Association of cyclin D1, p16 and retinoblastoma protein expressions with prognosis and metastasis of gallbladder carcinoma

AIM: To investigate the role of cyclin D1, p16 and retinoblastoma in cancerous process of gallbladder carcinomas and to assess the relation between cyclin D1, p16, Rb and the biological characteristics of gallbladder carcinoma. METHODS: Forty-one gallbladder carcinoma, 7 gallbladder adenoma and 14 chronic cholecystitis specimens were immunohistochemically and histopathologically investigated for the relation of cyclin D1, p16 and Rb with Nevin staging and pathologic grading. RESULTS: The expression rates of abnormal cyclin Dl in gallbladder carcinoma (68.3%)and gallbladder adenoma (57.1%) were significantly higher than those in chronic cholecystitis (7.1%) (P<0.05). No significant difference was found both among the pathological grades G1, G2 and G3 and among Nevin stagings S1-S2, S3 and S4-S5 of gallbladder carcinoma. The positive rates of p16 (48.8%) and Rb (58.5%) in gallbladder carcinoma were significantly lower compared to those in adenoma (100.0%) and cholecystitis (100.0%) (P<0.05). The positive rates of p16 and Rb in Nevin stagings S1-S2 (80.0% and 90.0%) and S3 (46.2% and 61.5%) gallbladder carcinomas were significantly higher than those in S4-S5(33.3% and 38.8%) (P<0.05), and those in pathologic grades G1(54.5% and 81.8%) and G2 (50.0% and 62.5%) gallbladder carcinoma were significantly higher than those in G3 (28.6% and 35.7%) (P<0.05). The protein expression of p16 and Rb had a negative-correlation in gallbladder carcinoma (r= -0.2993, P<0.05), and this negative-correlation was correlated with Nevin staging (P<0.05). Moreover, the protein expression of p16 and cyclin Dl had a negative-correlation in gallbladder carcinoma (r = -0.9417, P<0.05). CONCLUSION: Cyclin Dl may play a role in the early stage of gallbladder carcinoma. Mutation of p16 and Rb genes might be correlated with progression of gallbladder carcinoma. Analysis of p16 and Rb can estimate the prognosis of gallbladder carcinoma. Expression of p16 and Rb may be correlated with Nevin staging and pathologic grading in gallbladder carcinoma.

Expression of altered retinoblastoma protein inversely correlates with tumor invasion in gastric carcinoma

AIM: To investigate the clinical and pathological significance of altered retinoblastoma (Rb) encoding protein (pRb) in gastric carcinoma. METHODS: Expression of altered pRb was analyzed in 91 patients with gastric adenocarcinoma by immunohistochemistry. RESULTS: Sixty-five percent (59/91) of the tumors were positively stained and the staining in tumor nuclei of gastric carcinoma ranged 0%-90%. Moreover, strong expression of altered pRb was found in 35% (6/17), 24% (5/21), 17% (8/46) and 0% (0/7) of T1, T2, T3 and T4 gastric carcinomas, respectively. altered pRb expression was inversely correlated with the depth of tumor invasion (P = 0.047). Degree of immunoreactivity had no significant correlation with tumor grade, node metastasis and distant metastasis. In terms of prognostic significance, univariate analysis showed that poor differentiation [41 (66.1%) vs 34 (42.5%) P = 0.051], advanced tumor stage (P < 0.001) and weakly altered pRb expression [17 (80.5%) vs 58 (49.6%) P = 0.044] were associated with worse prognosis in these patients. However, multivariate analysis revealed that advanced tumor stage was the only independent poor prognostic factor (P < 0.001). CONCLUSION: The mutation of Rb gene is frequent in gastric carcinoma. The expression of altered pRb inversely correlates with tumor invasion and is not an independent prognostic marker in gastric adenocarcinoma.

