丙酸睾酮对大鼠胸腺细胞凋亡及胸腺中Bcl-2、Bax表达的影响

目的探讨丙酸睾酮在大鼠胸腺退化过程中的作用及其对大鼠胸腺中Bcl-2和Bax表达的影响。方法雄性大鼠去势后给予丙酸睾酮皮下注射,分别于注射后第1、4、7天取材,用半薄切片甲苯胺蓝染色检测胸腺细胞凋亡情况,免疫组织化学法检测胸腺组织中Bcl-2和Bax的表达情况,采用t检验进行统计学处理。结果丙酸睾酮处理组大鼠胸腺组织中可发现较多的凋亡细胞和凋亡小体;去势组大鼠胸腺组织中Bcl-2表达量明显高于假手术组（P〈0.001）,丙酸睾酮处理后Bcl-2表达量明显减少（P〈0.001）;而Bax的表达正好相反,去势组大鼠胸腺组织中Bax表达量明显低于假手术组（P〈0.001）,给予丙酸睾酮后Bax表达量明显增加（P〈0.001）;结论丙酸睾酮可诱导大鼠胸腺细胞凋亡,并抑制大鼠胸腺组织中Bcl-2的表达,促进Bax的表达。     

尼美舒利对乳腺癌MDA-MB-231细胞生长调控作用的研究

目的研究尼美舒利（nimesulide,NIM）体外对乳腺癌细胞MDA-MB-231生长抑制和诱导凋亡的作用,对其机制做进一步探讨。方法 MTT法检测不同浓度、时间NIM对乳腺癌MDA-MB-231细胞的生长抑制率;流式细胞技术分析细胞周期变化,计算细胞凋亡率;免疫组织化学检测bcl-2,bax,cox-2蛋白表达变化;RT-PCR半定量检测COX-2mRNA表达改变;ELISA法检测PGE2水平变化。结果 NIM可抑制乳腺癌MDA-MB-231细胞生长,呈浓度、时间依赖性;不同浓度NIM处理48h后,细胞内Bcl-2、COX-2蛋白及COX-2mRNA表达水平均明显降低,Bax蛋白水平明显升高（P〈0.05）。结论 NIM可抑制乳腺癌细胞MDA-MB-231细胞增殖,且呈时间和剂量依赖性。其机制与抑制COX-2有关外,还可能与调节bcl-2、bax等表达有关。     

熊果酸促进K562细胞凋亡

本研究旨在探讨熊果酸在体外对人红白血病K562细胞系的作和机制。采用MTT法和流式细胞术检测熊果酸对K562细胞的增殖抑制和杀伤效应;用慧星电泳分析熊果酸对K562细胞DNA的损伤;Hoechst33258荧光染色观察DNA片段化：细胞免疫化学染色检测Bcl-2和Bax的表达;Western blotting检测K562细胞内Caspase-3和磷酸化酪氨酸的表达和活性。结果显示熊果酸存在体外对K562细胞具有中度增殖抑制效应,并呈浓度和时间依赖性。在熊果酸作用下K562细胞呈现凋亡征象,同时细胞内Bcl-2表达下降,Bax表达升高,Caspase-3被激活,磷酸化酪氨酸表达下降,说明熊果酸存体外对K562细胞有诱导凋亡作用,而提高Bax的表达、诱导Caspase-3活化及降低BCR／ABL的酪氨酸激酶活性可能足其作用机制之一。     

