【作者】 王欢；

【导师】 李迎新；

【作者基本信息】 天津医科大学 ， 生物医学工程， 2007， 硕士

【摘要】 目的：利用激光的生物刺激效应研究低强度592 nm和630 nm激光对成年人及老年人皮肤成纤维细胞的增殖活性的作用,以及对前胶原基因和细胞内信号转导分子SMAD基因表达的影响,为低强度592 nm和630 nm激光用于改善皮肤老化提供细胞学依据和可供参考的治疗参数。方法：1.用体外培养的成年人和老年人真皮成纤维细胞作为细胞模型,分别以波长592 nm和630 nm的连续激光对细胞进行照射,照射功率为10 mw和30mw,功率密度为3.5mw/cm2和10.6mw/cm2,分别非重叠照射2 min、5 min和10 min,连续照射3 d,用于检测细胞增殖活性；功率密度为3.5 mw/cm2和10.6mw/cm2,分别非重叠照射5min和10min,连续照射3 d,用于检测前胶原基因和SMADs mRNA表达。2.激光照射前以及每次照射后24 h,用相差显微镜对细胞照相,比较细胞形态、数量的变化。3.用MTT比色法检测细胞增殖活性。4.应用RT-PCR技术检测细胞前胶原Ⅰ型α1、Ⅰ型α2、Ⅲα1以及SMADs mRNA的表达,用Bandscan5.0凝胶成像系统分析软件测定PCR产物电泳条带的光密度值。5.应用SPSS11.5软件进行统计分析。结果：1.592 nm和630 nm激光照射细胞后,增殖活性增强,在不同功率密度之间差异具有显著性统计学意义(p<0.05),细胞增殖活性由高到低依次为3.5mw/cm2水平、10.6 mw/cm2水平和未照射水平即对照组。2.细胞增殖活性在不同的照射时间之间差异具有显著性统计学意义(p<0.05),细胞增殖活性由高到低依次为照射5 min水平、照射2 min水平、照射10 min水平和未照射水平即对照组。3.592 nm和630 nm激光照射后,功率密度为3.5 mw/cm2、分别照射5 min和10 min时,与对照组相比,Ⅰ、Ⅲ型前胶原和SMAD2、3的mRNA表达增强,差异具有显著性统计学意义(p<0.05),基因表达依次为照射5 min水平、10 min水平和未照射水平即对照组。4.592 nm和630 nm激光照射后,功率密度为10.6 mw/cm2、照射5 min时,与对照组相比,Ⅰ、Ⅲ型前胶原和SMAD2、3的mRNA表达增强,差异具有显著性统计学意义(p<0.05)；在照射10min时与对照组相比,Ⅰ、Ⅲ型前胶原和SMAD2.3的mRNA表达增强不具有显著性统计学意义(p>0.05)。结论：1.低强度592 nm和630 nm激光照射成人和老年人真皮成纤维细胞,能够刺激细胞增殖和前胶原基因的表达,并且随照射功率密度和时间而改变,较低的功率密度(3.5 mw／cm2)对细胞的刺激作用比较高的功率密度(10.6mw／cm2)强,较短的照射时间(5 min)对细胞的刺激作用比较长照射的时间(10min)强。2.照射后细胞内信号转导分子SMADs的mRNA表达增强,提示通过SMAD转导信号的细胞因子TGF-β升高,低强度激光促进成纤维细胞自分泌TGF-β。3.由于592 nm和630 nm的激光能够穿透表皮到达真皮,低强度592nm和630 nm激光对体外培养的真皮成纤维细胞模型能够产生生物刺激效应,为592 nm和630 nm激光改善皮肤老化提供细胞学依据和可供参考的治疗参数,还需结合动物试验和临床试验验证。4.630 nm激光照射的细胞模型源自老年人皮肤,因此该结果对于低强度630 nm激光用于临床上改善皮肤老化意义较大,还需进一步进行在体研究加以证实。更多还原

