细胞周期素CyclinD1蛋白表达与发育小鼠心肌细胞增殖分化的关系

目的探讨细胞周期素CyclinD1蛋白表达与发育小鼠心肌细胞增殖分化的关系。为先天性心脏病提供理论依据。方法利用HE染色、免疫组织化学和图像分析方法检测胎鼠、出生1、3、6、9、12 d小鼠心肌细胞有丝分裂指数和增殖因子cyclinD1表达情况。结果胎鼠、1、6、12 d小鼠的有丝分裂指数是依次降低的,到12 d时有丝分裂相已经很少。免疫组化在胎鼠、出生1 d的小鼠心肌细胞CycinD1阳性颗粒主要表达在细胞核;出生3 d和出生6 d的小鼠心肌细胞CycinD1阳性颗粒主要分布于核周围的胞浆和细胞核内;9、12 d时CyclinD1颗粒染色较浅,阳性颗粒在整个细胞分散分布,以后逐渐减少。结论随着小鼠发育天数的增大,心肌细胞的分裂指数越来越小,到12 d时基本上停止了分裂。CyclinD1蛋白的表达主要在细胞核中,胞浆也有少许,且随发育时间的增加逐渐减少。     

Ang-（1-7）对AngⅡ诱导培养的乳鼠心肌细胞分泌bFGF的影响

目的研究血管紧张素-（1-7）[Ang-（1-7）]对血管紧张素Ⅱ（AngⅡ）诱导培养的乳鼠心肌细胞肥大和合成碱性成纤维细胞生长因子（bFGF）的影响，探讨Ang-（1-7）的细胞保护机制。方法分离出新生1～3d内的SD大鼠心肌细胞进行体外培养，4d后将细胞随机分为正常对照组、AngⅡ组和AngⅡ＋Ang-（1-7）组行加药干预，加药2d后显微镜下测量细胞表面积，用免疫细胞化学方法检测心肌细胞bFGF含量。结果AngⅡ组细胞平均表面积较正常对照组明显增大，AngⅡ＋Ang-（1-7）组细胞表面积较AngⅡ组明显减小，但仍大于正常对照组；AngⅡ组细胞bFGF表达灰度值较正常对照组明显增加，AngⅡ＋Ang-（1-7）组bFGF表达灰度值较AngⅡ组明显减小，但仍大于正常对照组。结论Ang-（1-7）可拮抗AngⅡ的促心肌细胞肥大和促bFGF合成，起到细胞保护作用。     

丹参酮ⅡA磺酸钠抑制人肾间质成纤维细胞体外增殖过程中PCNA和Cyclin E的表达

丹参酮ⅡA磺酸钠(DS-201)是丹参脂溶成分丹参酮ⅡA经磺化而成的水溶性钠盐,药理作用与丹参酮ⅡA相同,因其水溶性提高、疗效强于丹参酮ⅡA而在临床心血管疾病的治疗和预防中广泛应用.在肾脏间质纤维化的治疗方面,其具体作用机制尚不清楚.本实验拟采用DS-201体外干预人肾间质成纤维细胞(hRIF),观察药物对细胞PCNA和CyclinE表达的影响,揭示DS-201治疗肾间质纤维化的分子机制.     

pCDNA3.1-MT2A真核表达载体的构建及亚细胞定位

目的:构建pCDNA3.1-MT2A真核表达载体并观察其在293T和SMMC7721细胞株中的定位和表达情况。方法:使用基因合成法合成MT2A基因,在其N端添加Kozak序列及His标签序列,将扩增后的目的基因双酶切连接至pcDNA3.1(+)载体的BamHⅠ和NotⅠ之间。转化DH5α大肠杆菌后,挑取阳性克隆子进行质粒抽提电泳及测序鉴定。将鉴定正确的pCDNA3.1-MT2A质粒采用脂质体法转染293T和SMMC7721细胞株,激光共聚焦显微镜观察其在真核细胞中的表达和定位情况。结果:pCDNA3.1-MT2A重组质粒经酶切电泳及DNA测序证实,目的基因MT2A的序列完全正确,真核表达载体构建成功,激光共聚焦观察发现该重组质粒表达于293T和SMMC7721细胞的胞质中。结论:成功构建pCDNA3.1-MT2A融合基因并进行真核表达,发现MT2A主要定位于293T和SMMC7721细胞的细胞质中。本研究为探讨MT2A在肝癌细胞内的功能奠定了基础。     

