【作者】 伍斌；

【导师】 谢红付；

【作者基本信息】 中南大学 ， 皮肤与性病学， 2008， 博士

【摘要】 目的(1)探讨Ezrin,phos-Ezrin在正常皮肤(normal skin,NS)、脂溢性角化(seborrheic keratosis,SK)、基底细胞癌(basal cell carcinoma,BCC)、皮肤鳞癌(squamous cell carcinoma,SCC)中的表达及其与临床病理参数、预后之间的相关性。(2)研究黄芩素(Baicalein,BAI)对上皮鳞状细胞癌A431细胞的增殖、伪足及集落形成的影响,探索BAI对A431细胞无细胞毒性的最适抑制浓度范围。(3)探讨BAI抑制A431细胞增殖,影响A431细胞爬行运动及伪足形成的分子机制,明确BAI是否通过抑制Ezrin,phos-ezrin蛋白的表达来实现抗肿瘤侵袭转移的目的。方法(1)采用免疫组化法检测36例SCC、27例BCC和20例SK、10例正常人皮肤(normal skin,NS)中Ezrin及phos-Ezrin蛋白表达水平,COX回归分析二者与SCC预后的相关性。(2)以体外培养的人表皮鳞癌A431细胞为研究对象,用MTT法观察BAI对A431细胞的生长抑制作用;用光镜观察细胞生长的形态变化;扫描电镜观察siRNA及BAI对A431细胞伪足形成的影响;集落形成实验观察BAI对A431细胞的成瘤性的影响;细胞划痕实验检测BAI对A431细胞爬行运动的抑制作用;Trans-well检测BAI对A431细胞运动侵袭力的影响等细胞生物学行为的形态表现;(3)用流式细胞仪检测BAI处理A431细胞后,各期细胞比例和细胞凋亡率:RT—PCR检测BAI对A431细胞Ezrin蛋白mRNA表达量的影响;Western Blotting及免疫荧光技术检测BAI及siRNA对A431细胞Ezrin、phos-ezrin蛋白表达量的影响。结果1).Ezrin,phos-Ezrin在NS、SK、BCC、SCC中的阳性表达率分别为20.0%,10.0%;25.0%,15.0%;66.7%,77.7%和91.3%,97.2%,各组之间比较差异有显著性(P<0.01)。2).Ezrin,phos-Ezrin蛋白表达水平与皮肤肿瘤的性质、恶性程度(SCC的病理分级)、淋巴结转移密切相关(R1,r1=0.87,0.89;R2,r2=0.80,0.86;R3,r3=0.89,0.91,P<0.01,R1,R2,R3分别表示Ezrin与肿瘤性质,SCC病理分级,淋巴结转移的相关性;r1,r2,r3分别表示phos-ezrin与肿瘤性质,SCC病理分级,淋巴结转移的相关性)。3)MTT检测显示随着BAI作用时间的延长及剂量的增加对A431细胞增殖抑制作用增强,有明显的时间和剂量依赖性,与对照组比较差异有显著性(P<0.01);光镜观察显示随黄芩素浓度的增大细胞生长速度明显受到抑制,形态变圆;电镜观察显示A431细胞伪足明显缩短,数量减少;Transwell证实不同浓度BAI处理A431细胞48h后穿过人工基底膜的细胞数量明显减少,与对照组比较差异有显著性(P<0.01);同时BAI呈浓度依赖性地抑制A431细胞爬行运动。4)流式细胞仪检测显示随BAI浓度及作用时间的延长,细胞周期明显受到抑制,凋亡率显著增高,与对照组比较差异有显著性(P<0.05);BAI呈浓度依赖性地抑制A431细胞Ezrin,Phos-Ezrin及Ezrin-mRNA的表达,与对照组比较差异有显著性(P<0.01)。5)BAI呈浓度依赖地抑制A431细胞MMP2、MMP9蛋白的表达。各组两两比较差异有显著性(P<0.05)。结论1)Ezrin,phos-Ezrin表达水平在SK、BCC、SCC组织中与皮肤肿瘤的性质、恶性程度(病理分级)和淋巴结转移有关,两者呈平行表达关系,联合检测可能成为预测皮肤恶性肿瘤转移及预后的重要指标。2)BAI处理48h后,抑制A431细胞增殖而无细胞毒性的最适剂量范围为10μM-30μM。3)BAI通过直接或间接抑制Ezrin,phos-Ezrin表达而抑制A431细胞的增殖,侵袭运动,爬行运动,从而达到抗肿瘤增生及浸润转移的目的。更多还原

