【摘要】： 背景： 紫外線是與人體皮膚密切相關(guān)的一種重要的環(huán)境因素,根據(jù)波長(zhǎng)不同,紫外線可分為長(zhǎng)波紫外線(UVA,320-400nm)、中波紫外線(UVB,280-320nm)和短波紫外線(UVC,200-280nm)。能夠到達(dá)地球表面的紫外線僅包括UVA和UVB,與UVA相比,同等劑量的UVB對(duì)皮膚的損傷強(qiáng)度是UVA的1000倍左右。 成纖維細(xì)胞是真皮的主要組成細(xì)胞,經(jīng)紫外線照射后,皮膚成纖維細(xì)胞的生長(zhǎng)、分化和功能等方面會(huì)發(fā)生明顯的改變。本課題組在前期工作中通過(guò)雙向電泳結(jié)合質(zhì)譜鑒定和免疫印跡技術(shù)發(fā)現(xiàn),小劑量UVB照射會(huì)導(dǎo)致皮膚成纖維細(xì)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的酶切位點(diǎn),在細(xì)胞凋亡過(guò)程中,當(dāng)細(xì)胞中的波形蛋白被這類(lèi)蛋白酶破壞以后,其降解產(chǎn)物能夠促進(jìn)細(xì)胞凋亡。目的： 在UVB引起的皮膚成纖維細(xì)胞凋亡過(guò)程中,影響波形蛋白的胱天蛋白酶-8和-3是否能夠被UVB激活,與胱天蛋白酶-8相關(guān)的受體相互作用蛋白-1如何變化；成纖維細(xì)胞特有的細(xì)胞骨架波形蛋白是否被胱天蛋白酶破壞,并橫向比較了另外兩種普遍存在的細(xì)胞骨架蛋白即微管蛋白和肌動(dòng)蛋白在凋亡過(guò)程中的變化,從而探討UVB對(duì)皮膚成纖維細(xì)胞凋亡的機(jī)制。 方法： (1)體外培養(yǎng)人原代皮膚成纖維細(xì)胞并進(jìn)行傳代,用第3-6代細(xì)胞進(jìn)行實(shí)驗(yàn); (2)為了選擇合適的UVB照射劑量和觀測(cè)時(shí)間,先用噻唑藍(lán)(MTT)比色法比較了不同劑量UVB對(duì)成纖維細(xì)胞活力的影響,然后對(duì)比了150mJ/cm2 UVB照射細(xì)胞后,在12h、24h、36h和48h四個(gè)時(shí)間點(diǎn)細(xì)胞活力的變化； (3)用Hoechst染色法對(duì)細(xì)胞核染色,比較150mJ/cm2 UVB照射成纖維細(xì)胞后,在上述時(shí)間點(diǎn)觀察細(xì)胞核的形態(tài)變化并計(jì)算凋亡細(xì)胞的數(shù)量； (4)用熒光比色法檢測(cè)照射了150mJ/cm2 UVB的成纖維細(xì)胞中,不同時(shí)間點(diǎn)的胱天蛋白酶-8和-3的活性變化； (5)用細(xì)胞熒光和Western Blot技術(shù)檢測(cè)照射UVB誘導(dǎo)凋亡的成纖維細(xì)胞中波形蛋白的蛋白含量的變化； (6)用Western Blot技術(shù)檢測(cè)照射了150mJ/cm2 UVB的成纖維細(xì)胞中,α-微管蛋白、β-微管蛋白和β-肌動(dòng)蛋白的蛋白含量的變化； (7)用RT-PCR和Western Blot技術(shù)檢測(cè)照射了150mJ/cm2 UVB的成纖維細(xì)胞中受體相互作用蛋白-1的mRNA和蛋白含量的變化。 結(jié)果： (1)50-300mJ/cm2 UVB照射后24小時(shí),皮膚成纖維細(xì)胞的活力出現(xiàn)不同程度的下降；150mJ/cm2 UVB照射細(xì)胞以后,在12h、24h、36h和48h四個(gè)時(shí)間點(diǎn),細(xì)胞活力逐漸降低； (2)150mJ/cm2UVB能夠誘導(dǎo)皮膚成纖維細(xì)胞出現(xiàn)凋亡,當(dāng)胱天蛋白酶的活性被抑制時(shí),UVB誘導(dǎo)的凋亡細(xì)胞數(shù)量減少； (3) 150mJ/cm2UVB照射后,成纖維細(xì)胞的胱天蛋白酶-8和-3被激活,在照射后12h時(shí),兩者活性明顯升高,隨著時(shí)間的延長(zhǎng),其活性逐漸降低,在照射后48h時(shí),其活性最低； (4)當(dāng)抑制了胱天蛋白酶-8的激活時(shí),胱天蛋白酶-3的活化延遲；反之,當(dāng)胱天蛋白酶-3的活性被抑制時(shí),胱天蛋白酶-8的活化也出現(xiàn)延遲; (5)當(dāng)分別抑制胱天蛋白酶-8和-3的活性時(shí),凋亡細(xì)胞的數(shù)量明顯減少； (6)在UVB誘導(dǎo)皮膚成纖維細(xì)胞凋亡過(guò)程中,波形蛋白的含量增加,β-微管蛋白的含量減少,α-微管蛋白和β-肌動(dòng)蛋白的含量不變； (7)在凋亡的成纖維細(xì)胞中,受體相互作用蛋白-1的mRNA含量減少,其蛋白含量升高。 結(jié)論： (1)在UVB誘導(dǎo)皮膚成纖維細(xì)胞凋亡過(guò)程中,活化的胱天蛋白酶-8和-3互相影響,共同發(fā)揮促進(jìn)細(xì)胞凋亡的作用； (2)當(dāng)細(xì)胞凋亡時(shí),活化的胱天蛋白酶-8和-3不能通過(guò)水解其底物波形蛋白而影響細(xì)胞的凋亡； (3)在細(xì)胞凋亡過(guò)程中,細(xì)胞中減少的β-微管蛋白和升高的受體相互作用蛋白-1可能發(fā)揮著一定作用； (4)α-微管蛋白和β-肌動(dòng)蛋白不受UVB的影響,在研究UVB誘導(dǎo)皮膚成纖維細(xì)胞凋亡時(shí),它們可以作為免疫印跡的內(nèi)參蛋白使用。
[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.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R751

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