COX-2表达下调抑制人结肠癌细胞系HT-29增殖并促进凋亡

目的探讨环氧化酶-2(COX-2)表达对大肠癌细胞HT-29细胞的细胞活力、增殖和凋亡等生物学行为的影响。方法利用脂质体将siRNA COX-2及空载体转入HT-29大肠癌细胞中,并设立对照组。48 h后,real-time PCR法检测COX-2、Bcl-2、Bax和基质金属蛋白酶2(MMP2)mRNA表达;Western blot检测COX-2及p-AKT表达;CCK-8法检测细胞活力;Annexin V/PI流式双染检测细胞凋亡;流式细胞计量术检测细胞周期;划痕实验及Transwell实验分别检测细胞迁移和侵袭能力。结果与对照组及空载体组比较,COX-2抑制组COX-2蛋白及mRNA表达量显著降低(P0.01);细胞活力下降(P0.01);细胞凋亡率提高,细胞周期阻滞于G期,细胞迁移及侵袭能力降低(P0.01);Bcl-2及MMP2 mRNA表达下调(P0.01);Bax mRNA表达上调(P0.01);p-AKT蛋白表达下调(P0.01)。结论 COX-2表达量下调后能显著的抑制细胞增殖、迁移与侵袭,并诱导细胞凋亡,可能与下调p-AKT有关。     

雌激素对大鼠胸腺细胞凋亡及Bcl-2、Bax表达的影响

目的:探讨苯甲酸雌二醇对大鼠胸腺Bcl-2和Bax表达及细胞凋亡的影响及其机制.方法:雌性大鼠行卵巢切除术,给予苯甲酸雌二醇后,观察胸腺指数的变化,Hochest33342荧光染色及透射电镜标本观察胸腺细胞凋亡情况,免疫组织化学检测胸腺组织中Bcl-2和Bax的表达情况,原位杂交技术检测Bcl-2、Bax m RNA的表达情况.结果:双侧卵巢切除组大鼠胸腺指数较假手术组增加,双侧卵巢切除+雌激素组大鼠胸腺指数较双侧卵巢切除组减小;假手术组和双侧卵巢切除组大鼠胸腺组织中以正常胸腺细胞为主,偶见凋亡细胞或凋亡小体,双侧卵巢切除+雌激素组可见较多凋亡细胞和凋亡小体;双侧卵巢切除+雌激素组大鼠胸腺组织中Bcl-2表达较双侧卵巢切除组和假手术组增高明显降低,而Bax表达呈现相反趋势;Bcl-2 mRNA、Bax mRNA的表达与Bcl-2、Bax的表达呈一致性.结论:雌激素可以降低大鼠胸腺指数,抑制胸腺组织中Bcl-2的表达,促进Bax的表达,从而诱导大鼠胸腺细胞凋亡,促进雌性大鼠胸腺退化.     

人参皂苷Rg1调控癫痫大鼠海马Bcl-2和Bax的表达

目的:本研究通过建立氯化锂-匹罗卡品诱导大鼠癫痫模型,用人参皂苷Rg1(Rg1)进行干预,检测癫痫大鼠海马中的Bax、Bcl-2的表达及Rg1对其的调控作用,初步探讨Rg1对癫痫可能的神经保护机制。方法:健康成年雄性SD大鼠随机分为5组,即空白对照组,模型组,低、中、高剂量Rg1治疗组,每组20只,观察行为学变化,并于点燃后72 h取脑,分别通过免疫荧光显色、免疫印迹和RT-PCR检测Bax和Bcl-2的蛋白和RNA表达情况。结果:空白组未出现癫痫发作及死亡,不同剂量的Rg1预处理组与模型组相比,大发作潜伏期时间延长。匹罗卡品诱导大鼠癫痫发作后,海马神经元的胞质内凋亡因子Bax免疫阳性反应增强,蛋白产物表达增多,抗凋亡因子Bcl-2表达减少。Rgl预处理组促凋亡因子Bax及其mRNA表达减少,抗凋亡因子Bcl-2及其mRNA表达增加。结论:癫痫发作后可引起凋亡因子的表达变化,而人参皂苷Rg1可以通过下调促凋亡因子Bax的表达,上调抗凋亡因子Bcl-2的表达,从而发挥抗癫痫的神经保护作用。     

