Fas基因转导大肠癌细胞对化疗药物作用敏感性的实验研究

目的:观察表达Fas基因的大肠癌细胞株对化疗药物的敏感性,为今后大肠癌的基因治疗、临床化疗打下实验基础.方法:应用四甲基偶氮唑盐(MTT)微量酶反应观察Fas转导细胞株(Lovo Fas cells)对化疗药物作用的敏感性,实验分组包括:①非转导细胞组;②Fas转导细胞组;③Fas转导细胞+Fas抗体组(抗体结合组),Fas抗体浓度1∶100;各组分别培养3个复孔相应细胞株为对照组,未加化疗药物.将转导细胞和非转导细胞接种于96孔细胞培养板,各组分别加入顺铂(CDDP)、氟尿嘧啶(5-FU)、长春新碱(VCR)已知临床血浆高峰浓度的0.1倍、1.0倍及10倍的剂量,各设3个复孔,培养3天,加入MTT溶液,继续孵育4 h,弃上清,加入二甲基亚砜(DMSO),酶联免疫检测仪上选择590 nm波长测定各孔OD值.结果:各组细胞存活率比较,Fas转导细胞经过CDDP、5-FU、VCR作用细胞存活率在0.1倍、1.0倍、10倍剂量均低于非转导细胞,但无统计学意义(P＞0.05);抗体结合组经上述化疗作用,细胞存活率明显降低,与非转导细胞组和Fas转导细胞组比较,差异有非常显著性意义(P＜0.01).各组与未加化疗药物组比较,细胞存活率随药物浓度增加而明显下降(P＜0.01),以抗体结合组最为明显.结论:Fas转导细胞株与Fas抗体联合作用较非转导细胞株对化疗药物的敏感性明显增强.     

Fas基因转导大肠癌细胞对化疗药物作用敏感性的实验研究

目的:观察表达Fas基因的大肠癌细胞株对化疗药物的敏感性,为今后大肠癌的基因治疗、临床化疗打下实验基础.方法:应用四甲基偶氮唑盐(MTT)微量酶反应观察Fas转导细胞株(Lovo Fas cells)对化疗药物作用的敏感性,实验分组包括:①非转导细胞组;②Fas转导细胞组;③Fas转导细胞 Fas抗体组(抗体结合组),Fas抗体浓度1∶100;各组分别培养3个复孔相应细胞株为对照组,未加化疗药物.将转导细胞和非转导细胞接种于96孔细胞培养板,各组分别加入顺铂(CDDP)、氟尿嘧啶(5-FU)、长春新碱(VCR)已知临床血浆高峰浓度的0.1倍、1.0倍及10倍的剂量,各设3个复孔,培养3天,加入MTT溶液,继续孵育4 h,弃上清,加入二甲基亚砜(DMSO),酶联免疫检测仪上选择590 nm波长测定各孔OD值.结果:各组细胞存活率比较,Fas转导细胞经过CDDP、5-FU、VCR作用细胞存活率在0.1倍、1.0倍、10倍剂量均低于非转导细胞,但无统计学意义(P＞0.05);抗体结合组经上述化疗作用,细胞存活率明显降低,与非转导细胞组和Fas转导细胞组比较,差异有非常显著性意义(P＜0.01).各组与未加化疗药物组比较,细胞存活率随药物浓度增加而明显下降(P＜0.01),以抗体结合组最为明显.结论:Fas转导细胞株与Fas抗体联合作用较非转导细胞株对化疗药物的敏感性明显增强.     

衰老模型大鼠下颌下腺组织结构的改变

目的观察衰老大鼠下颌下腺组织结构形态学改变。方法D-半乳糖诱导大鼠衰老模型，取下颌下腺制备组织切片。光学显微镜下计算腺实质、脂肪和纤维间质的相对体积比。结果正常组腺实质相对体积比明显高于模型组（P〈0．01）；模型组脂肪和纤维间质含量明显高于正常组（P〈0．01）。结论D-半乳糖模型大鼠下颌下腺腺实质成分伴随衰老逐渐减少而被脂肪和纤维间质所替代。     

组织芯片结合图像分析技术研究兔死后DNA不同时间段的变化

目的:测量组织中DNA含量变化,研究不同时间段、不同组织DNA含量变化,与已知人体组织相比较,检验其用于推断死亡经过时间的可能。方法:利用高通量的组织芯片技术,对Feulgen染色后组织芯片细胞DNA用细胞图像分析技术定量分析。结果:兔子死亡后各组织的DNA发生降解,一定时间内DNA降解出现平台期,各组织DNA降解改变平均值与死后经过时间之间都存在显著相关性(P0.05),气管、延髓、软骨组织表现更明显,与人体组织相比较,确定兔组织DNA作为参照的可行性。     

