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  本文關(guān)鍵詞： 光動力療法 自噬 5-氨基酮戊酸 LC3 Beclin1　出處：《中南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 1.觀察ALA-紅光-PDT對體外培養(yǎng)人皮膚成纖維細(xì)胞形態(tài)及增殖活性的影響,探討在體外條件下模擬ALA-紅光-PDT嫩膚的合適處理參數(shù)。 2.觀察ALA-紅光-PDT對成纖維細(xì)胞自噬的影響,初步探討ALA-紅光-PDT嫩膚可能的機(jī)制。 方法 1.5-氨基酮戊酸(ALA)孵育成纖維細(xì)胞后,予以不同時間的紅光照射成纖維細(xì)胞,光學(xué)顯微鏡下觀察細(xì)胞形態(tài)學(xué)變化。 2.以不同濃度的ALA孵育成纖維細(xì)胞后以不同劑量照射成纖維細(xì)胞,四甲基偶氮唑鹽(MTT)法檢測各組細(xì)胞增殖活性的變化。 3.ALA-紅光-PDT處理成纖維細(xì)胞后,聚合酶鏈反應(yīng)(PCR)法檢測自噬相關(guān)基因LC3及Beclinl mRNA的表達(dá)情況。 4.ALA-紅光-PDT處理成纖維細(xì)胞后,MDC染色法檢測細(xì)胞內(nèi)自噬泡的變化。 結(jié)果 1.ALA+紅光10min組細(xì)胞照射后形態(tài)無明顯改變；ALA+紅光20min和ALA+紅光30min組細(xì)胞照射后細(xì)胞出現(xiàn)變形,皺縮,死亡。 2.紅光照射時間為20min時,ALA濃度為0.25mmol/l組細(xì)胞與對照組比較細(xì)胞活性無明顯變化(P0.05)；ALA濃度為0.5mmol/1、1.0mmol/l及2.0mmol/l組細(xì)胞增殖活性明顯下降(P＜0.05)。ALA孵育濃度為1.0mmol/l時,紅光照射時間為5min及10min組細(xì)胞與對照組比較增殖活性無明顯變化(P0.05)；紅光照射時間為15min、20min、30min組細(xì)胞增殖活性明顯下降(P0.05)。 3.ALA-紅光-PDT處理成纖維細(xì)胞后,隨著時間延長,LC3和Beclinl mRNA水平逐漸升高,24小時達(dá)到高峰,48小時出現(xiàn)回落(P0.05)；與對照組比較,ALA+紅光組成纖維細(xì)胞LC3和Beclinl mRNA水平明顯升高(P0.05)。 4.與對照組比,ALA+紅光組成纖維細(xì)胞內(nèi)自噬泡明顯增多(P0.05)。 結(jié)論 1.ALA-紅光-PDT在低劑量時對成纖維細(xì)胞的增殖活性無明顯影響,而在相對高劑量是則可使成纖維細(xì)胞增殖活性顯著下降。 2.ALA-紅光-PDT可促進(jìn)成纖維細(xì)胞自噬活性增強(qiáng),這可能是其嫩膚的機(jī)制之一。
[Abstract]:Purpose 1. To observe the effect of ALA-red-light PDT on the morphology and proliferative activity of cultured human skin fibroblasts in vitro, and to explore the appropriate treatment parameters of simulated ALA-red-PDT skin in vitro. 2. To observe the effect of ALA-red light-PDT on autophagy of fibroblasts, and to explore the possible mechanism of ALA-red light-PDT skin rejuvenation. Method The fibroblasts were incubated with 1.5 aminolevulinic acid (ALAA). The fibroblasts were irradiated with red light for different time. The morphological changes of the fibroblasts were observed under optical microscope. 2. Fibroblasts were incubated with different concentrations of ALA and irradiated with different doses. 3. The expression of autophagy related gene LC3 and Beclinl mRNA was detected by polymerase chain reaction (PCR) after the fibroblasts were treated with ALA-red light PDT. 4. The changes of autophagy in fibroblasts treated with ALA-red light PDT were detected by MDC staining. Results 1. There was no obvious change in the morphology of ALA cells in red light group after 10 min irradiation. The cells in ALA red light group and ALA red light group were deformed, shrinked and died after irradiation for 20 min and 30 min respectively. 2. When the red light irradiation time was 20min, there was no significant change in cell activity in the group of 0.25 mmol / L Ala concentration as compared with the control group (P 0.05). The concentration of ALA was 0.5 mmol / 1. The cell proliferation activity of 1.0 mmol / l and 2.0 mmol / l groups was significantly decreased when the concentration of Ala was 1.0 mmol / l. Compared with the control group, the proliferative activity of the cells in the 5 min and 10 min red light irradiation groups showed no significant change (P 0.05). The proliferation activity of red light irradiation group was significantly decreased after irradiation for 15 min or 20 min or 30 min (P 0.05). 3. After the fibroblasts were treated with ALA-red light PDT, the levels of LC3 and Beclinl mRNA increased gradually and reached the peak at 24 hours. After 48 hours, there was a drop of P0.05A; Compared with the control group, the levels of LC3 and Beclinl mRNA in Ala red fiber cells were significantly higher than those in the control group (P 0.05). 4.Compared with the control group, the number of autophagic vesicles in Ala red light cells increased significantly (P 0.05). Conclusion 1. ALA-red light PDT had no significant effect on the proliferation of fibroblasts at low dose, but decreased the proliferation activity of fibroblasts at relatively high dose. 2. Ala-red light-PDT can promote the enhancement of autophagy activity of fibroblasts, which may be one of the mechanisms of its skin rejuvenation.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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本文編號：1464363