小鼠乳鼠原代心肌细胞的改良培养

背景：大鼠心肌细胞原代培养技术日渐成熟,但小鼠心肌细胞在同样实验条件下不易获得,而小鼠基因组与人类基因组有更多相似之处,具有更高的研究价值。目的：改进小鼠乳鼠原代心肌细胞的培养方法,得到纯度高、活力强、保持心肌细胞原有结构和功能的心肌细胞。方法：将小鼠心肌组织利用酶消方法分离为单个的心肌细胞,实验步骤中将经典的胰酶、胶原蛋白酶混合酶消法分解开,先用胰酶消化心肌组织,使心肌组织变得松散,再用胶原蛋白酶,作用于细胞间质的胶原纤维,使心肌组织分离为单个的心肌细胞。调整胰酶和Ⅱ型胶原蛋白酶的浓度和作用时间,严格控制试剂pH值和各步骤的温度。结果与结论：心肌细胞在接种24 h后开始贴壁,48 h后可观察到部分心肌细胞出现自主搏动,72 h后彼此形成交联,96 h后可观察到心肌细胞形成细胞簇,并出现一致性搏动。心肌细胞的存活率和纯度均达95%以上。结果证实,实验采用改良方法成功培养出小鼠乳鼠心肌细胞,并且保留心肌细胞的结构和功能,纯度和成活率高,是可行的培养方法。中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

乳鼠心肌细胞可成片生长的分离和培养方法北大核心

背景:传统方法培养的心肌细胞活力、纯度仍不高,细胞多呈簇状生长,细胞与细胞之间的联系较少,细胞多呈单个搏动。目的:改进传统的乳鼠心肌细胞培养方法,获得纯度高、成片生长的心肌细胞。方法:运用0.1%胰蛋白酶4℃过夜消化心肌组织后加入Ⅱ型胶原酶重复消化,二次差速贴壁分离法联合化学抑制剂纯化心肌细胞。倒置相差显微镜下动态观察心肌细胞形态变化,培养48 h后用抗心肌肌钙蛋白T抗体鉴定心肌细胞纯度,连续10 d隔日记录心肌细胞的搏动频率。结果与结论:(1)心肌细胞在24 h内贴壁,72 h后成簇状分布,5-7 d细胞连接成片生长;(2)细胞存活率为96%,免疫荧光鉴定心肌细胞纯度97%以上,连续10 d内心肌细胞搏动频率差异无显著性意义(P>0.05);(3)综上,通过这种改进的培养方式获得成片生长的心肌细胞纯度、活力更高,是一种更为有效的乳鼠心肌细胞培养方法。     

地塞米松干预大鼠星形胶质细胞周期素的表达及细胞周期进程

背景：有研究表明地塞米松可引起星形胶质细胞凋亡,但其机制不清,有关地塞米松干扰星形胶质细胞的周期素表达的作用未见报道。 目的：观察地塞米松引起大鼠星形胶质细胞凋亡与周期素表达的关系。 方法：将不同浓度的地塞米松(0,10-5,10-4,10-3 mol/L)与纯化培养的大鼠大脑皮质星形胶质细胞共同孵育24 h后,经流式细胞仪检测细胞周期,并用免疫细胞化学方法检测周期素A和E在星形胶质细胞内的表达。 结果与结论：地塞米松10-5,10-4 mol/L浓度组G1期细胞指数较对照组增加(P < 0.05,P < 0.01),10-3 mol/L组的S期细胞指数较对照组明显升高(P < 0.01);周期素A的表达随着地塞米松浓度的升高而减弱(P < 0.05)。周期素E的表达在地塞米松0,10-5,10-4 mol/L组随着地塞米松浓度的升高而减弱(P < 0.05),但在10-3 mol/L浓度组反而表达增强(P < 0.01)。说明地塞米松10-5,10-4 mol/L干预的星形胶质细胞周期进程受阻于G1期且与周期素A和E表达降低有关,地塞米松10-3 mol/L干预的星形胶质细胞周期进程受阻于S期主要与周期素A表达降低有关。       

甲醛熏染模型大鼠肝脏组织增殖细胞核抗原表达与三七复方合剂的干预

背景：甲醛作为一种有毒物质可给人体带来严重的损害,同时又缺少有效的防治方法。 目的：观察中药三七复方合剂对甲醛熏染大鼠肝细胞增殖的影响。 方法：将SD大鼠随机分成对照组、甲醛组和三七治疗组。对照组不做任何处理;甲醛组每天置于静态熏染箱内行甲醛熏染,连续8周;三七治疗组处理同甲醛组,但自第5周起,每日给予三七复方合剂灌胃1次。 结果与结论：苏木精-伊红染色结果显示,甲醛熏染8周甲醛组大鼠肝组织肝小叶形态结构紊乱、肝细胞呈现明显的增殖现象,窦间隙缩窄,而三七治疗组肝组织的形态结构明显恢复。免疫组织化学结果显示,甲醛组大鼠肝组织增殖细胞核抗原阳性细胞数明显多于对照组(P < 0.01),而三七治疗组与甲醛组相比则明显降低(P < 0.01)。结果证实,三七复方合剂能够降低由甲醛诱发的肝组织增殖细胞核抗原的表达,可有效减缓由甲醛诱发的大鼠肝细胞增殖。     

