津力达联合通心络减轻高糖环境下肾脏微血管内皮细胞内质网凋亡

目的:观察津力达联合通心络对高糖环境下肾脏微血管内皮细胞内质网凋亡途径的影响,探讨其减轻肾脏微血管内皮细胞损伤的作用机制。方法:将体外培养的肾脏微血管内皮细胞分为正常对照组、高糖组、通心络组、津力达组和通心络+津力达组。干预24 h后,CCK-8法检测细胞活力;流式细胞术检测细胞凋亡率和活性氧簇(ROS)水平。Western blot检测蛋白激酶R样内质网激酶(PERK)、磷酸化PERK(p-PERK)、葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)、caspase-12和Bcl-2的蛋白水平。结果:与正常对照组比较,高糖环境下肾脏微血管内皮细胞活力降低,细胞凋亡率增加,ROS生成增多,PERK、p-PERK、GRP78、CHOP和caspase-12的蛋白水平升高,而Bcl-2的蛋白水平下降(P<0.05)。与高糖组比较,通心络、津力达和通心络+津力达均能不同程度地增强高糖环境下肾脏微血管内皮细胞活力,减少细胞凋亡和ROS生成,抑制PERK、p-PERK、GRP78、CHOP和caspase-12蛋白的水平,提高Bcl-2蛋白的水平(P<0.05)。与津...     

小檗碱改善高糖诱导血管内皮细胞和微血管内皮细胞损伤

目的:探讨小檗碱对高糖引起的人脐静脉血管内皮细胞(HUVEC)和人心脏微血管内皮细胞(HCMEC)损伤的保护作用。方法:采用含5mM葡萄糖的完全培养基培养的HUVEC和HCMEC为对照组;含30、50、100、200mM葡萄糖的完全培养基培养的HUVEC和HCMEC为各高糖组;含100mM葡萄糖和0.01、0.1、1μM小檗碱的完全培养基培养的HUVEC和HCMEC为各小檗碱组,各组处理24h和48h后应用MTT法检测细胞活力。应用诱导型一氧化氮合酶(iNOS)试剂盒检测HUVEC细胞iNOS活性。结果:与对照组比较,30、50、100、200mM葡萄糖浓度依赖性引起HUVEC和HCMEC的细胞活力依次下降(P0.05)。同组细胞处理48h与24h比较,随着作用时间的延长细胞活力降低越明显。0.01μM、0.1μM和1μM小檗碱组较100mM高糖组细胞活力在一定程度上有所改善(P0.05)。与对照组比较,高糖组HUVEC的iNOS活性增加(P0.01),小檗碱组较高糖组iNOS活性明显降低(P0.01)。结论:30-200mM高糖可造成HUVEC和HCMEC损伤,小檗碱对损伤有一定的保护作用,其作用和下调iNOS活性有关。     

几种外用中药成份对离体人脐静脉内皮细胞增殖的影响

目的:体外观察几种常用外用中药有关成分对血管内皮细胞增殖作用的影响,初步探讨其对创伤愈合的可能作用。方法:采用MTT比色法观察中药人参皂苷Rg1、Rh1、黄芪多糖、鹿茸多肽、川芎嗪、麝香酮、桂皮醛和乳香水提物对人脐静脉内皮细胞(HUVEC)增殖的影响。结果:9.75mg/L-2.5g/L的黄芪多糖对HUVEC未表现出增殖促进作用;1.94mg/L-0.5g/L的人参皂苷Rh1促进HUVEC的增殖(P＜0.05或P＜0.01),31mg/L的人参皂苷Rg1抑制细胞增殖(P＜0.05);1mg/L-0.5g/L的鹿茸多肽明显促进HUVEC的增殖,以10mg/L作用最明显(P＜0.01),桂皮醛2g/L时促进HUVEC的增殖(P＜0.05);1g/L的麝香酮明显抑制HUVEC的增殖(P＜0.01);0.5kg/L-2.5kg/L(生药)乳香水提物明显抑制HUVEC的增殖(P＜0.01),抑制增殖率为35.56%-55.56%。川芎嗪0.125g/L-0.5g/L明显抑制HUVEC的增殖(P＜0.01)。结论:益气温阳药物人参(Rh1)、鹿茸(鹿茸多肽)、肉桂(桂皮醛)促进HUVEC增殖;通络活血药物麝香(麝香酮)、乳香(乳香水提物)、川芎(川芎嗪)抑制HUVEC增殖。     

