本文選題：UVB照射 + 黑素細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2011年碩士論文

【摘要】：人體皮膚的顏色主要受色素沉著的影響,它不僅決定皮膚、頭發(fā)和眼睛的顏色,還能夠保護(hù)皮膚免受紫外線的過(guò)度照射。其過(guò)程非常復(fù)雜,包括:1、黑素細(xì)胞內(nèi)黑素小體的裝配與黑素合成;2、成熟的黑素小體沿細(xì)胞樹(shù)突伸展方向向樹(shù)突遠(yuǎn)端轉(zhuǎn)移;3、而后黑素小體脫離黑素細(xì)胞進(jìn)入周圍的角質(zhì)形成細(xì)胞;4、黑素小體在角質(zhì)形成細(xì)胞內(nèi)再分布和降解。通常將黑素小體從核周向樹(shù)突末梢轉(zhuǎn)移并傳遞至鄰近角質(zhì)形成細(xì)胞的過(guò)程稱為黑素小體的轉(zhuǎn)運(yùn)[1]。研究表明黑素小體的轉(zhuǎn)運(yùn)在皮膚色素沉著過(guò)程中發(fā)揮極為重要的作用,但其機(jī)制尚未被完全認(rèn)識(shí)。紫外線是電磁波譜中波長(zhǎng)為0.01～0.40微米輻射的總稱。皮膚的色素沉著主要由UVB引起,UVB的生物學(xué)效應(yīng)強(qiáng)度是UVA的800～1000倍,是引起皮膚光損傷的最重要波段[2]。研究證明,UVB照射可以引起皮膚色素沉著,一定劑量的UVB照射可以促進(jìn)黑素細(xì)胞增殖,增加黑素合成~([3])。已有研究證實(shí)UVB照射體外重建表皮類似物時(shí)可以增強(qiáng)黑素小體的轉(zhuǎn)運(yùn)。發(fā)現(xiàn)在表皮類似物的基底層,及角質(zhì)層都有黑素小體~([4-5])。以上提示UVB照射可以促進(jìn)黑素小體的轉(zhuǎn)運(yùn)。但是對(duì)于UVB促黑素轉(zhuǎn)運(yùn)的機(jī)制并不清楚。因此,我們通過(guò)實(shí)驗(yàn)研究初步探討UVB照射促進(jìn)黑素小體向角質(zhì)形成細(xì)胞轉(zhuǎn)運(yùn)的機(jī)制。 實(shí)驗(yàn)?zāi)康?研究UVB照射對(duì)黑素小體向角質(zhì)形成細(xì)胞轉(zhuǎn)運(yùn)的影響及其機(jī)制。 實(shí)驗(yàn)方法:本研究使用上海希格瑪公司生產(chǎn)的紫外線光療儀,輻照度以UVB輻照度監(jiān)示器標(biāo)定,UVB劑量=UVB輻照度×?xí)r間(s)。研究?jī)?nèi)容有:①不同能量UVB照射對(duì)黑素細(xì)胞株P(guān)IG1增殖活性的影響;②UVB照射對(duì)PIG1細(xì)胞的形態(tài)學(xué)影響及細(xì)胞樹(shù)突變化;③UVB照射對(duì)酪氨酸酶Tyr的mRNA及蛋白影響的時(shí)間規(guī)律性;④gp100免疫熒光法觀察UVB照射對(duì)黑素合成的影響;⑤RT-PCR方法檢測(cè)UVB照射對(duì)F-actin、Rac、Rho及PAR-2的mRNA表達(dá)的影響;⑥Western Blot方法檢測(cè)UVB照射對(duì)黑素小體轉(zhuǎn)運(yùn)相關(guān)蛋白F-actin、Rac、Rho及PAR-2蛋白表達(dá)的影響。 實(shí)驗(yàn)結(jié)果: 1.不同能量UVB照射對(duì)黑素細(xì)胞株P(guān)IG1活性的影響; MTT結(jié)果表明:UVB照射后24h,照射能量在50、100mJ/cm~2時(shí),細(xì)胞增殖活性高于照射前水平(P0.05),50mJ/cm~2時(shí)細(xì)胞增殖活性最強(qiáng);而照射能量高于200mJ/cm~2時(shí),細(xì)胞增殖活性低于照射前水平,并隨著能量升高呈逐漸下降趨勢(shì)。UVB照射后48h,照射能量在100mJ/cm~2時(shí)細(xì)胞活性有一定程度的恢復(fù),而到400mJ/cm~2后呈下降趨勢(shì)。100mJ/cm~2時(shí)的細(xì)胞增殖活性較24h升高,200 mJ/cm~2時(shí)細(xì)胞活性達(dá)最大值,而24h則從200 mJ/cm~2時(shí)開(kāi)始下降。 2. UVB照射對(duì)PIG1細(xì)胞的形態(tài)學(xué)影響及樹(shù)突變化:由于UVB照射能量在100mJ/cm~2時(shí)能夠較穩(wěn)定增加細(xì)胞活性,因此選擇100mJ/cm~2 UVB照射細(xì)胞后48h觀察了細(xì)胞的形態(tài)變化。①選擇100mJ/cm~2 UVB照射PIG1后48h,光學(xué)顯微鏡下觀察細(xì)胞形態(tài)的變化。