蛋白酶体功能障碍通过激活P53抑制人骨髓间充质干细胞增殖

 目的：明确蛋白酶体活性下降导致人骨髓间充质干细胞（hBMSCs）增殖能力降低的机制,探讨P53在此过程中的调控作用,以期为组织工程提供数量充足且活性优良的种子细胞。方法：检测早期（第3~4代）与晚期（≥14代） hBMSCs蛋白酶体活性及P53表达变化。将早期hBMSCs分为DMSO对照组、蛋白酶体抑制剂MG132组（10 μmol/L MG132作用4 h）和P53抑制剂pifithrin-α（PFT-α）+ MG132组（20 μmol/L PFT-α预干预1 h,随后加入10 μmol/L MG132作用4 h）,通过BrdU掺入实验和流式细胞术观察细胞增殖能力及细胞周期分布。随后将PFT-α直接作用于晚期hBMSCs,通过BrdU掺入实验检测P53抑制剂对晚期细胞增殖能力的影响。结果：体外培养晚期hBMSCs伴随蛋白酶体活性降低,P53表达水平较早期细胞上调16.89%±4.44%（P<0.05）。给予MG132能够显著抑制hBMSCs增殖,BrdU阳性率降低至33.36%±2.24%（P<0.01）;但是若提前给予PFT-α则能部分拮抗MG132对hBMSCs增殖能力的影响,BrdU阳性率上升至49.23%±2.67%（P<0.05）。流式细胞术结果显示,PFT-α+ MG132组各期细胞分布与DMSO对照组相似,增殖指数显著高于MG132组（P<0.01）。抑制P53激活能够显著提高晚期hBMSCs增殖能力,BrdU阳性率较对照组升高24.73%±8.35%（P<0.01）。结论：传代晚期hBMSCs蛋白酶体活性下降可能通过激活P53、影响细胞周期进程进而导致hBMSCs增殖潜能下调。     

叔丁基对苯二酚激活蛋白酶体活性促进成年小鼠神经干细胞增殖与分化

目的:探讨叔丁基对苯二酚(tBHQ)对成年小鼠神经干细胞(aNSCs)增殖分化能力的影响,寻找维持aNSCs活力的有效手段。方法:成年BALB/c小鼠腹腔注射tBHQ(每日16.7 mg/kg)7 d,收集室管膜下区(SVZ)的脑组织,应用荧光酶标仪定量检测其蛋白酶体的活性;同时小鼠腹腔注射5-溴脱氧尿嘧啶核苷(BrdU),Brd U免疫荧光染色检测SVZ内a NSCs的增殖能力。分离培养SVZ aNSCs,tBHQ(20μmol/L)作用7 d,分析比较tBHQ组与DMSO组a NSCs的蛋白酶体活性、BrdU阳性率以及形成神经球的数量和直径。应用1%的胎牛血清诱导a NSCs分化,早期神经元标记物β-Ⅲ微管蛋白(Tuj1)免疫荧光染色并比较tBHQ组与DMSO组a NSCs向神经元分化的能力。结果:小鼠腹腔注射tBHQ,其SVZ蛋白酶体活性较DMSO对照组上调12.9%±4.6%(P0.05),并且BrdU标记实验显示tBHQ组阳性细胞数为31.3±6.3,与DMSO对照组(15.4±1.3)比较,aNSCs增殖能力显著提高(P0.05)。体外实验结果也显示tBHQ组aNSCs蛋白酶体活性较DMSO对照组上升10.1%±0.8%(P0.01)。tBHQ组Brd U阳性率(31.3%±3.2%)是DMSO对照组(20%±1.5%)的1.6倍(P0.05)。tBHQ组神经球的数量和直径分别是DMSO对照组的1.7倍(P0.05)和1.4倍(P0.05),提示tBHQ促进aNSCs的自我更新。此外,tBHQ组Tuj1阳性率为26.5%±1.6%,较DMSO对照组18.6%±2.1%显著提高(P0.05),提示tBHQ能够促进a NSCs向神经元方向分化。结论:tBHQ能够上调蛋白酶体活性,促进aNSCs的增殖与分化。     

