成釉细胞瘤中细胞周期素D1及其抑制因子的表达

目的 研究细胞周期素D1(cyclin D1)、周期素依赖激酶抑制因子p16INK4、p21WAF1 mRNA和p27KIP1 蛋白在人成釉细胞瘤(ameloblastomas,ABs)中的表达及其临床生物学意义.方法 用原住杂交和免疫组化(SP法),分别检测ABs中cyclin D1、p16INK4、p21WAF1 mRNA和p27KIP1 蛋白的表达.结果 cyclin D1 mRNA阳性率为42.6%(23/54);p16INK4、p21WAF1 mRNA和p27KIP1 蛋白的阳性率分别为31.5%(17/54)、22.6%(12/53)、16.7%(9/54).伴随ABs的复发与恶变cyclin D1 mRNA阳性率逐渐上升,p16INK4、p21WAF1 mRNA和p27LIP1 蛋白阳性表达丢失.经Kendall相关性分析得出,cyclin D1 mRNA与p21WAF1 mPRNA、p27KIP1 蛋白之间在AB中rk分别为-0.213、-0.284,均P<0.05.结论 AB的发生发展可能与cyclin D1过表达及p16INK4、p21WAF1 mRNA、p27KIP1 蛋白丢失等多种因子共同调控有关.     

p21WAF1、细胞周期蛋白E mRNA及p27KIP1蛋白在成釉细胞瘤中的表达

目的研究细胞周期蛋白E(cyclin E)、周期素依赖激酶抑制因子p21WAF1 mRNA和p27KIP1蛋白在人成釉细胞瘤(AB)中的表达,探讨其临床生物学意义.方法用原位杂交法分别检测了54例AB中cyclin E和 p21WAF1 mRNA,免疫组化SP法检测p27KIP1蛋白的表达.结果 cyclin E mRNA在AB浆核中有较强的表达,阳性率为66.7%(36/54),伴随AB的复发与恶变,cyclin E mRNA阳性率有所增加,原发AB、复发AB、恶性AB组间差异有统计学意义(P<0.05).牙源性角化囊肿(OKC)中cyclin E阳性率为50.0%(8/16).p21WAF1 mRNA在AB浆核中表达强度减弱且有较多的丢失,阳性率为22.6%(12/53),伴随AB复发与恶变阳性率表达下降.OKC p21WAF1阳性率为37.5%(6/16).p27KIP1蛋白在AB细胞核中大部分丢失,AB阳性率为16.7%(9/54),OKC 中p27KIP1阳性率为6.3%(1/16).AB中,cyclin E与p21WAF1 、p27KIP1均呈中度负相关(rk=-0.440, rk=-0.519, P=0.001).结论 AB的发生、浸润与细胞的增殖活性相关,且受cyclin E的高表达及p21WAF1 、 p27KIP1的低表达等多种因子共同调控.     

cyclin D1、p16蛋白在腮腺多形性腺瘤(PA)和癌在多形性腺瘤中(CPA)的表达及意义

目的：检测正常腮腺、PA和CPA中cyclin D1和p16的表达情况及其表达间的相关性。方法：免疫组化S－P法检测cyclin D1和p16的表达。结果：cyclin D1在PA和CPA中的表达率为56．8％和78．6％;p16在PA和CPA中的表达率分别为65％和28．6％。在多数病例（34／51）中cyclin D1和p16呈相反表达,关联系数r=-0.372。结论：cyclin D1与p16表达呈负相关,由它们异常所致细胞周期调控“pRb”通路的异常,在腮腺CPA的发生中可能具有一定的作用。     

