Hsp60基因在小鼠腭和肢正常与异常发生过程中的表达

背景与目的:研究热休克蛋白60基因(heat shock protein 60 gene,Hsp60)在小鼠胚胎腭、前肢正常和异常发育过程中的表达情况.材料与方法:将受孕后ICR小鼠随机分为实验组和对照组2组,每组各64只,于孕10 d(gestational day 10,GD10),分别经口一次给予实验组孕鼠80 mg/kg的全反式视黄酸,对照组孕鼠给予等体积的大豆油,并分别于GD11～GD18取2组胎鼠的前肢,于GD15～GD17取2组胎鼠的腭,利用实时荧光定量PCR检测Hsp60的表达丰度.结果:Hsp60在正常、异常肢和腭中均有表达.对照肢的表达丰度在GD14和出生前呈高表达,而实验肢的表达在各胚龄无明显差异(P＞0.05);实验肢Hsp60在GD11～GD18的表达水平高于同一胚龄的对照肢(P＜0.05).在正常腭中,Hsp60恒定表达,在异常腭中,Hsp60的表达随胚龄增大而降低;在GD15～GD17的表达丰度为实验腭低于同胚龄的对照腭(P＜0.05).结论:全反式视黄酸所致的短肢中,脚60的表达呈应激性升高;而在异常腭中,Hsp60的表达受到抑制.     

未折叠蛋白反应关键基因在小鼠前肢正常发育和异常发生过程中的表达

背景与目的： 研究未折叠蛋白反应(unfolded protein response,UPR)关键基因在小鼠胚胎前肢正常发育和异常发生过程中的表达情况。 材料与方法： ICR小鼠受孕后,将其随机分为实验组和对照组,每组各64只。于孕第10 d(gestational day 10,GD10),经口灌胃1次给予实验组孕鼠80 mg/kg的全反式视黄酸、对照组孕鼠给予等体积的大豆油,并分别于GD11～GD18取两组胎鼠的前肢,利用实时荧光定量PCR检测UPR关键基因Atf6、Ire1、Perk、Grp78的表达丰度。 结果： 除Ire1在GD18未被检出外,UPR中的上述4个主要基因在GD11～GD18正常肢发育过程中均有表达,且在GD13、GD17时间点前后呈现两个表达峰;其中Grp78的表达丰度最高,Ire1的表达丰度最低。而在实验组异常前肢发育过程中GD15之前上述4个基因的表达丰度均高于对照组正常前肢发育过程中的表达丰度(P<0.05);而GD15后均低于对照组正常前肢的表达丰度(P<0.05),呈相对稳定的低水平状态;且在GD12～GD14上述4个基因有一非常明显的表达高峰,其表达时程比对照组正常肢长,且表达丰度远高于对照组正常肢(P<0.05)。 结论： 全反式视黄酸所致的短肢模型中, UPR关键基因的表达在GD11～GD14显著升高,而在GD16～GD18则受到抑制,推测UPR可能与致畸有关。     

全反式视黄酸致ICR小鼠胎鼠短肢模型的建立

背景与目的：探索建立发生率高、畸形类型明确且易于获得的化学物致小鼠胎鼠短肢畸形的模型的方法。材料与方法：ICR小鼠合笼后,查到阴栓当天为孕期第0天（GDO）,孕鼠共50只,随机分为GD9、GD10、GD11、GD12给全反式视黄酸（all-trans-retinoicacid,atRA）实验组和对照组,共5组,每组10只,实验组经口灌胃1次给予孕鼠80mg／kgatRA,对照组则给予等体积的大豆油,于GD18将孕鼠处死并取出胎鼠,观察不同时间给予孕鼠atRA的胎鼠的短肢畸形情况。结果：于GD10给予孕鼠atRA,可致胎鼠双前肢短小,肱骨、桡骨、尺骨均较正常组短（P均〈0．05）;于GD11给予孕鼠atRA,可致胎鼠双前肢和双后肢短小,肱骨、桡骨、尺骨、股骨、胫骨均较正常组短（P均〈0．05）,腓骨常缺失;于GD12给予孕鼠atRA,可致胎鼠尺骨较正常组短（P〈0．05）。结论：成功建立了全反式视黄酸致小鼠短肢畸形的模型,为进一步研究肢体畸形的分子机制奠定了基础。     