蛋白酶体活性对小鼠神经干细胞活性氧水平的影响

目的:探讨蛋白酶体活性对小鼠神经干细胞(NSCs)活性氧(ROS)水平和增殖能力的影响,寻求维持NSCs活力的有效方法。方法:分离培养新生(P0)和成年(P90)小鼠室管膜下区(SVZ)神经干细胞。比较P0和P90 NSCs成球数量和增殖能力,荧光酶标仪检测蛋白酶体活性,DCFH-DA法测定ROS水平。应用蛋白酶体抑制剂MG132和激活剂18α-GA分别作用于P0和P90 NSCs,CCK-8法、DCFH-DA法和JC-1染色检测蛋白酶体活性改变对NSCs增殖能力、ROS水平和线粒体膜电位的影响。结果:P90 NSCs神经球直径和数量较P0明显减少,增殖活性较P0 NSCs明显下降(P0.001)。此外,P90 NSCs蛋白酶体活性较P0 NSCs降低0.55±0.03(P0.05),但ROS水平却较P0 NSCs升高13.25±0.12倍(P0.001)。MG132作用后P0 NSCs ROS水平呈浓度依赖性升高,其中10μmol/L组较对照组显著升高131%±8.4%(P0.001),增殖活性却降低54.4%±7.8%(P0.001);MG132组NSCs线粒体膜电位较对照组下降。相反,18α-GA作用后P90 NSCs ROS水平呈浓度依赖性降低,其中6和8μg/ml组分别下降2.77±0.20和2.78±0.32(P0.01),增殖活性却是对照组的3.76和5倍(P0.001);18α-GA组NSCs线粒体膜电位较对照组升高。结论:蛋白酶体活性与NSCs ROS水平密切相关,激活蛋白酶体活性可减少ROS对成年NSCs线粒体功能的影响,提高NSCs增殖活力。     

抑制大鼠动脉损伤后早期中膜平滑肌细胞增殖可降低内膜肥厚

目的:探讨早期中膜平滑肌细胞增殖与内膜肥厚的关系。方法:球囊导管损伤大鼠动脉后,在不同时期投予血管紧张素转换酶抑制剂(ACEI)(temocapril-HCl,10mg·kg^-1·d^-1),观察平滑肌细胞BrdU(5-溴脱氧尿嘧啶尿苷)标记率和内膜面积。结果:损伤后动脉中膜平滑肌BrdU阳性细胞出现于术后24h,BrdU标记率呈双峰期变化,第1期在1-3d,峰值在2d为5.3%,第2期在3-7d,峰值在5d为2.7%。在第1个增殖期投予temocapril显著抑制第2个增殖期的BrdU标记率(0.05±0.02)%,对照组为(4.50±0.27)%(P＜0.01),同时也显著抑制了术后10d内膜面积的增加(10670.1μm²±7713.3μm²)(P＜0.01)。而仅在第2个增殖期投药,则不能抑制内膜面积的增加(83499.5μm²±31360.0μm²)(P＞0.05),其程度与未投药组相当。结论:球囊导管损伤后动脉中膜平滑肌细胞第1个增殖期是内膜形成所必需的。     

低氧诱导因子-1α基因促进大鼠局灶性脑缺血后神经干细胞的增殖和分化

目的观察低氧诱导因子-1α(HIF-1α)基因对大鼠局灶性脑缺血后内源性神经干细胞(NSCs)增殖和分化的影响,并探讨HIF-1α对内源性NSCs增殖分化的作用机制。方法建立大鼠大脑中动脉缺血再灌注模型,分为假手术组(sham)、生理盐水组(NS)、腺病毒空载体组(AD)及携带HIF-1α基因的重组腺病毒组(Ad-HIF-1α)。分别将NS、AD和Ad-HIF-lα注射到模型鼠缺血侧侧脑室,观察4组大鼠神经功能缺失评分;免疫组织化学法观察4组大鼠局灶性脑缺血后缺血灶周围促红细胞生成素(EPO)的表达;免疫荧光法计数再灌注不同时间点室管膜下区(SVZ)BrdU阳性细胞(3d、7d、14d、21d、28d)和皮层BrdU/NF200、BrdU/GFAP(28d)阳性双标细胞。结果Ad-HIF-lα组神经功能缺失评分与AD组和NS组比较,结果有统计学差异;EPO表达增强;Ad-HIF-lα组BrdU标记细胞数明显增加;新生细胞分化结果显示,28d时Ad-HIF-lα组BrdU/NF200(47.74±13.52)%、BrdU/GFAP(67.83±20.75)%,与其他组相比均有显著性差异(P<0.05)。结论低氧诱导因子-1α基因可促进大鼠局灶性脑缺血后内源性NSCs的增殖与分化,从而促进神经功能的恢复。     

