Bcl-2、PCNA基因在鸡、鹌鹑及其杂交种睾丸组织中的表达研究

为探讨鸡(♂)与鹌鹑(♀)属间杂交种睾丸生长发育不良的分子机理,试验采集新罗曼蛋用型公鸡、朝鲜公鹌鹑、鸡与鹌鹑的雄性杂交种不同发育时期的睾丸组织样共计84份,并用实时荧光定量PCR对公鸡、公鹌鹑及其杂交种各阶段睾丸组织中Bcl-2、PCNA基因的mRNA表达进行了检测。结果显示,鸡和鹌鹑不同生长发育期睾丸组织中Bcl-2、PCNA基因mRNA的表达具有相似性,Bcl-2基因整体呈波动性变化,下降到最低值后,迅速回升到较高水平,PCNA基因均有一个极显著的峰值;与鸡和鹌鹑相比,杂交种Bcl-2、PCNA基因mRNA表达均无明显变化,表明杂交种睾丸发育过程中,Bcl-2基因调控睾丸中细胞凋亡的模式不同于鸡和鹌鹑,也不存在明显的细胞增殖过程,从而推测鸡与鹌鹑的杂交种睾丸生长发育不良的分子影响因素与其睾丸组织中Bcl-2、PCNA基因mRNA的异常表达有关。本试验结果为进一步研究鸡与鹌鹑属间杂交种睾丸发育不良的分子机理提供了参考依据。

P^(53)、Cer-bB_2、PCNA基因在胃镜活检组织中的表达及其意义

胃癌的发生、发展和转移涉及多种基因的异常.本文着重研究胃镜活检组织伴有肠上皮化生、异型增生和癌变中P53、Cer-Bb2及PCNA的表达情况,探讨其多项指标联合检测在上述胃部良、恶性疾患中的表达意义及其相关性.

p53、nm23和PCNA在临床Ⅰ期乳腺癌中的表达及其临床病理意义

目的:研究Ⅰ期乳腺癌p53、nm23和PCNA的表达,探讨其与预后的关系。方法:采用免疫组化S-P法检测138例Ⅰ期乳腺癌中p53、nm23、PCNA的表达。结果:p53阳性表达率为59.4%(82/138),其中髓样癌表达高于其它类型84.6%(11/13)。与病理类型、组织学分级无关(P>0.05),但与术后腋淋巴结转移密切相关(P<0.05)。淋巴结阳性p53与淋巴结阴性表达与生存期有明显差异(P<0.05)。nm23阳性表达率为82.6%(114/138),其中髓样癌、导管内癌100%表达(13/13、4/4),其次为导管癌早期浸润、管状腺癌、浸润性小叶癌、浸润性导管癌等,与病理类型、组织学分级相关,与腋淋巴结转移率呈负相关趋势,但无统计学差异(P>0.05)。PCNA阳性率为85.5%(118/138),PCNA平均指数为66.43±23.95,PCNA指数与组织学分级、淋巴结转移及预后有密切关系(P<0.01)。p53、nm23和PCNA协同表达观察结果显示,nm23与PCNA共同表达94例,且呈负相关;p53和PCNA共同表达37例,呈正相关;p53和nm23共同表达71例,且呈负相关;p53、nm23和PCNA协同表达62例,但与淋巴结转移及预后无关。结论:p53、nm23和PCNA在Ⅰ期乳腺癌有不同的表达,可作为反映Ⅰ期肿瘤增殖活性、侵袭力、预测预后的重要指标。为临床治疗提供参考依据。三者协同表达在肿瘤进展转移中的作用有待进一步研究。

泉古菌Sulfolobus islandicus PCNA1的原核表达及其多克隆抗体的制备

泉古菌Sulfolobus islandicus中PCNA有3个同源类似蛋白,本研究选取PCNA1作为研究对象,为获得可溶性表达的PCNA1蛋白,制备多克隆抗体,深入了解PCNA1基因的功能,将PCNA1基因片段克隆到组氨酸标签融合的表达载体pET30a中,利用IPTG诱导,金属螯合亲和层析进行纯化分析表明,融合蛋白大部分可溶。紫外分光光度法测定纯化蛋白的纯度可达90%以上,浓度约为2 mg/mL。免疫家兔制备多克隆抗体,ELISA检测抗血清效价可达1∶10 000以上,Western印迹检测证明抗体特异性良好。

