【作者】 王珍；

【导师】 何春涤；

【作者基本信息】 中国医科大学 ， 皮肤性病学， 2007， 博士

【摘要】 维甲酸、γ-干扰素诱导皮肤鳞状细胞癌SCL-1细胞凋亡的机制研究前言与目的皮肤鳞状细胞癌(squamous cell carcinoma，SCC，简称鳞癌)是起源于表皮角质形成细胞或附属器鳞状上皮细胞的一种恶性肿瘤，是常见的皮肤恶性肿瘤之一，其发病率约占全部非黑素细胞癌的20％。目前，外科手术仍然是治疗皮肤鳞癌的首选方法，外科手术和术前术后的放疗、化疗是治疗皮肤鳞状细胞癌的标准模式。然而，皮肤鳞癌转移后的治疗是尚需解决的难题，因此，寻求有效的基因治疗技术具有重大意义。基因治疗的关键就是靶基因的选择。随着人类基因组计划的完成，许多基因被发现并克隆，为基因治疗提供了广阔的前景。生存素(Survivin)基因是新近发现的凋亡抑制基因，是凋亡抑制蛋白(inhibitor of apoptosis protein，IAP)家族的成员。研究发现生存素是一种凋亡抑制作用最强的IAP家族新成员，在抑制肿瘤细胞凋亡中发挥重要作用。新近报告，生存素在人类恶性肿瘤(胰腺癌，鼻咽癌等)中表达上调，并与肿瘤的凋亡、增殖、血管生成及其预后和耐药性的产生有密切关系。恶性肿瘤是细胞过度增殖和异常分化的结果，细胞分化常导致细胞增殖速度的减慢；细胞的凋亡与肿瘤的发生发展、临床治疗有密切的关系，是肿瘤生长的重要调节因素之一，凋亡也可能是细胞被诱导分化成熟以后的死亡形式，是诱导分化的结局。最近研究表明，维甲酸类化合物是良好的诱导分化剂，干扰素具有抗肿瘤和免疫调节作用，γ-干扰素(interferon-γ，γ-IFN)、全反式维A酸(all-trans retinoic acid，ATRA)通过调节某些抗凋亡基因(生存素、bcl-2等)，促进某些肿瘤细胞凋亡，γ-干扰素与维A酸联合应用可以增加抗肿瘤疗效，成为治疗恶性肿瘤新近研究热点之一。但皮肤鳞癌方面的研究未见报道。本研究旨在研究人皮肤鳞癌组织表达bcl-2、生存素的情况；探讨维甲酸、γ-干扰素在不同浓度、不同作用时间对皮肤鳞癌细胞形态分化及凋亡的影响；两者对SCL-1细胞表达生存素的影响。为γ-IFN、维A酸类药物诱导皮肤肿瘤细胞凋亡的分子基础研究及临床应用提供实验依据。方法1、免疫组化法检测了生存素、bcl-2在皮肤鳞癌中表达情况，探讨临床病理特征的关系。2、MTT比色分析法检测ATRA和γ-IFN对SCL-1细胞增殖影响。(1)不同浓度ATRA对SCL-1细胞增殖的影响；(2)不同浓度γ-IFN对SCL-1细胞增殖的影响；(3)联合应用ATRA和γ-IFN对SCL-1细胞增殖的影响。3、透射电镜及1％碘化丙啶染色检测γ-IFN及ATRA诱导SCL-1细胞发生细胞凋亡的形态变化。4、流式细胞仪(FCM)检测各组细胞早期凋亡率。5、倒置相差显微镜观察γ-IFN及ATRA对SCL-1细胞分化及形态的影响。6、细胞免疫化学及免疫印迹方法检测γ-IFN及ATRA诱导前后SCL-1细胞生存素蛋白表达情况。7、RT-PCR检测γ-IFN及ATRA诱导前后SCL-1细胞生存素mRNA表达。结果1、免疫组化法检测生存素、bcl-2蛋白在皮肤鳞癌组织中的表达生存素、bcl-2在SCC中呈阳性表达，胞浆均呈现黄色或棕黄色，而胞核不着色，而且阳性细胞集中在表皮基底层及真皮层；正常皮肤组织中未见bcl-2与生存素蛋白表达。按阳性标准判断，在皮肤鳞癌中，bcl-2蛋白的表达阳性率为70％，生存素蛋白表达阳性率为60％，两者的表达差异无显著性(p=0.339＞0.05)。2、MTT比色分析法检测处理因素对SCL-1细胞增殖的影响(相对抑制率)(1)ATRA组各组间差别具有显著性(p=0.000＜0.05)。进一步进行组间比较：0.1μmol／l ATRA组，0.5μmol／l ATRA组与对照组细胞相对抑制率无显著差别(p值分别为0.345，0.077＞0.05)；1μmol／l ATRA组，5μmol／l ATRA组，10μmol／l ATRA组与对照组细胞相对抑制率差别具有显著性(p＜0.05)。此外，1μmol／l ATRA组与0.5μmol／l ATRA，0.1μmol／l ATRA组细胞相对抑制率有显著差别(p=0.000＜0.01)；而与5μmol／l ATRA，10μmol／l ATRA组细胞相对抑制率差别无显著性(p值分别为0.079，0.055＞0.05)。上述结果说明，ATRA抑制SCL-1细胞的增殖，在一定范围内有剂量依赖性。(2)γ-IFN组各组间差别具有显著性(p=0.000＜0.01)。进一步进行组间比较，实验组组与对照组差别具有显著性(p值均＜0.01)。提示SCL-1细胞的增殖受γ-IFN浓度的影响，其中1000U／mlγ-IFN组与100U／mlγ-IFN组、500U／mlγ-IFN组差别具有显著性(p值分别为0.000，0.001＜0.01)。(3)相同浓度γ-IFN联合不同浓度ATRA组各组间差别具有显著性(p=0.000＜0.01)。进一步进行组间比较：实验组与对照组细胞相对抑制率差别均具有显著性(p=0.000＜0.01)。1000U／mlγ-IFN+0.1μmol／l ATRA组与1000 U／mlγ-IFN+0.5μmol／l ATRA组细胞相对抑制率差别无显著性(p=0.391＞0.05)。1000U／mlγ-IFN+1μmol／l ATRA组与1000U／mlγ-IFN+0.1μmol／l ATRA组、1000 U／mlγ-IFN+0.5μmol／l ATRA组细胞相对抑制率差异具有显著性(p=0.000＜0.01)；而与1000 U／mlγ-IFN+5μmol／l ATRA组、1000 U／mlγ-IFN+10μmol／l ATRA组细胞相对抑制率差异无显著性(p值分别为0.294，0.536＞0.05)。