姜黄素通过调控Notch通路促进大鼠脑缺血后神经干细胞增殖和迁移

 目的： 观察姜黄素对大鼠局灶性脑缺血后室管膜下区（SVZ）神经干细胞(NSCs)增殖和迁移的影响,及其与Notch信号通路的相关性。方法： 采用线栓法制备大鼠大脑中动脉缺血再灌注损伤模型,随机分成假手术组（sham）、缺血再灌注组（I/R）及姜黄素治疗组(I/R+curcumin)。动物在模型成功后1 h连续腹腔注射姜黄素7 d后处死。免疫荧光法检测各组梗死侧SVZ BrdU及BrdU/DCX标记NSCs的数目及迁移趋势。Western blotting方法检测各组Notch通路的中间产物Notch 细胞内域（NICD)的表达。结果： I/R+curcumin组的大鼠梗死侧SVZ BrdU及BrdU/DCX标记的NSC较I/R组显著增多（P＜0.05）,同时其较I/R组更广泛地分布在往病灶迁移的路径上。I/R+curcumin组较I/R组的NICD生成也增多（P＜0.05）。结论： 姜黄素能促进缺血性大鼠脑SVZ NSCs增殖和迁移,其机制可能是通过激活Notch信号通路产生的。     

蛋白酶体抑制剂MG132 对大鼠胶原诱导性关节炎的干预机制

目的：探讨蛋白酶体抑制剂MG132对大鼠胶原诱导性关节炎（Collagen induced arthritis,CIA）的干预效果及作用机制。方法：48只雌性SD大鼠被随机分为空白对照组、CIA模型组、MG132干预模型组,每组16只CIA模型组和MG132干预模型组注射牛Ⅱ型胶原建立CIA模型大鼠,初次免疫后第21天,干预组大鼠以1 mg/kg的剂量每天1次,连续14天皮下注射MG132。建模起每周观察大鼠关节肿胀程度,计算关节炎指数（Arthritis index,AI）,第42天后称重并处死大鼠;HE染色观察关节滑膜组织的病理改变;荧光底物测定法检测滑膜组织20S蛋白酶体的活性;蛋白质印迹法（Western blot）检测大鼠关节滑膜组织NF-κB/p65、IκBα蛋白的表达情况。结果：与CIA模型组比较,MG132干预模型组大鼠关节炎指数在注射MG132后一周明显降低（P<0.05）,关节滑膜组织未见明显增生,只伴有少量炎性细胞浸润。与空白对照组大鼠比较,CIA模型组大鼠关节滑膜组织20S蛋白酶体活性增高;与CIA模型组大鼠比较,MG132皮下注射干预后关节滑膜组织20S蛋白酶体活性降低（P<0.05）。与空白对照组比较,CIA模型组大鼠关节滑膜组织高表达NF-κB/p65蛋白,其中胞核NF-κB/p65表达增高更为明显（P<0.01）,注射蛋白酶体抑制剂MG132干预后,其滑膜组织胞浆及胞核NF-κB/p65蛋白表达均显著减少（P<0.01）。与空白对照组比较,CIA模型组大鼠关节滑膜低表达IκBα蛋白（P<0.01）,注射MG132干预后关节滑膜IκBα蛋白表达显著增多（P<0.01）。结论：大鼠CIA体内实验显示,蛋白酶体抑制剂MG132经皮下注射可明显改善大鼠关节炎症状,其作用机制可能与MG132降低大鼠CIA关节滑膜组织20S蛋白酶体活性,减少其底物蛋白IκBα的表达,从而抑制NF-κB活性有关。     