Retinoblastoma binding protein 4 up-regulation is correlated with hepatic metastasis and poor prognosis in colon cancer patients

Background: Retinoblastoma binding protein 4 (RBBP4) plays an essential role in the development of multiple cancers. However, its relationship with prognosis in colon cancer and colon cancer hepatic metastasis has not been elucidated. The aim of this study was to explore the relationship between RBBP4 expression and prognosis of colon cancer patients and to evaluate RBBP4 as a new prognostic marker in these patients. Methods: Eighty colon cancer patients underwent surgical resection of the colon were enrolled. Among them, forty colon cancer patients suffered with hepatic metastasis. The colon cancer tissues, para-colon cancer tissues, and hepatic metastatic cancer tissues were collected from the pathological department for further analysis. The expression of RBBP4 proteins was examined by immunohistochemistry and correlated with clinicopathological parameters. The Cancer Genome Atlas (TCGA) database was used to validate the expression and explore its relationship with clinical characteristics. Results: RBBP4 was up-regulated in the colon cancer tissues compared with the para-colon cancer tissues. The analysis of TCGA database verified the upregulation of RBBP4 in the colon cancer tissues and RBBP4 overexpression was correlated with nerve invasion and poor outcomes of chemotherapy. Moreover, the positive rate of RBBP4 expression in 40 colon cancer patients with hepatic metastasis was higher in the hepatic metastatic cancer tissues (39/40, 97.5%) than in the colon cancer tissues (26/40, 65.0%). Our clinicopathological analysis showed that RBBP4 expression was significantly correlated with vascular invasion, hepatic metastasis, and lymph node involvement (all P < 0.05). Additionally, the survival analysis demonstrated that RBBP4 over-expression was correlated with poor prognosis. Conclusions: RBBP4 was upregulated in the colon cancer. RBBP4 may be a novel predictor for poor prognosis of colon cancer and colon cancer hepatic metastasis.

Expression of multidrug-resistance associated proteins in human retinoblastoma treated by primary enucleation

AIM： To reveal the expression of multidrug-resistance associated proteins： glutathione-S-transferase π（GSTπ）, P-glycoprotein（P-gp） and vault protein lung resistance protein（LRP） in retinoblastoma（RB） without any conservative treatment before primary enucleation and to correlate this expression with histopathological tumor features. METHODS： A total of 42 specimens of RB undergone primary enucleation were selected for the research. Sections from the formalin-fixed, paraffin-embedded specimens were stained with HE and immunohistochemistry to detect the expression of GSTπ, P-gp and LRP.RESULTS： GSTπ was expressed in 39/42（92.86%） RBs and in 9/9（100%） well-differentiated RBs. P-gp/GSTπ was found in 30（71.42%） of 42 RBs. Totally 9（21.43%） tumors were well differentiated and 33（78.57%） were poorly differentiated. Totally 15（35.71%） eyes had optic nerve（ON） tumor invasion, 36（85.71%） had choroidal tumor invasion, and 14（33.33%） had simultaneous choroidal and ON invasion. There was no statistically significant relationship between P-gp, GSTπ, LRP positivity and the degree of ocular layer tumor invasion and ON tumor invasion（P〉0.05）. CONCLUSION： RB intrinsically expresses GSTπ, P-gp and LRP. GSTπ expression is positive in 100% welldifferentiation ones, so in which way it is correlated with differentiation. But the other two proteins expressions are not related to tumor differentiation and to the degree of tumor invasion. GSTπ may be a new target of chemotherapy in RB.

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.

Expression levels of autophagy related proteins and their prognostic significance in retinocytoma and retinoblastoma

·AIM: To discuss the prognostic significant of autophagy related proteins(ARPs) in retinoblastoma(RB)and to find the molecular marker to distinguish retinocytoma(RC) and RB by investigating the different expression profiling of microtubule-associated protein light chain 3(LC3B) and other ARPs in RC and RB.·METHODS: Specimens with retinocytoma region(RCR)or mainly composed with Flexner-Winterstein rosettes(FWR) were screen out from 219 paraffin-embedded RB samples and respectively taken as RCR group and FWR group. Others were taken as undifferentiated(UD) group.Immunochemistry(IHC) of LC3 B and electronic microscopy was used to identify autophagy. The IHC scores of LC3 B and other ARPs, such as Beclin, PTEN,p27, p16INK4 a, mTOR and BCL-2 were compared and correlation analysis was applied to find potential proteins which may involve in autophagy regulation. The prognostics significance of LC3 B was evaluated by comparing the high risk features(HRFs) in 3 groups of total 219 samples.·RESULTS: Twenty-one specimens with RCR and 36 specimens mainly composed with FWR were screen out.RCR cell had a high level of LC3 B and lots of autophagic vacuoles. Beclin, PTEN, p27 had positive correlation with LC3, and p16INK4 ahad negative correlation, while the expression of mTOR and BCL-2 in RCR and RB region did not show any difference. Cases with RCR had lower rate of HRFs than undifferentiated cases.·CONCLUSION: ARPs had different expression pattern between RCR and other pathological types of RB, and could be ideal markers to distinguish RC from RB. Our finding indicated cases with RCR had favorable prognosis just like those with FWR.