PCNA的过度表达在宫颈鳞癌诊断中的临床意义

目的 通过对 45例宫颈鳞癌中PCNA基因过度表达的研究 ,探讨其在子宫颈鳞形细胞癌诊断中的临床意义。方法 标本采用常规石蜡切片、HE染色及ABC法免疫组化染色 ,光镜观察 ,并对增殖细胞核抗原指数 (PCNALI)进行计数。结果 从正常宫颈鳞状上皮到CINⅡ级到子宫颈鳞癌 ,表达PCNA阳性的细胞范围和阳性强度呈明显增高的趋势 ,三者间PCNALI差异有显著性 (P <0 .0 5 )。在 45例鳞状细胞癌中PCNALI随着肿瘤分级增高而增高。鳞癌Ⅰ级、Ⅱ级和Ⅲ级之间PCNALI差异有显著性 (P <0 .0 5 )。局部淋巴结有转移癌者和无转移癌者PCNALI差异亦有显著性 (P <0 .0 5 )。结论 PCNALI对于检测宫颈鳞癌的发生与发展、早期诊断及判断肿瘤的生物学行为和预测患者的预后等方面具有重要的意义。

Expression of p57^(kip2) and its relationship with clinicopathology, PCNA and p53 in primary hepatocellular carcinoma

AIM: To investigate the expression of p57kip2 and its relationship with clinicopathology, PCNA and p53 in primary hepatocellular carcinoma (HCC). METHODS: Expression of p57kip2, PCNA and p53 in tumor tissues from 32 patients with HCC and 10 liver tissues of normal persons was detected with Elivision immunohistochemical technique. RESULTS: The p57kip2 protein positive-expression rate in HCC was 56.25%, lower than that in normal tissues (100%, P<0.05). The reduced expression of p57kip2 protein correlated significantly with moderate or low differentiation of tumor cells (P = 0.007 <0.05), high clinical stage (P= 0.041 <0.05) and poor prognosis (P= 0.036 <0.05), but did not correlate significantly with metastasis, tumor size, level of AFP and age (P>0.05). The PCNA positive-expression rate was 56.25%, which was correlated significantly with the expression of p57kip2 (P= 0.025<0.05). The p53 positive-expression rate was 46.88%, which was not correlated significantly with the expression of p57kip2 (P>0.05). CONCLUSION: There is a marked loss or absence of p57kip2 expression and high expression of PCNA in HCC, which are involved in carcinogenesis and development of HCC. The p57kip2 and p53 may induce apoptosis via different mechanisms.

Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA

AIM: To investigate the expression of NOS in gastric carcinoma, and to explore the relationship between the expression of nitric oxide synthases (NOS) and p53, PCNA,pathological features and clinical staging of gastric cancer.METHODS: The activity of NOS protein was investigated in 85 samples of human gastric carcinoma and 25 samples of normal gastric mucosal tissue by biochemical assay. We then examined the expression of NOS, p53, PCNA in 85 samples of human gastric cancer was examined by immunohistochemistry, and NOS mRNA expression in 85 gastric cancer tissue specimens by in situ hybridization.RESULTS: Biochemical assay showed that the activity of NOS was significantly higher in gastric carcinoma than in normal gastric mucosal tissues (t = 0.4161, P＜0.01).Immunohistochemistry revealed that endothelial nitric oxide synthase (eNOS) expressed in all samples of normal gastric mucosa, but only 6 cases of 85 gastric cancer specimens showed weak positive immunohistochemical reactions to eNOS (20%). Inducible nitric oxide synthase (iNOS) was expressed strongly in human gastric carcinoma (81.2%). In situ hybridization analysis showed that iNOS mRNA expression was significantly stronger than eNOS mRNA expression in gastric cancer tissue (x2 = 10.23, P＜0.01). The expression of iNOS in gastric cancer was associated with differentiation, clinical stages or lymph node metastases (r= 0.3426, P＜0.05). However,iNOS expression did not correlate with histological classifications and morphological types. The expression of iNOS was significantly correlated with p53 or PCNA expression (r = 0.3612, P＜0.05). The expression of neuronal nitric oxide synthase (nNOS) was not examined by immunohistochemistry and in situ hybridization in gastric cancer specimens and normal gastric mucosa.CONCLUSION: In human gastric cancer, there is an enhanced expression of iNOS, but not of eNOS. NOS promotes the proliferation of tumor cells and plays an important role in gastric cancer spread. Inactivation of antioncogene p53 and overexpression of iNOS might play a synergetic role in the process of carcinogenesis of human gastric carcinoma.