rhEGF激活ERK1/2对人胚胎羊膜细胞株Bcl-2、P53蛋白表达的影响

目的:探讨表皮生长因子(EGF)促人胚胎羊膜细胞(FL)增殖时对细胞外信号调节激酶(ERK1/2)通路的影响及其应用安全性。方法:不同浓度重组人EGF(rhEGF)对培养的FL细胞进行刺激,用MTT法检测细胞增殖、Western蛋白印迹法检测磷酸化ERK1/2及Bcl-2、P53蛋白水平。结果:剂量-效应关系示rhEGF浓度10μg/L时促细胞增殖率最高(42.4%,P<0.01)。rhEGF浓度在10-60μg/L时p-ERK1/2水平明显升高,各浓度组Bcl-2水平无变化,而60μg/L浓度组P53水平明显下降。结论:rhEGF最佳浓度10μg/L是安全的用量,但超大剂量rhEGF的应用可能会降低促细胞凋亡能力。     

脂多糖致HUVEC细胞凋亡和线粒体跨膜电位损伤影响

目的:探讨不同浓度脂多糖致HUVEC细胞凋亡和线粒体跨膜电位损伤相关性。方法:体外培养人脐静脉血管内皮细胞株(HUVEC),先将脂多糖(LPS)分为100、10、1、10-1、10-2、10-3、10-4、10-5μg·ml-18个浓度,刺激0.5、1、2、6、12、24h后用CCK-8法观察细胞增殖影响进行初筛,之后选择脂多糖(LPS)分为100、10、1、10-1、10-2μg·ml-15个浓度刺激HUVEC细胞株12h,Annexin-V/FITC双染后流式细胞仪检测细胞凋亡率,荧光显微镜观察Hochest33342/PI双染后细胞形态,罗丹明染色后荧光显微镜检测细胞线粒体跨膜电位变化。结果:同阴性对照组比较,LPS浓度≥10-2μg·ml-1刺激12、24h均可引起细胞活力明显下降(P0.05),其余浓度引起HUVEC细胞活力下降时间不等。LPS致细胞凋亡率随浓度增加而增加,分别为67.2%、43.5%、23.5%、17.3%、10.6%,呈剂量依赖性;Hochest33342/PI双染显示10-1μg·ml-1以上浓度组HUVEC即出现核浓缩和凋亡小体,甚至有少量死亡细胞;10-1μg·ml-1以上浓度组细胞荧光较强,线粒体跨膜电位降低。结论:10-1μg·ml-1以上浓度LPS刺激12h可致HUVEC细胞凋亡,线粒体跨膜电位下降;LPS10-1μg·ml-1刺激HUVEC 12h即可致理想的细胞凋亡模型。     

