mTOR通路调控肾间质成纤维细胞活化的机制

目的 探讨mTOR信号通路在肾间质成纤维细胞增生活化过程中的调控作用,并研究其抑制剂在抗肾纤维化治疗中的可行性.方法 用8周龄雌性C57BL/6小鼠构建单侧输尿管结扎(UUO)肾间质纤维化动物模型(n=30),按数字随机法分为雷帕霉素组(n=15)及UUO组(n=15).雷帕霉素组术前1d开始腹腔注射雷帕霉素(2 mg·kg-1·d-1)至实验结束;UUO组注射生理盐水.分别于术后1、3、7、14 d处死小鼠(n=3),留肾组织进行相关检测.同时,体外实验评估雷帕霉素对TGF-β诱导鼠成纤维细胞株(NIH3T3细胞)活化的干预作用.结果 UUO小鼠肾组织中活化的肌成纤维细胞[α肌动蛋白(α-SMA)阳性]高表达mTOR通路下游效应因子pS6K.雷帕霉素显著抑制pS6K表达及肾间质中肌成纤维细胞的活化,改善肾小管间质损伤及纤维化程度.实时荧光定量PCR结果提示雷帕霉素组小鼠肾皮质组织中成纤维细胞特异蛋白1 (FSP1)、转化生长因子β(TGF-β)、结缔组织因子(CTGF)及Ⅳ型胶原蛋白基因α1 (Col 4A1)的mRNA水平显著下降.体外实验结果示TGF-β诱导小鼠成纤维细胞株( NIH3T3)的mTOR通路显著活化,并大量合成α-SMA.雷帕霉素能够明显抑制mTOR通路活性,降低细胞的纤维化活性.结论 肾间质纤维化过程中成纤维细胞内的mTOR信号通路高度活化.抑制mTOR通路能够显著降低成纤维细胞的活性,改善肾间质纤维化程度.     

Hedgehog信号通路相关蛋白在人胰腺癌SW1990耐药株中的表达及意义

目的:探讨Hedgehog信号通路蛋白在人胰腺癌吉西他滨耐药细胞株SW1990中的表达,为克服胰腺癌获得性耐药提供实验基础。方法:采用浓度梯度递增法建立人胰腺癌SW1990耐药株,采用噻唑蓝法测定SW1990亲代与耐药细胞IC50。实时荧光定量PCR检测亲代与耐药细胞mRNA中hedgehog信号通路成员Shh、SMO、Gli-1的表达差异。Western印迹法检测亲代与耐药细胞中上述蛋白质的表达。结果:人胰腺癌耐药株SW1990的IC50从亲代的(3.1±0.2)μmol/L提高到(232.2±12.3)μmol/L。荧光定量PCR结果显示耐药株中Shh、Gli-1的表达提高了(12.07±1.71)倍和(4.15±0.42)倍。亲代SW1990中未检测到SMO表达,而耐药细胞中却可以检测到SMO的表达。Western印迹结果同样显示,人胰腺癌SW1990耐药细胞株中高表达上述蛋白质。结论:人胰腺癌耐药株中高表达部分hedgehog信号通路蛋白。针对hedgehog信号通路的靶向治疗可能为克服胰腺癌耐药提供新的理论基础。     

Hedgehog signaling displays a biphasic expression pattern during intestinal injury and repair

Background/Purpose

Gastrointestinal injury is common clinically. The exact mechanism by which gastrointestinal repair occurs has yet to be well defined. Hedgehog (Hh) signaling is known to be involved in gastrointestinal development and repair of tissues such as skin and heart. The present study aimed to investigate the role of Hh in the repair of the small intestine.

Methods

i) To study acute intestinal injury, we optimized a mouse model of 5-flurouracil (5-FU) induced injury of the small intestine. Ileal tissues were evaluated for injury and repair markers at day 0, 2, 5, and 9. ii) Immunohistochemistry (Sonic hedgehog, Shh), in situ hybridization (Shh), and Ptch/LacZ transgenic mice were carried out to localize hedgehog expression. A33CrPr × ShhTg knock-in mice were bred to study the effect of Shh over-expression. qPCR of Shh, Ihh, Ptch, Bmp4 was carried out to quantify hedgehog signaling. iii) 5FU treated mice were then treated with a hedgehog inhibitor or saline (control) and the effects of Shh inhibition including apoptosis, proliferation, and mitosis were then compared.

