Correlation and expression of COX-2 and P53 protein in basal cell carcinoma of eyelid

The correlation between the expression of COX-2 and p53 protein in basal cell carcinoma （BCC） of eyelid and apoptosis was investigated. Specimens of BCC were collected from 40 cases （aged 28-68 y） at the Department of Pathology, Renmin Hospital of Wuhan University, and Department of Pathology, Zhongnan Hospital of Wuhan University during from 1999 to 2006. Five specimens of paracancerous tissues served as control group. Immunohistochemical staining was performed to detect the expression of COX-2 and p53 in the tissues. The average absorbance （A） and the average positive area rate of COX-2 and p53 protein were measured by image analysis. The positive area rate of COX-2 and p53 protein was analyzed by linear correlation analysis. It was found that COX-2 and p53 proteins were highly expressed in BCC of eyelid, and weakly expressed in paracancerous tissues. Image analysis revealed that the expression of COX-2 and p53 proteins in BCC of eyelid was sig- nificantly higher than that in paracancerous tissues （P〈0.01）. Spearman rank correlation analysis demonstrated a positive correlation between the expression of COX-2 and p53 （r=0.113, P=0.421）. It was concluded that COX-2 can increase the expression of p53 protein, therefore suppressing apoptosis.     

Lymphocytes in patients with psoriasis promote proliferation of keratinocytes

Objective: To analyze the effect of lymphocytes on proliferation of keratinocytes in patients with psoriasis. Methods: Lymphocytes in lesion and peripheral blood were isolated and amplified, then cultured together with normal keratinocytes. By MTT method, the living cells were quantified in the mixed culture. Results: Compared with normal controls, lymphocytes from lesion and peripheral blood of psoriasis both promote the proliferation of keratinocytes (P<0. 01 and P<0. 05 respectively). The concentrations of IL-2 and IFN-7 in the mixture of lesion lymphocytes and keratinocytes were significantly higher than that of controls. Tripterygium glycosides inhibited this promotion. Conclusion: Lymphocytes in patients with psoriasis (mainly Th1 cell) play an important role in proliferation of keratinocytes. This psoriasis cell model is useful for studies on signal transduction in psoriasis.     

Ultraviolet B light-induced apoptosis in human keratinocytes enriched with epidermal stem cells and normal keratinocytes

Background  The stem-cell compartment is the primary target for the accumulation of oncogenic mutations. Overexposure to solar ultraviolet radiation is responsible for the development and progression of >90% of skin cancers. Ultraviolet B (UVB) light-induced keratinocyte apoptosis is a strong preventive mechanism against carcinogenesis. The aim of this study was to isolate keratinocytes enriched with putative human epidermal stem cells and to investigate their apoptotic induction by UVB.

Methods  Keratinocytes enriched with putative human epidermal stem cells were isolated by adherence to collagen IV and the expressions of β1-integrin and p63 were investigated. Keratinocytes enriched with putative human epidermal stem cells and normal keratinocytes were irradiated with UVB at 0–80 mJ/cm². The apoptotic response was investigated with phase-contrast microscopy, Hoechst 33342 staining, flow cytometry of annexin V/PI, and procaspase-3 Western blotting.

Results  Keratinocyte enriched with stem cells expressed high levels of p63 protein and β1-integrin and low level of pan-keratin (C11). In comparison to non-irradiated cells, significant apoptosis of keratinocyte enriched with stem cells was found with 40 and 80 mJ/cm² UVB. However, significant apoptosis of normal keratinocytes was only found for 80 mJ/cm² UVB.

Conclusions  Human epidermal stem cells can undergo apoptosis in response to UVB radiation and are more susceptible than other keratinocytes. The method could be used in vitro studies of human epidermal stem cells.

     

Expression and role of AQP1 in cervical squamous carcinoma and its precancerous lesions

Objective： To investigate the expression of aquaporin 1 in cervical squamous carcinomas （CSC） and cervical precancerous lesions, and the relationship between the tumor clinicopathological parameters, prognosis and the expression of AQP1. Methods： Immunohistochemical method （EliVision） was used to detect the expression of AQP1 in samples from 106 patients [20 with normal cervical tissue, 30 with cervical intraepithelial neoplasia （stage Ⅰ and Ⅱ） and 56 with CSC]. Survival analysis was performed by Kaplan-Meier method. Results： AQP1 protein was expressed in vascular endothelia of all samples. It showed upregulation of AQP1 expression in CSC. There was a significant difference between CSC and normal cervical tissues （P〈0.05）. AQP1 was expressed in some tumor cells and unexpressed in normal squamous epithelial cells. And APQl-expressing tumor cells were positively related to lymph node metastasis. Patients with APQl-expressing tumor cells had the lower survival rate than the ones without. Conclusion： Abnormal expression of AQP1 plays an important role in the development of CSC. Positive expression of AQP1 in tumor cells maybe enhances tumor metastasis and could be used as a marker for tumor prognosis.     