L-carnosine对NIT-1胰岛细胞的影响及机制

目的: 研究肌肽(L-carnosine)对高糖培养的NIT-1细胞胰岛素分泌、增殖和凋亡的影响及机制。方法:(1)正常和高糖培养细胞72 h,放射免疫法测定葡萄糖刺激的胰岛素分泌,然后分别换不同浓度葡萄糖和L-carnosine培养,测定胰岛素分泌;(2)细胞分为C组(11.1 mmol/L glucose)、H组 (33.3 mmol/L glucose)、H+A组(33.3 mmol/L glucose +1 mmol/L L-carnosine)和H+B(33.3 mmol/L glucose+ 20 mmol/L L-carnosine)组培养72 h,BrdU检测细胞增殖,流式细胞术检测凋亡,RT-PCR测定bcl-2和caspase-3 mRNA的表达,荧光法检测caspase-3活性。结果:(1)高糖组细胞胰岛素分泌减少; 20 mmol/L L-carnosine显著增加正常和高糖组胰岛素分泌(P<0.01),且呈正相关(P<0.01);(2)高糖组细胞增殖和凋亡均增加(均P<0.01),但总体数目减少;1 mmol/L L-carnosine使增殖增加,凋亡减少(P<0.01);(3) 高糖组caspase-3 mRNA表达明显增加,bcl-2 mRNA明显减少(P<0.05);1 mmol/L L-carnosine使前者明显减少(P<0.05),后者明显增加(P<0.01);不同浓度L-carnosine均明显降低caspase-3活性。结论: 高浓度L-carnosine可单独刺激细胞分泌胰岛素,低浓度的L-carnosine可增加NIT-1细胞增殖并减少高浓度葡萄糖所导致的凋亡。Caspase-3和Bcl-2可能参与了L-carnosine保护NIT-1细胞的过程。     

芥子酸对高糖诱导下大鼠血管平滑肌细胞增殖和凋亡的影响

目的:探讨芥子酸对高糖诱导下大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖和凋亡的影响及机制。方法:将培养的A7r5细胞随机分组处理,MTT法检测细胞活力,Brd U法检测细胞DNA合成,流式细胞术检测细胞周期进程和细胞凋亡,ELISA检测细胞活性氧簇(reactive oxygen species,ROS)生成,Western blot检测cyclin D1、P21和P27等蛋白的表达,以及蛋白激酶C(PKC)和P38的磷酸化水平。结果:与正常组比较,高糖组细胞活力显著升高,DNA合成加快,细胞周期加快,P21和P27表达降低,cyclin D1表达增加,ROS水平增加,细胞凋亡率降低,p-PKC和p-P38蛋白水平增加(P0.05)。而芥子酸(0.1、1和10μmol/L)处理引起细胞增殖活性降低,DNA合成减弱,细胞周期受阻,P21和P27表达增加,cyclin D1表达降低,ROS水平降低,细胞凋亡率升高,p-PKC和p-P38蛋白水平降低,且呈一定浓度依赖性(P0.05)。P38抑制剂SB203580和PKC抑制剂chelerythrine均显著抑制高糖诱导的PKC/P38活化和细胞活力(P0.05)。结论:芥子酸可通过抑制PKC/P38激活降低高糖诱导的VSMCs增殖,并促进细胞凋亡。     

盐酸克伦特罗对人类诱导多能干细胞来源心肌细胞的毒性作用

目的:探讨盐酸克伦特罗对人类诱导多能干细胞(iPSC)来源心肌细胞的毒性作用。方法:将人皮肤来源的iPSC成功定向分化为心肌细胞,加入不同浓度盐酸克伦特罗,使之分为对照组(0μg/ml)、1μg/ml组、10μg/ml组、20μg/ml组、50μg/ml组、100μg/ml组等6组(n均=3),相同条件共培养7天后,观察盐酸克伦特罗对心肌细胞形态大小、搏动频率及凋亡率的影响。结果:不同浓度盐酸克伦特罗均可使心肌细胞缩小(P0.05),50-100μg/ml盐酸克伦特罗可致心肌细胞骨架断裂,甚至呈颗粒状。各种浓度盐酸克伦特罗均致心肌细胞搏动频率加快(P0.05),且有浓度越高搏动越快的趋势。10μg/ml组和50μg/ml组所致心肌细胞凋亡率显著高于对照组(P0.05)。结论:盐酸克伦特罗对人类皮肤来源iPSC分化的心肌细胞有毒性作用。     