(4)相同浓度ATRA联合不同浓度γ-IFN组各组间差别具有显著性(p=0.000＜0.01)。进一步进行组间比较：实验组与对照组细胞相对抑制率差别均具有显著性(p=0.000＜0.01)。1μmol／l ATRA+1000 U／mlγ-IFN组与前两者差异均具有显著性(p=0.000＜0.01)。3、透射电镜观察细胞凋亡形态正常培养的SCL-1细胞多呈长梭形，细胞核大，常染色质发达，有2-3个核仁，胞核内染色质均匀，核膜清晰。1μmol／l ATRA+1000 U／mlγ-IFN处理组可以观察到一系列凋亡特征性的形态学改变。ATRA和γ-IFN诱导初期(24h)细胞呈圆形或卵圆形，胞核变小，核膜皱褶内陷，细胞核内染色质边集，呈新月状附在核膜周边；诱导48h～72h，核染色质更加致密，浓缩分块，细胞核聚集成块。4、1％碘化丙啶染色检测细胞凋亡形态对照组细胞早期凋亡细胞较少，多表现为细胞形态规则，呈红色荧光，淡而均匀；1μmol／l ATRA+1000 U／mlγ-IFN处理组可见较多早期凋亡细胞，表现为细胞形态不规则，胞膜粗糙，表面有突起的小泡，细胞核呈黄红色荧光，浓缩成颗粒状，荧光强。5、γ-IFN及ATRA对SCL-1细胞分化及形态的影响对照组细胞呈菱形、多角形，镶嵌状排列，增殖旺盛。1μmol／l ATRA处理3天组细胞趋于圆形、卵圆形，略呈双极性改变，细胞分布较零散。1000 U／mlγ-IFN处理3天组细胞呈双极性改变，细胞狭长，脱壁悬浮细胞增多。两者联合应用组细胞生长缓慢，细胞数目减少，脱壁悬浮细胞明显增多，双极性细胞明显可见。6、FCM检测细胞凋亡率各组早期凋亡率总体差异有意义(p=0.000＜0.01)。进一步两两比较，1μmol／l ATRA处理3天组与对照组早期凋亡率有差异(p=0.046＜0.05)，说明1μmol／l ATRA可以诱导SCL-1细胞发生早期凋亡。1000 U／mlγ-IFN处理3天组、1μmol／l ATRA+1000 U／mlγ-IFN处理3天组与对照组差异显著(p=0.000＜0.01)。而且后两者之间的早期凋亡率的差别也具有显著性(p=0.000＜0.01)，提示γ-IFN与ATRA联合应用诱导SCL-1细胞早期凋亡，具有协同作用。悬浮1μmol／l ATRA+1000U／mlγ-IFN组与贴壁1μmol／l ATRA+1000 U／mlγ-IFN组差异显著(p=0.002＜0.01)。7、细胞免疫化学方法检测γ-IFN及ATRA诱导前后SCL-1细胞生存素表达情况生存素在DAB染色下呈棕黄色，对照组细胞生存素表达较强，呈深棕黄色；1000U／mlγ-IFN处理3天组细胞生存素表达减弱，呈浅棕黄色；1000U／mlγ-IFN+1μmol／mL ATRA处理3天组细胞生存素表达最弱。8、RT-PCR检测γ-IFN及ATRA诱导前后SCL-1细胞生存素mRNA表达取三个时间点的等量cDNA通过RT-PCR来检测生存素mRNA表达量，γ-IFN及ATRA作用后，生存素mRNA表达率有不同程度的降低，且随着作用时间的延长，生存素mRNA表达逐渐降低。此外，在相同作用时间下，联合使用ATRA后，生存素mRNA表达率比单独应用γ-IFN时明显减少。方差分析结果，各组均数间差异具有显著性(P=0.000＜0.05)，作两两比较，对照组与1、2组间差异无显著性(分别为P=0.159＞0.05；P=0.053＞0.05)；1、2组间差异无显著性(P=0.386＞0.05)；3、4组间差异无显著性(P=0.586＞0.05)；8、9组间差异无显著性(P=0.072＞0.05)。9、免疫印迹方法检测γ-IFN及ATRA诱导前后SCL-1细胞生存素表达情况对照组生存素呈高表达(131.60±1.52)。方差分析结果，各组均数间差异具有显著性(P=0.000＜0.05)，作两两比较，其中0、1组间差异无显著性(P=0.171＞0.05)；3、4组间差异无显著性(P=0.130＞0.05)，其余各组均数间差异均有意义(P＜0.05)。结论1、生存素在皮肤鳞状细胞癌组织中表达增高，与年龄、性别、发病部位、病理分级无明显关系，与有无淋巴结转移密切相关；生存素表达与bcl-2表达无统计学相关性，提示两者在皮肤鳞状细胞癌的发生发展中不起协同作用，两者可能通过不同的抗凋亡路径发挥作用；生存素可能与鳞癌的易于转移特性相关。2、ATRA和γ-IFN可促进皮肤鳞癌细胞系SCL-1细胞分化，抑制其增殖，两者具有协同作用，在一定范围内有剂量依赖性；两者可促进皮肤鳞癌细胞系SCL-1细胞发生细胞凋亡。3、生存素在皮肤鳞癌细胞系SCL-1中表达增高；γ-IFN能下调SCL-1细胞表达生存素，且有时间依赖性和剂量依赖性；ATRA可以增强γ-IFN对生存素的抑制作用；γ-IFN联合ATAR诱导皮肤鳞状细胞癌细胞发生细胞凋亡的机制之一可能是通过下调生存素的表达实现的。更多还原