THE EXPRESSION OF C-erbB-1 AND C-erbB-2 ONCOGENES IN BASAL CELL CARCINOMA AND SQUAMOUS CELL CARCINOMA OF SKIN

The expression of c-erbB-1 and c-erbB-2 oncogenes were investigated by immunohistochemistry using monoclonal antibodies to c-erbB-1 and c-erbB-2 protein in 43 cases of basal cell carcinoma (BCC) and 26 cases of squamous cell carcinoma (SCC). We found that the expression of c-erbB-1 oncogene in all BCC increased by different degrees and the expression of c-erbB-2 oncogene in BCC was significantly reduced or lost when compared to that in normal epidermal cells.Furthermore,apparent negative and positive relationships were observed respectively between the tumor differentiation and the expression of c-erbB-1 and c-erbB-2 oncogenes in SCC.It is suggested that the abnormal expression of c-erbB-1 and c-erbB-2 oncogenes in BCC and SCC may play a role in the development of skin tumors.The pattern of the c-erbB-1 and c-erbB-2 oncogenes expression in SCC may assist in distinguishing the biological behavior and prognosis of SCC.     

Expression of Survivin, p53 and its relationship with apoptosis, proliferation in hepatocellular carcinoma（HCC）

Objective:To investigate the expression of Survivin p53 and its relationship with apoptosis、proliferation in hepatocellular carcinoma(HCC). Methods:The expression of Survivin, p53 and the proliferation of tumor cells marked by proliferation cell nuclear antigen (PCNA) in 42 cases of HCC were assessed by immunohistochemical method. TUNEL was used to detect apoptosis. Results:Survivin protein was expressed in 30 of 42 cases of HCC(71.4%) and in 4 of 34 cases of adjacent cirrhosis tissues(11.8%). Expression of Survivin protein was negative in 10 cases of normal tissues. Survivin protein positive expression rate in HCC was significantly higher than adjacent cirrhosis tissues and normal tissues(P < 0.001). The increased Survivin protein expression in cancer was significantly associated with histological grade(P = 0.003), p53 protein(P = 0.013), survival time(P < 0.05) and the ratio of proliferative index to apoptotic index(P < 0.01), but was not significantly correlated with age, sex, clinical stage, tumor size, metastasis, AFP, HBs-Ag(P > 0.05).Conclusion:There is a marked increased expression of Survivin in HCC, which may play an important role in breaking the balance of proliferation and apoptosis of HCC cells. The correlation between Survivin and p53 expression in HCC indicates that cooperation between Survivin and p53 plays a certain role in occurrence and /or development of HCC.     

An in Vitro Tissue-Culture Evaluation of the Cell Toxicity of Platelet-Rich Plasma and Silver Dressings

Objective： To establish whether platelet-rich plasma （PRP） and silver-impregnated dressings, used individually are cytotoxic to human-skin keratinocytes, dermal-fibroblasts and adipose derived stem cells （ADSC） cultured in vitro. Method： Cultures of human keratinocytes, fibroblasts and ADSC were established in vitro. All test-cells were raised initially on RegenPRP~-Kit coated, testing petri-dishes. Group-I served as cultures without PRP or silver-stimulation. Group-II consisted of continuous culture of the 3-cell lines on the PRP-base without any further additives. Group-Ill cells initially raised on PRP were now exposed to and co-cultured with small nanocrystalline silver implants placed on the established cell monolayers （Acticoat~）. Diagnostic inverted-microscopy, trypan-blue staining, cell-testing and the Rosdy and Clauss cell-toxicity scoring system were used to identify cell-toxicity. Results： Cultures consisting of keratinocytes, dermal fihroblasts and ADSC （group-II） were established in 95% of all explants on platelet-rich plasma coated test-wells. In group-Ill or cell-recipients treated with silver implants, marked cell toxicity developed within days, with high non-viability staining and cell-scoring counts compared to Group-II （p〈 0.05）. Diluted PRP has a strong proliferative effect on keratinocytes, dermal fibroblasts and adipose derived stem cells （ADSC）. While silver impregnated implants interferes with newly cultured cells, epidermal cell proliferation and migration, and has notable cytotoxic properties in vitro. Conclusion： Diluted Regen-PRP shows good cytocompatibility with tissue-culture including keratinocytes, dermal fibroblasts and ADSC and favours cell proliferation of skin epidermal and mesodermal components ex vivo. Silver dressing explants placed on established cell-lines ex vivo, induce consistent cell-toxicity in the same cell lines when cultured under controlled conditions.     