Expression of multidrug resistance proteins in retinoblastoma

AIM： To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance.METHODS： Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy（PCNC） were also prepared. Their chemosensitivity to chemotherapeutic agents（vincristine, etoposide and carboplatin） were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells.RESULTS： Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein（P-gp） was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1（Mrp-1） expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associatedprotein（Lrp） was observed in the drug resistant Y79 cells as well as in PCNC.CONCLUSION： Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

Tumor-specific lytic path“hyperploid progression mediated death”:Resolving side effects through targeting retinoblastoma or p53 mutant

A major advance was made to reduce the side effects of cancer therapy via the elucidation of the tumor-specific lytic path“hyperploid progression-mediated death”targeting retinoblastoma(Rb)or p53-mutants defective in G1 DNA damage checkpoint.The genetic basis of human cancers was uncovered through the cloning of the tumor suppressor Rb gene.It encodes a nuclear DNA-binding protein whose self-interaction is regulated by cyclin-dependent kinases.A 3Dstructure of Rb dimer is shown,confirming its multimeric status.Rb assumes a central role in cell cycle regulation and the“Rb pathway”is universally inactivated in human cancers.Hyperploidy refers to a state in which cells contain one or more extra chromosomes.Hyperploid progression occurs due to continued cell-cycling without cytokinesis in G1 checkpoint-defective cancer cells.The evidence for the triggering of hyperploid progression-mediated death in RBmutant human retinoblastoma cells is shown.Hence,the very genetic mutation that predisposes to cancer can be exploited to induce lethality.The discovery helped to establish the principle of targeted cytotoxic cancer therapy at the mechanistic level.By triggering the lytic path,targeted therapy with tumor specificity at the genetic level can be developed.It sets the stage for systematically eliminating side effects for cytotoxic cancer therapy.

肌纤维母细胞瘤伴黏液样变性1例并文献复习

肌纤维母细胞瘤(myofibroblastoma,MFB)伴黏液样变性是一种少见的间叶源性良性肿瘤。1例42岁女性MFB患者,发现右侧腹股沟无痛性肿块1年,肿块表面光滑,切面灰褐、实性、质中,呈结节状。镜下见肿瘤背景黏液丰富,瘤细胞圆形或短梭形排列成不规则条束状,胞质丰富、嗜酸性,部分细胞核空泡明显。免疫组织化学染色示:肿瘤细胞白细胞分化抗原34(cluster of differentiation 34,CD34)、结蛋白(desmin)呈弥漫阳性,平滑肌肌动蛋白(smooth muscle actin,SMA)、白细胞分化抗原56(cluster of differentiation 56,CD56)、成红细胞转化特异性相关基因(erythroblast transformation-specific related gene,ERG)部分阳性,细胞核相关抗原Ki-67增殖指数约为5%。荧光原位杂交(fluorescence in situ hybridization,FISH)检测示:视网膜母细胞瘤蛋白1(retinoblastoma protein 1,RB1)基因缺失阳性,核受体亚家族4成员3(nuclear receptor subfamily 4 group a member 3,NR4A3)基因和尤文肉瘤断裂区域1(Ewing sarcoma breakpoint region 1,EWSR1)基因阴性。手术切除后随访3~24个月,患者无肿瘤局部复发和远处转移。MFB伴黏液样变性的诊断及鉴别诊断主要依靠临床病理特征、免疫组织化学及基因检测,治疗以单纯手术切除为主,预后好,无明显的复发风险。

p16^(INK4a)在细胞衰老调控中的作用

细胞衰老的原因通常包括外在和内在两个方面。外源性因素主要是细胞外基质的改变、炎症反应和衰老相关分泌表型的表达。内源性因素通常源于调节细胞周期基因的影响,如肿瘤抑制基因。p16^(INK4a)是一种肿瘤抑制因子和细胞周期调节因子,与细胞衰老密切相关。在衰老细胞和与年龄相关的病理过程中,p16^(INK4a)表达显著增加,p16^(INK4a)被认为是检测细胞衰老的标志物。p16^(INK4a)高表达是防止受损细胞继续分裂的保护机制,与细胞周期停滞相关,有助于防止癌症发展。因此,p16^(INK4a)的表达调控在衰老细胞的积累、细胞周期的控制和肿瘤抑制中发挥重要作用。

