中波紫外线诱导皮肤成纤维细胞凋亡的机制研究

发布时间：2018-09-18 10:07

【摘要】： 背景：紫外线是与人体皮肤密切相关的一种重要的环境因素,根据波长不同,紫外线可分为长波紫外线(UVA,320-400nm)、中波紫外线(UVB,280-320nm)和短波紫外线(UVC,200-280nm)。能够到达地球表面的紫外线仅包括UVA和UVB,与UVA相比,同等剂量的UVB对皮肤的损伤强度是UVA的1000倍左右。成纤维细胞是真皮的主要组成细胞,经紫外线照射后,皮肤成纤维细胞的生长、分化和功能等方面会发生明显的改变。本课题组在前期工作中通过双向电泳结合质谱鉴定和免疫印迹技术发现,小剂量UVB照射会导致皮肤成纤维细胞特有的骨架蛋白波形蛋白(vimentin)含量增加。波形蛋白含有胱天蛋白酶-8和-3的酶切位点,在细胞凋亡过程中,当细胞中的波形蛋白被这类蛋白酶破坏以后,其降解产物能够促进细胞凋亡。目的：在UVB引起的皮肤成纤维细胞凋亡过程中,影响波形蛋白的胱天蛋白酶-8和-3是否能够被UVB激活,与胱天蛋白酶-8相关的受体相互作用蛋白-1如何变化；成纤维细胞特有的细胞骨架波形蛋白是否被胱天蛋白酶破坏,并横向比较了另外两种普遍存在的细胞骨架蛋白即微管蛋白和肌动蛋白在凋亡过程中的变化,从而探讨UVB对皮肤成纤维细胞凋亡的机制。方法： (1)体外培养人原代皮肤成纤维细胞并进行传代,用第3-6代细胞进行实验; (2)为了选择合适的UVB照射剂量和观测时间,先用噻唑蓝(MTT)比色法比较了不同剂量UVB对成纤维细胞活力的影响,然后对比了150mJ/cm2 UVB照射细胞后,在12h、24h、36h和48h四个时间点细胞活力的变化； (3)用Hoechst染色法对细胞核染色,比较150mJ/cm2 UVB照射成纤维细胞后,在上述时间点观察细胞核的形态变化并计算凋亡细胞的数量； (4)用荧光比色法检测照射了150mJ/cm2 UVB的成纤维细胞中,不同时间点的胱天蛋白酶-8和-3的活性变化； (5)用细胞荧光和Western Blot技术检测照射UVB诱导凋亡的成纤维细胞中波形蛋白的蛋白含量的变化； (6)用Western Blot技术检测照射了150mJ/cm2 UVB的成纤维细胞中,α-微管蛋白、β-微管蛋白和β-肌动蛋白的蛋白含量的变化； (7)用RT-PCR和Western Blot技术检测照射了150mJ/cm2 UVB的成纤维细胞中受体相互作用蛋白-1的mRNA和蛋白含量的变化。结果： (1)50-300mJ/cm2 UVB照射后24小时,皮肤成纤维细胞的活力出现不同程度的下降；150mJ/cm2 UVB照射细胞以后,在12h、24h、36h和48h四个时间点,细胞活力逐渐降低； (2)150mJ/cm2UVB能够诱导皮肤成纤维细胞出现凋亡,当胱天蛋白酶的活性被抑制时,UVB诱导的凋亡细胞数量减少； (3) 150mJ/cm2UVB照射后,成纤维细胞的胱天蛋白酶-8和-3被激活,在照射后12h时,两者活性明显升高,随着时间的延长,其活性逐渐降低,在照射后48h时,其活性最低； (4)当抑制了胱天蛋白酶-8的激活时,胱天蛋白酶-3的活化延迟；反之,当胱天蛋白酶-3的活性被抑制时,胱天蛋白酶-8的活化也出现延迟; (5)当分别抑制胱天蛋白酶-8和-3的活性时,凋亡细胞的数量明显减少； (6)在UVB诱导皮肤成纤维细胞凋亡过程中,波形蛋白的含量增加,β-微管蛋白的含量减少,α-微管蛋白和β-肌动蛋白的含量不变； (7)在凋亡的成纤维细胞中,受体相互作用蛋白-1的mRNA含量减少,其蛋白含量升高。结论： (1)在UVB诱导皮肤成纤维细胞凋亡过程中,活化的胱天蛋白酶-8和-3互相影响,共同发挥促进细胞凋亡的作用； (2)当细胞凋亡时,活化的胱天蛋白酶-8和-3不能通过水解其底物波形蛋白而影响细胞的凋亡； (3)在细胞凋亡过程中,细胞中减少的β-微管蛋白和升高的受体相互作用蛋白-1可能发挥着一定作用； (4)α-微管蛋白和β-肌动蛋白不受UVB的影响,在研究UVB诱导皮肤成纤维细胞凋亡时,它们可以作为免疫印迹的内参蛋白使用。
[Abstract]:Background:
Ultraviolet radiation is an important environmental factor closely related to human skin. According to different wavelengths, ultraviolet radiation can be divided into long-wave ultraviolet radiation (UVA, 320-400 nm), medium-wave ultraviolet radiation (UVB, 280-320 nm) and short-wave ultraviolet radiation (UVC, 200-280 nm). The ultraviolet radiation that can reach the earth's surface includes only UVA and UVB. Compared with UVA, the same dose of UVB on the skin. The damage intensity is about 1000 times that of UVA.
Fibroblasts are the main components of dermis. After ultraviolet irradiation, the growth, differentiation and function of skin fibroblasts will be significantly changed. In our previous work, we found that low doses of UVB irradiation can cause specific changes in skin fibroblasts by two-dimensional electrophoresis, mass spectrometry and immunoblotting. Vimentin (vimentin) content increased. Vimentin contains the digestion sites of cystatin-8 and cystatin-3. In the process of cell apoptosis, the degradation products of vimentin can promote cell apoptosis when it is destroyed by these proteases.
In the process of UVB-induced apoptosis of skin fibroblasts, whether vimentin-8 and vimentin-3 can be activated by UVB, how the receptor-interacting protein-1 associated with cystatin-8 changes, and whether the cytoskeleton vimentin specific to fibroblasts is destroyed by cystatin are compared horizontally with the other two. The ubiquitous changes of cytoskeleton proteins, tubulin and actin, in the process of apoptosis, were investigated to explore the mechanism of UVB-induced apoptosis of skin fibroblasts.
