水，0.75、1.5、3.0 g/kg扶正化瘀溶液（扶正化瘀胶囊药物粉末与蒸馏水配制）灌胃1次/d，连续3 d，取血制备空白血清（对照组）和含药血清。以大鼠HSC-T6细胞为研究对象，分别用5%、10%、20%体积分数的各组含药血清培养细胞。CCK-8检测细胞存活率，选取细胞存活率在50%左右的体积分数重新分为对照组及Fzhy-低、中、高剂量组。流式细胞术检测细胞周期及凋亡率、线粒体膜电位变化和活性氧（ROS）水平；实时荧光定量聚合酶链反应检测细胞中activinA、smad3、samd7、核因子（NF）-κB的mRNA水平；蛋白质印迹法检测细胞中activinⅡA受体（ActRⅡA）、smad3、NF-κB p65、胱天蛋白酶（caspase）-3的蛋白水平。结果　各扶正化瘀含药血清组的细胞存活率均低于对照组（P＜0.05），选择体积分数为10%的含药血清进行后续实验。对照组和各扶正化瘀方含药血清组细胞周期和凋亡率差异均无统计学意义。对照组及Fzhy-低、中、高剂量组细胞内ROS水平及线粒体膜电位水平降低比例逐次升高（P＜0.05）。与对照组比较，Fzhy-中、高剂量组smad3 mRNA表达水平降低，smad7 mRNA升高，Fzhy-低、中、高剂量组NF-κB、activinA mRNA，smad3、NF-κB p65、ActRⅡA蛋白表达水平降低，Fzhy-低剂量组、中剂量组caspase-3蛋白表达水平升高（P＜0.05）；与Fzhy-低剂量组相比，Fzhy-中、高剂量组smad3 mRNA表达水平降低，Fzhy-中剂量组activinA及smad7 mRNA升高（P＜0.05）。结论　扶正化瘀方含药血清可通过影响HSC-T6细胞增殖、参与细胞的氧化应激以及调节细胞activinA/smad信号转导通路来实现抗纤维化作用。     

放疗干预对宫颈癌荷瘤小鼠的抑瘤作用及对Th1/Th2免疫细胞比例的影响

目的探讨放疗干预对宫颈癌荷瘤小鼠的抑瘤作用及其对辅助性T细胞1(Th1)/Th2细胞比例的影响。方法建立宫颈癌荷瘤小鼠模型,随机分为荷瘤组和放疗组,每组各10只,另设对照组10只。放疗组进行放疗干预14天,荷瘤组和对照组不治疗。放疗后4、6、8、10、12、14天测量肿瘤体积;末次治疗后,ELISA法测定血清干扰素-γ(IFN-γ)、白介素-2(IL-2)、IL-4、IL-10含量;计算抑瘤率、胸腺指数和脾脏指数;HE染色观察肿瘤组织学变化;TUNEL染色观察肿瘤组织细胞凋亡情况;流式细胞术检测脾脏Th1/Th2细胞比例;RT-qPCR和Western blot检测脾脏T盒子转录因子(T-bet)、GATA结合蛋白3(GABA-3)mRNA和蛋白表达。结果与荷瘤组比较,放疗组小鼠4、6、8、10、12、14天肿瘤体积及瘤质量减小,血清IL-2、IFN-γ升高,IL-4、IL-10降低,胸腺指数、脾脏指数升高,Th1细胞比例、Th1/Th2增加,Th2细胞比例减少,T-bet mRNA和蛋白及T-bet/GATA-3表达升高,GATA-3 mRNA和蛋白表达降低(P<0.05)。HE染色显示,荷瘤组肿瘤细胞数量较多,核大深染,无明显坏死;放疗组肿瘤细胞数量减少,出现大量坏死组织。TUNEL染色显示,荷瘤组TUNEL阳性细胞较少,放疗组TUNEL阳性细胞明显增多。结论放疗对宫颈癌荷瘤小鼠具有明显抑瘤作用,可能是通过调节T-bet/GATA-3表达,促进Th1/Th2分化平衡,增强机体免疫功能发挥作用。     