p53对体外神经干细胞增殖能力的影响

目的:探讨p53对神经干细胞(neural stem cells,NSCs)增殖能力的影响,寻求维持NSCs活力的有效途径。方法:分离出生后0 d(postnatal day 0,P0)和生后90 d(postnatal day 90,P90)小鼠前脑室管膜下区(subventricular zone,SVZ)组织,培养NSCs。比较P0和P90 NSCs增殖能力并通过q PCR和Western Blot检测衰老相关分子p53和p21在新生和成年NSCs的表达变化。应用p53抑制剂PFT-α(20μmol/L)连续作用于P90 NSCs 72 h,通过Brd U掺入实验和CCK-8实验,分析抑制p53对NSCs增殖能力的影响。结果:随年龄增加,NSCs的增殖能力降低,P90组神经球的数量和直径分别是P0组的22.9%±1.2%(P0.01)和63.5%±3.7%(P0.05)。P90NSCs p53和p21 mRNA表达水平分别较P0 NSCs显著增高了1.4±0.05和1.2±0.04(P0.01)。Western Blot结果证明P90 NSCs p53蛋白的表达水平比P0 NSCs增加了1.2±0.01倍(P0.05)。经p53抑制剂PFT-α连续处理P90 NSCs 72 h后,CCK-8结果显示PFT-α组吸光度值1.1±0.02高于DMSO组0.8±0.03(P0.05),Brd U掺入实验也显示PFT-α组Brd U阳性细胞率为43.3%±4.0%显著高于DMSO组24.8%±3.1%(P0.05)。结论:p53信号通路激活可能是导致成年小鼠NSCs增殖能力下降的重要因素,应用p53抑制剂PFT-α能够增加成年NSCs的增殖能力。     

成年小鼠室管膜下区神经干细胞分离及培养方法改良

目的:改良成鼠神经干细胞(NSCs)分离培养方法,优化培养条件,为系统研究成体干细胞增殖与分化特性以及利用成体干细胞进行细胞治疗奠定基础。方法:视交叉前1.5 mm处离段脑组织,分离前脑室管膜下区(SVZ),通过机械性消化与化学性消化相结合的操作方法制备细胞悬液,应用改良无血清培养基悬浮培养NSCs。nestin免疫荧光染色鉴定NSCs,1%胎牛血清诱导分化后免疫荧光染色鉴定NSCs多向分化潜能。系列稀释实验和5-溴脱氧尿核苷(BrdU)掺入实验比较改良培养与常规培养NSCs自我更新能力和增殖潜能。结果:改良培养条件,NSCs 7～9 d可形成nestin阳性神经球。应用1%FBS诱导后,NSCs分化为形态各异的细胞,包括神经元、星形胶质细胞和少突胶质细胞,分化比例分别是(18.6±3.5)%、(73.2±5.2)%和(3.6±0.4)%。系列稀释实验结果显示当细胞接种数量为500、1000和2000个,改良培养NSCs形成次代神经球数量较常规培养对照组明显增多(P<0.05),并且8 h BrdU掺入率也显著增高达(42.4±6.2)%(P<0.05),表明改良培养条件下NSCs自我更新能力和增殖活力良好。结论:本研究建立了一种简便、操作性强、重复性高的成鼠NSCs分离培养方法。     

~(60)Co-γ射线照射对小鼠SVZ神经干细胞增殖的影响

目的:探讨60Co-γ射线照射对成体小鼠侧脑室室管膜下区(SVZ)神经干细胞增殖的影响。方法:28只昆明小鼠随机分成4组,每组7只,分别以0 Gy、5 Gy(小剂量)、15 Gy(中剂量)、30 Gy(大剂量)剂量进行照射,于照射后1周观察侧脑室室管膜下区(SVZ)的5-溴脱氧核苷尿嘧啶(BrdU)阳性细胞形态和数目。结果:照射后1周,各照射组SVZ的BrdU阳性细胞形态与对照组无差别,呈圆形、椭圆形以及梭形等;15 Gy、30 Gy照射组SVZ的BrdU阳性细胞数明显降低(P<0.01),5 Gy照射组SVZ的BrdU阳性细胞数无差别(P>0.05)。结论:大中剂量60Co-γ照射会抑制SVZ神经干细胞的增殖分裂能力。     

18α-甘草次酸通过抗氧化作用促进成年小鼠室管膜下区神经干细胞增殖

目的 探讨18 α-甘草次酸(18α-GA)对成年小鼠室管膜下区(SVZ)神经干细胞(NSCs)增殖的影响及其机制.方法 100只6月龄BALB/c小鼠随机平均分为18α-GA组(腹腔注射18α-GA 40 mg/kg两个月,以DMSO为溶解介质)和DMSO对照组(腹腔注射含等体积DMSO溶解介质的PBS溶液),每组5...     