Results

i) Immunohistochemistry and in situ hybridization of Shh, qPCR of hedgehog signaling pathway genes, and Ptch/LacZ staining results consistently showed down-regulation during the injury phase (P < 0.05) followed by up-regulation during the repair phase (P < 0.005). ii) Hh signaling inhibition following 5-FU induced injury augmented apoptotic activity (P < 0.05), suppressed mitotic activity (P < 0.005) in intestinal crypts, and reduced Paneth cell hyperplasia (P < 0.005). iii) Shh over-expression in conditionally knock-mice led to increased mitotic, Paneth, and goblet cells.

Conclusion

Hedgehog signaling pathway displays a biphasic expression pattern during the injury/repair of small intestine. It may play an important regulatory role in intestinal repair.     

异氟醚预处理对局灶性脑缺血再灌注损伤大鼠缺血半暗带TLR4-MyD88信号通路的影响

目的 探讨异氟醚预处理对局灶性脑缺血再灌注损伤大鼠缺血半暗带TLR4-MyD88信号通路的影响.方法 成年雄性SD大鼠54只,体重250 ～ 280 g,采用随机数字表法,将其随机分为3组(n=18):假手术组(S组)、脑缺血再灌注组(I/R组)和异氟醚预处理组(IP组).S组仅分离血管不留置线栓;I/R组采用线栓法制备右侧局灶性脑缺血再灌注损伤模型,缺血2h,再灌注24 h;IP组吸入2.0％异氟醚2h,预处理结束后24h时制备右侧局灶性脑缺血再灌注损伤模型.于再灌注24 h时行神经功能缺陷评分,随后处死大鼠,每组随机抽取5只大鼠,取脑组织,测定脑梗死体积,采用Westernblot法和RT-PCR法检测大鼠右侧脑缺血半暗带区HSP60、TLR4、MyD88蛋白及mRNA的表达情况;每组剩余的3只大鼠,采用TUNEL法检测大鼠右侧脑缺血半暗带区细胞凋亡情况.结果 与S组比较,I/R组和IP组神经功能缺陷评分升高,脑梗死体积增大,右侧脑缺血半暗带区凋亡指数升高,HSP60、TLR4、MyD88蛋白及mRNA表达均上调(P＜0.05);与I/R组比较,IP组神经功能缺陷评分降低,脑梗死体积减小,右侧脑缺血半暗带区凋亡指数降低,HSP60、TLR4、MyD88蛋白及mRNA表达均下调(P＜0.05).结论 异氟醚预处理可保护脑缺血再灌注大鼠缺血半暗带,其机制可能与抑制大鼠脑缺血半暗带TLR4-MyD88信号通路有关.     

Notch信号通路在鱼藤酮诱导的PC12细胞凋亡中的作用

目的 探讨Notch信号通路在鱼藤酮诱导PC12细胞凋亡中的作用机制.方法 用鱼藤酮(5 μmol/L)处理PC12细胞,检测PC12细胞的凋亡和Notch信号通路的活化状态.同时以Notch信号抑制剂(2S)-N-N-(3,5-二氟苯乙酰基)-L-丙氨酰-2-苯基甘氨酸叔丁酯(DAPT,10 μmol/L)处理上述PC12细胞,观察Notch信号通路抑制后PC12细胞对鱼藤酮处理的凋亡反应,分析Notch信号通路与鱼藤酮诱导PC12细胞凋亡之间的关系.结果 鱼藤酮显著诱导PC12细胞凋亡[(35.6±5.4)％,P＜0.05]和半胱氨酰天冬氨酸特异性蛋白酶-3(Caspase-3)活性升高(0.52±0.15,P＜0.05),并活化其Notch/Jagged1信号通路.DAPT显著抑制鱼藤酮诱导的Notch/Jagged1信号通路活化并降低PC12细胞凋亡率[(9.8±3.1)％,P＜0.05]和Caspase-3活性(0.16 ±0.06,P＜0.05).结论 Notch信号通路活化是鱼藤酮诱导PC12细胞凋亡的主要机制之一.     

TGF-β/Smad Signaling in Kidney Disease

Chronic progressive kidney diseases typically are characterized by active renal fibrosis and inflammation. Transforming growth factor-β1 (TGF-β1) is a key mediator in the development of renal fibrosis and inflammation. TGF-β1 exerts its biological effects by activating Smad2 and Smad3, which is regulated negatively by an inhibitory Smad7. In the context of fibrosis, although Smad3 is pathogenic, Smad2 and Smad7 are protective. Under disease conditions, Smads also interact with other signaling pathways, such as the mitogen-activated protein kinase and nuclear factor-κB pathways. In contrast to the pathogenic role of active TGF-β1, latent TGF-β1 plays a protective role in renal fibrosis and inflammation. Furthermore, recent studies have shown that TGF-β/Smad signaling plays a regulating role in microRNA-mediated renal injury. Thus, targeting TGF-β signaling by gene transfer of either Smad7 or microRNAs into diseased kidneys has been shown to retard progressive renal injury in a number of experimental models. In conclusion, TGF-β/Smad signaling plays a critical role in renal fibrosis and inflammation. Advances in understanding of the mechanisms of TGF-β/Smad signaling in renal fibrosis and inflammation during chronic kidney diseases should provide a better therapeutic strategy to combat kidney diseases.     

