【摘要】：Gadd45α是細胞生長停滯與DNA損傷誘導(dǎo)蛋白45(Gadd45)家族成員之一,在細胞生長抑制、DNA損傷后誘導(dǎo)細胞凋亡及腫瘤抑制中具有重要作用,參與細胞生長的負性調(diào)控,瞬時轉(zhuǎn)染Gadd45α可誘導(dǎo)腫瘤細胞發(fā)生凋亡,具有維護基因組穩(wěn)定性的功能。有研究表明：Gadd45α是保護表皮不被紫外線誘導(dǎo)轉(zhuǎn)化的關(guān)鍵因子,對上皮源性腫瘤中的原癌基因起負性調(diào)節(jié)作用。本課題通過誘導(dǎo)正常角質(zhì)形成細胞(HaCaT)及人表皮癌細胞株(A431)上調(diào)Gadd45α表達及對各細胞生物學行為的影響,初步探討Gadd45α在皮膚鱗狀細胞癌中的作用。 1窄譜中波紫外線對HaCaT細胞Gadd45α表達及細胞增殖、凋亡的影響 HaCaT細胞分別經(jīng)100mJ/cm2、200 mJ/cm2、400 mJ/cm2 311nm NB-UVB照射后,應(yīng)用半定量RT-PCR、western blot法檢測發(fā)現(xiàn)：HaCaT細胞表達Gadd45α隨照射劑量增加,Gadd45αmRNA及蛋白表達升高；不同劑量311nm NB-UVB照射后對HaCaT細胞有抑制作用,其抑制增殖作用呈量效及時效關(guān)系；照射后HaCaT細胞周期出現(xiàn)G2期阻滯,并促進細胞凋亡 2 HaCaT和A431細胞中Gadd45α表達差異 應(yīng)用RT-PCR、western blot及細胞免疫化學法檢測HaCaT和A431細胞Gadd45α表達。結(jié)果顯示：HaCaT細胞表達Gadd45α,Gadd45α表達在維持HaCaT細胞的增殖和分化中可能有一定作用；A431細胞表達減弱,下調(diào)Gadd45α表達可能參與了其惡性生物學行為。 3曲古菌素A對A431細胞Gadd45α表達及對細胞增殖、凋亡的影響 應(yīng)用不同濃度曲古菌素A處理A431細胞。結(jié)果顯示：曲古菌素A對細胞有抑制作用,其抑制增殖作用呈量效及時效關(guān)系；不同濃度曲古菌素A處理A431細胞后,細胞周期發(fā)生G1期阻滯,細胞凋亡增加；不同濃度曲古菌素A處理后,A431細胞Gadd45αmRNA及蛋白表達升高。Gadd45α可能參與了A431細胞增殖抑制、周期阻滯,并可誘導(dǎo)細胞凋亡。 4 Gadd45α蛋白在皮膚鱗狀細胞癌組織中的表達 應(yīng)用免疫組織化學方法檢測發(fā)現(xiàn)：在高分化皮膚鱗狀細胞癌組,Gadd45α蛋白表達陽性表達率較中低分化組表達上調(diào)。 以上研究結(jié)果說明：誘導(dǎo)HaCaT、A431細胞上調(diào)Gadd45α表達后均可抑制細胞增殖,Gadd45α可能參與了細胞周期阻滯并促進凋亡；皮膚鱗狀細胞癌組織中存在Gadd45α表達異常,Gadd45α可能參與了皮膚鱗狀細胞癌的發(fā)生過程。
[Abstract]:Gadd45 偽 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 偽 can induce apoptosis of tumor cells and maintain genomic stability. Some studies have shown that Gadd45 偽 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 偽 in cutaneous squamous cell carcinoma (SCC), the expression of Gadd45 偽 was up-regulated by inducing normal keratinocytes (HaCaT) and human epidermal carcinoma cell line (A431). Effect of narrow-band ultraviolet B on Gadd45 偽 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 偽 in HaCaT cells increased with the increase of irradiation dose, and the expression of Gadd45 偽 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 偽 in 2 HaCaT and A431 cells was promoted. The expression of Gadd45 偽 in HaCaT and A431 cells was detected by RT-PCR,western blot and immunocytochemistry. The results showed that the expression of Gadd45 偽 and Gadd45 偽 in HaCaT cells might play a role in maintaining the proliferation and differentiation of HaCaT cells, while the expression of Gadd45 偽 in A431 cells was weakened, and the down-regulation of Gadd45 偽 expression might be involved in the malignant biological behavior of A431 cells. (3) the effects of tricuccinin A on the expression of Gadd45 偽 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 偽 mRNA and protein in A431 cells increased after treatment with different concentrations of tragomycin A. Gadd45 偽 may be involved in the inhibition of proliferation, cell cycle arrest and apoptosis of A431 cells. 4The expression of Gadd45 偽 protein in squamous cell carcinoma of skin was detected by immunohistochemical method. The positive expression rate of Gadd45 偽 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 偽 expression in HaCaT,A431 cells can inhibit cell proliferation, and Gadd45 偽 may participate in cell cycle arrest and promote apoptosis. There is abnormal expression of Gadd45 偽 in cutaneous squamous cell carcinoma, and Gadd45 偽 may be involved in the pathogenesis of cutaneous squamous cell carcinoma.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R739.5

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