本文關(guān)鍵詞： 熱 UVB 黑素細(xì)胞 黑素合成 酪氨酸酶活性 細(xì)胞周期　出處：《第四軍醫(yī)大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的 白癜風(fēng)是一種常見(jiàn)的以局部或全身皮膚黏膜色素脫失為特征的皮膚病。人群患病率約為0.1～2%,多于幼年起病，無(wú)明顯種族及性別差異。皮損可發(fā)生于任何部位，皮膚病理顯示皮損處表皮黑素細(xì)胞明顯減少或消失。該病病因不明，目前認(rèn)為可能與遺傳、自身免疫異常、氧化應(yīng)激、神經(jīng)精神等因素有關(guān)。白癜風(fēng)臨床治療較為棘手，主要包括藥物治療、光療、手術(shù)治療等。光療，特別是波長(zhǎng)為311～313nm的中波紫外線，由于療效確切、安全可靠，現(xiàn)已成為白癜風(fēng)臨床治療的首選方法之一。 近年來(lái)，人們逐漸發(fā)現(xiàn)熱處理(42℃,1h）與紫外線相似，能夠增加黑素細(xì)胞的酪氨酸酶活性及黑素合成，促使黑素細(xì)胞和角質(zhì)形成細(xì)胞共培養(yǎng)體系中酪氨酸酶相關(guān)蛋白-1陽(yáng)性的黑素細(xì)胞明顯增多；同時(shí)，以熱效應(yīng)為主的紅外線可明顯減少大劑量UVB輻射誘導(dǎo)的角質(zhì)形成細(xì)胞凋亡。以上結(jié)果提示我們熱與UVB的聯(lián)合治療可能將成為白癜風(fēng)治療的重要手段。本實(shí)驗(yàn)研究熱處理對(duì)UVB輻射后人表皮黑素細(xì)胞的增殖活性、酪氨酸酶活性、黑素合成及細(xì)胞周期的影響，并初步探討其機(jī)制，為今后熱療與UVB聯(lián)合應(yīng)用治療白癜風(fēng)等色素脫失性皮膚病提供理論依據(jù)。 方法 1.以體外培養(yǎng)的正常人黑素細(xì)胞作為研究對(duì)象，分別以20、30、50、70、90、120、180mJ/cm~2NB-UVB輻射，采用四甲基偶氮唑藍(lán)比色法（MTT）檢測(cè)細(xì)胞存活率，并選擇最佳照射劑量進(jìn)行后續(xù)實(shí)驗(yàn)。 2.以42℃,1h為熱處理劑量，將黑素細(xì)胞分為四組：正常對(duì)照組、UVB組、加熱組、UVB+熱組，，分別給予不同干預(yù)，連續(xù)3d，末次干預(yù)24h后以MTT法檢測(cè)細(xì)胞增殖活性，左旋多巴(L-Dopa)為底物測(cè)定酪氨酸酶活性，NAOH法測(cè)定黑素含量，流式細(xì)胞儀檢測(cè)細(xì)胞周期變化。 結(jié)果 1.分別以20、30、50、70、90、120、180mJ/cm~2UVB連續(xù)照射黑素細(xì)胞3d后，細(xì)胞存活率隨著劑量的增加而降低，選擇50mJ/cm~2作為最佳照射劑量。 2.黑素細(xì)胞經(jīng)不同干預(yù)后比較細(xì)胞增殖活性，UVB組較對(duì)照組減少7.1%（P0.05），加熱組、UVB+熱組細(xì)胞較對(duì)照組分別增加25.3%和58.1%（P0.05）。熱處理與UVB共同干預(yù)具有協(xié)同作用。 3.黑素細(xì)胞經(jīng)不同干預(yù)后，UVB組、加熱組、UVB+熱組細(xì)胞的酪氨酸酶活性較對(duì)照組分別增長(zhǎng)3.8%、11.5%和31.9%（P0.05），三組細(xì)胞的黑素含量較對(duì)照組分別增加17.5%、31.6%和67.3%（P0.05）。熱處理與UVB輻射聯(lián)合干預(yù)具有協(xié)同作用。 4.黑素細(xì)胞給予不同干預(yù)后，UVB組、加熱組、UVB+熱組處于G1期的細(xì)胞較對(duì)照組分別下降24.61%、13.16%和32.02%（P0.05）；處于S期的細(xì)胞較對(duì)照組分別增加15.02%、8.58%和17.14%（P0.05），處于G2期的細(xì)胞分別增加12.93%、7.52%、23.60%（P0.05）。熱處理與UVB輻射聯(lián)合干預(yù)具有協(xié)同作用。 結(jié)論 1.熱處理（42℃,1h）能有效促進(jìn)黑素細(xì)胞增殖，與UVB輻射（50mj/cm2）聯(lián)合干預(yù)具有協(xié)同作用。 2.熱處理（42℃,1h）和UVB輻射（50mj/cm2）均可顯著增加黑素細(xì)胞內(nèi)酪氨酸酶活性及黑素合成，兩者聯(lián)合干預(yù)具有協(xié)同作用。 3.熱處理（42℃,1h）及UVB輻射（50mj/cm2）干預(yù)均可促使細(xì)胞周期阻滯在S期和G2期，減少處于G1期的細(xì)胞數(shù)量，兩者聯(lián)合干預(yù)具有協(xié)同作用。
[Abstract]:objective
Vitiligo is a common skin disease to local or generalized depigmentation of skin and mucosa characterized. The prevalence is about 0.1 ~ 2%, more than the juvenile onset, no obvious racial and gender differences. The lesions can occur in any part of the skin lesions, pathological examination showed epidermal melanocytes decreased or disappeared. The cause of this disease currently unknown, it may be related to genetic, autoimmune disorders, oxidative stress and related neuropsychiatric factors. The clinical treatment of vitiligo is difficult, mainly including drug therapy, phototherapy, phototherapy. Surgical treatment, especially at the wavelength of 311 ~ 313nm due to ultraviolet ray, curative effect, safe and reliable, has become one of the preferred methods of clinical the treatment of vitiligo.
