本文選題：Q開關(guān)Nd:YAG + 1064nm激光　； 參考：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 近年來隨著“選擇性光熱理論”的提出,醫(yī)用激光技術(shù)在臨床治療和皮膚美容領(lǐng)域的發(fā)展十分迅速,其中Q開關(guān)Nd:YAG1064nm激光在治療太田痣、黃褐斑、雀斑等色素增加性皮膚病取得了比較滿意的效果。然而,目前Q開關(guān)激光治療表皮色素增加性皮膚病還存在一定的副作用,究其原因一方面可能是表皮色素增加性皮膚病病因復(fù)雜,另一方面是由于Q開關(guān)激光對表皮黑素細胞黑素合成的影響及其分子機制目前尚不十分清楚,從而限制了Q開關(guān)激光的廣泛應(yīng)用。目前人們對Q開關(guān)Nd:YAG1064nm激光的治療原理的認識尚僅局限于黑素顆粒對光的選擇性吸收,而除此之外對激光照射引起的光生物學(xué)作用并不清楚。因此,我們通過實驗研究初步探討Q開關(guān)Nd:YAG1064nm激光照射對人表皮黑素細胞的生物學(xué)及黑素合成的影響。 實驗?zāi)康?研究Q開關(guān)Nd:YAG1064nm激光照射對人表皮黑素細胞的生物學(xué)及黑素合成的影響。 實驗方法:在本研究中,我們選擇目前最先進的MedliteC6Q開關(guān)Nd:YAG激光照射體外培養(yǎng)的人表皮黑素細胞,參數(shù)選擇為:波長1064nm,光斑直徑6mm,頻率2Hz。研究內(nèi)容有:①光學(xué)顯微鏡下觀察Q開關(guān)Nd:YAG1064nm激光照射后細胞形態(tài)的變化;②MTT法檢測Q開關(guān)Nd:YAG1064nm激光照射對人表皮黑素細胞細胞活性的影響;③流式細胞儀檢測Q開關(guān)Nd:YAG1064nm激光照射對人表皮黑素細胞周期與凋亡的影響;④多巴染色法測定Q開關(guān)Nd:YAG1064nm激光照射后人表皮黑素細胞樹突數(shù)量及長度的變化;⑤多巴氧化法檢測Q開關(guān)Nd:YAG1064nm激光照射后酪氨酸酶(Tyr)活性的變化;⑥熒光定量反轉(zhuǎn)錄(RT)-PCR和Westenblot分別檢測Q開關(guān)Nd:YAG1064nm激光照射后Tyr和酪氨酸酶相關(guān)蛋白(TRP-1和TRP-2)基因轉(zhuǎn)錄和蛋白表達水平的變化。 實驗結(jié)果:1.不同能量密度的Q開關(guān)Nd:YAG1064nm激光照射對人表皮黑素細胞活性及形態(tài)的影響:①MTT結(jié)果表明:人表皮黑素細胞對Q開關(guān)Nd:YAG1064nm激光照射的能量密度有一定的耐受范圍,在本實驗中不影響細胞存活率的最高照射能量密度為3J/cm2,即人表皮黑素細胞可以耐受的照射能量密度閾值為3J/cm2。我們以此為依據(jù)將細胞分為對照組,1J/cm2照射組(代表本實驗中不影響細胞生存率的最低照射能量密度)與5J/cm2照射組(代表本實驗中超出黑素細胞可以耐受的閾值范圍的最高照射能量密度),繼續(xù)下面的實驗。②不同能量密度的Q開關(guān)Nd:YAG1064nm激光照射后人表皮黑素細胞活性的時效性分析結(jié)果表明:與對照組比較,1J/cm2組黑素細胞活性在各時間點均無明顯差異(P0.05),而5J/cm2組人表皮黑素細胞活性分別在照射后0h時、24h時與48h時這三個時間點明顯降低(P0.05)。5J/cm2照射后,人表皮黑素細胞增殖曲線在48h內(nèi)逐漸下降至最低點后又顯著上升。③我們在光學(xué)顯微鏡下觀察到各組細胞的形態(tài)在Q開關(guān)Nd:YAG1064nm激光照射后即刻無明顯改變,但接受照射后24h時與對照組比較1J/cm2照射組黑素細胞大小無明顯變化,但是樹突長度增加;而3J/cm2照射組黑素細胞體積明顯變小,樹突數(shù)量減少并且長度縮短。 2.Q開關(guān)Nd:YAG1064nm激光照射對人表皮黑素細胞周期與凋亡的影響:①在不影響細胞存活率的能量密度范圍內(nèi),將黑素細胞分為對照組、1J/cm2照射組(代表本實驗中不影響細胞生存率的最低照射能量密度)與3J/cm2照射組(代表在本實驗中不影響細胞存活率的最高照射能量密度,即黑素細胞可以耐受的照射能量密度閾值)②。細胞周期結(jié)果表明:1J/cm2照射組人表皮黑素細胞S期細胞數(shù)量輕度增加(P0.05),而3J/cm2照射組人表皮黑素細胞S期細胞數(shù)量則顯著減少(P0.05)。③細胞凋亡結(jié)果表明:與對照組細胞凋亡率相比,1J/cm2照射組無明顯差異(P0.05),而3J/cm2照射組細胞凋亡率明顯增加(P0.05)。 3.Q開關(guān)Nd:YAG1064nm激光照射對人表皮黑素細胞黑素合成的影響:①在不影響黑素細胞存活率的范圍內(nèi),能量密度為1J/cm2的激光照射可以刺激Tyr活性,增加黑素細胞樹突的數(shù)量和長度;上Ty調(diào)r與TRP-1的mRNA表達,而TRP-2mRNA表達并無明顯變化,其蛋白水平的變化與轉(zhuǎn)錄水平相符。②在不影響黑素細胞活性的范圍內(nèi),能量密度為3J/cm2的激光照射能明顯抑制Tyr活性,減少黑素細胞樹突的數(shù)量和長度;同時也不同程度的抑制了Try、TRP-1和TRP-2的mRNA表達與蛋白合成。 主要結(jié)論: 1.人表皮黑素細胞對Q開關(guān)Nd:YAG1064nm激光照射的能量密度存在一定的耐受范圍。當能量密度大于一定值(人表皮黑素細胞能耐受的值)閾時,Q開關(guān)Nd:YAG1064nm激光的照射對細胞活性的抑制作用會隨著能量密度的增加而逐漸增強,但這種抑制作用并不持續(xù)存在,黑素細胞的活性經(jīng)過一段時間后可以逐漸恢復(fù)。 2.在不影響黑素細胞存活率的范圍內(nèi),其中較低的能量密度的Q開關(guān)Nd:YAG1064nm激光照射能夠刺激人表皮黑素細胞DNA合成,表現(xiàn)出生物刺激作用。當選擇人表皮黑素細胞可以耐受的能量密度閾值照射時,Q開關(guān)Nd:YAG1064nm激光可以明顯抑制人表皮黑素細胞DNA合成并促進其凋亡。 3.在不影響黑素細胞存活率的范圍內(nèi),Q開關(guān)Nd:YAG1064nm激光照射可以影響黑素合成。其中較低的能量密度的激光照射能夠通過刺激酪氨酸酶家族蛋白(TRPs)從而促進黑素的合成,而選擇較高能量密度(人表皮黑素細胞可以耐受的能量密度閾值)的Q開關(guān)Nd:YAG1064nm激光照射則起著抑制黑素合成的作用。 4.在不影響黑素細胞存活率的范圍內(nèi),Q開關(guān)Nd:YAG1064nm激光照射可以影響人表皮黑素細胞樹突的形成。 5.Q開關(guān)Nd:YAG1064nm激光照射對人表皮黑素細胞存在光生物學(xué)作用。