巨噬细胞泡沫化对炎症反应的影响

目的:观察巨噬细胞泡沫化对促炎因子水平的影响。方法:取6-8周龄C57BL/6J雄性小鼠骨髓细胞,采用L929培养分化成巨噬细胞。在巨噬细胞悬液中加入不同浓度氧化型低密度脂蛋白(ox-LDL),分为空白对照组(0μg/ml)、10μg/ml组和25μg/ml组,再培养24h后观察各组细胞泡沫化程度,检测分析各组泡沫细胞中促炎因子白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)mRNA的表达水平。结果:所取骨髓细胞被成功分化为巨噬细胞。不同浓度ox-LDL均能诱导巨噬细胞的泡沫化,25μg/ml组巨噬细胞泡沫化程度高于10μg/ml组(P0.01)。与空白对照组相比,25μg/ml组的IL-1β和TNF-αmRNA表达水平均显著降低(P0.05)。结论:巨噬细胞泡沫化可以抑制炎症反应。     

雷公藤内酯醇对AD细胞模型海马神经元凋亡的影响

目的:研究雷公藤内酯醇对阿尔茨海默病(AD)细胞模型海马神经元凋亡的影响,探讨雷公藤内酯醇治疗AD的可能机制。方法:用凝聚态Aβ1-40(20μg/ml)刺激的小胶质细胞条件培养液(MCM)作用于培养的大鼠海马神经元,建立AD细胞模型,应用MTT法和TUNEL染色,观察不同剂量的雷公藤内酯醇(5μg/ml和25μg/ml)在不同时程(2 h和24 h)内对AD细胞模型海马神经元凋亡的影响。结果:加药后2 h除模型MCM组海马神经元凋亡数高于正常对照组和正常MCM组(P<0.05)外,其余各组之间海马神经元凋亡数无明显差异。加药后24 h,模型MCM组海马神经元凋亡数较正常对照组和正常MCM组显著增多(P<0.01);低剂量用药MCM组和高剂量用药MCM组海马神经元凋亡数与模型MCM组比较显著降低(P<0.05,P<0.01);高剂量用药MCM组海马神经元凋亡数较低剂量用药MCM组明显降低(P<0.01)。结论:雷公藤内酯醇对AD细胞模型海马神经元的凋亡具有抑制作用。     

Langerhans Cells and Extra-Epidermal Dendritic Cells

S-100 protein was demonstrated in the cytoplasm of dendritic cells (DCs) in normal and pathologic lymphoid tissues and epidermis in man and several other species. The presence of S-100 protein served to distinguish these cells from other mononuclear cells, most importantly from those of macrophage/histiocyte lineage. Fractionation procedures to isolate and enrich suspensions of DCs were coupled with immunocytochemical techniques to identify S-100-positive cells. Langerhans cells in the epidermis and in aural cholesteatomata and nodal, splenic, and thymic interdigitating cells were S-100-positive. Lymph node and splenic follicular dendritic cells (except in rats) were negative, indicating that this DC may be a separate cell type.     

Label Retaining Cells and Cutaneous Stem Cells

This is a comprehensive review on label retaining cells (LRC) in epidermal development and homeostasis. The precise in vivo identification and location of epidermal stem cells is a crucial issue in cutaneous biology. We discuss here the following problems: (1) Identification and location of LRC in the interfollicular epithelium and hair follicle; (2) The proliferative potential of LRC and their role in cutaneous homeostasis (3); LRC phenomenon and the Immortal Strand Hypothesis, which suggests an alternative mechanism for retention of genetic information; (4) Significance of LRC studies for development of stem cell concept. Now, it seems evident that LRC are a frequent feature of stem cell niches and revealing highly dormant LRC may be used for identification of stem cell niches in different tissues. LRC were used for screening specific markers of epidermal stem cells. Within a given tissue stem cells have different proliferative characteristics. There are more frequently cycling stem cells which function primarily in homeostasis, while LRC form a reserve of dormant, may be ultimate, stem cells, which are set aside for regeneration of injury or unforeseen need. The authors suggest that LRC dormancy described in Mammalia has much in common with developmental quiescence found in some other animals. For example in C. elegans reproductive system, vulval precursor cells have developmentally programmed cell-cycle arrest in the first larval stage, and then undergo an extended period of quiescence before resuming proliferation. Another example of developmental quiescence is the diapause, a widespread phenomenon exhibited by animals ranging from nematodes to mammals, often occurring at genetically predetermined life history stage.     

