BMAL1/CLOCK在UVB照射下对角质细胞凋亡、DNA损伤和免疫的调控作用

发布时间：2023-05-14 01:22

　　背景24小时生物节律控制生物进程的多样性。系统生物钟位于大脑的视交叉神经核,在很多的外周器官和组织中都表现出生物节律变化,例如:肝脏、肺、肾脏、心脏和皮肤。在分子水平上,生物节律的中心调控由4个重要的核心生物钟蛋白组成,包括brain and muscle ARNT-like(BMAL1),circadian locomotor output cycles kaput(CLOCK),period(PERs)和cryptochrome(CRYs)。这4种转录因子的表达通过转录翻译反馈回路调节。其中BMAL1和CLOCK形成异质二聚体并通过结合在其下游基因启动子区域的E-box(CACGTG)序列来正向调节基因表达,从而作为生物钟蛋白中正向分枝来起作用。CRYs和PERs则相反,它们属于BMAL-CLOCK的下游基因。当其表达积累到一定水平后,二者形成异质二聚体,并由胞浆移动至细胞核内,从而抑制BMAL-CLOCK的表达,产生所谓的负反馈调节回路。除此之外,BMAL1的表达还受另外两个孤核受体RORs和REV-ERBs的调节。RORs能够促进BMAL1的表达,被认为是生物节律的正向调节因子...

【文章页数】：115 页

【学位级别】：硕士

【文章目录】：
摘要
abstract
Chapter1 Introduction
    1.1 Introduction of Human Skin
        1.1.1 Introduction of the morphology and functions of human skin
    1.2 Introduction of the Circadian Rhythm in the skin
        1.2.1 The molecular components of circadian clock
        1.2.2 Circadian Clock biological functions in skin
        1.2.3 Skin diseases related to abnormal circadian rhythm
    1.3 Circadian rhythm controls UV-induced DNA damage in the skin
        1.3.1 UV Rays in the skin
        1.3.2 UV-induced DNA damage in the skin
        1.3.3 Circadian clock and UV-induced DNA damage
        1.3.4 Circadian clock and DNA damage repair response
    1.4 Retinoid-related orphan nuclear receptorα,RORα
        1.4.1 Structure characteristic of RORα
        1.4.2 Biological functions of RORα
    1.5 RORαand Circadian clock
    1.6 Hypothesis and Aims
Chapter2 Experimental Materials and Methods
    2.1 Experiment materials
        2.1.1 Cell lines
        2.1.2 Antibody and reagent
        2.1.3 Preparation of the solutions
        2.1.4 Instruments and equipment
    2.2 Method
        2.2.1 Cell culture
        2.2.2 Small RNA interference the expression of Circadian Clock genes Bmal1 and Clock in cells
        2.2.3 Using shRNAs to establish RORαknockout stable cell line
        2.2.4 UVB irradiation
        2.2.5 Real time PCR for cellular mRNA levels detection
        2.2.6 BCA Protein Quantification
        2.2.7 Western Blot for cellular protein level detection
        2.2.8 Immunofluorescence Microscopy detect target genes expression in cells
        2.2.9 Cell viability assay
        2.2.10 Cellular Apoptosis detection
        2.2.11 Data analysis
Chapter3 Result
    3.1 Alterations in the expression of circadian clock genes in the immortal human adult low calcium temperature(HaCaT)keratinocytes following low-dose UVB exposure
        3.1.1 Low dose of UVB induces cycling of Circadian Clock genes in Hacat keratinocytes
        3.1.2 Low dose of UVB induces cycling of cytokines release in Hacat keratinocytes
    3.2 Circadian Clock genes are essential for regulates HaCat keratinocytes Proliferation under UVB irradiation condition
        3.2.1 BMAL1 and CLOCK proteins are critical for regulates HaCat keratinocytes Proliferation
    3.3 Clock genes are essential for regulates HaCat keratinocytes Apoptosis under UVB exposure condition
        3.3.1 The Core Circadian Clock genes BMAL1 and CLOCK are critical for UVB-induced HaCat keratinocytes Apoptosis
    3.4 The molecular mechanisms of clock genes regulating UVB induced HaCat keratinocytes DNA damage responses
        3.4.1 BMAL1 and CLOCK are not indispensable for UVB-induced CPDs formation. ..
        3.4.2 BMAL1 and CLOCK are not involved in activation of DNA damage checkpoints in HaCat keratinocytes
        3.4.3 BMAL1 and CLOCK are involved in activation of histone H2A.X in HaCat keratinocytes
    3.5 BMAL1 and CLOCK control primary human keratinocytes proliferation and apoptosis under UVB irradiation condition
        3.5.1 BMAL1 and CLOCK control primary human keratinocytes growth under5 m J/cm2 of UVB irradiation
        3.5.2 BMAL1 and CLOCK control primary human keratinocytes apoptosis under low dose of UVB irradiation
    3.6 Effects of BMAL1 and CLOCK depletion on UVB-induced DNA damage responses in primary human keratinocytes
        3.6.1 BMAL1 or CLOCK depletion has no effect on UVB-induced DNA damage responses at early time
        3.6.2 CLOCK protein depletion attenuates UVB-induced p53 activity in primary human keratinocytes
    3.7 Depletion of BMAL1 or CLOCK triggers terminal differentiation in HKCs
    3.8 Clock genes are essential for regulating cytokines release under UVB irradiation in HaCat keratinocytes
        3.8.1 BMAL1 and CLOCK control UVB induced cytokines release in HaCat cells. ..
    3.9 RORA is essential for UVB-induced Apoptosis in HaCat keratinocytes
    3.10 RORA is critical for UVB induced cytokines release in HaCat cells
        3.10.1 RORA knockdown receded UVB-induced cytokines release in HaCat cells
        3.10.2 Overexpression of RORA slightly increased UVB-induced cytokines release in HaCat cells
    3.11 RORA controls the core clock gene BMAL1 expression under UVB irradiation in HaCat keratinocytes
Chapter4 Discussion and Prospect
    4.1 Discussion and Conclusion
        4.1.1 Discussion
        4.1.2 Conclusion
    4.2 Prospect
References
Published papers
Scientific research
Acknowledgement



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