丹皮酚抑制小鼠乳腺癌细胞生长作用及机制的研究

目的:研究丹皮酚抑制小鼠EMT6乳腺癌细胞生长作用及分子机制.方法:建立小鼠乳腺癌移植瘤模型,随机分为4组:阴性对照组、阳性对照组(环磷酰胺25 mg/kg体重)、丹皮酚低剂量组(150 mg/kg体重)、高剂量组(300 mg/kg),每组10只.小鼠EMT6细胞株107个/ml接种后24小时给予不同处理,每日1次,腹腔注射.14天后,检测各组肿瘤重量、计算生长抑制率;HE染色、特异性凋亡TUNEL染色对肿瘤组织形态学进行观察,RT-PCR技术检测肿瘤组织中Bcl-2和Bax的mRNA表达.结果:丹皮酚能明显抑制肿瘤细胞生长,低、高剂量丹皮酚组肿瘤重量明显低于对照组(P＜0.01),肿瘤生长抑制率分别为42.64％和52.33％.丹皮酚治疗组肿瘤细胞形态出现核型不规则、核碎裂及凋亡小体.TUNEL染色测得丹皮酚高剂量组凋亡率达到21.24％,与对照组相比具有显著性差异(P＜0.01);肿瘤组织中Bcl-2 mRNA表达明显减弱,而Bax表达却明显增强.结论:丹皮酚通过诱导细胞凋亡抑制乳腺癌细胞生长,其机制可能与启动线粒体凋亡途径有关.     

bFGF对卵巢癌CAOV3细胞Bcl-2、Bcl-xl、Bax、Bad表达的影响

目的研究bFGF调控卵巢癌CAOV3凋亡的信号通路及对Bcl-2、Bcl-xl、Bax、Bad表达的影响。方法无血清饥饿诱导细胞凋亡。分为饥饿对照、bFGF、bFGF PD98059、bFGF Wortmannin组。流式细胞术、DNA Ladder检测细胞凋亡;Western印迹法检测ERK、PKB、Bad活性以及Bcl-2、Bax表达,RT- PCR检测Bcl-2、Bcl-xl mRNA变化。结果bFGF促进p-ERK、p-PKB、p-Bad、Bcl-2表达,抑制Bax表达及饥饿诱导的细胞凋亡。激酶抑制剂PD98059可抑制bFGF对ERK、Bcl-2、Bax的调节作用,Wortmannin可抑制bFGF对PKB、Bad、Bax的调控作用,二者均可阻断bFGF对凋亡的抑制作用。bFGF对Bcl-xl表达无影响。结论bFGF可能通过激活MEK/ERK、P13K/PKB信号途径通路调节Bcl-2、Bax、Bad表达,抑制饥饿诱导的卵巢癌CAOV3细胞凋亡。     

槲皮素对INH诱导的大鼠肝损伤及肝组织Bcl-2和Bax表达的影响

目的探讨槲皮素对异烟肼(INH)致大鼠肝损伤的保护作用。方法将动物随机分为正常对照组、INH组、槲皮素低剂量组、槲皮素高剂量组,灌胃染毒,每天1次,14d后测定大鼠血清中天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)的活性,肝匀浆中丙二醛(MDA)含量、谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)活性,用Westernblot法检测肝组织中Bcl-2和Bax蛋白的表达。结果与正常对照组比较,INH组大鼠血清AST和ALT的活性增强,肝组织的MDA含量升高,SOD和GSH-Px活性降低,Bcl-2蛋白表达减少,而Bax蛋白表达增加;高剂量槲皮素可减轻INH所致的损伤。结论槲皮素对INH所致的大鼠肝损伤具有保护作用,可能与其抗脂质过氧化作用及调节凋亡相关蛋白Bcl-2和Bax的表达有关。     

