视网膜母细胞瘤增殖细胞核抗原表达的研究

目的 :探讨增殖细胞核抗原 (proliferatingcellnuclearantigen ,PCNA)在视网膜母细胞瘤 (retinoblastoma ,Rb)中的表达及其与Rb病理类型和侵犯视神经情况的关系。方法 :用免疫组化技术检测PCNA在 3 7例Rb组织标本中的表达 ,并应用多功能真彩色病理图像分析系统 (CMIAS系列 )来分析PCNA染色的阳性细胞个数 ,采用积分光密度值定量分析其增殖指数 ,并将各指标进行SPSS统计软件相关性分析。结果 :本组 3 7例Rb标本中PCNA的阳性表达为 97 3 0 % ,而在 2例正常视网膜组织内 (对照组 )的表达均为阴性。PCNA的表达水平与Rb不同的分化、增殖程度有显著的统计学差异 (P <0 0 1) ,Rb侵犯视神经情况越严重、分化程度越差 ,PCNA的表达越强。结论 :Rb中PCNA的高表达可作为判断Rb恶性及其程度的新型标记物。PCNA与Rb分化类型、视神经侵犯情况密切相关 ,为PCNA表达水平反映Rb分化程度和增殖能力提供理论依据     

HSP27、增殖细胞核抗原在视网膜母细胞瘤中的表达

目的探讨热休克蛋白(heat shock proteins,HSP)27、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在视网膜母细胞瘤(retinoblastoma,Rb)中的表达及其与Rb分化程度和视神经浸润的关系。方法应用鼠抗人HSP27及PCNA的单克隆抗体,采用PV9000二步法免疫组织化学方法,对36例Rb患儿和2例正常对照视网膜组织常规石蜡标本行HSP27及PCNA表达的测定,并分析它们与肿瘤分化程度及视神经浸润的关系。结果HSP27及PCNA在Rb中的表达阳性率分别为69.4%及83.3%;两指标在未分化组阳性表达高于分化组(P<0.05),两指标在视神经浸润组的阳性表达高于未浸润组(P<0.05)。Rb中HSP27的表达与PCNA的表达呈正相关(P<0.01)。结论Rb中HSP27和PCNA的表达与Rb的分化程度、视神经浸润有关。     

端粒酶与视网膜母细胞瘤组织病理学特征相关性的初步研究

目的　探讨端粒酶基因 (hTR)和端粒酶催化亚单位基因 (hTRT)在视网膜母细胞瘤 (RB)组织标本中的表达 ,研究端粒酶与RB增殖和分化的关系。方法　应用原位杂交方法检测hTR、hTRT在 37例RB和 2例正常组织标本中的表达 ;并对标本中的增殖细胞核抗原 (PCNA)进行免疫组化染色 ,经过量化 ,作为肿瘤的增殖指数 ;应用SPSS统计软件对各指标进行相关性分析。结果　RB标本中hTR和hTRT的阳性表达分别为 83 8%、89 2 % ,2例正常视网膜组织内的表达均为阴性。hTR、hTRT的表达水平与RB不同的增殖、分化程度的统计学差异均有显著意义 (均P <0 0 5 ) ,RB侵犯视神经情况越严重、分化程度越差 ,hTR、hTRT的表达越强。此外 ,RB的端粒酶hTR、hTRT指标间有一定相关性。结论　RB中端粒酶基因hTR和催化亚单位基因hTRT的高表达可作为判断RB恶性及其程度的新型标记物。hTR、hTRT与RB分化类型、视神经侵犯情况密切相关 ,为端粒酶表达水平反映RB分化程度和增殖能力提供理论依据。端粒酶在RB中的存在为开辟新型临床治疗方法 ,即通过抑制RB细胞端粒酶活性达到治疗目的提供了新思路。     

视网膜母细胞瘤基因对视网膜母细胞瘤移植瘤细胞周期的影响

目的 观察外源性视网膜母细胞瘤基因(Rb基因)对视网膜母细胞瘤(retinoblastoma,RB)移植瘤细胞周期的影响。 方法 在建立裸鼠眼玻璃体腔RB移植瘤模型及构建Rb基因的逆转录病毒表达载体PBabe-Rb的基础上,用脂质体Dosper介导法将Rb基因导入裸鼠RB移植瘤,流式细胞仪检测RB移植瘤细胞周期变化。 结果 Rb基因在RB移植瘤内的表达使RB移植瘤G₁期细胞数明显增加,DNA指数及S期百分比值有所降低。 结论 外源性Rb基因可部分抑制RB移植瘤细胞周期的进程。（中华眼底病杂志,2000,16：1-70）     