對(duì)照組PIG1細(xì)胞形態(tài):細(xì)胞貼壁后很快進(jìn)入對(duì)數(shù)生長(zhǎng)期,生長(zhǎng)狀況良好,細(xì)胞呈多分支樹(shù)突狀,輪廓清楚折光性好。照射組形態(tài):PIG1細(xì)胞數(shù)量明顯增多,樹(shù)突顯著延長(zhǎng)。③UVB照射對(duì)PIG1細(xì)胞樹(shù)突的影響統(tǒng)計(jì)學(xué)方法計(jì)數(shù)PIG1細(xì)胞的樹(shù)突數(shù)量及長(zhǎng)度。用100mJ/cm~2的UVB照射PIG1后48h,細(xì)胞樹(shù)突相對(duì)于未照射組明顯增多延長(zhǎng),具有統(tǒng)計(jì)學(xué)意義。 3. UVB照射對(duì)酪氨酸酶Tyr的mRNA及蛋白影響的時(shí)間規(guī)律性:用100mJ/cm~2的UVB照射PIG1后48h,酪氨酸酶Tyr的mRNA和蛋白表達(dá)在UVB照射后24h后顯著增加,并呈上升趨勢(shì)。 4. gp100免疫熒光法觀察UVB照射對(duì)黑素合成的影響:UVB能量為50 mJ/cm~2時(shí),照射后48h時(shí)熒光強(qiáng)度最強(qiáng)。UVB能量為100mJ/cm~2時(shí)同樣在照射后48h時(shí)熒光強(qiáng)度最強(qiáng)。 5.RT-PCR方法檢測(cè)UVB照射對(duì)F-actin、Rac1、RhoA及PAR-2的mRNA表達(dá)的影響:100mJ/cm~2 UVB照射48h后Rac1、F-actin和PAR-2的mRNA表達(dá)相對(duì)于未照射組明顯增加,而RhoA的表達(dá)下降。6.Western Blot方法檢測(cè)UVB照射對(duì)黑素小體轉(zhuǎn)運(yùn)相關(guān)蛋白F-actin、Rac1、RhoA及PAR-2蛋白表達(dá)的影響:100mJ/cm~2 UVB照射48h后Rac1、F-actin和PAR-2的表達(dá)升高,而RhoA的表達(dá)下降。 主要結(jié)論: 1. UVB照射能量在100mJ/cm~2時(shí)能夠較穩(wěn)定增加細(xì)胞活性。提示一定劑量的UVB照射有刺激PIG1細(xì)胞增殖的作用。而選擇超過(guò)100mJ/cm~2的能量照射時(shí),PIG1細(xì)胞的活性受到抑制。而這種抑制作用并不是持續(xù)性的,細(xì)胞活性會(huì)在一段時(shí)間后恢復(fù),因此在這種情況下UVB對(duì)PIG1細(xì)胞表現(xiàn)出的損傷作用是可逆性的。 2.一定劑量的UVB照射有促進(jìn)PIG1細(xì)胞增殖和刺激樹(shù)突生長(zhǎng)的作用。 3.UVB照射可以影響酪氨酸酶Tyr的mRNA及蛋白表達(dá),在UVB照射后24h后顯著增加,并呈上升趨勢(shì)。 4. UVB照射后用gp100這一黑素小體特異性抗體進(jìn)行免疫熒光染色,可以觀察黑素合成情況。黑素合成在100mJ/cm~2 UVB照射后48h時(shí)達(dá)到最大值。 5.UVB照射可以促進(jìn)樹(shù)突形態(tài)調(diào)控因子Rac1、絲狀肌動(dòng)蛋白F-actin及蛋白酶活性受體PAR-2的mRNA及蛋白表達(dá),抑制RhoA的表達(dá)。
[Abstract]:The color of the human skin is mainly affected by the pigmentation, which not only determines the color of the skin, hair and eyes, but also protects the skin from excessive ultraviolet radiation. The process is very complex, including: 1, the assembly of melanosomes in melanocytes and melanin synthesis; and 2, the mature melanosomes extend to the distal dendrites along the dendritic direction. 