【Abstract】 Objective(1) To investigate Ezrin, phosphate-Ezrin expression in normal skin(NS), seborrheic keratosis(SK), basal cell carcinoma(BCC) and squamous cell carcinoma(SCC), and the relationship between Ezrin, phosphate-Ezrin and clinic pathological parameter and prognosis.(2) To deterimine the effect of baicalein in proliferation, pseudopodia and colony formation of epithelial squamous cell carcinoma-A431 cells and find out the optimal inhibitory concentration range of baicalein in A431 cell without cytotoxicity .(3) To probe into the molecular mechanism of baicalein inhibiting the proliferation, wound-healing and pseudopodia formation and then identify whether baicalein suppressing tumor-invasive action through inhibiting the expression of Ezrin, phosphate-ezrin.Methods(1) The expression of Ezrin, phos-ezrin was detected in 36 cases of SCC, 27 cases of BCC, 20 cases of SK and 10 cases of NS using immunohistochemistry, and the relationship between ezrin, phos-ezrin and prognosis of SCC was analyzed using COX regression.(2) Proliferation-inhibitory effect of baicalein on A431 cells was determined by MTT assay. Cell growth and their Morphologic change were observed under normal-optic-microscope. The pseudopod formation of A431 cells treated by baicalein and siRNA using scanning electron microscope, and colony formation of A431 was observed after baicalein treatment. The suppression effect of baicalein was determined on creeping movement of A431 cells using wound healing assay .The invasiveness of A431 cells was detected after baicalein and siRNA treatment by Trans-well assay.(3) Cell cycle analysis was performed by flow cytometry to determine cell percentage of each stage and apoptosis rate in A431 cells treated by baicalein. RT- PCR analysis to show the expression of Ezrin-mRNA of A431 cell line treated by baicalein, Western Blotting and immunofluorescence were used to investigate Ezrin and phos - ezrin expression in A431 cells treated by baicalein and siRNA.Results1) The expression of Ezrin and phos-Ezrin in NS, SK, BCC, SCC was 20.0% and 10.0%, 25.0% and 15.0%, 66.7% and 77.7%, 91.3% and 97.2% respectively. There was significant difference between each group (P<0.01).2) Ezrin, phos-Ezrin expression was closely related to skin-tumor type, clinic grade of SCC and lymph node metastasis. (R1, r1=0.87, 0.89 ; R2, r2=0.80, 0.86; R3, r3=0.89, 0.91, P<0.01. R1, Ezrin and cancer type, r1 phos-Ezrin and cancer type; R2, Ezrin and SCC grade; r2, phos-Ezrin and SCC grade; R3, Ezrin and lymph node metastasis; r3, phos-Ezrin and lymph node metastasis ).3) Methylthiazolyl tetrazolium (MTT) data showed that the inhibitory effect of baicalein on proliferation of A431 cell increased at time-dependent and dose-dependent, and it was significance to compare with control group(P <0.01). The growth index of A431 decreased along with baicalein concentration-increasing under optic-microscope and morphous became round and curtate. The pseudopodiao of A431 cells obviously decurtated and its amount reduced under scanning electron microscope. 48 hours after being treated by baicalein, A431 cell numbers permeating the artificial basement membrane largely decreased through Transwell assay, it was significant difference compared with control group (P < 0.01). Baicalein also suppressed the wound-healing of A431 cells depending on it’s dosage.4) Cell cycle results showed that cell cycle was obviously inhibited by baicalein at dosage and time dependent and apoptosis rate was also increased, it was significant compared with control (P < 0.05). Baicalein inhibited the expression of ezrin, phos-ezrin and ezrin -mRNA of A431 cells depending on it’s dosage (P < 0.01).5) Baicalein restrained the expression of MMP2, MMP9 proteins in A431 cells depending on it’s dosage.Conclusions1) The expression of Ezrin, phos-Ezrin in SK, BCC and SCC is closely related to skin tumor-type, clinic grade, and whether lymph node metastasis or not, both of them were expressed in a parallel way. Combined-detection of Ezrin, phos-Ezrin may be one of the important way of predicting skin malignant tumor metastasia and prognosis.2) The optimal dosage range of baicalein was 10μM to 30μM, which inhibited A431 cell proliferation and without cytotoxicity after 48 hours treated by baicalein.3) Baicalein inhibited A431 cell proliferation, invasion, wound -healing through direct or indirect inhibiting the expression of Ezrin, phos-Ezrin, so as to achieve anti-tumor hyperplasia and infiltrating-metastasis.更多还原