mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响

目的　探讨mTOR通路抑制剂雷帕霉素对真菌性角膜炎小鼠角膜瘢痕化的影响。方法　取96只SPF级C57BL/6J雄性小鼠,随机分为雷帕霉素组和对照组，每组各48只。两组小鼠同时建立真菌性角膜炎模型。雷帕霉素组模型制作前1 d按6.0 mg·kg^-1雷帕霉素对小鼠进行腹腔注射预处理，之后按0.2 g·L^-1浓度在结膜下注射5 μL，持续3 d；对照组注射PBS溶液。造模后对各组小鼠进行角膜临床评分，Western blot和实时荧光定量PCR分别检测造模后各组小鼠不同时间角膜LC-3Ⅱ、α-SMA和 TGF-β1表达情况。结果　造模后24 h、48 h、72 h、96 h、120 h、144 h、336 h，对照组小鼠角膜临床评分均明显高于雷帕霉素组，差异均有统计学意义(均为P＜0.05) 。造模后144 h、216 h雷帕霉素组小鼠角膜LC-3Ⅱ表达上调，LC-3Ⅱ蛋白和mRNA相对表达量与对照组相比，差异均有统计学意义（均为P<0.05）；而在造模后72 h及336 h两组LC-3Ⅱ蛋白和mRNA相对表达量差异均无统计学意义（均为P>0.05）。与对照组相比，造模后144 h、216 h雷帕霉素组小鼠角膜α-SMA蛋白及 mRNA相对表达量下降，差异均有统计学意义（均为P<0.05）；造模后144 h、336 h雷帕霉素组TGF-β1 mRNA相对表达量亦下降，与对照组相比，差异均有统计学意义（均为P＜0.05）。结论　雷帕霉素通过促进自噬作用下调角膜瘢痕化相关因子的表达，减轻了真菌性角膜炎模型小鼠角膜瘢痕化程度。     

Sorafenib,rapamycin, and venetoclax attenuate doxorubicin-induced senescence and promote apoptosis in HCT116 cells

Emerging evidence has shown that the therapy-induced senescent growth arrest in cancer cells is of durable nature whereby a subset of cells can reinstate proliferative capacity. Promising new drugs named senolytics selectively target senescent cells and commit them into apoptosis. Accordingly, senolytics have been proposed as adjuvant cancer treatment to cull senescent tumor cells, and thus, screening for agents that exhibit senolytic properties is highly warranted. Our study aimed to investigate three agents, sorafenib, rapamycin, and venetoclax for their senolytic potential in doxorubicin-induced senescence in HCT116 cells. HCT116 cells were treated with one of the three agents, sorafenib (5 µM), rapamycin (100 nM), or venetoclax (10 µM), in the absence or presence of doxorubicin (1 µM). Senescence was evaluated using microscopy-based and flow cytometry-based Senescence-associated-β-galactosidase staining (SA-β-gal), while apoptosis was assessed using annexin V-FITC/PI, and Muse caspase-3/-7 activity assays. We screened for potential genes through which the three drugs exerted senolytic-like action using the Human Cancer Pathway Finder PCR array. The three agents reduced doxorubicin-induced senescent cell subpopulations and significantly enhanced the apoptotic effect of doxorubicin compared with those treated only with doxorubicin. The senescence genes IGFBP5 and BMI1 and the apoptosis genes CASP7 and CASP9 emerged as candidate genes through which the three drugs exhibited senolytic-like properties. These results suggest that the attenuation of doxorubicin-induced senescence might have shifted HCT116 cells to apoptosis by exposure to the tested pharmacological agents. Our work argues for the use of senolytics to reduce senescence-mediated resistance in tumor cells and to enhance chemotherapy efficacy.     

雷帕霉素药物洗脱支架直接置人术治疗急性冠脉综合征的临床研究

目的 评价直接置入雷帕霉素药物洗脱支架(CYPHER^TM,codis治疗急性冠脉综合征(ACS)罪犯病变的安全性、可行性。方法 32例接受CYPHERTM支架直接置入的患者(直接支架组)和一般情况匹配的34例球囊扩张后行冠脉支架术的患者(常规支架组),比较两组的一般情况,冠脉造影及介入治疗即刻和临床随访结果。结果 直接支架组和常规支架组的成功率分别为97％和100％,直接支架组1例支架无法通过病变,球囊预扩张后成功置入原支架。两组无一例发生介入治疗相关的的严重心脏事件。随访期间两组严重心脏不良事件发生率无明显统计学差异。结论 CYPHER^TM支架直接置入术治疗ACS罪犯病变安全可行,成功率高。     