胃肠道间质瘤组织中PDGFR-α、Ki-67和细胞周期素D1的表达及临床意义

目的:探讨血小板衍生生长因子受体-α(PDGFR-α)、Ki-67、细胞周期素D1(Cyclin D1)在胃肠道间质瘤(GISTs)中的表达,分析其与胃肠道间质瘤临床病理特征的关系.方法:收集本院普外科2000-01/2008-12收治的胃肠道间叶源性肿瘤手术切除患者196例,应用免疫组织化学法检测196例GISTs组织中PDGFR-α、Ki-67及Cyclin D1的表达情况,并分析上述指标与肿瘤临床病理学特征、危险度的关系.结果:不同部位和不同病理类型组间PDGFR-α阳性表达率有统计学差异(P<0.05),不同危险度组间Ki-67 LI、Cyclin D1表达差异有统计学意义(P<0.05).50例CD117阴性或弱阳性病例中,PDGFR-α同为阴性或弱阳性病例23例(46.0%),PDGFR-α中度以上阳性27例(54.0%);146例CD117中度以上阳性病例中,PDGFR-α阴性或弱阳性118例(80.8%),PDGFR-α中度以上阳性28例(19.2%),比较有统计学意义(P<0.05).结论:PDGFR-α联合CD117对胃肠道间质瘤的诊断及鉴别诊断具有重要的临床意义,但其不能作为胃肠道间质瘤分化程度的评定指标,而Ki-67和CyclinD1可作为判断GISTs恶性程度的参考因素.     

灯盏花素干预糖尿病模型大鼠睾丸组织增殖细胞核抗原和c-fos的表达

背景：有研究表明灯盏花素可影响2型糖尿病大鼠的生殖能力,但其作用机制少有报道。 目的：探讨灯盏花素对2型糖尿病大鼠睾丸增殖细胞核抗原和原癌基因c-fos表达的影响作用。 方法：选取36只健康雄性大鼠随机分为对照组、模型组和灯盏花素组各12只。模型组和灯盏花素组采用链脲佐菌素连续腹腔注射建立2型糖尿病大鼠模型,大鼠血糖监测达到16.7 mmol/L作为建模标准,对照组大鼠予以同等容积的柠檬酸缓冲液单次腹腔注射。灯盏花素组采用灯盏花素注射液10 mg/(kg•d)连续4周腹腔注射,其他2组在相同时间注入等量生理盐水。 结果与结论：干预4周后,血清睾酮检测、免疫组织化学染色和PCR检测结果显示,血清睾酮水平、增殖细胞核抗原、c-Fos蛋白及mRNA表达：对照组>灯盏花素组>模型组(P < 0.05);血糖水平：对照组<灯盏花素组<模型组(P < 0.05)。结果证实,灯盏花素可以通过增强增殖细胞核抗原和c-fos的表达,保护2型糖尿病大鼠的生殖功能。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

胃癌中hMSH2、PTEN和PCNA表达的相关性及意义

目的：探讨胃癌中错配修复基因 hMSH2与 PTEN、PCNA 表达的关系及意义。方法：采用免疫组织化学 SP 法检测胃癌、癌旁和胃炎粘膜中 hMSH2、PTEN 和 PCNA 的表达。结果：(1)hMSH2和 PCNA 在癌组织的表达阳性率均显著高于非癌组织;PTEN 在癌组织的表达阳性率显著低于非癌组织。(2)PTEN 在低分化癌和有淋巴结转移的病例表达阳性率显著低于高分化癌和无转移者。PCNA 在低分化癌和有淋巴结转移的病例表达阳性率显著高于高分化癌和无转移者。(3)hMSH2与 PCNA 蛋白表达呈显著性正相关;PTEN 与 PCNA 蛋白表达呈显著性负相关。结论：hMSH2的高表达及 hMSH2与 PCNA 表达的相互影响与胃癌的发生可能有关;检测3种蛋白有助于判断胃癌的恶性程度和生物学行为。     