黄芪多糖对人鼻咽癌CNE-1细胞的放疗增敏及上皮间质转化的作用

目的:采用黄芪多糖(APS)与放射治疗(IR)联用,研究APS对人鼻咽癌CNE-1细胞的放疗敏感性及上皮间质转化(EMT)的作用机制。方法:采用细胞计数(CCK-8)法检测不同质量浓度APS(0,6.25,12.5,25,50,100,200 g·L-1)对CNE-1细胞的细胞毒性;克隆形成实验计算12.5 g·L-1APS与不同放射剂量(0,2,4,6 Gy)联用后对CNE-1细胞的存活分数(SF),利用线性二次方程数学模型(LQ)根据SF值绘制放射敏感曲线;细胞划痕和transwell小室实验分别检测各组细胞的迁移和侵袭能力;流式细胞仪检测各组细胞的凋亡情况;蛋白免疫印迹法(Western blot)检测各组细胞的EMT标记物、凋亡标记物以及蛋白激酶B/细胞外调节蛋白激酶(Akt/ERK)通路蛋白的表达水平。结果:克隆形成实验和放射敏感曲线结果表明,非毒性剂量12.5 g·L-1APS与4 Gy放射剂量联合给药可以明显增加CNE-1细胞的放疗敏感性;与空白组及IR组比较,APS与IR联用可以抑制CNE-1细胞的迁移和侵袭能力(P0.05),明显增加CNE-1细胞的凋亡率(P0.05)。与空白组及IR组比较,APS与IR联用可以显著下调间质型钙黏蛋白(N-cadherin),p-Akt和p-ERK蛋白表达水平,明显上调上皮型钙黏蛋白(E-cadherin),B淋巴细胞瘤-2相关X蛋白(Bax)和半胱天冬氨酸蛋白酶-3(Caspase-3)蛋白表达水平(P0.05)。结论:APS与IR联用可以抑制CNE-1细胞的迁移和侵袭,增加放疗引起的细胞凋亡,其可能通过抑制EMT和Akt/ERK通路有关。     

miR-145调控肝癌腹水模型Th9细胞升高的机制研究

目的:探讨miR-145调控肝癌腹水模型Th9细胞升高的作用机制。方法:构建小鼠H22肝癌腹水模型。造模两周后处死小鼠并分离出脾脏组织，流式细胞术分析肝癌腹水组（MA组）与正常对照组（Control 组）小鼠脾脏Th9细胞表达水平。ELISA法检测脾脏IL-9表达水平。RT-PCR法检测脾脏miR-145表达水平。Western Blot法检测脾脏中PI3K/Akt/mTOR/P70S6K/HIF-1α相关蛋白表达水平。分选小鼠脾脏CD4+T细胞，随机分为miR-145 mimics组和NC组，分别应用miR-145-5P mimics、阴性对照寡核苷酸（negative control，NC）进行转染，RT-PCR检测各组miR-145、HIF-1α mRNA及IL-9 mRNA表达水平。结果:与Control 组相比，MA组Th9细胞及其细胞因子IL-9表达均升高（P<0.05），miR-145表达降低（P<0.05），p-PI3K、p-Akt、mTOR、p-mTOR、p-P70S6K、HIF-1α蛋白均升高（P<0.05）。与NC组相比，miR-145 mimics组miR-145显著升高，HIF-1α mRNA及IL-9 mRNA表达下降。结论:肝癌腹水中Th9细胞升高可能与miR-145下降导致的PI3K/Akt/mTOR/P70S6K/HIF-1α通路激活有关。     

七氟烷预处理对大鼠骨髓间充质干细胞在缺氧复氧环境下的作用和机制初探

目的:研究七氟烷预处理对大鼠骨髓间充质干细胞（MSCs）在缺氧复氧环境下的作用和机制初探。方法:分离培养SD大鼠骨髓间充质干细胞，通过流式细胞仪和诱导分化培养鉴定其免疫表型和分化潜能。细胞随机分为3组:组1空白对照组；组2缺氧复氧组（缺氧24 h，复氧24 h）；组3七氟烷预处理组（2%七氟烷预处理2 h后，缺氧24 h，复氧24 h）。处理后收集各组细胞，用流式细胞仪测定其凋亡率，线粒体膜电位变化及细胞周期。Western blot测定细胞外调节蛋白激酶（ERK）的蛋白表达。结果:与组2相比，组3 MSCs凋亡率明显下降，处于S期细胞增多，细胞增殖能力增强。Western blot结果显示:与组2相比，组3磷酸化ERK蛋白表达上升。结论:七氟烷预处理能提高缺氧复氧环境下骨髓间充质干细胞存活率，降低MSCs凋亡数量，并能增强MSCs的增殖能力，这一机制与七氟烷促进MSCs细胞p-ERK蛋白表达相关。     

Suppression of Rotenone-Treated Human Breast Cancer Stem Cell Survival Using Survivin Inhibitor YM155 is Associated to Oxidative Stress Modulation