Comparison of Telomere Length between Buccal Cells and Blood Cells

Bulletin of Experimental Biology and Medicine - Telomere length (TL) in blood cells is commonly used as a proxy for TL in other tissue types. The source of DNA of adequate quality and quantity is...     

Thymic Dendritic Cells and B Cells: Isolation and Function

The thymus is the primary organ in which T cells undergo rearrangement of T cell receptor α and β genes, positive selection for affinity to self MHC products, and elimination (negative selection) of reactivity to self antigens. These events require an interaction of the developing T cell with other cell types in the thymus. The latter include epithelial cells, macrophages, dendritic cells, and the recently described thymic B cells the majority of which are CD5⁺. Here we review the identification and isolation of thymic dendritic cells and CD5⁺ B cells. We consider phenotype, ontogeny, and function, including possible contributions to the induction of self tolerance. Thymic dendritic cells are similar to spleen dendritic cells, but are larger and exhibit a few differences in phenotype. Dendritic cells from both organs are equally potent accessory cells for the MLR and lectin-induced, T cell proliferation. Thymic dendritic cells have higher levels of Fc receptors and support anti-CD3 dependent mitogenesis. Thymic CD5⁺ B cells share phenotypic features with peritoneal CD5⁺ B cells. However thymic B cells neither proliferate nor form antibody producing cells in response to the stimulation with LPS or anti-IgM plus IL-4, but do respond to stimulation with MHC class II-restricted helper T cells. Thymic dendritic cells and CD5⁺ B cells both appear at a similar time in ontogeny, about 14 d of gestation, which is the time T cell differentiation begins to take place. Dendritic cells from spleen, which are potent activators for peripheral T cells, are also potent inactivators for thymic-derived cytotoxic T cells. A correlation between reactivity to MIs products and the expression of TCR-Vβ genes is well documented, and B cells are the primary APC for this antigen. Therefore, thymic CD5⁺ B cells may be a good tool for the investigation of tolerance to Mls products.     

Intrathyroidal Dendritic Cells, Epitheloid Cells, and Giant Cells in Iodine Deficient Goiter

Immunohistochemistry and immunofluorescence were performed on thyroid sections of 44 consecutive patients undergoing thyroid surgery for goiter due to iodine deficiency. Sections were compared with specimens from ten individuals without goiters from the same endemic area, with specimens from ten sporadic nontoxic goiter patients, and with specimens from an area with sufficient iodine supply from nine healthy subjects. Cells were characterized using monoclonal antibodies to the CR3 receptor (CD11b) and the p150/95 antigen (CD11c) present on macrophages, to HLA-DR, to antigen presenting cells (RFD1), to T helper (CD4) and to T suppressor/cytotoxic cells (CD8), and with a polyclonal antibody to human cytokeratin. In iodine deficient goiters, focal aggregates were found of RFD1-positive dendritic cells. Furthermore, RFD1-positive epitheloid cells were seen. In 27% of cases, these epitheloid cells completely filled the thyroid follicles. Within the epitheloid cell clusters, multinucleated giant cells could be detected that carried the macrophage markers. Dendritic cells, epitheloid cells, and giant cells were strongly HLA-DR positive. In nongoitrous thyroids from the endemic area such aggregates could also be seen but they were more sparse and were RFD1 negative. Giant cells were absent there. In normal thyroids with sufficient iodine supply, only a few isolated dendritic cells were seen. All except RFD1, which was negative, showed the same marker pattern. In sporadic nontoxic goiters from an area with sufficient iodine supply, dendritic cells occurred in much higher numbers than in the normal thyroids from that area, and they were RFD1 positive. They never aggregated as in iodine deficiency, and giant cells were not observed. These observations on iodine deficient goiter strongly suggest involvement of active antigen-presenting cells in this disorder. However, the immunohistologic difference between this disease and sporadic goiter suggests different underlying mechanisms.     

