中波紫外线及Gadd45 α在皮肤鳞状细胞癌发病中的作用研究
发布时间：2018-11-08 18:17
【摘要】：Gadd45α是细胞生长停滞与DNA损伤诱导蛋白45(Gadd45)家族成员之一,在细胞生长抑制、DNA损伤后诱导细胞凋亡及肿瘤抑制中具有重要作用,参与细胞生长的负性调控,瞬时转染Gadd45α可诱导肿瘤细胞发生凋亡,具有维护基因组稳定性的功能。有研究表明：Gadd45α是保护表皮不被紫外线诱导转化的关键因子,对上皮源性肿瘤中的原癌基因起负性调节作用。本课题通过诱导正常角质形成细胞(HaCaT)及人表皮癌细胞株(A431)上调Gadd45α表达及对各细胞生物学行为的影响,初步探讨Gadd45α在皮肤鳞状细胞癌中的作用。 1窄谱中波紫外线对HaCaT细胞Gadd45α表达及细胞增殖、凋亡的影响 HaCaT细胞分别经100mJ/cm2、200 mJ/cm2、400 mJ/cm2 311nm NB-UVB照射后,应用半定量RT-PCR、western blot法检测发现：HaCaT细胞表达Gadd45α随照射剂量增加,Gadd45αmRNA及蛋白表达升高；不同剂量311nm NB-UVB照射后对HaCaT细胞有抑制作用,其抑制增殖作用呈量效及时效关系；照射后HaCaT细胞周期出现G2期阻滞,并促进细胞凋亡 2 HaCaT和A431细胞中Gadd45α表达差异 应用RT-PCR、western blot及细胞免疫化学法检测HaCaT和A431细胞Gadd45α表达。结果显示：HaCaT细胞表达Gadd45α,Gadd45α表达在维持HaCaT细胞的增殖和分化中可能有一定作用；A431细胞表达减弱,下调Gadd45α表达可能参与了其恶性生物学行为。 3曲古菌素A对A431细胞Gadd45α表达及对细胞增殖、凋亡的影响 应用不同浓度曲古菌素A处理A431细胞。结果显示：曲古菌素A对细胞有抑制作用,其抑制增殖作用呈量效及时效关系；不同浓度曲古菌素A处理A431细胞后,细胞周期发生G1期阻滞,细胞凋亡增加；不同浓度曲古菌素A处理后,A431细胞Gadd45αmRNA及蛋白表达升高。Gadd45α可能参与了A431细胞增殖抑制、周期阻滞,并可诱导细胞凋亡。 4 Gadd45α蛋白在皮肤鳞状细胞癌组织中的表达 应用免疫组织化学方法检测发现：在高分化皮肤鳞状细胞癌组,Gadd45α蛋白表达阳性表达率较中低分化组表达上调。 以上研究结果说明：诱导HaCaT、A431细胞上调Gadd45α表达后均可抑制细胞增殖,Gadd45α可能参与了细胞周期阻滞并促进凋亡；皮肤鳞状细胞癌组织中存在Gadd45α表达异常,Gadd45α可能参与了皮肤鳞状细胞癌的发生过程。
[Abstract]:Gadd45 伪 is a member of cell growth arrest and DNA damage inducible protein 45 (Gadd45) family. It plays an important role in cell growth inhibition, apoptosis induced by DNA injury and tumor inhibition, and participates in the negative regulation of cell growth. Transient transfection of Gadd45 伪 can induce apoptosis of tumor cells and maintain genomic stability. Some studies have shown that Gadd45 伪 is a key factor to protect epidermis from UV induced transformation, and plays a negative role in regulating proto-oncogene in epithelial-derived tumors. In order to explore the role of Gadd45 伪 in cutaneous squamous cell carcinoma (SCC), the expression of Gadd45 伪 was up-regulated by inducing normal keratinocytes (HaCaT) and human epidermal carcinoma cell line (A431). Effect of narrow-band ultraviolet B on Gadd45 伪 expression and cell proliferation and apoptosis in HaCaT cells after 100mJ/cm2200 mJ/cm2400 mJ/cm2 311nm NB-UVB irradiation, semi-quantitative RT-PCR, was applied to HaCaT cells. Western blot assay showed that the expression of Gadd45 伪 in HaCaT cells increased with the increase of irradiation dose, and the expression of Gadd45 伪 mRNA and protein increased with the increase of irradiation dose. Different doses of 311nm NB-UVB could inhibit the proliferation of HaCaT cells in a dose-dependent and time-dependent manner. After irradiation, the cell cycle of HaCaT cells was arrested in G2 phase, and the differential expression of Gadd45 伪 in 2 HaCaT and A431 cells was promoted. The expression of Gadd45 伪 in HaCaT and A431 cells was detected by RT-PCR,western blot and immunocytochemistry. The results showed that the expression of Gadd45 伪 and Gadd45 伪 in HaCaT cells might play a role in maintaining the proliferation and differentiation of HaCaT cells, while the expression of Gadd45 伪 in A431 cells was weakened, and the down-regulation of Gadd45 伪 expression might be involved in the malignant biological behavior of A431 cells. (3) the effects of tricuccinin A on the expression of Gadd45 伪 and the proliferation and apoptosis of A431 cells were studied. The results showed that the inhibition of mycocytomycin A on cell proliferation was dose-dependent and time-dependent, and the cell cycle of A431 cells was blocked in G1 phase and apoptosis was increased after treatment with different concentrations of trabeculin A. The expression of Gadd45 伪 mRNA and protein in A431 cells increased after treatment with different concentrations of tragomycin A. Gadd45 伪 may be involved in the inhibition of proliferation, cell cycle arrest and apoptosis of A431 cells. 4The expression of Gadd45 伪 protein in squamous cell carcinoma of skin was detected by immunohistochemical method. The positive expression rate of Gadd45 伪 protein in highly differentiated skin squamous cell carcinoma group was higher than that in middle and low differentiation group. The above results suggest that up-regulation of Gadd45 伪 expression in HaCaT,A431 cells can inhibit cell proliferation, and Gadd45 伪 may participate in cell cycle arrest and promote apoptosis. There is abnormal expression of Gadd45 伪 in cutaneous squamous cell carcinoma, and Gadd45 伪 may be involved in the pathogenesis of cutaneous squamous cell carcinoma.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.5