视网膜母细胞瘤结合锌指蛋白1调控肥胖和肿瘤信号通路研究综述

视网膜母细胞瘤结合锌指蛋白1(retinoblastoma-interacting zinc finger protein 1,RIZ1)基因,又称PRDM2(positive regulatory domain 2)基因,是PRDM基因家族一员,其蛋白序列包含1个PR结构域、1个核激素受体结合基序、8个锌指结构域和1个视网膜母细胞瘤蛋白(retinoblastoma protein,Rb)相互作用基序。RIZ1主要定位于细胞核内,在核内发挥转录抑制因子、基因调控、蛋白质-蛋白质相互作用等功能。RIZ1是代谢通路的重要参与者,其通过调控代谢相关基因影响基础代谢,抑制肥胖的形成;RIZ1功能突变或含量不足与多种肿瘤发生发展相关,其通过激活下游致癌基因或调控代谢参与肿瘤进程。RIZ1通过v-akt鼠科胸腺瘤病毒癌基因同源物Ⅲ(v-akt murine thymoma viral oncogene homolog 3,AKT3)、胰岛素样生长因子-1(insulin-like growth factor 1,IGF-1),以及作为协同激活剂等方式分别调控AKT/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、IGF-1、雌激素这3条肿瘤和肥胖相关分子信号通路。3条分子通路功能有差异且其下游分子存在交叉,提示RIZ1在不同年龄、性别和器官中的作用可能不同。详细研究RIZ1与RIZ2在代谢进程中的调控作用有助于全面了解RIZ1参与肥胖和肿瘤形成的机制。未来基于RIZ1靶点进行诊断研究或功能恢复可能对代谢性疾病和肿瘤的诊断和治疗有重要意义。

棕榈酸酯通过调控p16INK4a/Rb信号通路诱导AC16人心肌细胞发生衰老表型改变

目的探讨棕榈酸酯(palmitic acid,PA)在AC16人心肌细胞衰老表型改变中的作用及机制。方法AC16细胞分为(1)对照组PA浓度(0.08、0.19、0.3、0.8 mmol/L)组;(2)对照组,时间(24、36、48、72 h)刺激组。细胞计数试剂盒-8法测细胞总体活性,流式细胞术测细胞周期;活性氧(reactive oxygen species,ROS)试剂盒测细胞内ROS水平;β-半乳糖苷酶(SA-β-Gal)染色测染色阳性细胞率;酶联免疫吸附测定法(enzyme-linked immuno⁃sorbent assay,ELISA)测衰老相关分泌表型(senescence-associated secretory phenotype,SASP)相关因子白细胞介素(interleukin,IL)-1A、IL-6、IL-8和干扰素(interferon,IFN)-γ浓度;实时定量聚合酶链反应(quantitative real time polymerase chain reaction,qRT-PCR)及Western blot测SASP及p16^(INK4a)/视网膜母细胞瘤抑制蛋白(retinoblasto⁃ma protein,Rb)通路表达。为观察p16^(INK4a)/Rb通路对心肌衰老表型影响,细胞分为4组:对照组、PA组、si-con组和si-p16^(INK4a)组,qRT-PCR测p16^(INK4a) mRNA表达;Western blot测p16^(INK4a)、p-Rb、Rb及IL-1A、IFN-γ蛋白表达;β-半乳糖苷酶染色测阳性细胞率。结果随PA浓度及刺激时间增加,细胞活性不断下降,差异有统计学意义(P<0.05)。与对照组相比,0.19 mmol/L PA组在G1期比例显著升高,S期显著降低,差异有统计学意义(P<0.05)。0.08~0.3 mmol/L PA组ROS水平高于对照组,PA组中β-半乳糖苷酶染色阳性率均高于对照组,差异有统计学意义(P<0.05)。0.19 mmol/L PA组IL-1A、IL-6、IL-8、IFN-γ浓度显著高于对照组,差异有统计学意义(P<0.05)。0.19 mmol/LPA组IL-1A、IL-6、IL-8、IFN-γ及p16^(INK4a)、Rb mRNA及蛋白表达明显高于对照组,而p-Rb蛋白表达下降,差异有统计学意义(P<0.05)。si-p16^(INK4a)组IL-1A、IFN-γ蛋白表达及SA-β-Gal阳性率低于si-con组,差异有统计学意义(P<0.05)。结论0.19 mmol/L PA可能通过调控p16^(INK4a)/Rb通路诱导人心肌细胞衰老表型改变。