【Abstract】 The mechanisms of apoptosis of skin squamous cell carcinoma induced by IFN-γand ATRAIntroduction and objectiveSkin squamous cell carcinoma (SCC) is a malignant neoplasm derived from epidermal keratinocyte and squamous cells of skin appendant organ .It is one of the most common skin carcinoma, and the incidence rate of SCC is 20% in malignant non-melanoma. It is a common disease that seriously harms health of people in our country, so we should pay more attention to it.At present, surgical operation is still a preferred method of treatment of SCC of skin,and surgical operation and assisted preoperative and postoperative radiotherapy and chemotherapy have become a standard mode for the treatment of skin SCC. But skin SCC at the progressive stage is still a difficult problem that needs to be solved in the treatment, so the search for promising gene therapy technology has an important significance.The key to gene therapy is the selection of the target gene. With the completion of the Human Genome Project, many genes have been discovered and cloned, giving great promise to the gene therapy of tumors. Survivin is a recently discovered apoptosis inhibiting protein, belonging to the family of apoptosis protein inhibiting factors, with unique structure, only expressed in the embryonic tissue and most tumorous tissues, such as colon cancer, lung cancer, breast cancer, stomach cancer and liver cancer, and is related to the progress of disease and bad prognosis. The specific distribution of surviving makes it become a gene therapy target.The aim of this research was to approach the expression of survivin and bcl-2 in human skin squamous cell carcinoma, and the effect of ATRA andγ-IFN on the growth,apoptosis,differentiation and the expression of suvivin.Methods1. Expression of survivin in the skin SCC tissue by the immunohistochemical method and its clinical significance.2. Detection of cell growth inhibition with the MTT analysis method.3. To detect the morphous of the apoptosis cell by transmission electron microscope(TEM) and iodinate tritroche stain.4. To detect the apoptosis rate by FCM.5. To detect the expression of survivin before and afer the treatment of ATRA andγ-IFN by immunohistochemical method and Western blot.6. To detect the expression of survivin mRNA before and afer the treatment of ATRA andγ-IFN by RT-PCR method.Results1. Survivin and bcl-2 expression in the skin SCC tissueIn SCC, the positive rates of bcl-2 and survivin were 70% and 60%,respectively(P＞0.05). Survivin expression was not related to clinical staging of patients, but related to the lymph node metastasis.2. To detect growth inhibition rate of SCL-1 cells by MTT:ATRA andγ-IFN could significantly inhibit the proliferation of SCL-1 cells and showed dose dependence.