ALA-红光-PDT对成纤维细胞增殖活性及自噬的影响

发布时间：2018-01-26 02:14

  本文关键词： 光动力疗法 自噬 5-氨基酮戊酸 LC3 Beclin1　出处：《中南大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的 1.观察ALA-红光-PDT对体外培养人皮肤成纤维细胞形态及增殖活性的影响,探讨在体外条件下模拟ALA-红光-PDT嫩肤的合适处理参数。 2.观察ALA-红光-PDT对成纤维细胞自噬的影响,初步探讨ALA-红光-PDT嫩肤可能的机制。 方法 1.5-氨基酮戊酸(ALA)孵育成纤维细胞后,予以不同时间的红光照射成纤维细胞,光学显微镜下观察细胞形态学变化。 2.以不同浓度的ALA孵育成纤维细胞后以不同剂量照射成纤维细胞,四甲基偶氮唑盐(MTT)法检测各组细胞增殖活性的变化。 3.ALA-红光-PDT处理成纤维细胞后,聚合酶链反应(PCR)法检测自噬相关基因LC3及Beclinl mRNA的表达情况。 4.ALA-红光-PDT处理成纤维细胞后,MDC染色法检测细胞内自噬泡的变化。 结果 1.ALA+红光10min组细胞照射后形态无明显改变；ALA+红光20min和ALA+红光30min组细胞照射后细胞出现变形,皱缩,死亡。 2.红光照射时间为20min时,ALA浓度为0.25mmol/l组细胞与对照组比较细胞活性无明显变化(P0.05)；ALA浓度为0.5mmol/1、1.0mmol/l及2.0mmol/l组细胞增殖活性明显下降(P＜0.05)。ALA孵育浓度为1.0mmol/l时,红光照射时间为5min及10min组细胞与对照组比较增殖活性无明显变化(P0.05)；红光照射时间为15min、20min、30min组细胞增殖活性明显下降(P0.05)。 3.ALA-红光-PDT处理成纤维细胞后,随着时间延长,LC3和Beclinl mRNA水平逐渐升高,24小时达到高峰,48小时出现回落(P0.05)；与对照组比较,ALA+红光组成纤维细胞LC3和Beclinl mRNA水平明显升高(P0.05)。 4.与对照组比,ALA+红光组成纤维细胞内自噬泡明显增多(P0.05)。 结论 1.ALA-红光-PDT在低剂量时对成纤维细胞的增殖活性无明显影响,而在相对高剂量是则可使成纤维细胞增殖活性显著下降。 2.ALA-红光-PDT可促进成纤维细胞自噬活性增强,这可能是其嫩肤的机制之一。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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本文编号：1464363