过表达miR-515-5p的视网膜母细胞瘤细胞增殖、凋亡、侵袭能力变化及其机制

目的探讨过表达miR-515-5p对视网膜母细胞瘤细胞增殖、凋亡、侵袭能力的影响及其机制。方法利用StarBase数据库进行生物信息学分析并预测miR-515-5p的靶基因为CBX4,并经双荧光素酶报告基因实验验证。选择视网膜母细胞瘤细胞系SO-RB50、Y79、HXO-RB-44细胞中miR-515-5p表达降低最明显的SO-RB50细胞进行实验,分为miR-515-5p NC组、miR-515-5p mimics组、miR-515-5p mimics+CBX4组、miR-515-5p mimics+CBX4-NC组,分别转染miR-515-5p阴性对照、miR-515-5p mimics、miR-515-5p mimics+CBX4过表达载体、miR-515-5p mimics+CBX4空载体。采用实时荧光定量PCR法检测各组miR-515-5p及CBX4 mRNA表达,Western blotting法检测CBX4蛋白表达,CCK-8法检测转染12、24、48、72 h的细胞增殖能力(OD值),TUNEL法检测细胞凋亡率,Transwell小室实验检测细胞侵袭能力。结果miR-515-5p mimics组、miR-515-5p mimics+CBX4组、miR-515-5p mimics+CBX4-NC组miR-515-5p相对表达量均高于miR-515-5p NC组(P均<0.05)。miR-515-5p mimics组、miR-515-5p mimics+CBX4-NC组、miR-515-5p mimics+CBX4组CBX4 mRNA相对表达量及培养48、72 h的OD值均低于miR-515-5p NC组,且miR-515-5p mimics组、miR-515-5p mimics+CBX4-NC组降低更明显(P均<0.05)。培养12 h,各组细胞OD值比较P>0.05。培养24 h,miR-515-5p mimics组、miR-515-5p mimics+CBX4-NC组OD值均低于miR-515-5p NC组(P均<0.05)。与miR-515-5p NC组、miR-515-5p mimics+CBX4组比较,miR-515-5p mimics组、miR-515-5p mimics+CBX4-NC组细胞凋亡率均升高,CBX4蛋白相对表达量及侵袭细胞数均降低(P均<0.05)。结论过表达miR-515-5p能够抑制视网膜母细胞瘤细胞SO-RB50的增殖、侵袭能力并促进其凋亡,其机制可能与抑制CBX4表达有关。

大肠癌组织中CyclinD1和p16及Rb的表达及其临床意义

目的:探讨细胞周期调控因子在大肠癌组织中的表达及其与大肠癌临床病理特征的关系。方法:应用免疫组化SP法对70例大肠癌组织及距癌灶3cm以外的癌旁组织和10cm以外的正常组织中CyclinD1、p16和Rb进行检测。结果:CyclinD1在大肠癌中过度表达为51.4%(36/70),并与肿瘤分化程度呈反比,有淋巴结转移的大肠癌,CyclinD1阳性率为70.0%,无淋巴结转移的大肠癌阳性率为44.0%,两者相比差异有统计学意义(P=0.049)。p16在大肠癌中为低表达47.1%(33/70),癌旁组织和正常组织中p16的表达分别为57.1%和71.4%。Rb在大肠癌、癌旁及正常组织中的表达分别为65.7%、78.6%和85.7%。CyclinD1与p16表达呈负相关(P=0.000,r=-0.514),p16与Rb表达呈负相关(P=0.001,r=-0.403)。结论:CyclinD1过度表达与肿瘤的分化程度、淋巴结转移密切相关;p16表达可能受Rb蛋白的负调控;CyclinD1过度表达和p16低表达在大肠癌发生中起协同作用;大肠癌发生机制涉及CyclinD1、p16和Rb调节环路中多个基因的异常。