3, the melanosomes are separated from the melanocytes into the surrounding keratinocytes; 4, the melanosomes are redistributed and degraded in the keratinocytes. Usually the process of transferring melanosomes from the nucleus to the dendritic cells and transferring to the adjacent keratinocytes is called the transport of melanin corpuscle in the transport of [1].. The mechanism of skin pigmentation plays a very important role, but its mechanism has not been fully understood. Ultraviolet radiation is the general name of 0.01 to 0.40 microns in the electromagnetic wave spectrum. The pigmentation of the skin is mainly caused by UVB, and the biological effect of UVB is 800~1000 times that of UVA, and it is the most important band of [2]. to cause skin light damage. It has been shown that UVB irradiation can cause pigmentation in the skin, and a certain dose of UVB can promote the proliferation of melanocytes and increase melanin synthesis ~ ([3]). It has been proved that UVB irradiation can enhance the transport of melanosomes in the reconstruction of epidermal analogues in vitro. It is found that there are melanosomes in the basal layer of the epidermal analogues and in the cuticle layer ~ ([4-5]). These findings suggest that UVB irradiation can promote the transport of melanosomes. However, the mechanism for the transport of melanin in UVB is not clear. Therefore, we have preliminarily explored the mechanism of UVB irradiation promoting the transport of melanosomes to keratinocytes.
Objective: To study the effect of UVB irradiation on the transport of melanosomes to keratinocytes and its mechanism.
Experimental method: This study used the UV light therapy instrument produced by Shanghai Hege Ma company, irradiance was calibrated with UVB irradiance monitor, and UVB dose =UVB irradiance x time (s). The study contents were: (1) the effect of different energy UVB irradiation on the proliferation of PIG1 in melanocyte strain; the morphological influence of UVB irradiation on PIG1 cells and the change of cell dendrites (3) the time regularity of the effect of UVB irradiation on the mRNA and protein of tyrosinase Tyr; (4) the effect of UVB irradiation on the synthesis of melanin by gp100 immunofluorescence; and (5) RT-PCR method to detect the effect of UVB irradiation on F-actin, Rac, Rho and PAR-2 mRNA expression. And the effect of PAR-2 protein expression.