血氧深入图像分析对儿童组织缺氧的判断作用

90年代初期国外发展起来的血氧深入图像分析技术扩展了传统血气分析的概念 ,除了常规指标测定外 ,又特别强调氧在摄入、运送、释放全过程中氧状态的测定与分析 ,采用Ｐｘ氧效应分析综合判断组织氧供状况 ,使血氧分析从动脉血的氧供分析深入到组织内的氧供分析[1,2 ] ,为危重病人的抢救提供重要依据。一、血氧深入图像分析原理与传统的血气分析相比 ,血氧深入图像分析除了测定 ｐＨ、ＰａＣＯ2 、ＰａＯ2 参数外 ,还新增加了反映氧在摄入、运送、释放全过程中的血氧参数与分析图形。新增加的血氧参数有肺内分流 (Ｓｈｕｎｔ)、肺泡动脉氧压差…     

链脲佐菌素糖尿病鼠早期肾脏组织图像分析的定量研究*

目的：在糖尿病肾病早期尿蛋白排泄与肾脏组织学变化相关关系研究的基础上,进一步探讨早期糖尿病肾病肾脏体积肥大的潜在机制。方法：通过ＳＴＺ诱导糖尿病动物模型,ＥＬＩＳＡ法测定其２４小时尿白蛋白排泄率（ＵＡＥＲ）,光电镜观察糖尿病刚成模组及１、２、３、４周不同病程组的糖尿病大鼠肾脏组织学改变,用图像分析肾小球基底膜厚度、肾小球面积、肾小球细胞总数、平均细胞面积。结果：不同病程糖尿病鼠肾小球面积、肾小球细     

促凋亡蛋白Bim基因在甲状腺乳头状癌中的表达及图像分析

目的:探讨促凋亡蛋白Bim基因在甲状腺乳头状癌组织中的表达及其意义。方法:收集武汉大学人民医院和武汉大学中南医院病理2000～2006年手术切除及活检的甲状腺乳头状癌标本40例和甲状腺腺瘤标本20例。采用免疫组织化学方法检测各组组织内促凋亡蛋白Bim基因的表达。利用HPIAS-2000图像分析系统测定促凋亡蛋白Bim基因在甲状腺乳头状癌及甲状腺腺瘤中表达的平均光密度和平均阳性面积率。结果:甲状腺乳头状癌组织中Bim基因呈低表达,甲状腺腺瘤中Bim基因呈高表达。图像分析结果显示两组间差异有显著性意义(P<0.05)。结论:Bim基因在状腺乳头状癌组织中呈低表达,提示Bim基因与甲状腺乳头状癌的发生、发展有一定关系。     

毒性病理学及人工智能数字组织图像分析质量控制概述

随着人工智能和机器学习的快速发展,人工智能对全切片图像的诊断几乎可以媲美病理学家,建立人工智能算法需要大量的数字组织图像训练集数据。数字组织图像分析是通过各种算法分析全切片图像,并从其中提取大量复杂的定量数据集。数字组织图像分析的质量控制不仅非常重要,而且是确保建立高质量数据集和AI算法的基础和前提。本文简要概述了全切片图像的质量控制策略、数字组织图像分析的影响因素和质量控制方法、数字组织图像分析结果的质量控制方法、毒性病理学家在数字组织图像分析中的作用、数据解释和报告以及数字组织图像用于毒性病理学诊断及AI的挑战,以期为我国药物非临床安全性评价毒性试验中使用全切片图像进行毒性病理学诊断及建立AI各种算法提供一定参考。     