温和灸肾俞穴、关元穴对肾阳虚老年人衰老症状及外周血Rb蛋白表达的影响

目的观察温和灸对肾阳虚老年人外周血成视网膜母细胞瘤(retinoblastoma,Rb)蛋白表达以及衰老症状的影响,探讨温和灸的抗衰老作用。方法将60～75岁肾阳虚老年人81例随机分为温和灸治疗组41例和衰老对照组40例。温和灸治疗组采用温和灸肾俞穴、关元穴,18次为一个疗程,连续治疗两个疗程。采用积分表法观察温和灸对老年人治疗后综合情况的影响和症状改善情况,并用流式细胞术检测外周血Rb蛋白表达情况。结果 (1)经温和灸治疗,肾阳虚老年人衰老症状积分较衰老对照组疗效显著降低(P<0.01);(2)温和灸第二疗程末疗效较第一疗程末显著提高(P<0.01);(3)温和灸治疗组老年人外周血单个核细胞Rb蛋白阳性表达率显著低于衰老对照组(P<0.01)。结论温和灸可能通过干预肾阳虚老年人外周血Rb表达,改善衰老症状,降低衰老见证积分。

抑癌基因产物Rb蛋白在乳腺癌中的表达及其临床意义

目的:研究乳腺癌组织中Rb蛋白的表达及其临床意义。方法:采用S-P免疫组织化学染色法对52例乳腺癌组织和20例良性乳腺病变组织中Rb蛋白的表达进行检测。结果:Rb蛋白阴性表达率在乳腺癌组中为76.9%(40/52),显著高于良性乳腺病变组织[15.00%(3/20)](P<0.01);Rb蛋白的阴性表达与乳腺癌临床病理参数间无关联(P>0.05)。随访46例乳腺癌患者,38例Rb蛋白阴性表达者局部复发或远处转移为10.5%,而8例Rb蛋白阳性表达者则为0,两者比较差异有显著性(P<0.01)。结论:Rb蛋白的阴性表达与乳腺癌的发生有关;作为抑癌基因产物,Rb蛋白可以在蛋白质水平估计乳腺癌的病情,为临床制定治疗方案、估计病情及预后提供一个有意义的客观指标。

视黄酸阻滞胎鼠腭间质细胞周期进程并诱导凋亡

目的探讨全反式视黄酸(atRA)对小鼠胚胎腭间质细胞(MEPM)细胞增殖和细胞周期的影响及其分子生物学作用机制.方法MEPM细胞取材于孕13 d胎鼠腭组织.MTT比色法检测atRA对MEPM细胞增殖活力的影响;流式细胞术分析atRA对细胞周期分布的影响,并同时观察有无细胞凋亡现象发生;Western-blot检测atRA对细胞周期蛋白D和E表达的影响,并观察对视网膜母细胞瘤蛋白(Rb)磷酸化的影响.结果与溶剂对照组相比,在5～10μmol/L浓度范围内,atRA能够显著性抑制MEPM细胞增殖活力(P＜0.05),并将细胞周期滞留在G0/G1期(P＜0.05).atRA对细胞周期蛋白D和E的表达及相应酶活性均表现为抑制效应.观察Rb的变化也显示,atRA降低Rb的磷酸化作用.结论atRA对腭器官发育敏感期腭间质细胞增殖活力及细胞周期的抑制作用可能是atRA诱导诱导唇腭裂的机制之一.

细胞周期素D1、视网膜母细胞瘤样蛋白2及微小染色体维持蛋白7在肝细胞癌中的表达及对预后的意义

目的:研究细胞周期素D1(cyclin D1)、视网膜母细胞瘤样蛋白2(RBL2/p130)及微小染色体维持蛋白7(MCM7)在肝细胞癌(以下简称肝癌)中的表达及对预后诊断的意义。方法:用免疫组织化学法检测44例肝癌组织、26例癌旁硬化肝组织及18例正常肝组织中cyclin D1、RBL2/p130及MCM7的表达情况,并分析其与肝癌患者临床参数间的关系。结果:肝癌组织中cyclin D1和MCM7的阳性表达率分别是68.2%和72.7%,显著高于正常肝组织及癌旁硬化肝组织(P<0.01);RBL2/p130的阳性表达率为34.1%,显著低于正常肝组织及癌旁硬化肝组织(P<0.01),MCM7与cyclin D1的表达呈正相关(r=0.349,P<0.05),与RBL2/p130的表达呈负相关(r=-0.421,P<0.01);cyclin D1与RBL2/p130的表达呈负相关(r=-0.435,P<0.01)。Cyclin D1及MCM7的表达与肿瘤包膜的完整性、肿瘤分化程度及肝癌临床分期有关,RBL2/p130的表达与有无门脉癌栓、肿瘤分化程度及肝癌临床分期有关(P<0.05)。MCM7还与肿瘤大小及甲胎蛋白(AFP)值的大小有关。多因素分析显示肿瘤大小、MCM7及cyclin D1与肝癌预后相关(P<0.05)。MCM7及cyclin D1阳性表达的患者、RBL2/p130阴性表达的患者预后差(P<0.05)。结论:Cyclin D1、RBL2/p130和MCM7的异常表达在肝癌形成过程中发挥了重要作用。监测其在肝癌中的表达情况将有助于判断肝癌患者的预后。

