IL-6通过Sirt1/p53/caspase-3通路介导胰岛β细胞凋亡

目的 探讨炎性因子IL-6是否通过Sirt1/p53/caspase-3通路介导胰岛β细胞凋亡.方法 Western 印迹检测Sirt1在小鼠各组织器官和胰岛β细胞系NIT-1细胞中的表达,免疫荧光法检测Sirt1在细胞中的定位.IL-6（10 ng/ml）处理NIT-1细胞48 h,Hoechst3334染色及流式细胞仪检测细胞凋亡,Western印迹检测细胞内Sirt1、P53、乙酰化P53（acety-P53）、caspase-3和cleaved caspase-3的水平变化.结果 Sirt1在小鼠各组织器官和胰岛β细胞中均有表达,主要定位于细胞核.IL-6处理NIT-1细胞后,伴随Sirt1表达的显著减少,acety-P53明显上调,p53/caspase-3通路活化,NIT-1细胞凋亡增加.结论 IL-6通过下调Sirt1进而激活p53/caspase-3信号通路引起胰岛β细胞凋亡.     

雷诺嗪对高糖高脂诱导的NIT-1细胞凋亡的保护作用

目的 研究雷诺嗪对高糖高脂诱导的NIT-1胰岛β细胞凋亡的保护作用及Cleaved caspase-3表达的影响,探讨雷诺嗪保护胰岛β细胞的机制.方法 采用CCK-8法测定不同浓度的雷诺嗪对体外培养及高糖高脂诱导的NIT-1胰岛β细胞的增殖能力的影响,同时应用流式细胞术检测NIT-1细胞凋亡,Western blot检测凋亡因子Caspase-3活化片段Cleaved caspase-3蛋白的表达.结果 不同浓度的雷诺嗪对NIT-1胰岛β细胞保护作用呈剂量依赖性:低浓度雷诺嗪对细胞凋亡无明显保护作用,随浓度升高保护作用明显.在培养基中加入高脂高糖及高浓度的雷诺嗪(5μmol/L)共同培养24h,雷诺嗪组细胞凋亡率明显低于高脂高糖单独作用组(P＜0.01),同时相对于高糖高脂组,激活型Caspase3表达明显降低(P＜0.05).结论 雷诺嗪能抑制高糖高脂诱导的NIT-1胰岛β细胞凋亡,其分子机制可能是雷诺嗪对Caspase-3的激活作用.     

氟对小鼠胰岛β细胞增殖和胰岛素分泌的影响

目的:观察不同剂量氟化钠(NaF)对体外培养的小鼠胰岛β细胞增殖活力和胰岛素分泌的影响。方法:选用小鼠胰岛β细胞株Beta-TC-6作为实验对象,分别以0、0.1、0.5、1.0、2.0、4.0、8.0、16.0 mg/LNa F干预24 h、48 h、72 h、96 h观察对β细胞形态学的影响,采用四唑蓝[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT]比色法,检测不同剂量NaF对β细胞增殖活力的影响;用酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法测定不同剂量NaF对β细胞胰岛素分泌的影响。结果:0.5 mg/L、1.0 mg/L的NaF作用72 h时,可使胰岛β细胞增殖活力和胰岛素分泌较对照组明显增强(P0.05);≥8.0 mg/L时随着NaF剂量的增加和作用时间的延长,胰岛β细胞的增殖活力和胰岛素分泌明显减弱(P0.05)且随着NaF剂量的增加和时间的延长,细胞生长缓慢,数量减少,不易贴壁或融合成片,多边形细胞减少,可见较多椭圆或圆形细胞。结论:NaF对胰岛β细胞的增殖和胰岛素分泌呈剂量效应关系,随着剂量的增大和时间的延长对细胞增殖活力和胰岛素分泌能力的抑制逐渐增强。     

TGFB信号通路调节胰岛β细胞增殖的功能研究

该研究主要探讨了转化生长因子β(transforming growth factor β,TGFB)信号通路对胰岛β细胞增殖的影响。以Min6为细胞模型,使用TGFB信号通路激活剂TGFβ1、抑制剂SB-431542分别激活和阻断TGFB信号通路,采用CCK-8细胞活性检测法检测Min6细胞活性,并用流式细胞分选术(fluorescence activated cell sorting,FACS)检测Min6细胞中Ki-67阳性(Ki-67~+)细胞比例。最后用TGFβ1、SB-431542体外处理C57BL/6J小鼠胰岛48 h,免疫荧光检测小鼠胰岛中胰岛素、Ki-67双阳性(Insulin~+Ki-67~+)细胞数量。结果显示,使用TGFβ1处理Min6细胞、小鼠胰岛,Min6细胞活性下降,Ki-67~+ Min6细胞数量减少,小鼠胰岛中Insulin~+Ki-67~+细胞数量减少;使用SB-431542处理Min6细胞、小鼠胰岛,Min6细胞活性上升,Ki-67~+ Min6细胞数量增加,小鼠胰岛中Insulin~+Ki-67~+细胞数量增多。综上所述,抑制TGFB信号通路可促进胰岛β细胞的增殖能力,该研究为胰岛β细胞再生疗法的发展提供了理论支持。     