口腔鳞癌中p16/CDK4/cyclinD1/pRb通路的表达及其意义

目的分析口腔黏膜鳞状细胞癌(OSCC)中pRb、CDK4c、yclinD1、p16INK4a等细胞周期调控因子的表达状况和相互间的联系及其临床意义。方法采用免疫组化SP法,研究47例OSCC及10例正常口腔黏膜中pRb、CDK4c、yclinD1和p16INK4a的表达情况,并结合随访资料进行相关性分析。结果47例OSCC中,pRb、CDK4、cyclinD1和p16INK4a阳性表达率分别为55%、60%、74%和38%,与正常口腔黏膜中的表达有显著差异(P<0.05)。cyclinD1的表达和淋巴结转移有密切关系(P<0.05),p16和pRb呈负相关(r=-0.312)。结论p16/Rb通路蛋白的异常表达和OSCC的发生有密切的关系;cyclinD1可作为OSCC预后的辅助性指标之一。     

抑癌基因p16在成釉细胞瘤及牙源性角化囊肿中表达的研究

目的 研究成釉细胞瘤（Ameloblastoma,AB）中 p16蛋白及 p16 mRNA表达,探讨其生物学意义。方法 应用免疫组化方法（SP）检测40例 AB及牙源性角化囊肿（Odontogenic keratocyst,OKC）7例,基底细胞癌（Basal cell carcinoma,BCC）8例 p16蛋白的表达,同时用原位杂交法检测 p16 mRNA在20例 AB中表达。结果 40例AB,7例 OKC,8例 BCC,p16蛋白缺失表达分别为70％,85.7％,100％。p16mRNA缺失率80.5％,与蛋白缺失表达一致率53.8％。结论 （1）AB与 OKC的发生与p16蛋白缺失有关。（2）AB中原发与复发比,原发与恶变比 p16蛋白缺失率不同（P<0.05）。（3）AB及 OKC,BCC三种不同病变经统计学处理 P>0.05,末见明显相关性。（4）在 AB中p16蛋白与 p16 mRNA表达 P<0.05,具有一定相关性。     

Cyclin D1反义寡核苷酸对Tca8113/CDDP细胞基因转录表达的影响

目的：构建细胞周期蛋白D1(cyclin D1)的反义寡核苷酸真核表达载体,并检测其转染Tca8113／CDDP细胞系后的表达情况。方法：以Tca8113／CDDP细胞总RNA为模板,应用RT-PCR法扩增cyclinD1全长．通过克隆至pUCm-T载体中,测序完全正确;经IPrG诱导,可正确表达蛋白。上下游分别利用HindIII和EcoRI的酶切位点插入真核表达载体pcDNA3．1,测序鉴定;并构建表达cyclin D1反义寡核苷酸的2个重组载体,通过Lipofectamine将其导入Tca8113／CDDP细胞系中,G418筛选获得阳性稳定表达细胞系：通过RT-PCR、免疫组化检测cyclin D1基因转录和蛋白表达。结果：成功构建pcDNA3．1-cyclin D1真核表达载体,分别命名为pcDNA3．1空载体（Co）、cyclin D1 mRNA 5’端为靶区的反义载体(C5’)、cyclin D1 mRNA 3’端为靶区的反义载体(C3’)、cyclinD1正义载体(CDl)。3’端反义寡核苷酸转染细胞后,cyclin D1 mRNA表达水平降低58．8％,mRNA5’端反义寡核苷酸组无明显抑制。免疫组化结果显示,转导正义寡核苷酸组cyclin D1阳性表达为最高,转导mRNA3’端、mRNA5’端反义寡核苷酸组为弱阳性表达。结论：cyclin D1 mRNA3’端反义寡核苷酸能明显抑制cyclin D1在Tca8113／CDDP中的表达,为进一步研究逆转Tca8113／CDDP耐药性提供了实验工具。     

p53、p21、p27、p16在口腔鳞癌中的表达及与预后的关系

目的:研究细胞周期相关蛋白p53、p21、p27、p16在口腔鳞癌组织中的表达及其与预后的相关性,寻找预后判断的有效生物标志物。方法:用免疫组织化学的方法检测随访资料完整的110例口腔鳞癌患者术后石蜡切片中p53、p21、p27、p16的表达,通过目标蛋白阳性染色细胞比例和阳性细胞中蛋白的染色强度判定各蛋白的表达水平,应用SAS 9.0软件中的Kaplan-Meier分析p53、p21、p27、p16蛋白表达水平与口腔鳞癌预后的相关性,筛选和确定与口腔鳞癌预后相关的细胞周期蛋白。结果:110例口腔鳞癌标本蛋白检测和统计分析结果表明,p53、p27、p16蛋白的表达水平与口腔鳞癌术后生存时间并无显著的相关性;p21表达与口腔鳞癌的术后生存率呈显著的相关性(P<0.05)。结论:口腔鳞癌患者癌组织标本的p21表达水平与预后呈正相关,p21有望成为口腔鳞癌预后判断的候选生物标志物。     