人参、三七、川芎提取物对血管平滑肌细胞衰老相关β半乳糖苷酶及p16-cyclinD/CDK-Rb通路的影响

目的:观察人参、三七、川芎提取物延缓自发性高血压大鼠(spontaneously hypertensive rat,SHR)血管平滑肌细胞(vascular smooth muscle cell,VSMC)衰老的作用及其可能的细胞周期调控机制。方法:取大鼠胸主动脉用于分离VMSC,采用原代细胞培养的方法建立SHR大鼠VMSC模型,实验分为Wistar-Kyoto(WKY)大鼠对照组、SHR模型组、缬沙坦组、中药低剂量组和中药高剂量组。采用衰老相关β半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-gal)染色法观察细胞衰老情况,流式细胞仪分析细胞周期分布,逆转录聚合酶链式反应和蛋白质印迹法检测各组细胞p16、细胞周期蛋白D1(cyclin D1)、细胞周期蛋白依赖性激酶4(cyclin-dependent kinase4,CDK4)和成视网膜母细胞瘤蛋白(retinoblastoma protein,Rb)的表达。结果:SA-β-gal染色显示,与WKY组比较,SHR组SA-β-gal阳性细胞数明显增多(P<0.01),人参、三七、川芎提取物可使SHR VSMC SA-β-gal阳性细胞数明显减少(P<0.01);流式细胞仪检测显示,与WKY组比较,SHR组VSMC在G1期明显减少,S期细胞明显增加,中药干预后SHR的VSMC G1期细胞明显增多,S期细胞明显减少(P<0.05);实时荧光定量聚合酶链式反应检测显示,与WKY组比较,SHR组VSMC的p16和Rb mRNA表达量明显减少,cyclin D1和CDK4mRNA表达增加;人参、三七、川芎提取物干预后SHR VSMC p16和Rb mRNA表达量增加,cyclin D1和CDK4mRNA减少。蛋白质印迹法显示,与WKY组比较,SHR组VSMC的p16蛋白表达量明显减少,cyclin D1、CDK4和磷酸化Rb蛋白表达量明显增加(P<0.05);中药干预后SHR VSMC p16蛋白表达增加,cyclin D1、CDK4和磷酸化Rb蛋白表达量减少(P<0.05)。结论:人参、三七、川芎提取物通过改变p16-cyclin D/CDK-Rb通路因子的表达而发挥延缓SHR VSMC衰老的作用。

Cdk4/cyclin D1通路异常参与C型Niemann-Pic病小鼠神经元变性

以Balb/cnihnpc-1小鼠模型为对象,使用免疫组织化学和免疫印迹技术来研究cdk4/cyclinD1/Rb2通路的激活与神经元神经丝蛋白的过度磷酸化及神经元变性的相互关系,探讨C型Niemann-Pick病(NPC)病理性球状神经轴突形成的机制。实验表明,cdk4/cyclinD1在该小鼠模型脑白质球状神经轴突中异常聚集,其下游因子Rb2/p130呈现过度磷酸化并分布在上述病理性结构中。它们的分布随鼠龄增加而逐渐从脑干扩大到其他脑白质区域。球状神经轴突的主要成分之一为过度磷酸化神经丝蛋白,它与cdk4的分布在时空中具有高度的一致性。提示cdk4/cyclinD1/Rb2通路激活参与NPC神经元球状神经轴突形成,并与神经丝蛋白的异常磷酸化关系密切。该通路可作为干预神经元变性的一个新的靶点来挽救病损的神经元。