厄贝沙坦对链脲佐菌素和过氧化氢诱导的β细胞氧化损伤保护作用

目的 探讨厄贝沙坦对链脲佐菌素(STZ)和过氧化氢(H₂O₂)诱导的β细胞损伤影响。方法 (1)将NIT-1细胞分为对照组及1、2、5 mmol/L STZ和300、500μmol/L H₂O₂组,处理30 min后,采用Hoechst 33342法检测各组细胞凋亡形态变化,流式细胞术检测细胞凋亡率,RT-qPCR检测Caspase3 mRNA水平。(2)将NIT-1细胞分为STZ及0.001、0.01、0.1 mmol/L厄贝沙坦组,作用24、48和72 h。(3)将NIT-1细胞分为H₂O₂及0.001、0.01、0.1 mmol/L厄贝沙坦组,作用24、48和72 h。采用流式细胞术检测细胞凋亡率和活性氧(ROS)含量,RT-qPCR检测血管紧张素II型1受体(AT1R)mRNA表达。结果 Hoechst 33342染色显示,5 mmol/L STZ组相较于1、2 mmol/L STZ组,500μmol/L H₂O_2<...     

顺铂诱导人鼻咽癌细胞CNE1、CNE2后的辐射敏感性

为研究鼻咽癌细胞CNE1、CNE2多药耐药与辐射敏感性的关系,通过体外逐渐增加顺铂浓度的方法诱导鼻咽癌细胞CNE1、CNE2,MTT法分析环孢霉素A、IFN单药及联合应用对多药耐药的逆转倍数,克隆形成实验分析顺铂诱导鼻咽癌细胞CNE1、CNE2多药耐药前后辐射敏感性的改变,同时研究环孢霉素A、IFN逆转耐药处理对辐射敏感性的影响。结果显示,环孢霉素A、IFN联合应用逆转倍数明显高于单药应用,鼻咽癌CNE1细胞经顺铂诱导后辐射敏感性无明显改变,鼻咽癌CNE2细胞经顺铂诱导后辐射敏感性下调,环孢霉素A、IFN逆转耐药处理可以部分恢复其辐射敏感性。     

TGF-β1对卵巢癌细胞A2780增殖及迁移侵袭的影响

目的:探讨TGF-β1对卵巢癌细胞A2780增殖、迁移及侵袭能力的影响。方法:以体外培养的卵巢癌细胞A2780为研究对象,给予不同浓度(0、2、4…20 ng/m L)TGF-β1处理不同时间(12、24…72 h)。采用CCK-8法检测不同的浓度TGF-β1处理不同时间对卵巢癌细胞A2780增殖的影响。根据增殖实验结果选择合适的TGF-β1作用浓度及处理时间,采用细胞划痕实验测定细胞的迁移能力,Transwell实验检测细胞的侵袭及迁移能力。结果:相较于空白对照组,TGF-β1可以剂量和时间依赖性显著促进卵巢癌细胞A2780的增殖(P0.05)。细胞划痕实验结果显示TGF-β1处理组ΔS%/h明显高于空白对照组(P0.05);Transwell迁移实验结果显示:TGF-β1处理组OD570明显高于空白对照组,差异具有统计学意义(P0.05)。Transwell细胞侵袭实验结果显示:与空白对照组相比,TGF-β1处理组OD570明显升高(P0.05)。结论:TGF-β1可以明显促进卵巢癌细胞系A2780的增殖、迁移及侵袭能力,其促增殖效应呈剂量/时间依赖效应。     

竹节参皂苷Ⅳa通过Akt/mTOR通路保护胰岛β细胞损伤

目的:研究竹节参皂苷Ⅳa(CHS)对高糖诱导的胰岛β细胞损伤的保护作用及其作用机制。方法:采用高糖建立胰岛β细胞损伤模型,分为正常组、模型组、CHS给药低、中和高剂量组(25、50和100μM)。MTT法检测CHS对胰岛细胞存活率的影响,胰岛素释放实验检测CHS对胰岛β细胞功能的影响,试剂盒检测Caspase 3和细胞色素c的水平,蛋白印迹法检测Bax、Bcl-2、Akt、m TORC1、S6K蛋白表达和磷酸化水平变化。结果:与正常组比较,高糖使INS-1细胞存活率降低,胰岛素释放减少,同时Caspase-3,细胞色素c,Bax蛋白表达增加,Bcl-2蛋白表达减少;与模型组比较,CHS可以明显逆转这一趋势(P 0.05)。此外,CHS可剂量依赖性的促进Akt,m TORC1和S6K磷酸化水平,进一步研究发现,CHS保护胰岛INS-1细胞的作用及对m TORC1和S6K磷酸化的作用被si Akt抵消。结论:CHS可以对抗胰岛β细胞的糖毒性,降低胰岛INS-1细胞凋亡,增加胰岛素释放水平,其作用机制可能与激活Akt/mTOR信号通路有关。     