AY245441基因在小鼠腭突发育和腭裂形成中的动态表达

背景与目的： 观察AY245441基因在正常腭组织和全反式视黄酸诱导的小鼠腭裂形成过程中的表达变化,探讨AY245441基因的表达在腭突正常发育和腭裂发生中的作用。 材料与方法： 利用Real-time PCR和原位杂交方法动态检测正常腭组织和全反式视黄酸诱导的腭裂组织中AY245441基因的表达情况。 结果： Real-time PCR结果表明,AY245441基因在GD11～16正常腭突和腭裂组织中均有表达,在正常腭中GD11,GD13和GD14时AY245441表达丰度最高,与其它胚龄相比较差异具有统计学意义(P<0.05);腭裂组中AY245441的表达丰度明显低于正常腭 (P<0.05),而在腭裂组织中各胚龄AY245441的表达丰度差异无统计学意义(P>0.05)。原位杂交结果显示,AY245441基因在GD13正常腭板前部的上皮细胞,GD14腭板前部上皮细胞和间充质细胞中均有表达 ;在GD14正常腭板后部上皮细胞中有少许表达,但间充质细胞中无表达。在GD13腭裂前部上皮细胞中表达量明显下降,在GD14腭裂前部上皮细胞和间充质细胞中表达受到明显抑制,几乎未检出阳性信号;在GD14腭裂后部上皮细胞和间充质细胞中均无表达。 结论： AY245441基因在小鼠腭突组织的生长发育中行使重要功能,与腭裂的发生有密切的关系。     

先天性腭裂形成中胚胎腭器官的电镜观察

背景与目的： 建立先天性腭裂模型观察其形成过程中胚胎腭突细胞的超微结构改变。 材料与方法： 于NIH雌性小鼠受孕第12.5 d(GD12.5)通过腹腔注射磷酸地塞米松(50 mg/kg)诱发NIH小鼠胚胎先天性腭裂模型,同时设正常对照组注射生理盐水相同条件下饲养。实验组和对照组母鼠分别于GD13.5、GD14.5、GD15.5脱颈处死,剖腹取出胎鼠,切取胎鼠头部制作光镜和电镜样本,采用扫描电镜和透射电镜观察胚胎腭发育及先天性腭裂形成过程中腭突细胞的超微结构。 结果： 随着胚胎发育,对照组腭突双侧裂隙逐渐变小,上皮基底膜破坏,腭突中嵴上皮细胞(MEE)细胞核染色质呈块状并边集,并可见上皮细胞内出现分泌物质,胚腭间充质细胞(EPM)细胞之间基质丰富,胚胎GD15.5腭突完全融合;实验组腭突生长缓慢,体积较同期对照组小,随着腭突的发育,腭突表层MEE细胞仍然连接紧密,基底膜完整,上皮细胞多层化,细胞表面出现纤毛,EPM细胞之间基质较少,在GD15.5形成裂隙。 结论： 地塞米松作用后胚胎腭突细胞的正常发育分化受到影响,从而导致腭裂形成。     

维生素A缺乏对小鼠胚胎Hox-3.5 mRNA表达与胚胎发育关系的研究

目的 :探讨Vit.A缺乏对胚胎Hox-3.5mRNA表达的影响及其与胚胎发育的关系。 方法 :初断乳的昆明种雌鼠 ,随机分为正常对照组(N)、Vit.A缺乏组(A)、妊娠第0d补充Vit.A组(B)和妊娠第7d补充Vit.A组(C)。按AOAC方法建立孕鼠Vit.A缺乏模型。在妊娠第12d ,一半孕鼠剖腹取出胎鼠 ,采用原位杂交方法检测小鼠胚胎Hox-3.5mRNA的表达 ,同时检测孕鼠血清Vit.A水平。另一半孕鼠在妊娠第19d剖腹取胎 ,用于检查胚胎发育。 结果:孕鼠血清Vit.A缺乏时 ,孕12d胚胎组织Hox-3.5mRNA的含量明显减少 ;妊娠第0d补充Vit.A ,Hox-3.5mRNA的含量与N组无显著性差异 ;妊娠第7d补充 ,Hox -3.5mRNA的含量虽比A组增加 ,但仍明显低于N组。此外 ,Vit.A缺乏使胎鼠的体重、身长、尾长明显小于正常对照组(P<0.05) ,且出现脑膨出及细小肾等畸形。GD7补充Vit.A组明显好于Vit.A缺乏组 ,但与正常对照组相比仍有差异。交配后0d补充Vit.A组与正常对照组无差异。 结论 :Vit.A缺乏对胚胎发育的影响可能与Vit.A在转录水平调控Hox-3.5mRNA的表达有关。     

