强脉冲光照射对培养人皮肤成纤维细胞的影响

发布时间：2018-05-18 05:07

本文选题：强脉冲光 + 长波紫外线　；参考：《第四军医大学》2011年博士论文

【摘要】：皮肤老化分为自然老化和光老化。后者主要由日光中的紫外线照射引起,特别是长波紫外线UVA的照射。对于光老化皮肤的治疗经历了两个阶段:有创性治疗和无创性治疗,由于前者副作用较多,故其应用逐渐减少。强脉冲光(Intense Pulsed Light IPL)技术是近年来产生的一种治疗光老化皮肤的新的无创性方法,在临床研究中己证实能有效地改善光老化皮肤的许多症状,所以在临床得以普遍应用。在强脉冲光照射对皮肤光老化的研究方面,虽然取得了一定的成绩,但也存在分歧,并且以前的研究都是在正常组织和细胞上进行。正常细胞与光老化细胞的情况相差甚远。在细胞生物学研究方面,目前文献上还未见关于强脉冲光照射对细胞端粒影响的报道。本实验旨在用UVA作为对照,观察单次IPL照射对人皮肤成纤维细胞增殖活性和培养细胞上清液中MMP-1的表达情况。多次IPL照射,对细胞老化指标的影响。IPL照射对PUVA诱导的老化成纤维细胞的影响。从而更好的理解光老化的分子机制和IPL光子嫩肤机制。实验一:细胞分成7组,没有任何照射的一组作为对照组,三组分别接受IPL5J/cm~2,15J/cm~2和20J/cm~2照射,其它三组接受UVA9J/cm~2,11J/cm~2和13J/cm~2照射。观察不同剂量IPL和UVA照射后第二天细胞的形态和细胞的增殖活性,细胞上清液中MMP-1的表达水平。结果发现,IPL照射后,细胞形态未见明显改变,细胞增殖活性明显增加,但不存在剂量依赖性,以临床常用剂量15J/cm~2组细胞活性最高。UVA照射后,细胞变圆,皱缩,部分死亡,细胞活性下降,并且有剂量依赖性。UVA9J/cm~2照射后,细胞活性大约在80%。IPL照射后,MMP-1的分泌水平下降,以IPL15J/cm~2组下降最明显。UVA照射后,MMP-1的分泌水平上升,但不存在剂量依赖性,以UVA9J/cm~2照射后,水平最高。我们接着把细胞分成四组:即对照组, UVA9J/cm~2照射组, IPL15J/cm~2照射组和UVA9J/cm~2+IPL15J/cm~2照射组(成纤维细胞接受9J/cm~2UVA照射后随即给予15J/cm~2IPL照射),观察IPL照射能否降低UVA导致的MMP-1的上升。结果发现UVA照射后再进行IPL照射,MMP-1的水平回复到接近正常水平。实验二:细胞分成三组,一组未接受照射作为对照组,其它两组分别接受IPL15J/cm~2和UVA9J/cm~2照射,每天一次,连续照射5天。在第6天,收集细胞,分别进行β-半乳糖苷酶染色,细胞周期,细胞内活性氧和端粒长度的测定。结果发现:连续5天的IPL照射后,β-半乳糖苷酶染色和端粒长度与正常对照相比,未见明显变化(p0.05)。细胞周期中G1期所占百分比下降,细胞内活性氧下降(p0.05);连续5次的UVA照射后,与正常对照相比,β-半乳糖苷酶染色阳性细胞的百分数增加,细胞周期中G1期所占百分比未见明显变化,细胞内活性氧上升和端粒长度缩短(p0.05)。实验三:细胞分成三组:一组用非毒性的8-甲氧补骨脂素孵育细胞16-18小时,然后用UVA9J/cm~2照射(PUVA组),一组PUVA处理后,随即进行IPL15J/cm~2照射,以后每天进行一次,连续5天,即PUVA+IPL组,同时设正常对照组,除每天更换培养液后,未给予任何处理。在细胞处理后1,3,5天后用MTT法检测细胞活性。在处理5天后,收集细胞进行β-半乳糖苷酶染色,细胞内活性氧和端粒长度的测定。结果发现:和对照组细胞相比,PUVA组和PUVA+IPL组β-半乳糖苷酶活性明显增加。PUVA+IPL组β-半乳糖苷酶阳性成纤维细胞数明显低于PUVA组(p0.05)。与对照组细胞活性相比,PUVA组和PUVA+IPL组的细胞活性呈时间依赖性下降,并且两组之间未见明显差别。与对照组细胞相比,PUVA处理组端粒长度缩短,细胞内活性氧水平增加(p0.05),但与PUVA处理组相比,在PUVA处理后随即给予多次的IPL照射可阻止端粒缩短和降低细胞内活性氧水平(p0.05)。结论: (1) IPL和UVA单次照射人皮肤成纤维细胞后,细胞形态,细胞活性和MMP-1的表达不同。UVA和IPL的联合照射提示IPL可缓解UVA诱导的MMP-1的上调,因此降低细胞外基质蛋白的破坏作用。 (2)多次的UVA照射可诱导细胞出现老化表现,如β-半乳糖苷酶活性增加,细胞内活性氧增加,端粒长度缩短等。但多次IPL照射不会诱导细胞老化。 (3) PUVA处理可诱导培养的成纤维细胞出现细胞老化。但IPL照射通过降低活性氧和阻止端粒缩短而保护PUVA诱导的细胞老化。
[Abstract]:Skin aging is divided into natural aging and photoaging . The latter is mainly caused by ultraviolet irradiation in sunlight , especially the irradiation of long wave ultraviolet UVA . The treatment of photoaging skin has undergone two stages : invasive treatment and non - invasive treatment .

The purpose of this study was to observe the effect of single IPL irradiation on the proliferation activity of human skin fibroblasts and the expression of MMP - 1 in cultured cells by using UVA as control . The effects of IPL irradiation on the aging index of PUVA were observed . The molecular mechanism of photoaging and IPL photon rejuvenation mechanism were better understood .

The expression level of MMP - 1 and MMP - 1 were observed after irradiation with UVA9J / cm ~ 2 . After irradiation with UVA9J / cm ~ 2 , the activity of MMP - 1 increased , and the level of MMP - 1 was highest after irradiation with UVA9J / cm ~ 2 .

The results showed that the percentage of 尾 - galactosidase positive cells increased and the percentage of 尾 - galactosidase positive cells increased and the percentage of G1 phase in the cell cycle was not changed significantly after 5 consecutive days of UVA irradiation .

The activity of 尾 - galactosidase positive in PUVA group and PUVA + IPL group was significantly higher than that in control group ( P < 0.05 ) .

Conclusion :

( 1 ) After single exposure of IPL and UVA to human skin fibroblasts , the cell morphology , cell activity and expression of MMP - 1 were different . The combination of UVA and IPL suggested that IPL could alleviate the up - regulation of UVA - induced MMP - 1 , thus reducing the destruction of extracellular matrix protein .

( 2 ) Multiple doses of UVA irradiation could induce aging of cells , such as increased 尾 - galactosidase activity , increased intracellular reactive oxygen , shortened telomeric length , etc . However , multiple IPL irradiation did not induce cell aging .

( 3 ) PUVA treatment can induce cell aging in cultured fibroblasts , but IPL irradiation protects PUVA - induced cell aging by reducing reactive oxygen and preventing shortening of telomeric shortening .
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R751

【参考文献】