[Abstract]:Gadd45 伪 is a member of cell growth arrest and DNA damage inducible protein 45 (Gadd45) family. It plays an important role in cell growth inhibition, apoptosis induced by DNA injury and tumor inhibition, and participates in the negative regulation of cell growth. Transient transfection of Gadd45 伪 can induce apoptosis of tumor cells and maintain genomic stability. Some studies have shown that Gadd45 伪 is a key factor to protect epidermis from UV induced transformation, and plays a negative role in regulating proto-oncogene in epithelial-derived tumors. In order to explore the role of Gadd45 伪 in cutaneous squamous cell carcinoma (SCC), the expression of Gadd45 伪 was up-regulated by inducing normal keratinocytes (HaCaT) and human epidermal carcinoma cell line (A431). Effect of narrow-band ultraviolet B on Gadd45 伪 expression and cell proliferation and apoptosis in HaCaT cells after 100mJ/cm2200 mJ/cm2400 mJ/cm2 311nm NB-UVB irradiation, semi-quantitative RT-PCR, was applied to HaCaT cells. Western blot assay showed that the expression of Gadd45 伪 in HaCaT cells increased with the increase of irradiation dose, and the expression of Gadd45 伪 mRNA and protein increased with the increase of irradiation dose. Different doses of 311nm NB-UVB could inhibit the proliferation of HaCaT cells in a dose-dependent and time-dependent manner. After irradiation, the cell cycle of HaCaT cells was arrested in G2 phase, and the differential expression of Gadd45 伪 in 2 HaCaT and A431 cells was promoted. The expression of Gadd45 伪 in HaCaT and A431 cells was detected by RT-PCR,western blot and immunocytochemistry. The results showed that the expression of Gadd45 伪 and Gadd45 伪 in HaCaT cells might play a role in maintaining the proliferation and differentiation of HaCaT cells, while the expression of Gadd45 伪 in A431 cells was weakened, and the down-regulation of Gadd45 伪 expression might be involved in the malignant biological behavior of A431 cells. (3) the effects of tricuccinin A on the expression of Gadd45 伪 and the proliferation and apoptosis of A431 cells were studied. The results showed that the inhibition of mycocytomycin A on cell proliferation was dose-dependent and time-dependent, and the cell cycle of A431 cells was blocked in G1 phase and apoptosis was increased after treatment with different concentrations of trabeculin A. The expression of Gadd45 伪 mRNA and protein in A431 cells increased after treatment with different concentrations of tragomycin A. Gadd45 伪 may be involved in the inhibition of proliferation, cell cycle arrest and apoptosis of A431 cells. 4The expression of Gadd45 伪 protein in squamous cell carcinoma of skin was detected by immunohistochemical method. The positive expression rate of Gadd45 伪 protein in highly differentiated skin squamous cell carcinoma group was higher than that in middle and low differentiation group. The above results suggest that up-regulation of Gadd45 伪 expression in HaCaT,A431 cells can inhibit cell proliferation, and Gadd45 伪 may participate in cell cycle arrest and promote apoptosis. There is abnormal expression of Gadd45 伪 in cutaneous squamous cell carcinoma, and Gadd45 伪 may be involved in the pathogenesis of cutaneous squamous cell carcinoma.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.5
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