miR-31-5p对牙髓干细胞HIF-1α/BNIP3信号通路及成骨相关因子表达的影响

目的: 探讨微小RNA-31-5p（miR-31-5p）对牙髓干细胞（DPSCs）低氧诱导因子1α（HIF-1α）/Bcl-2/腺病毒E1B 19-kDa相互作用蛋白3（BNIP3）信号通路及成骨相关因子表达的影响。方法: 体外培养人DPSCs,分为对照组（不转染）、mimic NC组（转染negative control-miR-31-5p）、miR-31-5p mimic组（转染hsa-miR-31-5p mimic）、siRNA NC组（转染nonsense siRNA）和miR-31-5p siRNA组（转染miR-31-5p siRNA）。实时荧光定量PCR（qRT-PCR）检测各组DPSCs细胞中miR-31-5p、HIF-1α、BNIP3、碱性磷酸酶（ALP）、Runt相关转录子2（Runx2）mRNA的表达情况,MTT法检测各组DPSCs细胞增殖情况,ALP活性测定试剂盒检测各组DPSCs细胞ALP活性,蛋白印迹（WB）法检测各组DPSCs细胞中HIF-1α、BNIP3、Runx2蛋白的表达情况。采用SPSS 24.0软件包对数据进行统计学分析。结果: 与对照组、mimic NC组相比,miR-31-5p mimic组DPSCs细胞A值、ALP mRNA表达水平及活性、Runx2 mRNA及蛋白表达水平显著降低（P<0.05）,ALP染色明显减弱,miR-31-5p mRNA、HIF-1α、BNIP3 mRNA及HIF-1α、BNIP3、Beclin1蛋白表达水平显著升高（P<0.05）。与对照组、siRNA NC组相比,miR-31-5p siRNA组DPSCs细胞A值、ALP mRNA表达水平及活性、Runx2 mRNA及蛋白水平显著升高（P<0.05）,ALP染色明显增强,miR-31-5p mRNA、HIF-1α、BNIP3 mRNA及HIF-1α、BNIP3、Beclin1蛋白表达水平显著降低（P<0.05）。结论: miR-31-5p可以激活HIF-1α/BNIP3信号通路,抑制DPSCs细胞的成骨相关因子表达。     

人成釉细胞瘤中端粒酶活性与细胞周期素A的表达

目的　研究端粒酶hTERT基因在人成釉细胞瘤 (ameloblastoma,AB)中的表达及其与细胞周期素A (cyclinA)和相关因子 p53蛋白、增殖细胞核抗原 (proliferationcellnuclearantigen ,PCNA)的关系 ,探讨其临床生物学意义。方法　用免疫组化 (SP法 )和原位杂交技术分别检测了AB中细胞周期素A、p53蛋白和PCNA ,hTERTmRNA的表达 ,同时以正常口腔粘膜和牙源性角化囊肿(odontogenickeratocyst,OKC)为对照。 结果　在正常口腔粘膜中只有 1例hTERT阳性 (1 / 7) ,cyclinA、p53蛋白、PCNA三者之间表达有着相似共性。OKC中表达在基底或副基底层细胞核中 ,有1 4例hTERT阳性 (1 4 / 1 6) ,4例cyclinA阳性 (4/ 32 )、1 1例p53蛋白阳性 (1 1 / 2 5) ,5例PCNA阳性(5/ 9)。hTERTmRNA在AB中表达率为 94 4% (51 / 54) ,cyclinA为 66 7% (40 / 60 ) ,p53蛋白为85 7% (42 / 4 9) ,PCNA为 78 1 % (2 5/ 32 ) ,hTERT与cyclinA、p53蛋白、PCNA之间经Kendall相关分析 ,rs 分别为 0 91 4、0 848、0 80 4 ,P <0 0 5。结论　随着组织学分级增高 ,hTERTmRNA与cyclinA、p53蛋白、PCNA阳性率及信号强度均增高。在AB中端粒酶的活性表达与p53蛋白的蓄积有关 ,与细胞的增殖、分化有关 ,并受cyclinA调节 ,在S期中有较高表达     