雷帕霉素对烫伤后金黄色葡萄球菌脓毒症大鼠白细胞介素10表达的影响

目的探讨脓毒症时应用雷帕霉素(RPM)干预对肝脏内源性白介素10(IL10)表达和急性肝损伤的影响。方法采用烫伤后金黄色葡萄球菌感染致严重脓毒症大鼠模型,50只大鼠随机分为正常对照组(n=5)、烫伤对照组(n=5)、烫伤脓毒症组(n=30)和RPM处理组(n=10)。采用逆转录多聚酶链式反应测肝组织IL10mRNA表达,并应用ELISA方法分别检测肝组织、血中IL10蛋白水平,同时观察肝功能指标的改变。结果与烫伤脓毒症组比较,RPM干预后0.5h肝组织中IL10mRNA表达和血中IL10蛋白水平明显升高(P<0.05),干预后2h则有不同程度的下降,但仍显著高于伤前水平(P<0.05或P<0.01)。同时,RPM干预组肝功能酶学指标明显改善。结论烫伤脓毒症早期应用RPM有助于保护IL10的内源性抗炎效应,从而减轻失控性炎症反应和急性肝损伤。     

原发免疫性血小板减少症树突状细胞异常免疫反应机制

目的 探讨影响原发免疫性血小板减少症(ITP)患者树突状细胞功能异常的基因,为ITP治疗寻求新方法。 方法 随机选取ITP患者(ITP组)8例和同期入院体检健康者8例正常对照组为研究对象,分离外周单个核细胞并在体外诱导分化为单核细胞源性树突状细胞(moDCs),选取ITP组和正常对照组moDCs样本各3例,使用Illumina Hiseq平台进行转录组测序,并完成生物信息学分析。其他moDCs样本分为对照组、ITP组和ITP+雷帕霉素处理组,采用Western blotting法检测哺乳动物雷帕霉素靶蛋白复合体1(mTORC1)信号通路激活情况,采用流式细胞术检测moDCs表面分子表达,采用酶联免疫吸附(ELISA)法检测moDCs细胞因子分泌能力。 结果 差异性表达分析显示,与正常对照组相比,ITP组患者moDCs中有161个基因表达上调,320个基因表达下调,差异有统计学意义(P<0.05)。利用基因本体论(GO)和京都基因与基因组百科全书(KEGG)数据库对差异基因进行功能和分子通路注释,结果表明差异基因主要集中在T细胞分化、T细胞共刺激、T细胞活化等生物学过程和T细胞受体信号通路等信号途径。基因集富集分析(GSEA)显示,ITP组患者树突状细胞中mTORC1信号通路基因表达上调。进一步验证发现,ITP组患者moDCs中磷酸化哺乳动物雷帕霉素靶蛋白(mTOR)和mTORC1活化标志物磷酸化核糖体蛋白S6激酶(S6K)相对含量升高,同时ITP组患者moDCs共刺激分子CD80、CD86和促炎因子白介素6(IL-6)、白介素12(IL-12)的表达增加,而白介素10(IL-10)、转化生长因子-β(TGF-β)表达降低。使用mTORC1抑制剂雷帕霉素可以抑制moDCs共刺激分子CD80、CD86的表达和促炎因子IL-6、IL-12的分泌,并上调IL-10的表达,而对TGF-β的分泌无明显影响。 结论 ITP患者moDCs中mTORC1信号通路高度激活,使用mTORC1抑制剂雷帕霉素可以改善moDCs的免疫调节能力。因此,mTORC1信号通路可能是调节ITP患者moDCs功能异常的新靶点。     

RTP801 immunoreactivity in retinal ganglion cells and its down-regulation in cultured cells protect them from light and cobalt chloride

RTP801, a stress-related protein, is activated by adverse environmental conditions and inhibits the activity of mammalian target of rapamycin (mTOR) in promoting oxidative stress-dependent cell death. RTP801 exists both in the mammalian retina and the lens of the eye. Here, we observed RTP801 immunoreactivity in some retinal ganglion cells. Intravitreal injection of cobalt chloride (CoCl₂) to mimick hypoxia influenced retinal GFAP (glial fibrillary acidic protein) and heme oxygenase-1 (HO-1) levels, but did not affect RTP801 immunoreactivity or mRNA content relative to GAPDH. However, RTP801 mRNA was elevated when compared with Brn3a mRNA, suggesting that RTP801 is activated in stressed Brn3a retinal ganglion cells. In cultures of RGC-5 cells, RTP801 immunoreactivity was located in the cytoplasm and partly present in the mitochondria. An insult of blue light or CoCl₂ increased RTP801 expression, which was accompanied by cell death. However, in cultures where RTP801 mRNA was down-regulated, the negative influence of blue light and CoCl₂ was blunted. Rapamycin nullified the CoCl₂-induced up-regulation of RTP801 and attenuated cell death. Moreover, rapamycin was non-toxic to RGC-5 cells, even at a high concentration (10 μM). The protective effect of rapamycin on RGC-5 cells caused by the inhibition of RTP801 suggests that rapamycin might attenuate retinal ganglion cell death in situ, as in glaucoma.     