(1)In group ATRA The difference of inhibition rate among the six groups was significant (p=0.000＜0.05). There has no difference between the control and the group 0.5μmol/1, 0.1μmol/1 ATRA (p= 0.345, 0.077＞0.05).The difference of inhibition rate between the control and the grouplμmol/1, 5μmol/1ATRA and 10μmol/1 ATRA was significant (p=0.000＜0.05). (2)In groupγ-IFN The difference of inhibition rate among the four groups were significant (p=0.000＜0.01). There has difference between the control and the experiment groups (p＜0.01).The difference of inhibition rate between the group 1000 U/mlγ- IFN and the group 100 U/ml or 500U/mlγ- IFN, was significant (p=0.000,0.001＜0.01).(3)In group combination of ATRA andγ-IFN:①Different concentration of ATRA+1000 U/mlγ- IFNThe difference of inhibition rate among the six groups were significant (p=0.000＜0.01) There has no difference between 1000 U/mlγ- IFN+0.1μmol/1 ATRA and the group 1000 U/mlγ- IFN+0.5μmol/1 ATRA(p=0.391＞0.05).The difference of inhibition rate between the group 1000 U/mlγ- IFN+ 1μmol/1 ATRA and group 1000 U/mlγ- IFN+0.1μmol/1 ATRA,1000 U/mlγ- IFN+0.5μmol/1 ATRA was significant (p=0.000＜0.01), but no difference between the group 1000 U/mlγIFN+1μmol/1 ATRA and group 1000 U/mlγ- IFN+5μmol/1 ATRA,1000 U/mlγIFN+10μmol/1 ATRA (p=0.294, 0.536＞0.05).②Different concentration ofγ- IFN+1μmol/1 ATRAThe difference of inhibition rate among the five groups were significant (p=0.000＜0.01). There has difference between control group and experiment groups (p=0.000＜0.01).3. To survey the morphous of the cell apoptosis by transmissionelectron microscope(TEM):The shape of the cells in control were as follow lower tracing: the intranuclear chromoplasma were well-distributed,the nuclear membrane were clear, mitochondria ribosome,solvent can be seen.The shape of experiment were as follow lower tracing:The nuclus were pycnosis,cell membrane were damaged,the nucleolemma were shrinkage.Some of the cells were as followed:chromatic agglutination in the intranuclear,the chromatin were side,looking like crescent,the intermembrancee were not identical.Some of the cells were as followed:nuclus chromatins were side,nuclear type had several notches,nuclear membranes were clear.4. The cells that are stained by 1% iodinate tritroche can be observed by fluorescence microscope:Control group:the conformation of nucleous is satiation and present a yellow red color of fluorescence. 