Background: Despite recent progress in molecular-targeted therapies, breast cancer remains the primary leading cause of cancer related death among women worldwide. Breast cancer stem cells (BCSCs) are believed to be responsible for therapy resistance and cancer recurrence. We recently demonstrated that human BCSCs (CD24-/CD44+) could survive better than their counterpart non-BCSCs (CD24-/CD44-) when treated with rotenone, possibly due to lower levels of reactive oxygen species (ROS) production, high expression of antioxidant manganese superoxide dismutase (MnSOD), and anti-apoptotic survivin. The aim of this study was to verify the role of survivin on human BCSCs survival under oxidative stress modulation by suppressing its expression using YM155, a survivin inhibitor. Methods: Human BCSCs (ALDH+ cells) were treated with YM155 for 24 h prior to treatment with rotenone for a further 6 h. We determined intracellular superoxide levels were determined using dihydroethidium assay, survivin and MnSOD expression using qRT-PCR, survivin protein level using ELISA, as well as cell viability using trypan blue exclusion and acridine orange/ethidium bromide apoptosis assay. Results: Suppression of survivin expression using YM155 could reduce the survival of rotenone-treated BCSCs, which may be associated with oxidative stress modulation, as shown by increased ROS levels and decreased MnSOD expression. We confirm that survivin is responsible for maintaining BCSCs survival under oxidative stress modulation. Furthermore, YM155 could modulate oxidative stress in BCSCs by reducing MnSOD expression and increasing ROS levels. Conclusion: YM155 treatment could be used to overcome BCSCs resistance to oxidative stress-based anticancer therapies.     

Role of Toll-like receptor 2 against Streptococcus uberis infection in primary mouse mammary epithelial cells

Mammary epithelial cells (MECs) play an important role against Streptococcus uberis infection which is one of the main causes of bovine mastitis and a potential threat to human health. Toll-like receptors (TLRs) and their mediated signaling pathways are critical in both innate and infection responses, yet their roles in anti-S. uberis infection in MECs remains poorly defined. In this work we investigated the regulatory mechanisms of TLR2 in inflammatory responses, where WT and TLR2^−/− mice were euthanized at 15–18 days gestation, and mammary gland tissues were collected aseptically. The mouse MECs (MMECs) were isolated by combined digestion with type I collagenase, hyaluronidase and trypsin. We challenged MMECs with S. uberis and quantified antioxidant capacity as well as reactive oxygen species (ROS), proinflammatory cytokines and cell damage at different times. The loss of TLR2 function in MMECs results in more serious cell damage, increased cell adhesion, and significantly decreased ROS and mitochondrial ROS (mROS) with bactericidal function in response to S. uberis infection. Moreover, it was observed that the antioxidant capacity declined, and the production of TLR2-mediated cytokines (except CXC ligand 15) also were reduced. We demonstrated that TLR2 can mediate cellular anti-infective processes in MMECs by regulating the production of ROS and mROS and the secretion of cytokines. The results suggest an unpredicted role of TLR2 in MMECs in response to S. uberis infection.     

急性乙型肝炎患者pDCs上CD86表达变化及意义

目的 探讨外周血浆细胞样树突状细胞(pDCs)在急性乙型肝炎(AHB）患者临床转归中的变化及作用。方法 纳入2015年6月至2017年5月收治的急性乙型肝炎(AHB组)患者40例， 给予退黄、降酶、保肝等药物，并加用恩替卡韦0．5 mg空腹口服，1次/d。另纳入健康体检者26例为健康对照组（ HC组）。HC组于体检当天、AHB组于治疗前及治疗后6周采集外周静脉全血，采用ELISA法检测2组血浆HBV DNA、HBsAg、HbeAg及肝功能指标水平，并应用流式细胞仪检测pDCs的频数及其功能分子CD86的表达水平。结果 治疗6周后，AHB组临床症状显著缓解，血清学指标和乙型肝炎标志物水平显著下降（P<0.01）。治疗6周后，AHB组HBV DNA转阴率为82.5%，HBsAg清除率为72.5%，HBeAg血清学转换率为75.0%，与治疗前比较差异均有统计学意义（P<0.01）, HC组pDCs频数显著高于AHB组治疗前。AHB组治疗后CD86+pDCs显著高于治疗前。HC组CD86ABC显著低于AHB组治疗前。结论 在急性乙型肝炎患者外周血pDCs的数量降低。急性乙型肝炎患者在治疗后CD86+pDCs增多。急性乙型肝炎患者pDCs上CD86表达上调。