Cyclin D1,Cyclin E,c-Myc在涎腺良恶性多形性腺瘤中的表达研究

目的：研究cyclin D1,cyclin E,c-myc在涎腺多形性腺瘤中的表达,与临床生物学特性和细胞增殖的关系以及对于涎腺多形性腺瘤病理学诊断的意义。方法：采用免疫组化方法检测30例良性多形性腺瘤,30例细胞丰富型多形性腺瘤和30例恶性多形性腺瘤中cyclin D1、cyclin E、c-myc蛋白的表达水平,并与30例癌旁正常涎腺组织中的表达对比。结果：cyclin D1、cyclin E和c-myc在恶性多形性腺瘤中的阳性表达率明显高于良性和细胞丰富型多形性腺瘤（P〈0.05）,而三者在良性多形性腺瘤中的阳性表达率与在细胞丰富型多形性腺瘤的阳性表达率无统计学差异。cyclin E、cyclin D1和c-myc蛋白的异常表达与患者性别、是否复发、发生部位、肿瘤的大小无关（P〈0.05）。在恶性肿瘤中,cyclin D1的异常表达与肿瘤的TNM分期相关（P〈0.05）,cyclin E蛋白的表达于肿瘤的浸润性相关（P〈0.05）。c-myc的过表达与cyclin D1和cyclin E的阳性表达呈正相关。结论：cyclin D1、cyclinE、c-myc共同参与调控细胞周期,可作为预测多形性腺瘤恶变和预后的分子生物学指标之一,是多形性腺瘤病理学分类的依据之一。     

p21WAF1/CIP1 expression is a marker of poor prognosis in oral squamous cell carcinoma

BACKGROUND: Previous research on the prognostic relevance of p21(WAF1/CIP1) in oral squamous cell carcinomas (OSCC) yielded inconclusive and contradictory data. OBJECTIVES: To investigate the prognostic significance of p21(WAF1/CIP1) expression, its relationship to p53 accumulation, proliferation-associated proteins Ki-67 and cyclin D1 in relation to survival and clinicopathological features in OSCC. METHODS: Surgical specimens taken from 106 randomly selected patients were studied by immunohistochemistry. Expression of the protein of interest was correlated with clinical data. RESULTS: p21(WAF1/CIP1) expression was found in 61.3% of OSCCs. Expression of p21(WAF1/CIP1) significantly correlated with tumor size (P = 0.005), lymph node involvement (P = 0.002), clinical stage (P < 0.001), and tumor site (P = 0.002). Patients with tumors showing p21(WAF1/CIP1) immunopositivity had decreased 2-year survival (P = 0.018). Expression of p21(WAF1/CIP1) was not related to age, gender, risk factors (tobacco, alcohol), dental status, or tumor differentiation grade. The p21(WAF1/CIP1) expression positively correlated with proliferation-related variables Ki-67 (P = 0.010) and cyclin D1 (P < 0.001), but not with p53 expression. CONCLUSIONS: The expression of p21(WAF1/CIP1) was found to be associated with poorer prognosis and tumor aggressivity in OSCC.     

Prognostic role of p21WAF1 expression in areca quid chewing and smoking-associated oral squamous cell carcinoma in Taiwan.