In Vivo Anergized T Cells Form Altered Immunological Synapses In Vitro

T cells contact allogeneic antigen presenting cells (APCs) and assemble, at their contact interface, a molecular platform called the immunological synapse. Synapse-based molecules provide directional signals for the T cell--either positive signals, resulting in T-cell activation, or negative signals causing T-cell inactivation or anergy. To better understand the molecular basis of in vivo T-cell anergy we analyzed the contacts made between in vivo anergized T cells and APCs, and determined which signaling molecules were included or excluded from their immunological synapses. Anergy was induced in TCR transgenic mice by the intravenous injection of semiallogeneic donor spleen cells. T cells from anergized mice were mixed with APCs, the T-cell/APC synapses imaged using deconvolution microscopy, and their molecular compositions were determined. T cells from anergic mice formed unstable immunological synapses in vitro with allogeneic APCs and failed to recruit the signaling proteins necessary to initiate T-cell activation. These findings suggest that T-cell anergy induced by exposure to semiallogeneic donor cells is associated with defects in the earliest events of T-cell activation, immunological synapse formation and recruitment of TCR-mediated signaling proteins.     

Annulus fibrosus cells express and utilize C-C chemokine receptor 5 (CCR5) for migration

Background Context

Disc degeneration is associated with the progressive loss of the proteoglycan content of the intervertebral disc, decreased matrix synthesis, higher concentrations of proteolytic enzymes, and increased levels of proinflammatory cytokines. In previous studies, we have shown that C-C chemokine ligand (CCL)2, CCL3, and CCL5 are highly expressed by cultured nucleus pulposus (NP) and annulus fibrosus (AF) cells that have been treated by interleukin-1. The major function of these chemokines is to recruit immune cells into the disc. It is unclear if disc cells can respond to these chemokines. Recent studies by Phillips et al. (2015) showed that NP cells express a number of cytokines and chemokine receptors.

BANCR对甲状腺乳头状癌TSHR表达调控的研究进展

目的本文通过综述相关文献并分析甲状腺乳头状癌(PTC)发生发展过程中TSH/TSHR/c AMP和Ras/Raf/MEK/ERK(MAPK通路)两条信号通路,探讨了BRAF激活的长链非编码RNA(BANCR)对PTC组织中促甲状腺激素受体(TSHR)表达调节表达调控的意义。BANCR表达上调可能会使PTC组织中TSHR的表达降低,从而促进了PTC的发生发展,并可能影响患者预后。因此,明确BANCR对PTC组织TSHR表达调控的作用机制,有助于进一步明确PTC的发生机制,并可能会为PTC的临床诊断和治疗提供新的靶标。     

Notch通路在高浓度葡萄糖腹膜透析液所致大鼠腹膜纤维化模型中的活化

目的 观察Notch通路在高浓度葡萄糖透析液所致大鼠腹膜纤维化模型中的变化并探讨其可能机制。 方法 给予SD雄性大鼠每日腹腔注射高浓度葡萄糖腹膜透析液，于实验后2周和4周杀检。取壁层腹膜组织行光镜检查；免疫印迹检测转化生长因子β1 （TGF-β1）、 E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA) 和I型胶原蛋白（Col I）的表达；RT-PCR检测Notch通路的下游靶基因Hes-1的表达；免疫印迹和RT-PCR检测Notch配体Jagged-1和Notch通路的负性调节因子Numb的表达。 结果 HE染色显示模型组腹膜明显增厚，间皮细胞减少；Masson染色显示壁层腹膜中可见明显的胶原沉积。与健康对照组相比，模型组的TGF-β1、α-SMA 和 Col I的表达增加而E-cadherin的表达下降（均P < 0.01）。4周模型组与对照组相比Jagged-1表达明显增加（P < 0.05），同时Hes-1的表达亦明显增加（P < 0.01），而Notch通路的负性调节因子Numb的表达下降（P < 0.01）。 结论 在高浓度葡萄糖腹膜透析液所致的大鼠腹膜纤维化模型中有Notch通路的活化，而该通路的活化可能与Notch通路的负性调节因子Numb表达的下调有关。高表达Notch通路的负性调节因子，如Numb，可能是治疗腹膜透析患者腹膜纤维化的新途径。