Expression and significance of PTEN and PCNA in human laryngeal squamous cell carcinoma

Objective: To elucidate the expression and significance of PTEN and PCNA in human laryngeal squamous cell carcinoma. Methods: Immunochemical method was used to study 60 cases of laryngeal carcinoma,20 cases of normal laryngeal tissues which were closely adjacent to carcinoma and 10 cases of normal laryngeal tissues. Results: It was showed that PTEN gene was expressed in 85% laryngeal carcinoma tissues. The percentage of lymph node metastasis of laryngeal carcinoma which were negative or positive of PTEN protein was 77.8% and 33.3% respectively, and the difference was significance (P<0.05). Conclusion: Expression of PTEN in laryngeal carcinoma was different from that of normal laryngeal tissues. It may play a role but not important in the tumorigenesis and development of laryngeal carcinoma.

Relationship Between Apoptosis and PCNA Expression of Keratinocytes in Condylomata Acuminata

Objecrive: To investigate the relationship between apoptosis and proliferating cell nuclear antigcn (PCNA)expression of keratinocytes in Condylomata acuminata (CA). Methods: PCNA expression was observed byimmunohistochemistry technique (ABC method) in 51 CAspecimens and 1 normal specimens of foreskin or vaginalmucosae. 55 specimens (40 in the CA group and 15 in thecontrol group) were randomly sampled for in situ labelingof apoptotic cells using the TUNEL method. Results: Positive expression of PCNA in CA and controlgroups were 90.2% and 77.8%, respectively, and theproliferation index in CA group was significantly higherthan that in the control group (P<0.001). The positive rateof apoptosis was 42.5% in the LA group and 53.3% in thecontrol group, and there were no significant differences inthe apoptotic index and apoptosis-proliferation ratiobetween two groups (P>0.05). The proliferation indexshowed a significant negativc correlation with theapoptosis-proliferation ratio (r=-0.62, P=0.01) in the CAgrp. Conclusion: It is suggested that the proliferativeappearance of CA could be due to the imbalance betweencell growth and cell death which is caused by moreproliferation and less apoptosis in keratinocytes.

PCNA基因在小鼠睾丸发育过程中的表达研究

为探讨PCNA基因在小鼠睾丸生后发育全过程中的表达规律,以16组出生后不同发育时期的昆明种正常小鼠睾丸组织为材料,在其石蜡组织切片上进行PCNA蛋白的免疫组化检测及PCNA基因的DNA-mRNA原位杂交检测.结果发现:PCNA蛋白仅在精原细胞及初级精母细胞中表达,其表达峰值出现在小鼠生后第11天;PCNA基因在初级精母细胞、次级精母细胞、圆形精子细胞以及少数精原细胞中均有表达,其中初级精母细胞中的表达信号相对强烈.上述结果表明,PCNA基因参与了小鼠睾丸发育过程中精原细胞及初级精母细胞的DNA复制过程,其mRNA表达和蛋白质表达存在着一定差异.