【Abstract】 Objects:To detect the biostimulation effects of Low Power Laser at wavelengths of 590 nm and 630 nm on proliferation and procollagen gene expression of human skin fibroblasts, as well as SMADs gene expression, so as to provide theoretical evidence and possible parameters for clinical improvement of Low Power Laser therapy in aging akin at wavelengths of 590 nm and 630 nm.Methods:1. Select cultured adult and aging skin fibroblasts as cell model. To test the stimulation effects on cell proliferation, cultured fibroblasts were treated in a controlled manner during three consecutive days, either with a yellow laser or with a red laser source emitting two wavelengths(592 nm,630 nm) and two level power density outputs(3.5mw/cm2,10.6mw/cm2).Treatment duration including 2min,5min and 10min varied in relation to varying surface power densities (radiant exposures). To test gene expression, cultured fibroblasts were treated in a controlled manner during three consecutive days, either with a yellow laser or with a red laser source emitting two wavelengths(592nm,630nm) and two level power density outputs(3.5 mw/cm2,10.6 mw/cm2). Treatment duration including 5min and 10min varied in relation to varying surface power densities (radiant exposures).2. Each time before and after 24 h of irradiation, cells were photographed using a phase-contrast microscope, to observe the variation of cell morphous and viabilities.3. Using MTT method to test cell proliferation status.4. Utilizing RT-PCR technology to detect gene expressions of procollagen typeⅠ-α1,Ⅰ-α2, typeⅢ-α2 and SMAD2,3. Measure the optical density values with an analysis software Bandscan5.0 of each PCR product after electrophoresis.5. Utilizing SPSS11.5 statistical analysis software to analyze data. Results:1.Statistical analysis revealed a higher rate of proliferation (p<0.005) in all irradiated cultures in comparison with the controls. Low power density of both 592 nm and 630 nm (3.5mw/cm2) lasers yielded a significantly higher number of cells, than high power density (10.6 mw/cm2) lasers (p<0.005).2. Statistical analysis revealed a higher rate of proliferation (p<0.005) in all irradiated cultures in comparison with the controls.5 minutes’ irradiation of both 592 nm and 630 nm lasers yielded a significantly higher number of cells, than 2 minutes’ irradiation and 10 minutes’by turns.3. Statistical analysis revealed a higher rate of gene express (p<0.005) in those irradiated cultures at low power density (3.5mw/cm2) in comparison with the controls. Both 592 nm and 630 nm lasers yielded a significantly higher expression of procollagen typeⅠ-α1,Ⅰ-α2, typeⅢ-α2 and SMAD2,3, than high power density(10.6mw/cm2) lasers.5 minutes’ irradiation of both 592 nm and 630 nm (3.5 mw/cm2) lasers yielded a significantly higher gene expression thanlO minutes’ irradiation.4. Statistical analysis revealed a higher rate of gene expression (p<0.005) in those irradiated cultures under high power density (10.6 mw/cm2) in comparison with the controls. High power density (10.6mw/cm2) of both 592 nm and 630 nm lasers yielded a significantly higher expression of gene after irradiation 5 min than 10 min. There is no statistical significance (p>0.05) of gene expression increase in comparison with the controls when irradiated for 10 min at high power density (10.6mw/cm2) lasers with wavelengths of 592 nm and 630 nm.Conclusions:1.590 nm and 630 nm low power lasers irradiation resulted in an increased human fibroblast proliferation in vitro, and the trend varied between different power densities and irradiation duration times. Low power density (3.5mw/cm2) lasers yielded higher proliferation rate than high power density (10.6mw/cm2) lasers. Shorter irradiation duration time (5min) lead to higher proliferation rate than longer irradiation duration time (10min).2. The increase of gene expression of SMADs suggesting that the secretions of TGF-βby fibroblasts were increased.3. Base on the evidence of stimulated proliferation and gene expression and their penetration depth of each wavelength at 592 nm and 630 nm, this study therefore postulates possible improvements on wrinkle in vivo at the applied dosimetric parameters.4. Since the fibroblasts irradiated by 630nm laser were derived from aging skin, there is greater clinical significance based on the result of this study.更多还原