Experimental results:
1. the effect of different energy UVB irradiation on the activity of PIG1 in melanocyte strain. The MTT results showed that the proliferation activity of cells was higher than before irradiation (P0.05) and 50mJ/cm~2 was the strongest when UVB irradiation was 50100mJ/cm~2, while the irradiation energy was higher than that of 200mJ/cm~ 2, and the cell proliferation activity was lower than that before irradiation, and with the energy of irradiation, the cell proliferation activity was lower than that before irradiation. When the amount of.UVB decreased gradually, the cell activity was recovered to a certain extent at 100mJ/cm~2, and the cell proliferation activity was higher than that of 24h at.100mJ/cm~2 after 400mJ/cm~2, and the cell activity reached the maximum at 200 mJ/cm~2, while 24h began to decline from 200 mJ/cm~2.
The morphological influence of 2. UVB irradiation on the morphology of PIG1 cells and the change of dendrites: because UVB irradiated energy at 100mJ/cm~2, the cell activity was more stable, so the morphological changes of the cells were observed after 100mJ/cm~2 UVB irradiated the cells. (1) the 100mJ/cm~2 UVB irradiated PIG1 48h, the morphological changes of the cells were observed under the light microscope. Group PIG1 cell morphology: the cells quickly entered the logarithmic growth period after being adhered to the wall. The growth condition was good, the cells showed a multi branch dendritic shape, and the contour clearly refracted. The number of PIG1 cells increased significantly and the dendrites were significantly prolonged. (3) the number and length of the dendrites of the PIG1 cells were counted by the effect of UVB irradiation on the dendrites of the PIG1 cells. The number and length of the dendritic cells of PIG1 cells were counted. When 00mJ/cm~2 was irradiated with UVB for PIG1, the 48h cell dendrites increased significantly compared with the unirradiated group, with statistical significance.
The time regularity of the effect of 3. UVB irradiation on the mRNA and protein of tyrosinase Tyr: 48h after PIG1 irradiation with 100mJ/cm~2, mRNA and protein expression of tyrosinase Tyr significantly increased after 24h after UVB, and showed an upward trend.
The effect of UVB irradiation on the synthesis of melanin was observed by 4. gp100 immunofluorescence. When the energy of UVB was 50 mJ/cm~2, the strongest.UVB energy of the fluorescence intensity was 100mJ/cm~2 after 48h, and the intensity of the fluorescence was the strongest at 48h after irradiation.
The effect of UVB irradiation on the mRNA expression of F-actin, Rac1, RhoA and PAR-2: 100mJ/cm~2 UVB irradiated 48h Rac1, F-actin and F-actin expression were significantly increased compared to those in the unirradiated group. The effect of 100mJ/cm~2 UVB and 48h on Rac1, F-actin and PAR-2 increased, while RhoA expression decreased.
The main conclusions are as follows:
1. UVB irradiate energy at 100mJ/cm~2 can increase cell activity more steadily. It suggests that a certain dose of UVB irradiation stimulates the proliferation of PIG1 cells. While the activity of PIG1 cells is inhibited when the energy exceeds 100mJ/cm~2, and this inhibition is not persistent, and the cell activity will recover after a period of time. Therefore, the activity of the cells will be recovered after a period of time. In this case, the damaging effect of UVB on PIG1 cells is reversible.
2. a certain dose of UVB irradiation can promote the proliferation of PIG1 cells and stimulate the growth of dendrites.
3.UVB irradiation could affect the expression of tyrosinase Tyr mRNA and protein, and increased significantly after UVB irradiation, and showed an upward trend.
4. UVB irradiated with gp100, a melanosomes specific antibody for immunofluorescence staining, can be used to observe the synthesis of melanin. Melanin synthesis reached the maximum value at 48h after 100mJ/cm~2 UVB irradiation.
5.UVB irradiation can promote the expression of mRNA and protein of dendritic morphology regulator Rac1, filamentous actin F-actin and protease active receptor PAR-2, and inhibit the expression of RhoA.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R758.14

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相關(guān)期刊論文 前10條

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本文編號(hào)：2092906