Clinical Significance of p53, Proliferating Cell Nuclear Antige and bcl-2 Expression in Bladder Transitional Cell Carcinoma

Objective: To correlate the frequency of p53 mutations, bcl-2 expression and the proliferation status (proliferating cell nuclear antigen, PCNA) in patients with bladder cancer with cell proliferation, apoptosis and their clinico-pathologic ?ndings. Methods: Para?n-embedded sections from 39 super?cial (T1G1-G3) and 23 invasive (T2-T4a G3 N0M0) primary transitional cell carcinomas (TCC) in the bladder were investigated immunohistochemically for p53, bcl-2 and PCNA. The median follow-up was 37 months; 24 had recurrences. The proliferation index (PI) was expressed as a percentage of the PCNA-positive cells in the tumor cells. Apoptosis was detected by terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of the TUNEL- positive tumor cells. Results: P53 mutation was identi?ed in 50 patients (80.6%). The mutation was most common in tumors of grade 3 (91.3%) as compared to grade 2 (78.5%) and grade 1 (72.7%, P<0.05). Stage pT2 tumors had a higher frequency of p53 mutation (95.7%) as compared to pTa-1 tumors (74.3%, P<0.01). Only 14 tumors (22.5%) expressed bcl-2; grade 3 tumors expressed bcl-2 signi?cantly more frequently (P<0.05); there was no correlation between bcl-2 and tumor stage. There was no interrelation between p53 mutation and bcl-2 expression (P>0.05). The PI ranged from 17.2% to 41.8% (median 22.4%) and the AI from 1.9% to 3.5% (median 2.9%) in bladder cancer. Statistical analyses revealed a close associations between PI, AI and tumor grade and stage of bladder cancer. Conclusion: P53 mutation correlates with invasion. P53 and PCNA overexpression may o?er valuable additional prognostic information in bladder tumors. With the progression of the tumor grade, cell proliferation may be accompanied by frequent apoptosis in bladder cancer, but the PI increased much more than the AI.

cDNA array screening differentially expressed gene in liver regeneration

Objective: To screen the differentially expressed gene in liver regeneration with cDNA array and gain insight of the pathogenesis of the acute hepatic failure. Methods: Acute liver failure model in Sprague-Dawley (SD) rat was induced by 95% hepatectomy. The differential expression profiles in regenerating rat liver were studied by a cDNA array representing 1176 cDNA clusters. Some genes were randomly selected from a pool of differentially expressed genes and subjected to RT-PCR to further confirm the result from microarray hybridization. Results: The alterations of transcription levels were noted in the expressions of 188 genes. There were 138 genes with their expression up-regulated such as growth factors, PCNA, ribosomal proteins, IL6 and CDKs which were associated with cell cycle regulation, stress, metabolism and proliferation. Conclusion: cDNA array is a powerful tool to explore the gene expression of acute liver failure and is potential for the diagnosis and treatment of liver regeneration.

RASSF1A和PCNA在非小细胞肺癌组织中的表达及意义

目的探讨RASSF1A和PCNA基因在非小细胞肺癌发生发展中的作用。方法非小细胞肺癌患者34例。其中男24例,女10例;年龄39～71(57.5±8.6)岁。鳞癌22例,腺癌12例。TNM分期:Ⅰ～Ⅱ期13例,Ⅲa期21例。采用荧光定量RTPCR和Western blot检测RASSF1A和PCNA在肺癌组织组和正常组织组中的表达水平。结果试验组RASSF1A表达水平(1.39±0.42)较对照组(5.28±2.25)明显降低(P<0.01);而PCNA表达水平(2.44±1.13)较对照组(0.77±0.33)明显增加(P<0.01)。随着病变进展,RASSF1A表达降低,PCNA表达增高(P<0.05)。≤50岁组RASSF1A表达水平低于>50岁组(P<0.05),而PCNA表达水平高于>50岁组(P<0.05)。肺癌组织中RASSF1A与PCNA基因的表达呈负相关(r=-0.84,P<0.01)。结论非小细胞肺癌RASSF1A降低而PCNA表达增强。两种基因异常表达在非小细胞肺癌的发生发展中具有协同作用。