急性减压缺氧对大鼠肺组织损伤的研究

目的：观察氧化应激相关指标在模拟急进高原缺氧大鼠肺组织的变化情况。方法成年健康雄性Wistar大鼠40只,随机分为正常对照组、缺氧1、3、5 d组,每组10只。观察缺氧不同时间大鼠肺组织病理学的改变情况,检测肺组织相关生化指标水平。结果缺氧各组大鼠肺组织均有明显损伤,且以缺氧3d组大鼠损伤尤为严重。与正常对照组比较,缺氧各组大鼠肺组织总抗氧化能力( total antioxidant capacity, TAOC)、还原型谷胱甘肽( reduced glutathione, GSH)、谷胱甘肽过氧化物酶( glutathione peroxidase, GSH-PX)、总一氧化氮合酶( total nitric oxide synthase, TNOS)和乳酸脱氢酶(lactate dehydrogenase, LDH)在缺氧3 d组显著降低(P<0．05,P<0．01),丙二醛(maleic dialde-hyde, MDA)、乳酸( lactate, LD)含量显著升高( P<0．01),且随缺氧时间的延长逐渐增加;总超氧化物歧化酶( total superoxide dismutase, TSOD)、Na+K+-ATP酶活力显著降低(P<0．05,P<0．01),一氧化氮(nitric oxide, NO)含量在缺氧1、3 d组明显降低(P<0．01)。结论急进高原缺氧大鼠肺损伤机制与氧化应激反应有关,机体抗氧化能力降低、自由基增加是导致肺组织损伤的重要因素,且损伤程度与缺氧暴露时间有关。     

肾组织中降钙素基因相关肽与糖尿病大鼠早期肾损害的相关性研究

目的：观察糖尿病大鼠肾组织中CGRP的含量变化与24h尿微量白蛋白的关系，为早期防治DN提供理论依据。方法：Wistar大鼠，体重160-200g，于造成糖尿病大鼠模型后第6、8、10、12、14周测定肾组织中的CGRP和24h尿微量白蛋白。结果：肾组织中的CGRP在造模后第6周无明显改变，第8周开始呈进行性下降，并与24h尿微量白蛋白呈显著负相关。结论：动态观察肾组织中CGRP的变化对于DN的早期发现及分析病情有重要意义。     

Formulation of fenbufen suppositories. II. selection of a suppository base using dissolution studies and histological studies in rats

The dissolution of fenbufen and ethanolamine fenbufen from Polyethylene glycol 1500, Witepsol H12 and Suppocire AP suppository bases was determined. The more soluble ethanolamine salt produced significantly faster dissolution than the parent drug, with the Witepsol H12 base giving the most rapid release. In vivo studies showed that the incorporation of ethanolamine fenbufen into Witepsol H12 increases the morphological change in the rectal tissue l h after insertion but there was no difference with the control tissue after 24 h. The data highlights the importance of parallel in vitro and in vivo studies in the development of rectal formulations.     

腺病毒载体介导VEGF cDNA和bFGF cDNA联合治疗缺血皮瓣的实验研究

目的 探讨以腺病毒载体局部应用血管内皮生长因子(Ad-VEGF)和碱性成纤维细胞生长因子(Ad-bFGF)基因治疗对大鼠缺血皮瓣生存的影响.方法 50只SD大鼠随机分成5组,每组10只.在其背部形成8 cm ×2 cm的缺血皮瓣.于皮瓣远端皮下分别注射Ad-VEGF和Ad-bFGF(目的基因组), Ad-VEGF(基因对照组),Ad-bFGF(基因对照组) Ad-GFP(绿色荧光蛋白基因对照组),PBS(磷酸盐缓冲液空白对照组).48 h后按原设计掀起皮瓣并原位缝合,术后7 d,测量皮瓣的成活面积及免疫组化,组织学检查等.结果 目的基因组皮瓣成活面积比及单位面积微血管密度与其它4个对照组有显著性差异.免疫组化目的基因组毛细血管周围VEGF、bFGF沉积.结论 以腺病毒为载体局部应用VEGF和bFGF基因能增加缺血皮瓣的成活面积.     

可卡因对性成熟期大鼠睾丸谷胱甘肽过氧化物酶基因表达的影响

目的：研究可卡因对大鼠睾丸谷胱甘肽过氧化物酶（glutathione peroxidase GSH-PX）基因表达的影响。方法：性成熟期健康SD雄性大鼠皮下注射可卡因制造吸毒动物模型。化学比色法检测睾丸氧化应激指标;半定量RT-PCR方法检测睾丸组织的GSH-PXmRNA表达。结果：可卡因处理第7天,实验组GSH-PX活性、MDA含量与对照组比较无显著性差异（P〉0．05）;而GSH-PXmRNA水平显著高于对照组（P〈0．05）。可卡因处理第14天,实验组GSH-PX活性显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）,而GSH-PXmRNA水平显著高于对照组（P〈0．05）。可卡因处理第21天,实验组GSH-PX活性及mRNA表达水平显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）。可卡因处理第28天,实验组GSH-PX活性及mRNA表达水平显著低于对照组（P〈0．05）,MDA含量显著高于对照组（P〈0．05）。     