BACKGROUND: Alterations in p21WAF1 protein expression have been observed in a wide variety of human cancers by immunohistochemistry, and both decreased and increased levels of p21WAF1 protein expression have been shown to correlate with poor prognosis. METHOD: To examine the relation between p21WAF1 protein expression and prognosis in oral squamous cell carcinomas (SCCs), we performed an immunohistochemical study with antip21WAF1 antibody on 43 oral SCCs. Immunostaining results were then correlated with p53 protein levels, clinicopathological parameters of the tumors and overall patient survival. RESULTS: Of the 43 patients, 31 (72%) had tumors with positive p21WAF1 nuclear staining and 27 (63%) had tumors with p53 nuclear staining. There was no significant correlation between p21WAF1 and p53 protein expressions and both mutant p53-containing oral SCCs overexpressed p21WAF1 protein. In addition, no significant correlation was found between the p21WAF1 expression and the patients' age, sex, oral habit, cancer location, or primary tumor TNM status at the time of initial presentation. The Kaplan-Meier analysis showed a significant correlation between p21WAF1 protein overexpression and poor patient overall survival (P = 0.049). When p53 and p21WAF1 were evaluated together, the 5-year overall survival was lowest in p53(+)-p21WAF1(+) patients and highest in p53(-)-p21WAF1(-) patients (P = 0.057). CONCLUSION: Combined evaluation of p21WAF1 and p53 expressions may be useful in estimating the prognosis of patients with oral SCCs in Taiwan.     

Detection of cell cycle-related factors in ameloblastomas

To determine whether cell cycle regulation or alteration plays a role in oncogenesis and cytodifferentiation of odontogenic epithelium, cell cycle-related factors, including cyclin D1, p16INK4a, p21(WAF1/Cip1) and p27Kip1 proteins, DNA topoisomerase IIalpha and histone H3 mRNA, were examined in 8 tooth germs and 31 ameloblastomas. Cyclin D1 was expressed in epithelial cells near the basement membrane in tooth germs and ameloblastomas, suggesting that this protein participates in cell proliferation in odontogenic epithelium. Immunoreactivity for p16 protein was observed in most epithelial cells in tooth germs and ameloblastomas. Expression of p21 protein was detected in most epithelial cells in tooth germs and ameloblastomas, but not in keratinizing or granular cells in variants of ameloblastomas. Expression of p27 protein was chiefly found in central polyhedral cells and keratinizing cells in tooth germs and ameloblastomas. These cyclin-dependent kinase inhibitors were well preserved in ameloblastomas as compared with tooth germs, suggesting that the odontogenic epithelium is strictly regulated by these factors. The cell cycle phase/cellular proliferation markers, DNA topoisomerase IIalpha and histone H3 mRNA, were localized in scattered epithelial cells attached to the basement membrane in tooth germs and ameloblastomas.     

Vigabatrin-induced modification of Ki-67 expression in gingival epithelium: immunohistochemical study of a short series

OBJECTIVE: To study the expression and role in vigabatrin (VGB)-induced gingival enlargement of Ki-67 antigen and p27KIP1, p21WAF1, and p53, proteins that activate or inhibit cell-cycle progression. MATERIALS AND METHODS: Six patients treated with VGB for partial epileptic seizures refractory to classic anticonvulsant treatment were studied. Gingival biopsies were taken from four of these patients for immunohistochemical studies; 10 control biopsies from individuals with healthy gingiva and 10 from patients with periodontal disease were also evaluated. RESULTS: Four of the six patients presented some degree of gingival enlargement (mild or moderate). Nuclear expression of Ki-67 was elevated (mean of 894 positive cells/mm2 in VGB-induced gingival enlargement vs. 391 cells/mm2 in controls with healthy gingiva and 425 cells/mm2 in controls with periodontal disease) (p < 0.01, analysis of variance: anova), and nuclear expression of cyclin-dependent kinase (cdk) inhibitors p27KIP1 and p21WAF1 was reduced. The patients with gingival enlargement presented inflammatory infiltrate in lamina propria, mainly composed of T lymphocytes (CD3+) and plasma cells (CD38+), which was even more intense than in the biopsies of patients with periodontal disease. CONCLUSION: The overexpression of antigen Ki-67 and slight underexpression of cdk-inhibitors p27KIP1 and p21WAF1 suggest that VGB induced an increase in cell proliferation and contributed, together with concomitant periodontal disease, to the gingival enlargement.     