人胚胎发育过程中肝细胞增殖细胞核抗原表达水平的研究

目的　研究人胚胎发育过程中肝细胞增殖细胞核抗原 (PCNA)的表达水平。方法　采用免疫组织化学方法 ,检测 2～ 8个月胚胎肝细胞PCNA表达水平。结果　胚胎肝细胞PCNA的表达水平随月份增强。结论　胚胎肝细胞PCNA的表达水平的与胚胎发育过程中肝细胞的功能有着密切联系     

增殖细胞核抗原在实验性脑出血大鼠脑组织中的表达及其意义

研究实验性脑出血时大鼠脑组织中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达及其意义。将成年SD大鼠随机分为正常组、假手术组和脑出血组;脑出血组大鼠通过立体定向术向脑内注入自体动脉血制成脑尾壳核出血模型,并按不同的再喂养时间(1、3、7、14及30d)分为5个亚组。运用RT-PCR检测PCNA mRNA表达水平,采用免疫组化单标和双标染色观察PCNA阳性细胞的分布,增殖水平及分化情况。RT-PCR结果显示,PCNA mRNA表达在脑出血后14d达高峰。免疫组化单标结果表明,PCNA阳性细胞主要分布于出血灶边缘、脉络丛、室管膜下层、胼胝体和额顶皮质等处。PCNA阳性细胞数在脑出血后1d开始增加,14d达高峰,随后渐下降,但30d时仍明显高于正常组和假手术组。免疫组化双标结果显示在脑出血侧纹状体和额顶皮质可见PCNA/GFAP双标阳性细胞,在额顶皮质可见PCNA/NF-200双标阳性细胞。以上结果提示,脑出血可诱导PCNA阳性细胞增殖、分化,并向出血灶周边区聚集,这可能是脑出血后神经功能恢复的重要物质基础。     

Preliminary results of prognostic significance of proliferating cell nuclear antigen expression in advanced primary larynx carcinomas and lymph node metastases

Introduction

The aim of this study was to investigate the prognostic significance of proliferating cell nuclear antigen (PCNA) expression in laryngeal carcinoma in relation to clinicopathological features. Special emphasis was placed on examining the relationship of PCNA expression in the primary tumour and PCNA expression in corresponding lymph node metastases obtained from the same patients.

Material and methods

The study included 60 patients with advanced larynx carcinoma who had received treatment and follow-up for at least 5 years. Sixty laryngeal carcinoma specimens and metastatic lymph nodes from 24 patients were examined for immunohistochemical PCNA expression.

Results

The percentages of PCNA positive cells were significantly higher in the primary tumours which developed lymph node metastases than in those without metastases. The fraction of PCNA immunolabelled cells in metastatic lymph nodes increased significantly when compared with the PCNA positive cell score in their corresponding primary tumours obtained from the same patient. There was a significant difference in PCNA index score in primary tumours between the group of patients who survived a 5-year period and those who died within 5 years after treatment.

Conclusions

Our data demonstrate that a high proliferation index in primary larynx tumours is retained and increased in corresponding lymph node metastases. Measurement of the fraction of cancer cells stained for PCNA in primary larynx carcinomas can be helpful in selecting tumours with high aggressiveness potential that are more likely to develop neck metastases and thereby in identifying patients who need elective lymph node dissection or additional treatment.     

红外线对萎缩性胃炎大鼠PCNA及Bcl-2表达的影响

目的 观察红外线照射在大鼠慢性萎缩性胃炎（chronic atrophic gastritis,CAG）中对增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）和B细胞淋巴瘤/白血病-2（B-cell lymphoma-2,Bcl-2）表达的影响,探讨红外线与胃黏膜癌前病变、细胞凋亡的关系.方法 应用水杨酸钠和酒精的混合溶液灌胃,并结合劳累、饥饱失常等多因素建立大鼠CAG模型,采用红外线灯照射CAG大鼠,免疫组化法检测各组大鼠PCNA及Bcl-2的表达.结果 PCNA及Bcl-2在模型对照组胃黏膜中均呈高表达（P〈0.05）,但在红外线组均无显著降低.结论 红外线照射未能降低PCNA及Bcl-2 的表达,细胞的增殖调节改善作用不明显.     