Expression of aspartyl beta-hydroxylase and its clinicopathological significance in hepatocellular carcinoma

The human aspartyl beta-hydroxylase is a highly conserved enzyme that hydroxylates epidermal growth factorlike domains in transformation-associated proteins. The aspartyl beta-hydroxylase gene is upregulated in many human malignancies. The purpose of this study was to investigate the expression of aspartyl beta-hydroxylase in hepatocellular carcinoma. Aspartyl beta-hydroxylase mRNA levels were measured in 161 hepatocellular carcinomas and paired nontumorous liver tissues by conventional and real-time RT-PCR. Immunohistochemical staining of aspartyl beta-hydroxylase was performed using EnVision Plus system. The results showed that aspartyl beta-hydroxylase was overexpressed in 150 of 161 hepatocellular carcinomas （93 %）, including 45 of 48 unifocal small hepatocellular carcinomas （94 % ）. Aspartyl beta-hydroxylase was highly expressed in hepatocellular carcinoma cells in contrast to its low level of expression in non-neoplastic liver cells. The protein expression level of aspartyl heta-hydroxylase in the hepatocellular carcinoma was parallel with the mRNA expression level （r=0. 659 4, P〈 0. 000 1）. A significantly higher tumor aspartyl beta-hydroxylase overexpression level was associated with the presence of intrahepatic metastasis and the progression of histological grades.     

The expression of P63 protein in some keratinocyte original tissues and cells

ObjectiveTo examine the expression patterns of p63 in tissues of particular keratinocyte original hyperproliferate diseases and variety cell types for determining if P63 is the marker of proliferative potential keratinocytes.MethodsP63 protein was detected and analyzed by immunoreactivity method and Western blot in biopsy specimens of keratinocyte original disorders including squamous cell carcinomas SCC, basal cell carcinomas BCC, Bowen's disease and other tissues or cells, such as psoriasis vulgaris, normal skin tissues, primary cultured keratinocytes, immortal HaCaT cells, and epidermoid carcinoma cells A431.ResultsP63 protein was expressed in the nuclei of basal and suprabasal layer of the epidermis, germinative cells of sebaceous glands in normal epidermal. P63 was strongly and diffusely detected in the majority of tumor cells in BCC and poorly-differentiated SCC. In Bowen's disease, p63 expresses are remarkable in all cell layers. In the psoriasis plaque epidermal, p63 expressed mainly in basal cells and part of spinous cells. P63 expressed more strongly in primary cultured keratinocytes than in A431 cells or HaCaT cells.ConclusionP63 is a nuclei marker of undifferentiated keratinocytes with the proliferative potential and may disrupt the terminal differentiation. The overexpression of p63 reflects immaturity of the tumor cells. The immunohistochemical staining of p63 may be useful for investigating the origin and differentiation of tumor cells.     

LCE3C在皮肤癌组织中分布和细胞定位及其调控机制的初步研究

目的 研究LCE3C蛋白在皮肤鳞状细胞癌( SCC)以及基底细胞癌( BCC)中的分布及其调控的分子机制. 方法免疫组织化学法检测LCE3C在SCC和BCC组织中的表达. 构建绿色荧光蛋白融合表达的 LCE3C 重组表达载体pLCE3C-GFP,转染人SCC细胞系A431,采用激光共聚焦显微镜分析LCE3C在A431细胞中的定位. Western blot法检测转录因子GRHL3对LCE3C表达的影响.结果 免疫组化结果显示,与正常皮肤组织比较,LCE3C在SCC和BCC组织中表达明显增加;在皮肤癌组织中LCE3C主要分布在表皮的角质层. 激光共聚焦结果显示,LCE3C主要分布在细胞外基质( ECM )中;Western blot 结果显示,与角质形成细胞( HaCaT)比较, A431 细胞中 GRHL3 表达量显著降低,而LCE3C表达量明显增高;过量表达 GRHL3 的细胞( A431/GRHL3)LCE3C表达量显著降低.结论 与正常皮肤组织比较, LCE3C在皮肤癌中的表达增加,主要分布在皮肤角质层的ECM;转录因子GRHL3与皮肤癌组织LCE3C的表达呈负相关性.     