Association of aberrant p53 and p21WAF1 immunoreactivity with the outcome of oral verrucous leukoplakia in Taiwan

The expression of p53 and p21WAF1 in 53 oral verrucous leukoplakias (OVLs), mostly non-dysplastic lesions, was investigated to ascertain the role of such events in malignant conversion. Immunohistochemical analysis revealed aberrant p53 and p21WAF1 immunoreactivity in 51% (27 cases) and 75% (40 cases), respectively. After an average follow-up period of three and a half years, histopathological examination revealed that 22 (42%) cases had developed oral squamous cell carcinoma (OSCC), 14 (26%) cases had undergone recurrence, and 17 (32%) cases were free of disease. The oncogenic potential of this subset of premalignant lesions warrants attention. A significant difference in the frequency of OSCC progression/recurrence was noted in lesions bearing aberrant immunoreactivity of either p53 (93% vs 42%; P=0.00008) or p21WAF1 (80% vs 32%; P=0.002) in comparison with lesions without immunoreactivity. This study suggested that the aberrant immunoreactivity of p53 and p21WAF1 may represent important alterations of OVL and could affect the outcome of this lesion.     

人成釉细胞瘤中端粒酶与抑癌基因p53的表达

目的探讨端粒酶hTR、hTERT基因在人成釉细胞瘤(Ameloblastoma,AB)中的表达,并与p53蛋白相比较,探讨其临床生物学意义。方法用原位杂交及免疫组化技术,检测54例AB的hTR和hTERT的表达及49例AB的p53蛋白,将二者进行分析,同时以7例口腔黏膜为对照。结果94.4%(51/54)的AB有hTR、hTERTmRNA的表达,81.2%(13/16)和87.5%(14/16)的牙源性角化囊肿(Odontogenickeratocyst,OKC)及2/7和1/7例的正常口腔黏膜分别有hTR和hTERTmRNA表达,3组相比差异有显著性(P<0.001)。hTR与hTERT在AB中表达具有相关性(rs=1.000,P<0.001)。p53蛋白在85.7%(42/49)AB、44%(11/25)OKC、33.3%(1/3)正常口腔黏膜上皮的细胞核为阳性。hTERT与p53蛋白的rs=0.754,P<0.001,hTR与p53蛋白的rs=0.536,呈正相关,P<0.001。结论hTR和hTERT在AB中的检出率高于p53蛋白,其间具有一定相关性(P<0.001),并与AB的生物学行为有关。     

Prognostic value of cyclin D1, p27, and p63 in oral leukoplakia

BACKGROUND: Studies on the expression of genes regulating cell proliferation and apoptosis are essential to help better understand the severity and possible malignant transformation of oral leukoplakia. METHODS: The characteristics of cyclin D1, p27, and p63 were investigated in this microscopic study, complementing our previous results with Ki67, p53, and the apoptosis index. Clinical and histologic as well as immunohistochemical studies were carried out on oral leukoplakia of 18 patients. Homogenous, or non-homogenous (nodular or speckled) and erythroleukoplakia were determined clinically. Pathologic classification was performed according to the degree of dysplasia. Immunoperoxidase reaction for cyclin D1, p27, and p63 was carried out on the biopsy specimens and the positivity of the reactions was calculated for 1000 epithelial cells. RESULTS: The expression of cyclin D1 increased in parallel with the severity of leukoplakia. The p27 index was 14-16% in homogenous and nodular leukoplakias but it was substantially lower to 1-2% in erythroleukoplakia. The p63 index was 10% in homogenous, 5% in nodular or speckled, but nearly 20% in erythroleukoplakia, on the average. CONCLUSION: These results suggest that the characteristic expression of cyclin D1, p27, and p63 in various forms of leukoplakia may be of prognostic value.