【摘要】：目的建立模擬日光紫外線B輻射誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化的病理模型,從Gadd45a、DNMTs、P16和RASSF1A基因甲基化的角度,研究扇貝多肽(PCF)抑制UVB誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化的分子機(jī)制,為扇貝多肽防治UVB輻射致皮膚癌提供理論依據(jù)。方法采用CCK8法篩選并確定UVB輻射誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化的單次輻射劑量,Gimsa染色觀察細(xì)胞形態(tài)初步確立UVB輻射誘導(dǎo)HaCaT細(xì)胞惡性轉(zhuǎn)化的終點(diǎn),軟瓊脂克隆形成實(shí)驗(yàn)、明膠酶譜法檢測(cè)MMP-9蛋白酶的分泌證明UVB輻射誘導(dǎo)HaCaT細(xì)胞惡性轉(zhuǎn)化模型的成功。實(shí)驗(yàn)設(shè)計(jì)分為4組：正常對(duì)照組、UVB模型組、UVB+2.84mmol/LPCF, UVB+2.84mmol/L維生素C陽(yáng)性對(duì)照組。經(jīng)平板集落形成實(shí)驗(yàn),軟瓊脂集落形成實(shí)驗(yàn),流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期確立PCF對(duì)UVB誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化的抑制作用；RT-PCR檢測(cè)甲基轉(zhuǎn)移酶DNMTs(DNMT1、DNMT3a, DNMT3b)mRNA經(jīng)時(shí)(UVB輻射4、8、16、20次)表達(dá)；蛋白印跡法檢測(cè)GADD45a, DNMT3b, P16及RASSFIA蛋白經(jīng)時(shí)(UVB輻射4、8、12、16、20次)表達(dá)。以高分辨率溶解曲線(MS-HRM)檢測(cè)PCF對(duì)處于UVB誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化各階段(4次、12次、20次)中P16、RASSF1A基因甲基化的程度。結(jié)果UVB(波長(zhǎng)峰值297nm)照射的輻照強(qiáng)度約在1luw/cm2,單次輻射劑量10mJ/cm2,輻射次數(shù)20次,總輻射劑量200mJ/cm2時(shí),成功建立UVB輻射誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化模型；2,84mmol/LPCF比VC更明顯抑制UVB輻射誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化(P0.05)。DNMT1和DNMT3amRNA的經(jīng)時(shí)表達(dá)顯示UVB輻射HaCaT細(xì)胞DNMT1和DNMT3a的表達(dá)量與輻射前相比差異無統(tǒng)計(jì)學(xué)意義(I0.05); DNMT3b的mRNA和蛋白的表達(dá)呈現(xiàn)逐漸升高的趨勢(shì),UVB輻射20次后其表達(dá)量達(dá)到高峰(P0.05); Gadd45a蛋白經(jīng)時(shí)變化顯示UVB輻射4次時(shí)即可檢測(cè)表達(dá)量達(dá)到高峰,隨后表達(dá)量逐漸下降,UVB輻射20次后蛋白表達(dá)到低谷(P0.05)；P16和RASSF1A蛋白經(jīng)時(shí)變化顯示UVB輻射后表達(dá)量逐漸下降,UVB輻射20次后蛋白表達(dá)到低谷(P0.05)；PCF和VC均可抑制P16和RASSFIA基因的甲基化(P0.05),促進(jìn)P16, RASSFIA和GADD45a蛋白的表達(dá)(P0.05),抑制DNMT3b蛋白的表達(dá)(P0.05)且與VC相比,PCF效果更顯著(P0.05)。結(jié)論國(guó)際上首次建立模擬日光紫外線B輻射誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化模型,揭示P16和RASSFIA基因的高甲基化與HaCaT細(xì)胞惡性轉(zhuǎn)化密切相關(guān)。證明扇貝多肽通過調(diào)節(jié)DNMT3b和GADD45a基因表達(dá)而抑制P16和RASSF1A基因的高甲基化,阻上UVB輻射誘導(dǎo)的HaCaT細(xì)胞惡性轉(zhuǎn)化。
[Abstract]:Objective to establish a pathological model of malignant transformation of HaCaT cells induced by simulated solar ultraviolet B radiation, and to study the molecular mechanism of the inhibition of UVB induced malignant transformation of HaCaT cells by chlamyfish polypeptide (PCF) from the point of view of Gadd45a,DNMTs,P16 and RASSF1A gene methylation. It provides a theoretical basis for the prevention and treatment of skin cancer induced by UVB radiation. Methods CCK8 method was used to screen and determine the single dose of malignant transformation of HaCaT cells induced by UVB radiation. The terminal point of malignant transformation of HaCaT cells induced by UVB radiation was preliminarily established by Gimsa staining. Detection of the secretion of MMP-9 protease by gelatinase assay proved that the model of malignant transformation of HaCaT cells induced by UVB radiation was successful. The experimental design was divided into 4 groups: normal control group, UVB model group, UVB 2.84 mmol / L PCF, UVB 2.84mmol/L vitamin C positive control group. The inhibitory effect of PCF on the malignant transformation of HaCaT cells induced by UVB was determined by flow cytometry, and the expression of DNMTs (DNMT1,DNMT3a, DNMT3b) mRNA (20 times UVB radiation) was detected by RT-PCR. The expression of GADD45a, DNMT3b, P16 and RASSFIA protein was detected by Western blot. High resolution dissolution curve (MS-HRM) was used to detect the methylation of P16 RASSF1A gene in the malignant transformation of HaCaT cells induced by UVB (4 times, 12 times, 20 times) by PCF. Results the malignant transformation model of HaCaT cells induced by UVB radiation was successfully established when the irradiation intensity of UVB (wavelength peak 297nm) was about 1luw / cm ~ (2), the single dose was 10 mg / cm ~ (2), the number of times was 20 times, and the total radiation dose was 200mJ/cm2. (2) 84 mmol / L LPCF inhibited the malignant transformation of HaCaT cells induced by UVB radiation more significantly than VC (P0.05). The expression of DNMT1 and DNMT3a in HaCaT cells irradiated with DNMT1 and DNMT3amRNA was not significantly different from that in HaCaT cells irradiated with UVB (I0.05), and the mRNA and protein expressions of DNMT3b were observed. After 20 times of UVB radiation, the expression of Gadd45a protein reached the peak (P0.05), and the expression of Gadd45a protein reached the peak after 4 times of UVB radiation. The expression of P16 and RASSF1A decreased gradually after 20 times of UVB irradiation (P0.05), and the expression of P16 and RASSF1A decreased gradually after 20 times of UVB irradiation (P0.05). Both PCF and VC could inhibit the methylation of P16 and RASSFIA genes (P0.05), promote the expression of P16, RASSFIA and GADD45a (P0.05), inhibit the expression of DNMT3b (P0.05), and PCF was more effective than VC (P0.05). Conclusion the model of malignant transformation of HaCaT cells induced by simulated ultraviolet B radiation is established for the first time in the world. It is revealed that the hypermethylation of P16 and RASSFIA genes is closely related to the malignant transformation of HaCaT cells. It was demonstrated that the polypeptide of chlamys farreri inhibited the hypermethylation of P16 and RASSF1A genes by regulating the expression of DNMT3b and GADD45a genes and prevented the malignant transformation of HaCaT cells induced by UVB radiation.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.5

【共引文獻(xiàn)】

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本文編號(hào)：2281779