Type 1 Regulatory T Cells and Regulatory B Cells Induced by Tolerogenic Dendritic Cells

Dendritic cells (DC) are professional antigen‐presenting cells that are capable of both activating immune responses and inducing tolerance. Several studies have revealed efficiency of therapeutic vaccination with tolerogenic DC (tolDC) in inhibition of experimental autoimmunity. The purpose of this study was to compare four different protocols for generation of tolDC – the antidiabetic drug troglitazone (TGZ DC), NF‐κB inhibitor BAY 11‐7082 (BAY DC), prostaglandin D2 metabolite 15d‐PGJ₂ (PGJ DC) and a combination of dexamethasone and 1α,25‐dihydroxyvitamin D3 (DexVD3 DC) regarding phenotype, cytokine production and T cell stimulatory capacity. TGZ DC and BAY DC had a phenotype comparable to immature DC, while DexVD3 DC were more macrophage like. Analysis of cytokine production using cell culture supernatants from all DC populations revealed that DexVD3 DC were efficient producers of IL‐10 and produced less pro‐inflammatory cytokines. T cells primed with DexVD3 DC showed reduced proliferation, and further analyses of these T cells revealed that functionally effective type 1 regulatory T cells (Tr1) but not FoxP3⁺ Treg were induced. Furthermore, DexVD3 DC promoted the induction of regulatory B cells (Breg). Together, these results indicate that DexVD3 DC have the best potential to be used in a tolerogenic antigen‐presenting cell‐based immunotherapy setting.     

Morphological and Functional Comparison of Subcapsular Small Cells, Subcapsular Large Cells and Inner Layer Cells of Bovine Adrenal Cortex

Subcapsular small cortical cells (SC cells) and subcapsular large cortical cells (LC cells) of bovine adrenal cortex were cultured separately after purification by unit gravity sedimentation, and then compared with inner half layer cells (IL cells) prepared by the same method. Both SC and LC cells were polygonal in shape and their mitochondria were elongated with lamellar cristae. SC cells became as large as LC cells on day 6 of culture with increased cytoplasmic lipid droplets, whereas IL cells showed no change in size. IL cells were spindle-shaped and had mitochondria with tubulovesicular cristae. Both SC and LC cells produced 11β deoxycorticosterone, corticosterone, aldosterone and small amounts of 17α hydroxy progesterone (17 α OH Prog) and Cortisol (F). IL cells produced much more 17α-OH Prog and F than SC or LC cells. When stimulated with ACTH, cortical cells in each group showed cellular retraction and their mitochondria became spherical. The amounts of 17. OH Prog and F increased in all groups, especially in IL cells. These results show that LC cells have similar characteristics to SC cells in both morphology and function, and that they differ from IL cells, which correspond to classical fasciculata reticularis cells. Acta Pathol Jpn 41: 428–436, 1991.     

辅酶Q对血管内皮细胞及心肌细胞的保护作用

辅酶Q(CoenzymeQ,CoQ)又名泛醌、癸烯醌 ,是一种存在于动植物细胞中的小分子醌类化合物 ,主要参与线粒体内膜呼吸链的组成,并作为一类递氢体参与细胞的氧化磷酸化过程 ,在ATP合成中具有重要作用,与机体的能量代谢密切相关。此外 ,CoQ还具有生物膜稳定作用和抗氧化的特性 ,在生理和病理状态下发挥重要的生物学功能。近年来CoQ在内皮细胞及心肌细胞相关疾病的治疗方面日益受到人们的关注。1CoQ的概述1.1CoQ的发现CoQ首先由FrederickCrane于1957年在牛的心肌细胞线粒体中分离并提取,同年 ,Morto从缺乏维生素的小鼠肝脏中得到同一种物…