青蒿琥酯对人表皮鳞癌A431细胞增殖和凋亡的影响

目的 初步探讨青蒿琥酯(artesunate,ART)对人表皮鳞癌细胞增殖和凋亡的影响及其相关机制.方法 以人表皮鳞癌细胞系A431为研究对象,人永生化角质形成细胞系HaCaT为正常对照,顺铂(diamminedichloroplatinum,DDP)为药物阳性对照,通过MTT法和流式细胞术观察ART对A431细胞增殖和凋亡的影响,以及铁离子抑制剂去铁敏(desferrioxamine.DFOM)部分阻断ART对A431细胞的生长抑制作用.结果 ART对A431细胞有较明显的增殖抑制作用,对HaCaT细胞的损害显著低于DDP;药物组ART60 μmol/L(IC50)作用A431细胞48 h,A431细胞阻滞于S期[(67.60±4.12)%,P<0.05],同时凋亡率上调[(19.87±0.03)%,P<0.01];药物阻断组DFOM 60μmol/L预处理A431细胞4 h、加入ART60 μmol/L作用钙h,凋亡率显著下调[(7.37±0.02)%,P<0.01],而周期检测未发生变化.结论 ART通过阻滞A431细胞停滞于S期和诱导A431细胞凋亡发挥增殖抑制作用,此作用在一定范围内与药物剂量呈正相关;ART对A431细胞的诱导凋亡作用与Fe2+介导有关;ART毒副作用明显低于DDP.     

银屑病患者皮损间充质干细胞对HaCaT细胞增殖的影响

目的探讨银屑病患者皮损间充质干细胞（mesenchymal stem cells,MSCs）对HaCaT细胞增殖的影响,揭示银屑病可能的免疫发病机制。方法寻常性银屑病患者8例,进行期4例,静止期4例,银屑病皮损面积和严重程度（PASI）评分范围为1．6—18．0,平均11．64。健康对照组8例,取自我院泌尿外科和整形外科手术切除的正常皮肤。分离、培养患者皮损与健康人皮肤MSCs,流式细胞术进行细胞鉴定;将第5代皮肤MSCs与HaCaT细胞共培养,并设自然增殖组,采用实时细胞分析系统进行HaCaT细胞增殖检测,培养74h后用细胞计数法检测HaCaT细胞的数量。结果倒置显微镜下,银屑病组与健康对照组皮肤MSCs的细胞形态相似,细胞表面抗原均高表达CD29、CD44、CD73、CDg0、CDl05,而CD34、CD45及HLA—DR表达阴性。皮肤MSCs可抑制HaCaT细胞的增殖（P〈0．05）,与健康对照相比,银屑病患者皮损MSCs抑制HaCaT细胞增殖的能力显著减弱[患者组细胞数为（2．35±0．254）×105,对照组细胞数为（2．04±0．122）×105,P〈0．05]。结论银屑病患者皮损MSCs抑制角质形成细胞增殖的能力减弱,这可能是银屑病的发病机制之一。     

COX-2、p53和c-myc在皮肤肿瘤中的表达及意义

目的利用组织芯片检测Cox-2、p53和c-myc在皮肤鳞状细胞癌、基底细胞癌和Bowen病中的表达,探讨它们在上述皮肤肿瘤发病中的意义。方法手工制作组织芯片,用免疫组织化学技术检测30例皮肤SCC,21例BCC,20例BD的石蜡包埋组织及10例正常皮肤组织中COX-2、p53和c-myc的表达。结果 COX-2蛋白在皮肤肿瘤组织中阳性率明显高于正常皮肤对照组,两者差异有统计学意义（P〈0.01）;p53蛋白在皮肤肿瘤组织中阳性率高于正常皮肤对照组,两者差异有统计学意义（P〈0.05）;c-myc蛋白在皮肤肿瘤组织中阳性率高于正常皮肤对照组,两者差异有统计学意义（P〈0.05）;COX-2、p53和c-myc相互之间在所检测皮肤肿瘤中的表达无相关性（P均〉0.05）。结论组织芯片适合于样本量较大的研究。COX-2在皮肤SCC、BCC、BD中呈高表达,其与皮肤SCC、BCC、BD的发生关系密切。p53在皮肤SCC、BCC、BD中呈高表达,其与皮肤SCC、BCC、BD的发生关系密切。c-myc在皮肤SCC、BCC中呈高表达,与皮肤BD的发生无明显关系。三者均可作为判定上皮源性皮肤肿瘤恶性程度的一种标志。COX-2、p53和c-myc相互之间在所检测皮肤肿瘤中的表达水平无相关性,具体机制尚不清楚。     