[Abstract]:In recent years, with the "selective photothermal theory", medical laser technology has developed rapidly in the field of clinical treatment and skin beauty, in which Q switched Nd:YAG1064nm laser has achieved satisfactory results in the treatment of pigmented dermatosis such as nevus of Ota, chloasma, freckles and other pigmented dermatosis. However, the Q switch laser is currently used to treat epidermal pigments. There is also some side effects of additive dermatosis, which may be due to the complicated cause of epidermal pigmented dermatosis, and the other is that the effect of Q switched laser on melanin synthesis in epidermal melanocytes and its molecular mechanism are still not very clear, thus limiting the wide application of Q switched laser. At present, people have opened Q. The understanding of the principle of the treatment of Nd:YAG1064nm laser is still limited to the selective absorption of light by melanin particles, but it is not clear that the effect of laser irradiation is not clear. Therefore, we have preliminarily studied the biological and melanogenesis of human surface melanocytes by the Q switch Nd:YAG1064nm laser irradiation. Ringing.
Objective: To study the effects of Q switch Nd:YAG1064nm laser irradiation on the biological and melanin synthesis of human epidermal melanocytes.
Experimental methods: in this study, we chose the most advanced MedliteC6Q switched Nd:YAG laser to irradiate human epidermal melanocytes in vitro. The parameters were as follows: wavelength 1064nm, spot diameter 6mm, frequency 2Hz. research content: (1) observation of cell morphology changes after Q switch Nd: YAG1064nm laser irradiation under optical microscope; (2) MTT method detection The effect of Q switch Nd:YAG1064nm laser irradiation on the cell activity of human epidermal melanocytes; and (3) flow cytometry to detect the effect of Q switch Nd:YAG1064nm laser irradiation on the cell cycle and apoptosis of human epidermal melanocytes; (4) DOPA staining method was used to determine the number and length of dendritic cells of human epidermal melanocytes after Q switch Nd:YAG1064nm laser irradiation; 5 The changes of tyrosinase (Tyr) activity after Q switch Nd:YAG1064nm laser irradiation were detected by Ba. (6) fluorescence quantitative reverse transcription (RT) -PCR and Westenblot were used to detect the changes in the transcriptional and egg white expression of Tyr and tyrosinase related proteins (TRP-1 and TRP-2) after Q switch Nd:YAG1064nm laser irradiation, respectively.