D-半乳糖衰老大鼠下颌下腺超微结构的改变

目的探讨实验性衰老大鼠下颌下腺超微结构的改变。方法用D-半乳糖制造衰老大鼠模型。2组大鼠经4%多聚甲醛＋2.5%戊二醛灌注固定,常规方法制备下颌下腺超薄切片,透射电镜下观察主要细胞的超微结构。结果正常组大鼠颗粒曲管（GCT）及腺细胞内粗面内质网、线粒体、高尔基复合体发达,粗面内质网网腔均匀、表面附有较多核糖体颗粒;核膜清晰,染色质分布均匀,可见丰富的质膜内褶。模型组GCT及腺细胞核固缩,染色质浓缩,部分细胞膜断裂,可见线粒体肿胀及嵴断裂,粗面内质网减少、有核糖体脱落现象。结论D-半乳糖模型大鼠下颌下腺超微结构发生明显衰老改变。     

转人α1,2岩藻糖苷转移酶基因猪动脉内皮细胞的活化抑制实验

目的探讨人α1,2岩藻糖苷转移酶(HT)基因转移抑制猪动脉内皮细胞(PAEC)Ⅱ型活化的作用和机制。方法用不同条件刺激转人HT基因和正常的PAEC,在不同时段分别采用细胞酶联免疫吸附测定方法检测细胞间黏附分子(ICAM)-1在细胞表面的表达。采用免疫细胞化学方法检测细胞接受刺激12h后2种PAEC核因子(NF)-κB阳性细胞百分数。结果3种刺激均可以活化PAEC,使其表面ICAM-1的表达逐步升高,并在12h左右达到峰值。实验组和对照组细胞在接受刺激4h后ICAM-1的表达差异有统计学意义(P值均<0.05),实验组细胞ICAM-1的表达量均低于对照组,细胞的活化受到抑制。在不同条件下刺激12h后2种细胞的NF-κB阳性细胞百分数差异有统计学意义(P值均<0.05),表达人HT的PAECNF-κB阳性细胞百分数比正常猪动脉内皮细胞低。结论表达人HT的PAECⅡ型活化受到抑制,人HT基因转移可能一定程度抑制异种移植急性血管排斥反应(AVR),并且可能通过NF-κB发挥作用,PAECⅡ型活化的条件是多样的。     

大黄素抑制脂多糖诱导性动物牙周炎的组织学研究

目的 用脂多糖（LPS）诱导性实验牙周炎模型观察大黄素抑制LPS对动物牙周组织的毒性效应。方法 建立LPS诱导性鼠牙周炎模型。18只大鼠随机分为T组（实验组）和C组（对照组）各9只，T组牙周炎部位局部注射0.1mg/ml大黄素溶液0.4ml,C组局部注射等量生理盐水，均连续3天。分别在用药后0、3、7、14天处死取样，用光镜，透射电镜观察。结果 用药后3，7，14天，C组牙周组织炎症反应明显重于T组，透射电镜下，T组用药后7天牙龈成纤维细胞内的线粒体轻度肿胀，粗面内质网扩张，胶原排列正常，而C组牙龈成纤维细胞的线粒体肿胀，基质空化，严重者线粒体膜破裂，粗面内质网扩张，囊泡化，胶原崩解。结论 大黄素能阻断LPS对动物牙周组织损害。因此作为临床牙周炎的局部用药是可行的。     

基因转移FasL诱导人脑胶质瘤细胞凋亡的体外实验研究

目的：探讨体外基因转移Fas配体（Fas-Ligand,FasL)对恶性人脑胶质瘤细胞凋亡的影响，方法：用携带人FasL cDNA的缺陷型重组腺病毒载体（Ad-FL)，在体外转导5株人脑胶质瘤细胞，并使其表达，通过流式细胞仪，RT－PCR，TUNEL法及荧光显微镜分别进行Fas/FasL表达检测和相关凋亡检测，分析，结果：RT－PCR和流式细胞仪检测5株胶质瘤细胞表达均表达Fas，不表达FasL,而Ad-FL转导的5株胶质瘤细胞均能表达FasL,Fas表达水平与抗Fas抗体诱导胶质瘤细胞凋亡敏感性无相关性，Ad-FL能显著诱导5株胶质细胞瘤在体外凋亡或抑制其生长，结论：重组腺病毒FasL在体外诱导人脑胶质瘤凋亡效果显著。     

Arginine vasopressin gene expression changes within the nucleus accumbens during environment elicited cocaine-conditioned response in rats