1000 U/mlγ- IFN+1μmol/1 ATRA group: the nucleous of most cells are presenting karyopyknosis,becoming punctuate or massiveness and concentrating at one side of the cell to form crescent shape or granular shape.The nucleous/plasma is decreading.5. To observe the differentiation of SCL- 1 cell:Cells in the control group were well grew adhesively, most in shuttle shape, moderately sized, clear nucleolus, nuclear division phase found.ATRA-stimulated cells displayed a somewhat bipolar appearance.γ- IFN- stimulated cells showed the number of bipolar cells increased.The cells treated by ATRA andγ-IFN were present in the supematant.The density of adherent cells was diminished when compared with the control.6. To detect the cell apoptosis rate by FCM:The difference of the early apoptosis rate among the five groups were significant (p=0.000＜0.01).7. To detect the expression of survivin protein by cell immunochemistryThe expression of survivin protein was positive in SCL-1 cells untreated.It was weak stained after treatment of 1000 U/mlγ- IFN.We can’t detect the te expression of survivin protein after treatment of 1000 U/mlγ- IFN and 1μmol/1 ATRA.8. Survivin mRNA analysis by RT-PCR: The results showed that in the ATRA andγ-IFN treatment group, survivin mRNA cells were significantly lower than that in control.It shows the dependence of the time-dose.9. To detect the expression of survivin protein by western blot:γ- IFN could significantly inhibit the expression of survivin(131.60±1.52 in the control group)in dose-dependent and time-dependent manner.γ-IFN combined with ATRA could enhance the inhibition of survivin’s expression.Conclusions1. High survivin expression in the skin SCC tissue is related to the lymph node metastasis, suggesting that survivin plays an important role in the incidence.Survivin and bcl-2 may induce cell apoptosis through different pathways.2. Survivin might play an important role in the apoptosis of squamous cell carcinoma cell induced byγ- IFN and ATRA.3. ATRA andγ-IFN induce cell apoptosis of SCL-1 dependent of time-dose.The role can be enhanced by the combination of the drugs.4. One of the mechanisms of apoptosis of skin squamous cell carcinoma induced by IFN-γand ATRA may be through decreasing the expression of survivin.更多还原