Method:
(1) primary human dermal fibroblasts were cultured in vitro and passaged by 3-6 generation cells.
(2) In order to select the appropriate dose and observation time of UVB irradiation, the effects of different doses of UVB on the viability of fibroblasts were compared by MTT colorimetry, and then the changes of cell viability at 12, 24, 36 and 48 hours after 150 mJ/cm 2 UVB irradiation were compared.
(3) After 150 mJ/cm 2 UVB irradiation, the morphological changes of the nuclei were observed and the number of apoptotic cells was calculated.
(4) The activity of cystatin-8 and cystatin-3 in fibroblasts irradiated with 150 mJ/cm2 UVB was detected by fluorescence colorimetry.
(5) The changes of vimentin content in fibroblasts induced by UVB irradiation were detected by cell fluorescence and Western Blot assay.
(6) The contents of alpha-tubulin, beta-tubulin and beta-actin in fibroblasts irradiated with 150 mJ/cm2 UVB were detected by Western Blot technique.
(7) The changes of receptor-interacting protein-1 mRNA and protein content in fibroblasts irradiated with 150 mJ/cm2 UVB were detected by RT-PCR and Western Blot.
Result:
(1) 24 hours after 50-300 mJ/cm2 UVB irradiation, the viability of skin fibroblasts decreased in varying degrees; after 150 mJ/cm2 UVB irradiation, the viability of skin fibroblasts gradually decreased at 12, 24, 36 and 48 hours.
(2) 150mJ/cm2UVB could induce apoptosis of skin fibroblasts. When the activity of cystatin was inhibited, the number of apoptotic cells induced by UVB decreased.
(3) Cystatin-8 and cystatin-3 of fibroblasts were activated after 150 mJ/cm2 UVB irradiation. The activity of cystatin-8 and cystatin-3 increased significantly at 12 hours after irradiation, and decreased gradually with the prolongation of irradiation time. At 48 hours after irradiation, the activity of cystatin-8 and cystatin-3 was the lowest.
(4) When the activation of cystatin-8 was inhibited, the activation of cystatin-3 was delayed; otherwise, when the activity of cystatin-3 was inhibited, the activation of cystatin-8 was also delayed.
(5) when the activity of caspase -8 and -3 was inhibited respectively, the number of apoptotic cells decreased significantly.
(6) During UVB-induced apoptosis of skin fibroblasts, the content of vimentin increased, the content of beta-tubulin decreased, while the contents of alpha-tubulin and beta-actin remained unchanged.
(7) In apoptotic fibroblasts, the mRNA content of receptor-interacting protein-1 decreased and its protein content increased.
Conclusion:
(1) Activated cystatin-8 and cystatin-3 interact with each other in the process of UVB-induced apoptosis of skin fibroblasts, and play a role in promoting cell apoptosis.
(2) Activated cystatin-8 and cystatin-3 could not affect cell apoptosis by hydrolyzing vimentin.
(3) Reduced beta-tubulin and increased receptor-interacting protein-1 may play a role in apoptosis.
(4) Alpha-tubulin and beta-actin are not affected by UVB. They can be used as internal reference proteins for immunoblotting in the study of UVB-induced apoptosis of skin fibroblasts.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R751

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