咖啡酸锗对小鼠U14瘤的抑制作用及其诱导肿瘤细胞凋亡的机制研究

目的：研究咖啡酸锗对荷瘤U14小鼠的体内抑瘤作用及体外抗肿瘤活性，探讨其抗肿瘤作用机制。方法：观察咖啡酸锗对小鼠U14宫颈癌的抑制率；用MG-P染色和透射电镜的方法观察肿瘤细胞的凋亡情况；流式细胞术(FCM)观察瘤组织的细胞凋亡率并分析细胞周期；免疫组化方法观察肿瘤组织中Bax和Bcl-2蛋白表达情况；MTT法评价咖啡酸锗体外抑制U14肿瘤细胞活性。结果：咖啡酸锗低、中、高剂量组对宫颈癌U14的抑瘤率分别为38.50％、47.17％和64.04％（P<0.01）。MG-P染色及电镜观察可见，咖啡酸锗治疗组出现较多的凋亡细胞（P<0.05），可见典型的细胞凋亡的形态学特征。流式细胞术检测表明咖啡酸锗能诱导U14肿瘤细胞凋亡，在G₀-G₁前出现1个明显的凋亡峰，细胞被阻滞在S期。咖啡酸锗处理后肿瘤组织中Bcl-2蛋白表达下调，Bax蛋白表达上调。咖啡酸锗对体外培养的U14细胞增殖均有一定程度的抑制作用，48 h的IC₅₀值为48.57 mg/L。结论：咖啡酸锗在体内、外均能有效抑制小鼠U14瘤细胞的增殖，并诱导肿瘤细胞凋亡。咖啡酸锗上调U14细胞中Bax蛋白的表达，下调Bcl-2蛋白的表达，进而促进肿瘤细胞凋亡，这可能是其发挥抗肿瘤作用的机制之一。     

Colorectal cancer with and without microsatellite instability involves different genes.

There is evidence supporting a multistep genetic model for colorectal tumorigenesis. In familial adenomatosis polyposis (FAP), the inherited defect is a mutation in the APC gene. The vast majority of all sporadic colorectal cancers also show mutations in the APC gene, and the tumorigenesis in sporadic colorectal cancer and FAP is assumed to involve the same genes. Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in DNA mismatch repair genes and, as a result of defective mismatch repair, microsatellite instability (MSI) is frequently seen. Tumorigenesis in HNPCC was first thought to involve mutations in the same genes as in FAP and sporadic colorectal cancer. Recently, however, an alternative pathway to development of colorectal cancer has been suggested in colorectal tumors with MSI, compared to those tumors without the MSI phenotype. We used a consecutive series of 191 sporadic colorectal cancers to find out if there were any differences between the two groups of tumors regarding the prevalence of mutations in the APC, KRAS, TP53, and TGFbetaR2 genes. As expected, 86% (19/22) of MSI-positive tumors showed a mutation in TGFbetaR2, while only one of 164 (0.6%) MSI-negative tumors did. A highly statistically significant negative association was found between MSI and alterations in APC and TP53. The MSI-positive tumors were screened for mutations in exon 3 of beta-catenin, which has been suggested to substitute for the APC mutation in the genesis of colorectal cancer, without finding mutations in any of the 22 MSI-positive tumors. The number of mutations found in KRAS was lower in MSI-positive than in MSI-negative tumors but the difference was not statistically significant. Our results strongly support the idea that carcinogenesis in MSI-positive and MSI-negative colorectal cancer develops through different pathways.     

Lower frequency of allele loss on chromosome 18q in human breast cancer than in colorectal tumors

Inactivation of tumor suppressor genes is thought to be a critical step in tumorigenesis. TheDCC (deleted in colorectal carcinoma) gene, located on the long arm of chromosome 18, has been shown to be frequently deleted in colorectal tumors. To investigate the involvement of allelic deletions on chromosome 18q in breast cancer tumorigenesis we analyzed 28 primary breast tumors and 28 colorectal, tumors (24 carcinomas, 4 adenomas) with four different polymorphic DNA markers detecting RFLPs on chromosome 18q. In breast cancer we found loss of heterozygosity (LOH) in 4 of 27 (15%) informative cases whereas 15 of 25 (60%) colorectal tumors showed allelic deletions. In all cases of allelic loss theDCC locus or its proximal vicinity (locus SSAV1) were involved. LOH on chromosome 18q occurs both in breast and colorectal cancer, yet the frequency of these deletions in breast tumors is lower than in colorectal tumors. Moreover, in breast cancer these mutations were only detected in large and undifferentiated tumors.Abbreviations LOH Loss of heterozygosity     

Microsatellite instability as a predictor of a mutation in a DNA mismatch repair gene in familial colorectal cancer