In recent years, people gradually found that the heat treatment (at 42 1H) and ultraviolet similar, can increase the tyrosinase activity and melanin synthesis in black cells, promote the melanocytes and keratinocytes of tyrosinase related protein -1 positive cells co cultured black pigment cells increased significantly; at the same time, the thermal effect of the infrared ray reduce the dose of UVB radiation induced apoptosis of keratinocytes. The above results suggest that our combined treatment of heat and UVB will likely become an important means in the treatment of vitiligo. The experimental study on heat treatment of irradiation on Human Epidermal Melanocytes Proliferation and tyrosinase activity of UVB, effects of melanin synthesis and cell cycle, and to explore the mechanism, provide a theoretical basis for future hyperthermia combined with UVB treatment of depigmentation skin disease.
Method
1., the normal human melanocytes were cultured in vitro. The cell survival rate was detected by 20,30,50,70,90120180mJ/cm~2NB-UVB radiation and four MZR blue colorimetric assay (MTT). The best dose of radiation was selected for subsequent experiments.
2. to 42 DEG C, 1H heat treatment dose, melanocytes were divided into four groups: normal control group, UVB group, UVB+ group of heating, heat group, given different interventions, respectively for 3D, 24h after the last intervention by MTT was used to detect the proliferation activity of levodopa (L-Dopa) for determination of activity of casein substrate tyrosinase, melanin content was measured by NAOH assay, flow cytometry to detect the cell cycle.
Result
1., after continuously irradiating melanocytes 3D with 20,30,50,70,90120180mJ/cm~2UVB, the cell survival rate decreases with the increase of dose. 50mJ/cm~2 is chosen as the best dose.
2., compared with the control group, the proliferative activity of the melanocytes was decreased by 7.1% (P0.05) compared with the control group after different intervention. In the heating group, the UVB+ fever group increased by 25.3% and 58.1% (P0.05) respectively compared with the control group. The co intervention of heat treatment and UVB had synergistic effect.
3. melanocytes after different intervention, UVB group, UVB+ group, heat heating group, cell tyrosinase activity increased by 3.8% compared with the control group, 11.5% and 31.9% (P0.05), three groups of melanocytes was increased by 17.5% compared with the control group, 31.6% and 67.3% (P0.05). Heat treatment and UVB radiation combined with the intervention has a synergistic effect.
4. melanocytes give different intervention, UVB group, UVB+ group, thermal heating group, cells in G1 phase decreased by 24.61% respectively compared with the control group, 13.16% and 32.02% (P0.05); the cells at S phase increased by 15.02% respectively compared with the control group, 8.58% and 17.14% (P0.05), the cells at G2 phase increased respectively 12.93%, 7.52%, 23.60% (P0.05). The joint intervention of heat treatment and UVB radiation has a synergistic effect.
conclusion
1. heat treatment (42 centigrade, 1H) can effectively promote the proliferation of melanocytes and have synergistic effect with UVB radiation (50mj/cm2).
2. heat treatment (42, 1H) and UVB radiation (50mj/cm2) significantly increased tyrosinase activity and melanin synthesis in melanocytes, and the combined intervention had synergistic effects.
3., heat treatment (42 C, 1H) and UVB radiation (50mj/cm2) intervention can promote cell cycle arrest in S and G2 phase, reduce the number of cells in G1 phase, and the combination intervention has synergistic effect.

【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】：2012
【分類(lèi)號(hào)】：R758.41

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