微小RNA与磷酸化胞外信号调节激酶在视网膜母细胞瘤中的表达及其相关性分析

目的 探讨miRNA和磷酸化胞外信号调节激酶(phosphorylated extracellular signal regulated kinase,P-ERK)在视网膜母细胞瘤(retinoblastoma,Rb)中表达的变化及其相关性.方法 手术切除获取Rb组织标本经组织病理学检查分为低分化、中分化和高分化Rb组织,肿瘤外的正常视网膜组织作为对照组;应用逆转录聚合酶链反应(RT-PCR)技术检测正常视网膜组织和不同分化程度Rb组织中miRNA表达水平,Western blot检测正常视网膜组织和不同分化程度Rb组织中P-ERK蛋白表达情况.结果 Rb组织中miRNA基因表达(低分化0.214±0.140、中分化0.246±0.200、高分化0.256±0.180)、P-ERK蛋白表达(低分化0.367±0.270、中分化0.562±0.190、高分化0.609 ±0.110)明显高于癌旁正常视网膜组织(miRNA:0.200±0.190、P-ERK蛋白:0.211±0.260),差异均有统计学意义(均为P ＜0.05).miRNA、P-ERK蛋白表达与Rb组织分化程度有相关性;miRNA和P-ERK蛋白表达呈正相关.结论 miR-NA和P-ERK蛋白在Rb组织中表达均高于正常视网膜组织,并且二者表达呈正相关.     

基因表达调控物对HXO-Rb44细胞瘤株端粒酶活性的影响

目的　通过检测基因表达调控物全反式维甲酸对HXO Rb4 4 细胞端粒酶活性及细胞周期变化的影响 ,探讨治疗视网膜母细胞瘤的新途径。方法　将视网膜母细胞瘤株HXO Rb4 4 分为对照组及实验组 ,分别进行体外培养 2 4、48、72h后 ,以PCR ELISA方法检测其端粒酶活性及用流式细胞仪检测细胞周期变化。结果　实验组与对照组的端粒酶活性有显著差异 (P <0 .0 1) ,实验组的端粒酶活性与药物作用时间之间 ,呈显著负相关 (r =-0 7756,F =3 3 2 16,P <0 .0 1)。实验组细胞周期发生变动 ,即G1期比率上升 ,S期比率下降。结论　基因表达调控物全反式维甲酸可减弱HXO Rb4 4 细胞端粒酶活性 ,影响其周期变化 ,抑制其增殖     

视网膜母细胞瘤患者血T淋巴细胞亚群白细胞介素-2及肿瘤坏死因子含量变化

目的 探讨视网膜母细胞瘤(Rb)患者血T淋巴细胞(T--LC)亚群、白细胞介素一2(IL一2)及肿瘤坏死因子(TNF)含量变化与肿瘤分化程度的关系。方法 对19例分化型及21例未分化型Rb患者进行血T-LC亚群、CD4、CD8及IL-2、TNF含量检测，并与20例正常者作对照。结果 两型血T-LC亚群、CD4、IL-2及TNF含量均明显低于正常者(P＜0．01)；未分化型CD8高于正常者(P＜0．05)，而CD4／CD8比值则低于正常者；两型间CD4、IL-2及TNF含量比较均有显著性差异(P＜0．05—0．01)。结论 Rb患者存在免疫异常低下，以未分化型低下更为显著。     

视网膜母细胞瘤组织浸润程度临床相关危险因素研究

目的探讨视网膜母细胞瘤(Rb)对眼球组织浸润侵犯程度的相关临床危险因素。设计回顾性病例系列。研究对象经组织病理学确诊的Rb患者394例。方法收集1990-2005年北京同仁医院较完整的394例Rb患者的临床病理资料,术前观察主要临床体征,术后光镜下判读Rb眼球各组织浸润程度,通过logistic多元回归分析Rb眼球组织浸润程度的临床危险因素。主要指标Rb各临床体征与眼球组织浸润程度及两者之间的相关性。结果394例Rb中侵犯视盘315例(79．7％),侵犯筛板纤维203例(51．4％),肿瘤穿过筛板至球后视神经内102例(25．8％),侵犯脉络膜102例(25．8％),视神经切除断端受累者26例(6．6％)。统计分析显示:脉络膜组织受累与病程(P=0．032)、结膜充血(P=0．023)、眼球突出(P=0．026)及继发青光眼(P=0．027)有关;视神经切除断端浸润与结膜充血(P=0．008)、虹膜新生血管形成(P=0．033)、眼球突出(P=0．000)有关。结论本组Rb视神经侵犯率较高。病程长、结膜充血、眼球突出是眼球组织侵犯的主要因素。     