Germline alterations in human DNA mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer (HNPCC). Mutation analysis of the genes reveals carriers with a high risk of colorectal cancer, who will benefit from surveillance. We wanted to find the best predictive parameter of a germline mutation in those genes among patients with familial colorectal cancer. Affected members from a total of 83 unrelated colorectal cancer families previously analyzed for mutations in MSH2 and MLH1 were used to evaluate different parameters' ability to predict a germline mutation. We studied various clinical criteria such as family structure, age of onset, and prevalence of endometrial cancer, as well as microsatellite instability in the tumors from the families. In total, 124 tumors from 59 of the families were tested for microsatellite instability (MSI) using PCR-based mono- and dinucleotide markers to establish whether the families could be scored as MSI-positive or -negative. The finding of MSI-positive tumors in a family was the best predictor of a germline mutation, and was found in 73% of the MSI-positive, but in less than 3% of the MSI-negative families (P < 0.0001). In contrast, MSI in unselected colorectal cancer is not as useful, since most of these MSI-positive tumors are sporadic. The finding of microsatellite instability in colorectal tumors seems efficient enough even to select those with germline mutations among families fulfilling HNPCC Amsterdam criteria, once used in identification of the DNA mismatch repair genes. Genes Chromosomes Cancer 27:17-25, 2000.     

消化系恶性肿瘤患者血清Hcy测定的临床意义

目的:探讨恶性肿瘤患者血清同型半胱氨酸(Hcy)水平测定的临床意义.方法:选择了我院明确诊断、治疗的138例恶性肿瘤患者和35例正常健康体检者,采用化学发光免疫分析法测定了肿瘤患者和对照组人员的空腹血清Hcy、FA及VitB12三项指标水平.结果:5种肿瘤患者的血清Hcy水平均显著高于对照组(P均＜0.01);而FA水平则5种恶性肿瘤患者均显著低于对照组(P均＜0.01);VitB12测定结果与对照组比较仅有极轻微的下降,统计学处理无显著性意义(P均＞0.05).结论:5种消化系恶性肿瘤患者的血清Hcy及FA水平变化非常显著,有重要临床意义;VitaminB12测定临床价值似乎不大.     

散发性结直肠癌hMSH2的表达及与MIN的关系

目的：观察ｈＭＳＨ２表达改变和微卫星不稳定性（ＭＩＮ）在散发性结直肠癌发生中的作用。方法：应用免疫组化法检测７７例散发性结直肠癌及癌旁正常粘膜组织中ｈＭＳＨ２蛋白的表达,并用ＰＣＲ变性聚丙烯酰胺凝胶电泳－银染法检测肿瘤组织的ＭＩＮ状况。结果;肿瘤组织中ｈＭＳＨ２蛋白表达阳性率为５５％,远低于癌旁正常粘膜９４％的阳性率。     

CDX2和CYP24A1在大肠癌中的表达

目的 通过研究CDX2、CYP24A1在大肠癌中的表达研究,探讨CDX2和CYP24A1的相关性。方法 应用免疫组化S-P法检测100例大肠癌组织及20例正常大肠癌组织中CDX2和CYP24A1的表达。结果 大肠癌组织中的CDX2和CYP24A1的蛋白表达低于正常大肠癌组织中的表达（P<0.05）, 以正常大肠组织作为对照,CDX2 的蛋白水平表达量为100.00%, 癌旁组织和大肠癌组织的 CDX2 蛋白的相对表达量分别为77.12%、46.65%。CYP24A1 的蛋白水平表达量为 43.81%, 癌旁组织和大肠癌组织的 CYP24A1 蛋白的相对表达量分别为75.00%、96.82%。通过Pearson相关分析显示CDX2蛋白表达水平与CYP24A1蛋白表达水平之间呈显著负相关关系（P<0.05）。结论 CDX2和CYP24A1是具有较高敏感性的肠上皮特异性标志物,联合检测CDX2和CYP24A1的表达有助于鉴别良恶性大肠肿瘤。     

Predominance of normal karyotype in colorectal tumors from hereditary non-polyposis colorectal cancer patients

We report the cytogenetic study of 9 colorectal tumors arising in patients with hereditary non-polyposis colorectal cancer (HNPCC). According to the cytogenetic classification of colorectal tumors previously proposed by us, 2 cases were of the trisomic type, 2 were of the monosomic type, and 5 had a normal karyotype. This represents a significant excess of tumors with normal karyotype in HNPCC tumors (56%) compared to sporadic cases (10/184 = 5%).     