Temporal and histologic relationships of proliferating cell nuclear antigen and human papillomavirus type 11 in the mouse xenograft system

Proliferating cell nuclear antigen (PCNA) is an accessory protein of DNA polymerase delta. This protein is associated with cell cycle progression and can be detected in the replicating cells of normal tissues. Condylomata acuminata are benign epithelial tumors caused by infection with human papillomaviruses and are characterized by abnormal cell proliferation. The athymic mouse xenograft model of HPV 11 infection was used to test the hypothesis that PCNA is induced early in the course of HPV 11 infection and to study the temporal and histologic relationships between detection of PCNA and HPV DNA. Human foreskin tissue was infected with HPV 11 and implanted under the renal capsules of 10 athymic mice. Pairs of mice were sacrificed every week beginning four weeks after implantation. HPV DNA was detected in sections of foreskin implants by in situ hybridization. PCNA was as or more abundant in implants removed at earlier time points than at later time points, whereas HPV DNA became increasingly more abundant with time. PCNA was detected only in basal cells in areas of histologically normal epithelium that were also negative for HPV DNA. In contrast, PCNA was present throughout the epithelium in regions that were HPV DNA-positive. HPV DNA was detected only in differentiated epithelial cells in implants removed at all five time points, but in HPV DNA-positive regions, PCNA was detected with equal intensity in differentiated and undifferentiated cells. The foci of PCNA-positive cells were well demarcated and were larger than, but included, the foci of HPV DNA-positive cells. PCNA may be induced maximally in differentiated epithelium by HPV 11 prior to significant HPV DNA replication. © 1996 Wiley-Liss, Inc.     

Assessment of proliferating cell nuclear antigen expression in precursor stages of gastric carcinoma using the PC10 antibody to PCNA

Immunohistochemistry using the PC10 antibody to proliferating cell nuclear antigen (PCNA) was applied to archival material from mucosa adjacent to gastric carcinoma ('normal', hyperplasia, complete and incomplete intestinal metaplasia and dysplasia) and non-cancer controls (normal and complete intestinal metaplasia). Overall, increased PCNA indices, with expansion and altered location of the proliferative zones, were observed in carcinoma fields and compared with controls ( P ≤0.001). These differences were particularly significant in 'normal' mucosa far from carcinoma as compared with normal in controls ( P ≤0.001). In carcinoma 'fields' distinct patterns of PCNA expression were noted in complete and incomplete intestinal metaplasia. Similarly, in dysplastic lesions high PCNA indices were present either throughout the gland or found predominantly in the upper compartment. We conclude that these differences in PCNA index and staining patterns might prove useful in monitoring the evolution of the disease in the follow-up of patients at risk of developing gastric cancer.     

Expression and prognostic significance of cyclin B1 and cyclin A in non-small cell lung cancer

Aims:  Aberrant expression of cell cycle regulators has been implicated in the pathogenesis of many neoplasms, including non-small cell lung cancer (NSCLC). The aim was to examine the expression and prognostic value of cyclin B1 and cyclin A, key regulators of the G₂/M checkpoint of the cell cycle, in NSCLC and bronchial precursor lesions.
Methods and results:  Immunohistochemical expression of cyclin B1 and A was examined in 90 cases of stage I–II primary NSCLC and bronchial precursor lesions using tissue microarrays. Increased cyclin B1 and A expression was found in 40.9 and 58.9% of NSCLC cases, respectively, and was significantly higher in primary NSCLC, lymph node metastases and some bronchial precursor lesions compared with normal bronchial epithelium. Increased expression of cyclin A and cyclin B1 correlated with tumour type, poorly differentiated tumours and male gender. A significant association was found between increased cyclin B1 expression and reduced survival using Kaplan–Meier survival analysis. On multivariate analysis, cyclin B1 was not an independent prognostic factor ( P  = 0.067). Cyclin A expression was not associated with survival.
Conclusions:  Cyclin B1 and cyclin A are aberrantly expressed in NSCLC and some precursor lesions. Cyclin B1, but not cyclin A, shows some promise as a potential prognostic marker in NSCLC.     

Evaluation of cyclin D1 expression and its subcellular distribution in mouse tissues

Cyclin D1 is a key cell-cycle regulatory protein required for the cell to progress through G1 to S phase. We have shown by Western blot analysis that cyclin D1 has a wide distribution in adult mouse tissues, with its level of expression being tissue-dependent. Immunohistochemistry has also shown that cyclin D1 may be present in the cytoplasm, in the nucleus or in both these cell compartments: cytoplasmic staining was observed in both proliferating cells (e.g. kidney, intestine, stomach and salivary gland) and in the non-dividing cells (the mature neurons of adult brain), while nuclear staining was seen in the neurons of the embryonic nervous system. Immunoelectron microscopy results indicate that, in tissues where cyclin D1 is present in both compartments (e.g. intestinal enterocytes), it may move via nuclear pores from the nucleus to the cytoplasm, and vice versa. The findings as a whole suggest that cyclin D1 may play multiple roles within specific tissues, probably by interacting with different substrates, and that its transit between nuclear and cytoplasmic compartments may help maintain cell homeostasis.