HSP70、p53和c—myc在宫颈鳞癌中的表达及与HPV16／18感染的关系

目的探讨热休克蛋白70(heat shock protein 70,HSP70)、抑癌基因p53、原癌基因c-myc在宫颈鳞癌中的表达及其与人乳头瘤病毒(HPV)感染的关系。方法采用免疫组化技术检测45例宫颈鳞癌、12例宫颈原位癌和20例正常宫颈组织中HSP70、p53、c-myc蛋白的表达,同时采用聚合酶链反应(PCR)技术检测HPV16/18的感染情况。结果①宫颈原位癌和宫颈癌中HSP70、p53、c-myc蛋白表达均显著高于正常宫颈组织。②HPV16/18阳性组HSP70表达明显高于HPV16/18阴性组,HPV16/18阴性组p53表达明显高于HPV16/18阳性组,但c-myc表达差异无显著性。结论在HPV感染的应激状态下,HSP70在宫颈鳞癌中过表达,p53则表达降低,c-myc蛋白的表达与HPV感染无相关性。     

蒙药荜茇提取物荜茇宁的致畸试验研究

目的： 探讨蒙药荜茇提取物荜茇宁对大鼠的致畸作用。方法：将Wistar孕鼠75只随机分成5组,每组15只。受试组于妊娠第6～15天分别灌胃高 (500 mg/kg)、中 (100 mg/kg)、低 (20 mg/kg)剂量的荜茇宁混悬液,阴性对照组灌服等容量0.5%羧甲基纤维素钠水溶液,阳性对照组于妊娠第11天一次性腹腔注射环磷酰胺10 mg/kg。记录妊娠第0、3、7、10、13、16、20天孕鼠的体质量。于妊娠第20天处死,剖腹检查受孕情况及胎鼠外观和内脏、骨骼形态。结果：与阴性对照组相比,荜茇宁各剂量组对孕鼠外观,胎鼠外观、胎鼠生长指标和胎鼠顶骨、胸骨、肋骨等骨骼的骨化程度以及胎鼠主要脏器,均无明显影响 (P>0.05)。在实验过程中,500 mg/kg荜茇宁剂量组出现了2个吸收胎,但与阴性组相比,差异无统计学意义 (P>0.05)。结论：荜茇宁在本实验条件下,对孕鼠和胎鼠均无明显的胚胎毒性和致畸毒性。     

菊花浸膏对大鼠的致畸毒性试验

目的:研究菊花浸膏对SD大鼠的胚胎毒性与致畸毒性。方法:孕SD鼠分为4组,分别为菊花浸膏0.68、2.03、6.09g/kg剂量组和对照组,每组15～16只。实验组于受孕的第7～16天连续灌胃给予菊花浸膏,每天1次,对照组灌胃给予同体积纯净水,实验期间记录孕鼠的一般状况、体质量,于受孕第20天剖检孕鼠,检查孕鼠的体质量及妊娠情况,胎鼠的胎盘质量、体质量、身长与外观、骨骼、内脏的畸形情况。结果:菊花浸膏各剂量组孕鼠增重、活胎率、吸收胎率、活胎体质量及身长与对照组比较,差异均无统计学意义(P0.05);菊花浸膏各剂量组胎鼠的外观、骨骼及内脏未见畸形。结论:在本实验条件下,未发现菊花浸膏对SD大鼠的胚胎毒性和致畸毒性。     