The effect of proteasome inhibitor MG132 on experimental inflammatory bowel disease

Immunoproteasome up-regulation enhances the processing of nuclear factor-κB (NF-κB) and degradation of IκBα, which correlates with increased amounts of NF-κB in the various cells. Aberrant activation of NF-κB is involved in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to elucidate the effect of proteasome inhibitor MG132 on experimental IBD. We investigated the effects of MG132 on intestinal inflammation and epithelial regeneration in both interleukin-10-deficient (IL-10^−/−) mice and mice with dextran sulphate sodium (DSS)-induced colitis. Body weight, histological findings and tumour necrosis factor (TNF)-α mRNA expression, epithelial cell proliferation and NF-κB p65 activity in colonic tissues were examined. The effects of MG132 on cell proliferation, migration and multiple drug resistance 1 (MDR1) gene expression were determined in vitro. MG132 ameliorated intestinal inflammation of IL-10^−/− mice by decreasing TNF-α mRNA expression in the colonic tissues, which was associated with suppression of NF-κB activation, and reduced significantly the number of Ki-67-positive intestinal epithelial cells. On the other hand, MG132 did not reduce intestinal inflammation in mice with DSS-induced colitis, and delayed significantly the recovery of body weight and epithelial regeneration. MG132 also suppressed significantly epithelial cell proliferation, cell migration and MDR1 gene expression in vitro. Proteasome inhibition reduces T cell-mediated intestinal inflammation, but may interrupt both epithelial regeneration and barrier function of colonic mucosa. Optimal use of proteasome inhibitor should be kept in mind when we consider its clinical application for patients with IBD.     

Alcohol exposure inhibits adult neural stem cell proliferation

Alcohol exposure can reduce adult proliferation and/or neurogenesis, but its impact on the ultimate neurogenic precursors, neural stem cells (NSCs), has been poorly addressed. Accordingly, the impact of voluntary consumption of alcohol on NSCs in the subventricular zone (SVZ) of the lateral ventricle was examined in this study. The NSC population in adult male C57BL/6J mice was measured after voluntary alcohol exposure in a two-bottle choice task using the neurosphere assay, while the number of NSCs that had proliferated 2 weeks prior to tissue collection was indexed using bromodeoxyuridine (BrdU) retention. There was a significant decrease in the number of BrdU-retaining cells in alcohol-consuming mice compared with controls, but no difference in the number of neurosphere-forming cells that could be derived from the SVZ of alcohol-consuming mice compared with controls. Additionally, PCNA-labeled cells in the SVZ tended to be lower, but there was no difference in BrdU labeling in the dentate gyrus following alcohol exposure. To determine alcohol’s direct impact on NSCs and their progeny, neurospheres derived from naïve mice were treated with alcohol in vitro. Neurosphere formation was reduced by 100 mM alcohol without reducing cell viability. These findings are the first to assess the impact of moderate voluntary alcohol consumption on selective measures of adult NSCs and indicate that such exposure alters NSC proliferation dynamics in vivo and alcohol has direct but dissociable effects on the expansion and viability on NSCs and their progeny in vitro.     

Inhibition of the proteasome reduces transfer-induced diabetes in nonobese diabetic mice

Inhibition of the 26S proteasome reduces the severity of several immune-mediated diseases. Here, we report that the proteasome also regulates transfer-induced diabetes in nonobese mice. Treatment of recipient mice with the proteasome inhibitor N(alpha)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (MG132) resulted in a 76% reduction in transfer-induced diabetes. The closely related inhibitor carbobenzoxy-l-leucyl-l-leucinal that inhibits calpains but not the proteasome had no protective effect, suggesting that MG132 acted via inhibition of the proteasome. MG132 decreased proliferation of transferred T cells in the pancreatic lymph nodes in vivo and prevented their expansion in a dose-dependent manner in vitro, consistent with a direct effect by MG132 on the T cells. MG132 did not prevent migration of transferred T cells into the islets but reduced the number of mice with severe infiltration. We suggest that MG132 prevents transfer-induced diabetes by directly targeting the autoreactive T cells and lowering their diabetogenic potential.     