视网膜母细胞瘤组织芯片中PTEN、mdm2、p53表达及与组织病理特征关系的研究

目的 探讨视网膜母细胞瘤组织芯片中PTEN,murine double minute 2(mdm2)及p53蛋白的表达及与肿瘤临床组织病理特征的关系.设计实验性研究.研究对象 64例视网膜母细胞瘤和6例正常视网膜组织.方法 免疫组化方法检测视网膜母细胞瘤组织芯片和正常视网膜组织石蜡标本中PTEN,mdm2及p53的表达情况,将肿瘤分为球后视神经受侵袭组和球后视神经未受侵组,检测两组间mdm2及p53表达差异,并结合肿瘤的组织病理学特征进行综合分析.主要指标视网膜母细胞瘤组织芯片中PTEN,mdm2和p53表达率.结果 视网膜母细胞瘤中PTEN,mdm2与p53的阳性表达率分别为53.13%,48.43%和53.13%.球后视神经受侵袭组较球后视神经未受侵组mdm2(P=0.027)及p53(P=0.017)表达均明显增高.视网膜母细胞瘤中PTEN与p53表达呈负相关(r=-0.384,P=0.045),mdm2与p53蛋白表达呈正相关(r=0.281,P=0.018).结论 视网膜母细胞瘤中mdm2和p53的过度表达与视神经侵袭程度存在一定相关性.mdm2及p53蛋白的检测可能成为视网膜母细胞瘤恶性程度的参考指标.     

视网膜母细胞瘤和正常视网膜组织中基质调控蛋白表达及其意义(英文)

目的:研究视网膜母细胞瘤(RB)和正常视网膜组织中EMMPRIN、MMP1、MMP9和TIMP2表达水平及其临床病理意义和相互联系。方法:收集30例RB眼球摘除手术标本和15例正常视网膜组织标本。常规制作石蜡包埋切片,EMMPRIN、MMP1、MMP9和TIMP2染色方法均为Envision免疫组织化学法。结果:RB组织中EMMPRIN、MMP1、MMP9表达阳性率均高于正常视网膜组织(P<0.01),TIMP2则低于正常视网膜组织(P<0.01)。临床分期I期、分化型、未侵犯视神经及生存期≥2aRB病例EMMPRIN、MMP1、MMP9表达阳性率低于临床分期III期、未分化型、侵犯视神经和生存<2a病例(P<0.05或P<0.01);分化型、未侵犯视神经及生存期≥2aRB病例TIMP2表达阳性率明显高于未分化型、侵犯视神经和生存<2a病例(P<0.05或P<0.01);RB中,EMMPRIN、MMP1、MMP9表达呈高度一致性(P<0.05),TIMP2与EMMPRIN、MMP1、MMP9表达水平呈高度不一致性(P<0.05)。结论:EMMPRIN、MMP1、MMP9和TIMP2的表达水平可能是反映RB进展、侵袭能力及预后的重要标记物,它们之间存在着内在的相互调控关系。     

Effect on proliferation and apoptosis of retinoblastoma cell by RNA inhibiting high mobility group protein box-1 expression

AIM: To investigate the effect of high mobility group protein box-1 (HMGB1) siRNA on proliferation and apoptosis of retinoblastoma (Rb) cells. METHODS: The expression of HMGB1 in Rb cells were detected by real-time polymerase chain reaction (RT-PCR) and Western blot. Chemically synthesized HMGB1 siRNA was transfected into Y79 cells. The inhibitory rate was also examined by RT-PCR and Western blot. After HMGB1 siRNA transfection, the cell proliferation was analyzed by MTT, and cell apoptosis was detected by Caspase-3 active detection kit. Cell cycle distribution and apoptosis were detected by flow cytometry. RESULTS: The expression of HMGB1 significantly elevated in Rb cells (P<0.01). After transfected by siRNA, the HMGB1 protein level of Y79 cells was significantly reduced (P<0.01). After siRNA interference HMGB1, the proportion of proliferating cells reduced, and the proportion of quiescent cells increased (P<0.05). In addition, apoptosis rate of Y79 cells increased from 2.03% to 9.10% after interfering with HMGB1 siRNA (P<0.05). CONCLUSION: Specific HMGB1 siRNA can inhibit the expression of HMGB1. The effect may be attributed to inhibit the proliferation and promote cell apoptosis.     