热激蛋白47在人脑胶质瘤组织中的表达及其临床意义

目的：研究热激蛋白47（heat shock protein 47, HSP47）在人脑胶质瘤组织中的表达情况及其临床意义。方法：收集2008年1月至2009年12月沈阳军区总医院神经外科收治并手术的92例胶质瘤患者的肿瘤组织和同期手术切除病理诊断为胶质细胞增生的非胶质瘤组织10例,另有15例冻存的胶质瘤组织（收集于2014年5月至2014年6月）,应用免疫组织化学方法、Western blotting检测其中HSP47蛋白的表达部位及表达水平,并结合随访资料分析HSP47蛋白表达与临床病理指标、患者生存期之间的关系。结果：HSP47阳性主要定位于Ⅲ、Ⅳ级胶质瘤细胞胞质和血管内皮细胞,HSP47在胶质瘤组织中表达率为52.17%、在非胶质瘤组织中无表达, χ2检验非肿瘤组和胶质瘤组之间HSP47表达的差异有统计学意义(χ2=9.855, P=0.002)。不同年龄、性别胶质瘤患者间HSP47蛋白的表达差异无统计学意义（P=0.423,P=0.820）; HSP47蛋白随着WHO病理级别增高阳性表达逐渐增多（P<0.05）。HSP47蛋白低表达组患者中位生存时间明显长于高表达组患者\[43（95% CI,22.4～63.6）vs 17（95% CI,14.5～19.5）个月,P<0.01\]。结论：HSP47在胶质瘤中过表达,并与病理级别呈正相关,高表达患者预后不良;HSP47可以作为胶质瘤诊断和治疗的新靶点。     

Influence of regenerative capacity and innervation on oncogenesis in the adult frog (Rana pipiens).

Twenty-two sarcomas were induced in 19 adult frogs (Rana pipiens) treated with 3-methylcholanthrene pellets. Thirteen of these tumors arose first in a denervated forelimb, and only 2 arose first in normal or nerve-supplemented control forelimbs (P = 0.004). The remaining tumors developed either as a second tumor in a tumor-bearing frog or in hindlimbs. The critical role of innervation in regenerative capacity suggests that the predilection to tumor formation in the denervated limbs may have resulted from their lessened regenerative capacity.     

Alterations in neuroblastoma ganglioside synthesis by induction of GD1b synthase by retinoic acid

Recent findings link increased expression of the structurally complex 'b' pathway gangliosides GD1b, GT1b, GQ1b (CbG) to a favourable clinical and biological behaviour in human neuroblastoma (NB). Seeking a model to probe these observations, we evaluated four human NB cell lines. Very low CbG content (4-10%) in three of the four cell lines (LAN-5, LAN-1, SMS-KCNR) reflected the ganglioside pattern observed in the most aggressive NB tumours. Pharmacological alterations of complex ganglioside synthesis in vitro by a 5-7 day exposure to 5-10 microM retinoic acid, which is employed in maintenance therapy of disseminated NB, included markedly increased (i) relative expression of CbG (6.6+/-2.0-fold increase, P=0.037), (ii) relative expression of the analogous 'a' pathway gangliosides, termed CaG (6.4+/-1.4-fold increase in GM1a and GD1a; P=0.010), and (iii) total cellular ganglioside content (2.0-6.3-fold), which in turn amplified the accumulation of structurally complex gangliosides. Substantial increases (2.7-2.9-fold) in the activity of GD1b/GM1a synthase (beta-1,3-galactosyltransferase), which initiates the synthesis of CbG and CaG, accompanied the all-trans retinoic acid (ATRA)-induced ganglioside changes. Thus, increased CbG synthesis in NB cell lines is attributable to a specific effect of ATRA, namely induction of GD1b/GM1a synthase activity. Since the shift towards higher expression of CbG and CaG during retinoic acid-induced cellular differentiation reflects a ganglioside pattern found in clinically less-aggressive tumours, our studies suggest that complex gangliosides may play a role in the biological and clinical behaviour of NB.     

Targeting HSP90 Gene Expression with 17-DMAG Nanoparticles in Breast Cancer Cells

Background: Dysregulation of HSP90 gene expression is known to take place in breast cancer. Here we used D,L-lactic-co-glycolic acid-polyethylene glycol-17-dimethylaminoethylamino-17-demethoxy geldanamycin (PLGA-PEG-17DMAG) complexes and free 17-DMAG to inhibit the expression of HSP90 gene in the T47D breast cancer cell line. The purpose was to determine whether nanoencapsulating 17DMAG improves the anti-cancer effects as compared to free 17DMAG. Materials and Methods: The T47D breast cancer cell line was grown in RPMI 1640 supplemented with 10% FBS. Encapsulation of 17DMAG was conducted through a double emulsion method and properties of copolymers were characterized by Fourier transform infrared spectroscopy and H nuclear magnetic resonance spectroscopy. Assessment of drug cytotoxicity was by MTT assay. After treatment of T47D cells with a given amount of drug, RNA was extracted and cDNA was synthesized. In order to assess HSP90 gene expression, real-time PCR was performed. Results: Taking into account drug load, IC50 was significant decreased in nanocapsulated 17DMAG in comparison with free 17DMAG. This finding was associated with decrease of HSP90 gene expression. Conclusions: PLGA-PEG-17DMAG complexes can be more effective than free 17DMAG in down-regulating of HSP90 expression, at the saesm time exerting more potent cytotoxic effects. Therefore, PLGA-PEG could be a superior carrier for this type of hydrophobic agent.     