天马颗粒剂抑制人结肠癌细胞株SW480增殖机制研究

目的:探讨天马颗粒剂抑制人结肠癌细胞株SW480增殖的作用,并对其增殖抑制作用机制进行初步探讨。方法:采用不同质量浓度的天马颗粒剂(10^(-1)g·mL^(-1)、10^(-2)g·mL^(-1)、10^(-3)g·mL^(-1)及10^(-4)g·mL^(-1))处理人结肠癌细胞株SW480,同时设立空白对照组(RPMI 1640培养液处理)及阳性对照组(以5-氟尿嘧啶处理)。观察SW480细胞的形态学动态变化,以MTT法检测各组SW480细胞的增殖情况,另以速率法检测各组SW480细胞中碱性磷酸酶(alkaline phosphatase,AKP)比活性的变化情况。建立人结肠癌细胞株SW480免疫抑制小鼠肾包膜下移植瘤模型,同时设立天马颗粒剂组(天马颗粒剂灌胃),空白对照组(生理盐水灌胃)及阳性对照组(5-氟尿嘧啶腹腔注射),8 d后采集肿瘤组织,以免疫组织化学法及图像分析技术定量检测各组移植瘤SW480细胞中Ki-67及PCNA基因的表达情况。结果:随着天马颗粒剂质量浓度的增加,SW480细胞逐渐稀疏,散在生长,细胞变大,透明度差,颗粒增多;当天马颗粒剂质量浓度为10^(-4)g·mL^(-1)时,SW480细胞颜色加深,且部分细胞从瓶壁脱落,甚至部分细胞可见坏死崩解的碎片;实验组10^(-1)g·mL^(-1)、10^(-2)g·mL^(-1)、10^(-3)g·mL^(-1)及10^(-4)g·mL^(-1)的天马颗粒剂均可显著抑制SW480细胞增殖,且随天马颗粒剂作用时间延长及质量浓度增大,SW480活细胞计数逐渐降低(P<0.05);随着质量浓度的增加OD值逐渐降低,增殖抑制率逐渐升高(P<0.05);10^(-1)g·mL^(-1)、10^(-2)g·mL^(-1)、10^(-3)g·mL^(-1)及10^(-4)g·mL^(-1)的天马颗粒剂均会使SW480细胞的AKP比活性升高,且以10^(-2)g·mL^(-1)时的AKP比活性最高,且显著高于其他各组(P<0.05或P<0.01)。阳性对照组AKP比活性与空白对照组比较,差异无统计学意义(P>0.05);Ki-67及PCNA基因主要表达于SW480细胞的细胞核,天马颗粒剂组及阳性对照组Ki-67及PCNA基因阳性着色较空白对照组明显减少,其表达的面积均值及积分光密度均显著降低(P<0.01)。结论:天马颗粒剂可显著抑制人结肠癌细胞株SW480增殖,原因可能与其诱导SW480细胞成熟分化或抑制SW480细胞Ki-67及PCNA基因表达相关。

西罗莫司对移植动脉粥样硬化的预防作用

目的研究新型免疫抑制剂西罗莫司(Sirolimus)对大鼠移植动脉粥样硬化的预防作用。方法建立大鼠颈动脉移植模型,将受体鼠随机分为3组,即空白对照组、环孢素组及Sirolimus组,6周后对移植动脉行病理组织学检测观察移植动脉再狭窄程度、免疫组织化学检测转化生长因子(TGF)-β1和增殖细胞核抗原(PCNA)的表达。结果Sirolimus组血管内膜增厚程度显著低于对照组和环孢素组,其移植动脉再狭窄程度为(15.00±1.21)%,而对照组和环孢素组分别为(40.00±5.91)%、(34.00±6.55)%,差异均有统计学意义(P<0.01);PCNA在Sirolimus组表达明显低于空白对照组和环孢素组,分别为(1.10±0.30)%、(14.20±2.56)%及(4.10±0.62)%,差异有统计学意义;TGF-β1在Sirolimus组表达低于对照组和环孢素组,分别为(5.00±1.39)%、(18.00±1.52)%及(16.00±2.35)%(P<0.01)。结论Sirolimus可预防大鼠移植动脉粥样硬化,TGF-β1和PCNA基因表达的下调可能是Sirolimus预防移植动脉粥样硬化机制中重要环节。