全反式维甲酸对Rb细胞的生长及Cx43蛋白分子表达的影响

目的研究全反式维甲酸（ATRA）对视网膜母细胞瘤（Rb）细胞的生长及细胞间隙连接蛋白（Cx）43分子表达的影响，探讨其作用机制。方法设立3种浓度（10^-3mol／L、10^-6mol／L和10^-7mol／L）ATRA分别作用于Rb细胞，采用四甲基偶氮唑蓝（methyl thiazolyl tetrazolium，MTT）比色法检测细胞生长抑制率，流式细胞术检测细胞周期改变及凋亡率，免疫组织化学方法检测细胞Cx43蛋白的表达。结果经ATRA作用后，Rb细胞增生率下降，G0／G1期细胞比例增高而S期比例相对下降，细胞凋亡率升高，Cx43蛋白表达上调。上述效应均呈药物浓度及作用时间依赖性。结论ATRA通过将Rb细胞生长周期阻滞于G0／G1期并促进凋亡而抑制其生长，同时可诱导细胞Cx43分子表达上调。     

阿昔洛韦对体外培养人眼Tenon囊成纤维细胞增殖迁移的影响

目的:探索阿昔洛韦(ACV)对体外培养人眼Tenon囊成纤维细胞(HTFs)的增殖和凋亡的影响以及作用机制。方法:将HTFs分为ACV处理组和空白组;CCK8检测不同浓度梯度下的细胞增殖速率,划痕实验检测HTFs迁移能力,流式细胞术检测HTFs凋亡以及细胞周期。结果:与空白组相比,ACV处理组(终浓度分别为1.125、2.25、3.375、4.5mmol/L)HTFs增殖速率显著降低(P<0.05),且呈浓度依赖性。4.5mmol/L ACV处理组划痕细胞迁移率显著降低(P<0.05),细胞凋亡率显著增加(P=0.0005),细胞周期G0/G1期峰值升高(P=0.0011),S期下降(P=0.0006),细胞周期被阻滞在G0/G1期。结论:阿昔洛韦可以通过阻滞HTFs周期促进细胞凋亡,抑制HTFs的增殖和迁移。     

microRNA-127抑制葡萄膜黑色素瘤细胞增殖的表观遗传调控研究

目的：研究microRNA-127（miR-127）在人葡萄膜黑色素瘤细胞中的表达水平及其对细胞增殖的影响。方法：实验研究。通过实时荧光定量PCR（RT-qPCR）法检测miR-127在葡萄膜黑色素瘤细胞M23和SP6.5与正常葡萄膜黑色素细胞UM95中的表达水平。细胞实验通过阳离子脂质体介导的方法将miR-127和阴性对照（NC）转染入M23和SP6.5中,并应用细胞增殖实验法（MTS法）和流式细胞技术分别检测细胞增殖能力和细胞周期,采用Western Blot法检测转染miR-127后细胞周期相关蛋白的表达。另外,通过RT-qPCR检测用表观药物5-氮杂-2-脱氧胞苷（5-Aza-dC）和（或）曲古抑菌素A（TSA）处理后的M23和SP6.5细胞内miR-127的表达水平。MiR-127的表达量及细胞实验各参数在2组间比较采用独立样本t检验。结果：miR-127在M23和SP6.5中的表达水平低于UM95（t=72.2、591.5,P<0.001）。MTS结果显示,在M23和SP6.5细胞中,转染miR-127组相对细胞数目较NC组的100%分别减少至（62.3±4.2）%和（65.4±2.3）%,差异具有统计学意义（t=12.7、21.6,P<0.001）。流式细胞技术分析显示,转染miR-127组处于G0/G1期的M23和SP6.5细胞数量比例明显高于NC组（t=-6.7,P=0.003;t=-9.9,P<0.001）,同时处于S期的M23和SP6.5细胞数量比例明显低于NC组（t=8.6,P=0.001;t=12.7,P<0.001）。Western Blot结果表明miR-127可明显降低M23和SP6.5细胞内磷酸化Rb 蛋白的表达水平（t=22.2、15.6,P<0.001）。此外,miR-127在M23和SP6.5细胞中的表达可被5-Aza-dC和TSA诱导上调（P<0.05）。结论：miR-127通过抑制细胞周期而抑制人葡萄膜黑色素瘤细胞的增殖,并可被DNA甲基化和组蛋白乙酰化表观遗传机制调控。     