全反式维甲酸联合高三尖杉酯碱加阿糖胞苷诱导治疗高危急性早幼粒细胞白血病的临床疗效

目的:探讨维甲酸联合高三尖杉酯碱加阿糖胞苷诱导治疗高危急性早幼粒细胞白血病（APL）的临床疗效与安全性。方法:回顾性分析了2006年9月至2013年7月收治的17例高危APL患者的临床资料;全反式维甲酸剂量、高三尖杉酯碱及阿糖胞苷的中位平均剂量分别为27.2（10~60）mg/d、1（0.2~3）mg/d及50（10~200）mg/d;支持治疗包括连续输注纤维蛋白原、新鲜冰冻血浆、冷沉淀和/或单采血小板悬液。观察凝血功能状态对细胞毒药物化疗效果的影响。结果:17例患者中14例（82.3%）达完全缓解,缓解中位时间30 d,2例（11.8%）患者因脑出血而早期死亡,1例（5.9%）患者在维甲酸诱导治疗4 d后自动出院。结论:个体化剂量与疗程的维甲酸联合高三尖杉酯碱加阿糖胞苷是治疗高危APL的有效措施;化疗时的凝血机制改善是高危APL诱导治疗成功与否关键因素。     

9-cis retinoic acid — a better retinoid for the modulation of differentiation,proliferation and gene expression in human neuroblastoma

To date, the clinical success of 13-cis or all-trans retinoic acid in the treatment of neuroblastoma has been disappointing. In vivo, 13-cis will isomerise to both all-trans and 9-cis retinoic acid, believed to be the main biologically-active isomers. In vitro studies with an N-type neuroblastoma cell line, SH SY 5Y, show that 9-cis is better than other isomers at both inducing morphological differentiation and inhibiting proliferation. RAR-, a gene which may mediate retinoic acid responsiveness and be of prognostic significance, is also more-effectively induced by 9-cis retinoic acid. 9-cis and all-trans retinoic acid do not have synergistic effects on SH SY 5Y cell proliferation and gene expression. A retinoid X receptor (RXR)-specific analogue of 9-cis retinoic acid had similar effects on gene expression to 9-cis retinoic acid alone. In view of these results, 9-cis retinoic acid or stable analogues of this retinoid may have potential for the treatment of neuroblastoma.     

Effects of all-trans retinoic acid as a potential chemopreventive agent on the formation of azoxymethane-induced aberrant crypt foci: Differential expression of c-myc and c-fos mrna and protein

The main objectives were to determine the modulating effects of all-trans retinoic acid on the number, size and multiplicity of aberrant crypt foci as well as the in vivo expression of the genes c-myc and c-fos. These foci, which are hypothesized to be the pre-malignant lesions of colon cancer, were induced in Sprague-Dawley rats with a single injection of azoxymethane. Rats were fed either a control diet (AIN-76) or the control diet to which had been added 75 mg/kg or 150 mg/kg all-trans retinoic acid. Within 4 weeks, we observed that the diets containing all-trans retinoic acid reduced the total number and multiplicity of aberrant crypt foci in the colon. However, all-trans retinoic acid increased the size of the lesions that persisted, possibly due to a greater proportion of lesions with dilated crypts. In situ hybridization and immunohistochemistry were performed on the colons for the in vivo analysis of gene expression in these lesions. The expression of myc-specific mRNA and protein in aberrant crypt foci significantly decreased with both levels of all-trans retinoic acid. In contrast, fos-specific mRNA and protein in aberrant crypt foci significantly increased when 150 mg/kg all-trans retinoic acid was added to the diet. The most important findings of this investigation are that intervention with all-trans retinoic acid in the pre-malignant stage of colon carcinogenesis is effective in decreasing the number and growth of aberrant crypt foci and altering the expression of the c-myc and c-fas genes.