Frameshift mutations of human gastrin receptor gene (hGARE) in gastrointestinal cancers with microsatellite instability

Gastrointestinal tumors with DNA mismatch repair (MMR) defects show microsatellite instability (MSI) and harbor frameshift mutations in coding mononucleotide repeats of cancer-related genes (targets). We assessed MSI status in 233 sporadic gastrointestinal tumors. We classified as MSI-H (high-frequency microsatellite instability) 15 (10%) of 150 colorectal cancers and 13 (16%) of 83 gastric cancers. We searched for frameshift mutations in a coding poly(T)(8) tract within the gastrin receptor gene (hGARE), which has a potential role in gastrointestinal carcinogenesis. To this purpose, we screened 43 unstable tumors (including 15 hereditary nonpolyposis colorectal cancer cases previously classified as MSI-H), 98 stable tumors, as well as 3 MMR-deficient and 4 MMR-proficient gastrointestinal cancer cell lines. We found mutations in 8 (19%) of the 43 MSI-H tumors but in none of the 98 stable cancers. hGARE mutation frequency was similar in gastric (23%) and colorectal cancers, including sporadic (13%) and hereditary (20%) cases. All mutated tumors proved to harbor frameshift mutations in other cancer-related genes that are considered as targets in MSI tumorigenesis. The MMR-deficient and gastrin-sensitive LoVo colorectal cancer cells also showed a hGARE heterozygous frameshift mutation, but expressed only the mutated allele. All detected mutations can be predicted to generate a truncated protein carrying amino acid changes. On the basis of genetic findings, we propose hGARE as a new candidate target gene in MSI tumorigenesis. Functional studies are warranted to elucidate the mechanism by which the hGARE mutation might contribute to gastrointestinal carcinogenesis.     

Loss of nuclear p27 (CDKN1B/KIP1) in colorectal cancer is correlated with microsatellite instability and CIMP.

Downregulation of p27 (cyclin-dependent kinase inhibitor-1B, CDKN1B or KIP1) is caused by increased ubiquitin-mediated proteasomal degradation in colorectal cancer, and has been associated with poor prognosis. CpG island methylator phenotype (CIMP) is a phenotype of colorectal cancer with extensive promoter methylation, and associated with high degree of microsatellite instability (MSI-H) and BRAF mutations. We have recently shown that both CIMP and MSI-H are inversely associated with downregulation of p21 (CDKN1A or CIP1), another cyclin-dependent kinase inhibitor. However, no study to date has examined relationship between p27 and CIMP status in colorectal cancer. Using MethyLight assays, we measured DNA methylation in five CIMP-specific gene promoters {CACNA1G, CDKN2A (p16), CRABP1, MLH1 and NEUROG1} in 706 colorectal cancer samples obtained from two large prospective cohorts. Among the 706 tumors, 112 (16%) were CIMP-high tumors with >or=4/5 methylated promoters. We assessed p27 and p53 expressions by immunohistochemistry. Loss of nuclear p27 expression {observed in 231 tumors (33%)} was significantly associated with CIMP-high, MSI-H and BRAF mutations, and these associations were much more pronounced among p53-negative tumors than p53-positive tumors. When CIMP-high and non-CIMP-high tumors were stratified by MSI status (or KRAS and BRAF status), CIMP-high and MSI-H (but not BRAF mutations) were still significantly associated with nuclear p27 loss. Nuclear p27 loss did not appear to be directly related to CDKN2A (p16) methylation. We conclude that downregulation of nuclear p27 is associated with CIMP-high and MSI-H in colorectal cancer. These associations are stronger among p53 wild-type tumors, implying important interplay of p27 and p53 functions (or dysfunctions) in the development of various molecular subtypes of colorectal cancer.