Heterotopically transplanted CVO neural stem cells generate neurons and migrate with SVZ cells in the adult mouse brain

Production of new neurons throughout adulthood has been well characterized in two brain regions, the subventricular zone (SVZ) of the anterolateral ventricle and the subgranular zone (SGZ) of the hippocampus. The neurons produced from these regions arise from neural stem cells (NSCs) found in highly regulated stem cell niches. We recently showed that midline structures called circumventricular organs (CVOs) also contain NSCs capable of neurogenesis and/or astrogliogenesis in vitro and in situ (Bennett et al. [3]). The present study demonstrates that NSCs derived from two astrogliogenic CVOs, the median eminence and organum vasculosum of the lamina terminalis of the nestin-GFP mouse, possess the potential to integrate into the SVZ and differentiate into cells with a neuronal phenotype. These NSCs, following expansion and BrdU-labeling in culture and heterotopic transplantation into a region proximal to the SVZ in adult mice, migrate caudally to the SVZ and express early neuronal markers (TUC-4, PSA-NCAM) as they migrate along the rostral migratory stream. CVO-derived BrdU⁺ cells ultimately reach the olfactory bulb where they express early (PSA-NCAM) and mature (NeuN) neuronal markers. Collectively, these data suggest that although NSCs derived from the ME and OVLT CVOs are astrogliogenic in situ, they produce cells phenotypic of neurons in vivo when placed in a neurogenic environment. These findings may have implications for neural repair in the adult brain.     

BrdU标记法检测不同年龄阶段小鼠神经前体细胞的增殖潜能****

背景：BrdU是胸腺嘧啶核苷类似物,常用来标记前脑室管膜下区具有增殖潜能的神经前体细胞。 目的：建立不同年龄阶段BrdU标记方法,系统分析不同年龄阶段神经前体细胞增殖特点以及变化规律。 方法：选取胚胎期小鼠、新生小鼠、成年小鼠和老年小鼠各5只,腹腔注射BrdU,观察不同年龄阶段神经前体细胞增殖潜能及不同年龄阶段前脑衰老细胞,探讨微环境对神经前体细胞增殖潜能的影响。 结果与结论：胚胎期小鼠、新生小鼠、成年小鼠和老年小鼠前脑室管膜下区BrdU+细胞分别为(897.20±22.38),(262.17±26.26),(76.67±5.63),(43.8±2.61)个,随年龄增加细胞增殖潜能逐渐降低(P < 0.05)。成年小鼠和老年小鼠脑室周围有大量β-半乳糖苷酶染色阳性衰老细胞,说明随年龄增加脑内β-半乳糖苷酶染色活性增强,衰老细胞逐渐增多,神经前体细胞生存微环境失衡可能与神经前体细胞增殖潜能下降有关。     

高压氧对新生大鼠缺氧缺血性脑损伤后在体神经干细胞的影响

目的： 新生鼠的神经生发区-室管膜下区（SVZ） 和海马齿状回（DG）聚集了多种不同发育阶段神经干细胞（NSCs），它们可分化产生新的神经元和胶质细胞。本研究探讨缺氧缺血性脑损伤（HIBD）对脑生发区NSCs的损伤及高压氧（HBO）对此损伤的影响。从在体NSCs 探讨HBO对新生大鼠HIBD的保护作用机制。 方法： 新生7 d龄SD大鼠随机分为4组：① 正常对照组（CON，n=16）；② HIBD 模型组（HIBD，n=16）；③ 高压空气组（HBA，n=16）；④HBO治疗组（n=16）。Rice法复制HIBD模型，并予HBA或HBO治疗，每天1次共7 d。BrdU免疫组化显示在体NSCs。并取损伤侧脑SVZ区组织，体外NSCs培养并进行神经干细胞球计数。 结果： HIBD组生发区在体BrdU 阳性细胞和体外培养的神经干细胞球数目明显少于对照组。HBO组SVZ区的BrdU阳性细胞增多；体外培养的神经干细胞球增多。HBA组增加不明显。 结论： 新生大鼠HIBD后生发区NSCs减少，HBO治疗可以减轻HIBD后NSCs的死亡。HBA治疗无明显作用。