Efficacy of an autophagy-targeted DNA vaccine against avian leukosis virus subgroup J

Infection with the avian leukosis virus subgroup J (ALV-J) can lead to neoplastic disease in chickens, inflicting significant economic losses to the poultry industry. Recent reports have identified inhibitory effects of ALV-J on autophagy, a process involving in innate and adaptive immunity. Inspired by this connection between autophagy and immunity, we developed a novel DNA vaccine against ALV-J which includes co-administration of rapamycin to stimulate autophagy. To measure the efficacy of the developed prototype vaccine, five experimental groups of seven-day-old chickens was immunized three times at three-week intervals respectively with vector, pVAX1-gp85, pVAX1-gp85-LC3, pVAX1-gp85 + rapamycin and pVAX1-gp85-LC3 + rapamycin through electroporation. We then tested their antibody titers, cytokine levels and cellular immune responses. The immunoprotective efficacy of the prototype vaccines against the challenge of the ALV-J GD1109 strain was also examined. The results showed that the combination of pVAX1-gp85-LC3 and rapamycin was able to induce the highest antibody titers, and enhance interleukin(IL)-2, IL-10 and interferon (IFN)-γ expression, and the chickens immunized with the combination of pVAX1-gp85-LC3 and rapamycin showed the highest percentage of CD3+ CD8+ T lymphocytes. Based on our results, we suggest that stimulating autophagy can improve the efficacy of DNA vaccines and that our DNA vaccine shows the potential of being a candidate vaccine against ALV-J. This study provides a novel strategy for developing vaccines against ALV-J.     

Rapamycin prevents thoracic aortic aneurysm and dissection in mice

Objective

The purpose of this study was to investigate whether rapamycin inhibits the development of thoracic aortic aneurysm and dissection (TAAD) in mice.

雷帕霉素对去甲氧柔红霉素诱导急性髓系白血病THP-1细胞凋亡的影响

目的:探讨雷帕霉素（Rapa）对去甲氧柔红霉素（IDA）诱导急性髓系白血病THP-1细胞凋亡的影响及其分子机制。方法:分别用10、20、40、80 nmol/L Rapa处理THP-1细胞1 h，另设未经Rapa处理的细胞。采用蛋白质印迹法检测THP-1细胞自噬标志物LC3蛋白的转换情况（LC3Ⅱ/LC3Ⅰ），采用流式细胞术检测细胞凋亡，确定Rapa处理浓度。用不同浓度IDA作用THP-1细胞24 h，采用CCK-8法检测IDA对THP-1细胞的增殖抑制率，计算半数抑制浓度（ IC50）。以低于 IC50的IDA作用Rapa处理或未处理的THP-1细胞24 h，CCK-8法检测细胞增殖抑制率，流式细胞术检测细胞凋亡情况，实时荧光定量聚合酶链反应检测自噬相关基因Beclin-1、LC3和p62的表达变化，蛋白质印迹法检测自噬标志物LC3蛋白的转换情况。 结果:20 nmol/L Rapa处理的THP-1细胞LC3Ⅱ/LC3Ⅰ高于未处理的细胞（ P=0.002 4）；80 nmol/L Rapa处理的细胞凋亡率高于未处理的细胞（ P=0.007 3）。根据蛋白质印迹法和流式细胞术检测结果，选取20 nmol/L Rapa作为预处理浓度。IDA对THP-1细胞作用24 h的 IC50为59.874 nmol/L。50 nmol/L IDA作用24 h后，Rapa预处理的THP-1细胞增殖抑制率[（69.67±5.03）%比（41.67±3.51）%]和细胞凋亡率[（74.35±4.83）%比（41.25±5.24）%]均高于未预处理的细胞（均 P<0.05）；Rapa预处理的THP-1细胞Beclin-1、LC3 mRNA表达水平及LC3Ⅱ/LC3Ⅰ均高于未预处理的细胞，p62 mRNA表达水平低于未预处理的细胞（均 P<0.05）。 结论:Rapa能增强较低剂量IDA诱导的THP-1细胞凋亡，此效应可能是通过其引起THP-1细胞过度自噬实现的。