依法克生对大鼠胰岛细胞损伤的保护作用

目的探讨炎症因子对体外培养大鼠胰岛细胞的损伤情况及依法克生可能的保护作用。方法将分离、纯化的SD大鼠胰岛细胞置于体外培养,观察炎症因子IL-1β不同浓度(0.1~10 ng/ml)和不同作用时间(0~48 h)下对胰岛细胞分泌功能影响。胰岛细胞分为对照组、IL-1β组和依法克生组,用单因素方差分析比较各组在低糖和高糖环境下的胰岛素分泌,观察依法克生对胰岛素分泌的影响,对胰岛功能IL-1β损伤的保护作用,并比较三组间胰岛细胞凋亡率。结果高糖浓度下,随IL-1β浓度升高,胰岛素分泌下降(P0.001),随作用时间延长,胰岛素分泌亦减少(P0.001),IL-1β浓度超过5.0 ng/ml、时间超过24 h抑制作用较为明显。依法克生组胰岛素分泌较IL-1β组明显升高(P0.001),与对照组无差异。胰岛细胞凋亡在IL-1β组(49.7±15.5)﹪高于对照组(9.7±2.5)﹪(P0.01),在依法克生组(15.7±5.5)﹪低于IL-1β组(P0.01),提示干预后胰岛细胞凋亡明显减少。结论 IL-1β抑制高糖环境下胰岛素的分泌,并存在剂量和时间依赖关系。依法克生加入体外培养的胰岛细胞中,可有效防止IL-1β诱导胰岛细胞损伤,逆转被抑制的胰岛分泌功能,并减少胰岛细胞凋亡。     

慢性高剂量胰岛素刺激猪脂肪细胞脂肪分解

为研究慢性高剂量胰岛素对猪脂肪细胞脂肪分解的影响及其分子机制, 分化的猪脂肪细胞在PKA(Protein kinase A)或ERK(Extracellular signal-related kinase)抑制剂预处理或不处理的情况下, 再用不同浓度的胰岛素(0、200、400、800、1600 nmol/L)处理不同时间(24、48、72、96 h), 通过测定甘油释放量检测脂肪细胞的脂解率; 采用RT-PCR和Western blotting检测perilipin A和PPARg2的mRNA和蛋白表达。结果显示, 慢性高剂量胰岛素以剂量和时间依赖性的方式刺激猪脂肪细胞的脂肪分解, 并削弱脂肪细胞对异丙肾上腺素刺激的脂解应答; 同时显著下调perilipin A和PPARg2的mRNA及蛋白表达; 另外, PKA和ERK抑制剂均显著抑制胰岛素刺激的脂肪分解, 但仅ERK抑制剂显著逆转perilipin A基因表达的下调。由此推测, 慢性高剂量胰岛素通过ERK通路抑制perilipin A的表达, 进而刺激猪脂肪细胞的脂肪分解。     

微流控芯片技术在细胞生物学研究中的应用进展

20世纪90年代以来,微流控芯片技术得到了快速发展。由于具有小型化、集成化、高通量、低消耗、分析快速等特点,微流控芯片作为一种新型的生物学研究平台,能够提供传统方法不具备的精细和可控制的细胞研究条件,在细胞生物学研究领域中得到了广泛关注。该文主要介绍其在细胞培养、分选、裂解、计数、凋亡检测、迁移、单细胞捕获、细胞间作用等方面的研究进展。     

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

The confluence of protein engineering techniques and delivery protocols are providing new opportunities in cell biology. In particular, techniques that render the membrane of cells transiently permeable make the introduction of nongenetically encodable macromolecular probes into cells possible. This, in turn, can enable the monitoring of intracellular processes in ways that can be both precise and quantitative, ushering an area that one may envision as cellular biochemistry. Herein, the author reviews pioneering examples of such new cell‐based assays, provides evidence that challenges the paradigm that cell penetration is a necessarily damaging and stressful event for cells, and highlights some of the challenges that should be addressed to fully unlock the potential of this nascent field.     

Ultrastructural evidence for two-cell and three-cell neural pathways in the tentacle epidermis of the sea anemone Aiptasia pallida.