It is known that changes in gene expression within the nucleus accumbens (NAc) occur during cocaine dependence development. However, identification of specific genes involved in cocaine conditioning awaits further investigation. We conducted a high throughput gene expression profile analysis of the NAc, during different stages of the environment-elicited cocaine conditioning. Rats were assigned to two different environmental conditions. Cocaine conditioned group received a cocaine injection (10 mg/kg, i.p.) prior to being placed in the activity chambers. Control rats received saline injections before being exposed to their environment. Both groups received a saline injection in their home cage. Conditioning training lasted for 10 days. Animals were then re-exposed to their previously paired environments only on day 12 (test session). We found that the gene for arginine vasopressin (AVP) was differentially expressed on experimental subjects during all stages of environment-elicited cocaine conditioning. To further validate our molecular results, biochemical and immunolocalization experiments were conducted. We found the presence of AVP within accumbal fibers and changes in AVP protein levels following cocaine conditioning. Moreover, we tested the effects of accumbal microinfusions of either AVP receptor V_1A agonist [pGlu⁴, Cyt6, Arg⁸] AVP 4-9 1.0 ng/0.5 μl, or V_1A antagonist (CH2) 5[Tyr (Me) 2] AVP, 1.0 ng/0.5 μl or vehicle solution (0.9% saline solution) during different stages of the cocaine conditioning. Blockade of V_1A receptors within the NAc during acquisition interrupted the expression of the conditioned response, while activation leads to an increase in this response. Our findings propose a new role for AVP in cocaine addiction.     

Regional differences in expression of osteonectin mRNA after administration of cadmium to rats

 Osteonectin gene expression in relation to metallothionein mRNA expression was investigated in various tissues from Cd-treated rats. After a single 50 μmol/kg subcutaneous injection of CdCl₂, Cd predominantly accumulated in the liver and metallothionein gene expression significantly increased concomitantly with Cd accumulation, but no alteration of osteonectin gene expression was observed. In the kidney and lung, both metallothionein and osteonectin mRNA increased significantly but the elevation of metallothionein mRNA levels (1 h after Cd administration) preceded that of osteonectin (3 h after administration). A significant elevation of osteonectin mRNA levels was also observed in the testis after 3 h, but that of metallothionein mRNA occurred after 6 h. Not only accumulation of Cd but also increments in both osteonectin and metallothionein mRNA were minimal in the brain, but a significant increase in gene expression was observed after 1 h for osteonectin and after 3 h for metallothionein. Since, except in the testis, metallothionein gene expression preceded osteonectin gene expression, the induced metallothionein might transpose Cd and thereby affect its levels immediately, thus reducing the levels of Cd available for accumulation in other tissues. Hence, the osteonectin-Cd interaction might be secondary to the metallothionein-Cd interaction. However, the fact that osteonectin mRNA was predominantly induced by Cd administration in the target tissues of Cd toxicity, such as the lung, kidney and testis, suggests the possible involvement of osteonectin in Cd intoxication/detoxication mechanisms. Received: 29 September 1994 / Accepted: 11 January 1995     

Subacute effects of hexabromocyclododecane (HBCD) on hepatic gene expression profiles in rats

Hexabromoyclododecane (HBCD), used as flame retardant (FR) mainly in textile industry and in polystyrene foam manufacture, has been identified as a contaminant at levels comparable to other brominated FRs (BFRs). HBCD levels in biota are increasing slowly and seem to reflect the local market demand. The toxicological database of HBCD is too limited to perform at present a solid risk assessment, combining data from exposure and effect studies. In order to fill in some gaps, a 28-day HBCD repeated dose study (OECD407) was done in Wistar rats. In the present work liver tissues from these animals were used for gene expression profile analysis. Results show clear gender specificity with females having a higher number of regulated genes and therefore being more sensitive to HBCD than males. Several specific pathways were found to be affected by HBCD exposure, like PPAR-mediated regulation of lipid metabolism, triacylglycerol metabolism, cholesterol biosynthesis, and phase I and II pathways. These results were corroborated with quantitative RT-PCR analysis. Cholesterol biosynthesis and lipid metabolism were especially down-regulated in females. Genes involved in phase I and II metabolism were up-regulated predominantly in males, which could explain the observed lower HBCD hepatic disposition in male rats in this 28-day study. These sex-specific differences in gene expression profiles could also underlie sex-specific differences in toxicity (e.g. decreased thyroid hormone or increased serum cholesterol levels). To our knowledge, this is the fist study that describes the changes in rat hepatic gene profiles caused by this commonly used flame retardant.