The results were as follows: 1. the effects of different energy density Q switch Nd:YAG1064nm laser irradiation on the activity and morphology of human epidermal melanocytes: (1) MTT results showed that human epidermal melanocytes had a certain range of tolerance to the energy density of Q switched Nd:YAG1064nm laser irradiation, and the highest energy density of cell survival rate was not affected in this experiment. 3J/cm2, the threshold of radiation energy density that the human epidermal melanocytes can tolerate, is 3J/cm2.. We divide the cells into the control group based on this, and the 1J/cm2 irradiation group (representing the minimum irradiation energy density that does not affect the cell survival rate in this experiment) and the 5J/cm2 irradiation group (representing the threshold range beyond the tolerance of melanocytes in this test) The results of the aging analysis of the activity of human epidermal melanocytes after Q switch Nd:YAG1064nm laser irradiation with different energy density showed that there was no significant difference in the activity of melanocyte in the 1J/cm2 group at all time points compared with the control group (P0.05), while the activity of human epidermal melanocytes in the 5J/cm2 group was in the 5J/cm2 group, respectively. After 0h, the three time points of 24h and 48h decreased significantly (P0.05).5J/cm2 irradiation, and the proliferation curve of human epidermal melanocytes decreased gradually to the lowest point in 48h and then increased significantly. Compared with the control group, the size of the melanocytes in the 1J/cm2 irradiated group was not significantly changed, but the length of the dendritic cells increased, while the volume of melanocytes in the 3J/cm2 group decreased obviously, the number of dendrites decreased and the length shortened in the 3J/cm2 irradiation group.
The effect of 2.Q switch Nd:YAG1064nm laser irradiation on the cell cycle and apoptosis of human epidermal melanocytes: (1) the melanocytes were divided into the control group in the range of energy density that did not affect the cell survival rate, and the 1J/cm2 irradiation group (representing the minimum irradiation energy density that does not affect the cell survival rate in this experiment) and the 3J/cm2 irradiation group (represented in this experiment) The maximum radiation energy density that affects the cell survival rate, that is, the threshold of the radiation energy density that the melanocytes can tolerate. The cell cycle results show that the number of S cells in the human epidermal melanocytes in the 1J/cm2 irradiation group is slightly increased (P0.05), while the number of S cells in the 3J/cm2 irradiated group is significantly reduced (P0.05). (3) cell apoptosis The results showed that: compared with the control group, the apoptosis rate of 1J/cm2 group was not significantly different (P0.05), while the apoptosis rate of 3J/cm2 irradiation group increased significantly (P0.05).
The effect of 3.Q switch Nd:YAG1064nm laser irradiation on melanin synthesis in human epidermal melanocytes: (1) laser irradiation with energy density of 1J/cm2 can stimulate the activity of Tyr, increase the number and length of the dendrites of melanocytes in the range of the survival rate of melanocytes; the expression of R and TRP-1 in the upper Ty is expressed, and the expression of TRP-2mRNA is not significantly changed. The changes in the protein level are consistent with the transcriptional level. In the range of the activity of melanocytes, the laser irradiation with energy density of 3J/cm2 can significantly inhibit the activity of Tyr, reduce the number and length of the dendrites of melanocytes, and inhibit the mRNA expression and protein synthesis of Try, TRP-1 and TRP-2 to varying degrees.
The main conclusions are as follows:
1. human epidermal melanocytes have a certain range of tolerance to the energy density of Q switched Nd:YAG1064nm laser irradiation. When the energy density is greater than a certain value (the tolerance value of human epidermal melanocytes), the inhibitory effect of the Q switch Nd:YAG1064nm laser irradiation on the cell activity will gradually increase with the increase of the energy density, but this inhibition is inhibited. The production does not persist. The activity of melanocytes can recover gradually after a period of time.
2. in the range that does not affect the survival rate of melanocytes, the low energy density Q switch Nd:YAG1064nm laser irradiation can stimulate the DNA synthesis of human epidermal melanocytes and show biological stimulation. When the energy density threshold of human epidermal melanocytes can be tolerated, the Q switch Nd:YAG1064nm laser can obviously inhibit people. Epidermal melanocyte DNA synthesizes and promotes its apoptosis.
3. the Q switch Nd:YAG1064nm laser irradiation can affect melanin synthesis without affecting the survival rate of melanocytes, in which low energy density laser irradiation can stimulate the synthesis of melanin by stimulating the tyrosinase family protein (TRPs) and select the higher energy density (the energy density tolerable by human epidermal melanocytes). The Q switch Nd:YAG1064nm laser irradiation plays a role in inhibiting melanin synthesis.
4. the Q switch Nd:YAG1064nm laser irradiation can affect the formation of human epidermal melanocyte dendrites without affecting the survival rate of melanocytes.
5.Q switch Nd:YAG1064nm laser irradiation has a photo biologic effect on human epidermal melanocytes.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R454.2;R758.4

【參考文獻】

相關(guān)期刊論文 前1條

1 劉耿,劉燕,劉宏波,王永賢;Q開關(guān)Nd∶YAG激光治療顴部褐青色痣350例療效觀察[J];中華醫(yī)學(xué)美學(xué)美容雜志;2003年05期

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本文編號：1844046