Sensory and ganglion cells in the tentacle epidermis of the sea anemone Aiptasia pallida were traced in serial transmission electron micrographs to their synaptic contacts on other cells. Sensory cell synapses were found on spirocytes, muscle cells, and ganglion cells. Ganglion cells, in turn, synapsed on sensory cells, spirocytes, muscle cells, and other neurons and formed en passant axo-axonal synapses. Axonal synapses on nematocytes and gland cells were not traced to their cells of origin, i.e., identified sensory or ganglion cells. Direct synaptic contacts of sensory cells with spirocytes and sensory cells with muscle cells suggest a local two-cell pathway for spirocyst discharge and muscle cell contraction, whereas interjection of a ganglion cell between the sensory and effector cells creates a local three-cell pathway. The network of ganglion cells and their processes allows for a through-conduction system that is interconnected by chemical synapses. Although the sea anemone nervous system is more complex than that of Hydra, it has similar two-cell and three-cell effector pathways that may function in local responses to tentacle contact with food.     

The role of glutamine, serum and energy factors in growth of enterocyte-like cell lines

Background: Glutamine is routinely added to most cell cultures. Glutamine has been found to be the preferential nutrient to the rapidly replicating intestinal mucosa, but whether this is a metabolic effect or due to other properties of this amino acid is not determined. To study the importance of glutamine on the growth of two enterocyte-like cell lines, the effects of depriving the media or supplementing it with glutamine were assessed in media with different serum and energy supplements. Methods: CaCo-2 and HT-29 cells were grown in serum-free medium, with fetal bovine or synthetic serum, and with or without glucose or galactose. The glutamine content was varied between 0 and 4 mM. All growth assays were performed in triplicate by counting in a hemocytometer. Results: Both cell lines were dependent of serum factors for growth, but displayed distinct requirements on glutamine supplementation. Glutamine was an obligate supplement with dose-dependent correlation to growth (r=0.87, p<0.01) for CaCo-2 cells cultured in synthetic, but not in fetal bovine serum. In HT-29 cells, the correlation between glutamine and growth was significant (r=0,68, p<0,05) only in fetal bovine serum in the absence of galactose. Conclusion: This study shows that glutamine has different growth stimulating effects on two enterocyte-like cell lines studied. This could reflect different modes of action of glutamine on proliferation and differentiation in an enterocyte cell population.     

Of mice, frogs and flies: generation of membrane asymmetries in early development

Embryonic development begins with cleavage of the fertilized egg. Cleavage comprises two major processes: cytokinesis and formation of a polarized epithelial cell layer. The focus of this review is comparison of the generation of membrane polarity during embryonic cleavage in three different developmental model systems. In mammalian embryos, as exemplified by analysis of the mouse, generation of distinct membrane domains is uncoupled from cleavage divisions and is initiated in a specific developmental phase, called compaction. In Xenopus laevis embryos, generation of polarized blastomeres occurs simultaneously with cytokinesis. The origin of specific membrane domains of X. laevis polar blastomeres, however, can be traced back to oogenesis. Finally, in Drosophila melanogaster, generation of polarized cells occurs at cellularization. The relevance of cell adhesion, cell junctions and cytocortical scaffolds will be discussed for each of the model systems. Despite enormous morphologic differences, the three models share many common features; in particular, many important molecular interactions are conserved.     

哺乳动物体细胞核移植中供体细胞的研究进展

在哺乳动物体细胞核移植中,供体细胞是影响其效率的主要因素之一。供体细胞的类型、细胞周期、细胞的培养代数、冷藏与冷冻处理,以及供体动物的性别、年龄等都可能影响核移植胚胎的发育。根据现有资料,简要综述了在哺乳动物体细胞核移植中有关供体细胞的研究进展。     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

Engineering Cardiac Cell Junctions In Vitro to Study the Intercalated Disc

Abstract

This review article discusses a recent work using engineered cardiac cells to study the function of the intercalated disc putting emphasis on mechanical and electrical coupling.     

Mammalian testis: a target of in vivo electroporation

Mammalian spermatogenesis consists of three biologically significant processes: stem cell self-renewal and differentiation, meiosis, and haploid cell morphogenesis. Understanding the molecular mechanisms behind these processes might provide clues to the puzzle of species preservation and evolution, and to treatments for male infertility. However, few useful in vitro systems exist to investigate these processes at present. To elucidate these mechanisms, in vivo electroporation of the testis might be a convenient option. Since DNA solution can be injected into the seminiferous tubule via the rete testis, similar to germ cell transplantation, it is easy to transfect expression vectors into various differentiated germ cells and supporting Sertoli cells with adequate electric shock. Unfortunately, it is difficult to create transgenic animals using this method because of its low efficiency. However, gain- and loss-of-function assays, promoter assays, and tagged-protein behavior assays can be conducted with this technique, as in in vitro culture systems.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.