【In Press】Senescence Marker Protein 30 Promotes the Proliferation of Human Lens Epithelial Cells SRA01/04 and Inhibits its Apoptosis

AIM: To study the effect of senescence marker protein 30 (SMP30) on the proliferation and apoptosis of human lens epithelial cell (HLEC) SRA01/04. METHODS: SMP30 overexpression and knock down type cell lines were cultivated by using two groups RGN (SMP30) lentiviral vectors (LV-RGN, LV-RGN-RNAi) and the respective negative control virus infect SRA01/04 cells. Western blot and q-PCR analysis were used to determine RGN overexpression and knock down efficiency. We use Cell Counting Kit-8 (CCK8) assay to measure cell viability and 5-bromodeoxyuridine (BrdU) assay to test cell proliferation. Cell cycle was measured by PI FACS assay and Cell apoptosis was tested by Annexin-V APC assay through flow cytometry. We use western blot to measure the content of caspase-3 in SRA01/04. RESULTS: We used PCR and WB techniques to determine the successful transfection of SMP30 overexpression (OE) and knock down (KD) SRA01/04 cell lines. By CCK8, Brdu and PI FACS cell cycle assay, it was found that the SMP30 OE group promoted cell proliferation (P<0.05) compared with the control group, and the KD group inhibited cell proliferation (P<0.05).The results of Annexin V APC signal staining detection indicated that compared with respective control group, the cell apoptosis rate was higher in KD group (P<0.05) but lower in OE group (P<0.01). The expression of caspase-3 was down-regulated in OE group through Western Blot assay and up-regulated in KD group compared with respective control group. CONCLUSION: Proliferation of SRA01/04 was promoted by over-expressing SMP30 and apoptosis was suppressed. Increasing the expression of SMP30 may protect HLEC SRA01/04 against apoptosis in cataract.     

The effect of a selective cyclooxygenase-2 (COX-2) inhibitor on the proliferation rate of retinoblastoma cell lines

PURPOSE: To examine the effect of nepafenac, a selective cyclooxygenase-2 (COX-2) inhibitor, on the proliferation rate of two human retinoblastoma (Rb)cell lines. METHODS: Two human Rb cell lines (WERI-RB and Y79) were cultured. COX-2 expression in these cell lines was verified by immunocytochemical analysis of cytospin sections and Western blotting. An MTT-based proliferation assay was used to compare Rb cell growth with and without amfenac, the active metabolite of nepafenac. The averaged results per condition were recorded. The Student's t-test was used to compare results from the cells cultured with and without amfenac. RESULT: The Y79 cell line showed a higher proliferative rate than the WERI-RB cell line. Both cell lines were negative for COX-2 expression by immunocytochemical analysis; however, both cell lines were positive for COX-2 expression by Western blot. When amfenac was added to both of the cell lines, a statistically significant reduction in proliferation was observed in both cell lines. The two Rb cell lines were positive for COX-2 only in the Western blot, indicating that they probably express low levels of COX-2, which was undetectable by immunocytochemical analysis. CONCLUSION: The selective, anti-COX-2 molecule amfenac inhibited proliferation of both tested Rb cell lines. Further trials should be undertaken to study the effect of selective COX-2 inhibitors on Rb.     

Mechanisms of inhibition of elemene on human lens epithelial cell proliferation in vitro

AIM: To study the effects of elemene (Ele) on proliferation and cell cycle of human lens epithelial cells B3 (HLE-B3) and the mechanisms of its signal transduction. METHODS: Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 80mg/L Ele for 24 hours. The inhibitory effects of Ele on the proliferation of HLE-B3 cells were evaluated by MTT method. The effect of Ele on HLE-B3 cell cycle was analyzed by flow cytometry(FCM). The expressions of protein kinase A (PKA) and protein kinase G (PKG) of HLE-B3 were also analyzed by FCM. RESULTS: Ele altered the cell cycle of HLE-B3 and effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. Ele up-regulated PKA and down-regulated the expression of PKG in HLE-B3 cell. CONCLUSION: Ele inhibits HLE-B3 proliferation, making it an attractive potential agent in regimens to treat after- cataracts.