整合素β1在皮肤鳞状细胞癌组织中的表达作用

目的 检测整合素β1在皮肤鳞状细胞癌组织中的表达情况,探索干细胞相关因子与肿瘤的关系.方法 采用免疫组织化学及细胞学染色方法检测整合素β1在人皮肤鳞状细胞癌组织及人皮肤鳞状细胞癌细胞株A431中的表达,全反式维甲酸(ATRA)诱导鳞状细胞癌细胞分化,RT-PCR检测分化前后整合素β1变化水平.结果 在高分化鳞状细胞癌中,整合素β1在肿瘤团块外周基底样细胞呈连续性表达,内侧至团块中央可见散在或岛屿状阳性细胞;在低分化鳞状细胞癌组织中,岛屿状阳性细胞明显增多,呈弥漫分布;在A431细胞中,整合素β1散在表达于肿瘤细胞.ATRA诱导24h和48h后,ATRA处理组整合素β1与内参照β-actin的RT-PCR产物电泳光密度值之比分别为0.071±0.025和0.029±0.018,明显低于对照组的0.148±0.027和0.136±0.011(P<0.05).结论 整合素β1在皮肤鳞状细胞癌组织培养肿瘤细胞中具有异质性表达模式,并随肿瘤细胞分化程度增高而表达下降,提示整合素β1与皮肤鳞状细胞癌发生及可能存在的肿瘤干细胞具有密切关系.     

人表皮角质形成细胞Toll样受体表达

目的 研究人表皮角质形成细胞Toll样受体表达谱.方法 培养人永生化角质形成细胞系HaCaT细胞和正常人表皮角质形成细胞(NHEK),采用分散酶分离表皮.逆转录-聚合酶链反应(RT-PCR)法分别检测10种Toll样受体mRNA表达,流式细胞仪检测HaCaT细胞和NHEK表面Toll样受体2和4蛋白的表达.结果 HaCaT细胞、NHEK以及分离的表皮均有全部10种Toll样受体mRNA表达,其表达强弱各不相同.流式细胞仪检测显示HaCaT细胞和NHEK表面有Toll样受体4蛋白的表达,而Toll样受体2无明显表达.结论 人表皮角质形成细胞中有Toll样受体1～10 mRNA的组成性表达.     

P^53,Bcl—2在皮肤鳞状细胞癌、基底细胞癌脂溢性角化病中联合表达的意义

目的：探讨P^53蛋白,Bcl-2蛋白在皮肤鳞状细胞癌（SCC）,基底细胞癌（BCC）及脂溢性角化病（SK）中的表达及意义。方法：采用S－P法分别对12例SCC,BCC,SK进行免疫组化P^53蛋白,Bcl－2蛋白检测。结果：12例SCC,BCC中分别有9例,11例P^53蛋白表达阳性有10例,11例Bcl-2蛋白表达阳性,12例SK中有1例P^53表达阳性,11例Bcl-2表达阳性,P^53蛋白在SCC,BCC中表达显著高于SK（P＜0．01）。结论：P53蛋白基因突变和Bcl-2蛋白的过度表达（即细胞凋亡机制）在皮肤恶性肿瘤的发生,发展中起重要促进作用。     

两种老年性皮肤肿瘤Dsg1和E-cad的表达

目的比较两种老年人常见的皮肤肿瘤—鳞状细胞癌(BCC)与基底细胞癌(SCC)的生物学行为的异同。方法用免疫组化方法检测了26例SCC、30例BCC及10例正常皮肤组织中桥粒芯糖蛋白1(Dsg1)和E-钙黏着蛋白(E-cad)的表达。结果Dsg1在BCC和SCC中的表达均显著减弱或完全消失;E-cad在SCC中的表达显著减弱,而在BCC中的表达却为阳性。结论BCC和SCC侵袭性生长的特性与Dsg1的低表达有关,SCC易发生转移的特性与E-cad的低表达有关。