Investigation Of Skin Photoaging Mechanisms-Influences Of Ultraviolet Irradiation On The Telomere Length Of Chromosome In Primary Cultured Human Fibroblast

Skin aging includes intrinsic aging and photoaging. Aging can be induced by multiple mechanisms.Telomeres are the ends of linear chromosomes, several thousand bases in length that consist of tandem repetitive DNA sequences. The 3′strand has in mammals the sequence (TTAGGG)n,and forms special structure in space. Age-associated changes in pattern of gene expression and cellular proliferate capacity appear largely under the control of the telomeres. Telomere has tight association with skin aging, cancer and gene regulation, as demonstrated in the studies of skin aging in literature.The function of the telomeres is largely depend on the length of the telomere and its'normal structure. Telomere length in cells obtained directly from tissues in vivo is inversely related to the individual's physiologic age, being shorter in cells derived from older versus younger adults; telomere will lost about tens to hundreds base pair with every cell passage. When telomeres become"critically short", cells enter proliferate senescence. Furthermore, telomere length may influence the expression of age-related genes just proximal to it, suggesting a modulation effect on mechanism of skin aging.UV is the primary factor to cause the skin photoaging. UV irradiation can activate the transduction pathway of the cells directly and indirectly; it can also inhibits the synthesis of the collagen. Some verified information has shown that intrinsic aging and photoaging have the same basic molecular transduction pathway. Whether UV irradiation can affect the length of the telomere and lead to photoaging is a new subject that worth to explore.This experiment took the primary human fibroblast as object and made use of real-time PCR to approach the mechanisms of the UV caused shorten of the telomere, in order to further develop the theory of the phothoaging and provide a new target for the photoaging. The following works have been done in this research:1. The effect of the UV irradiation on proliferation of the fibroblast:Cells were irradiated by different doses of UV (290nm-400nm) and then the proliferations were detected by the WST-8. We found that the higher of the dose the lower of the proliferation of the cells.2. Constructing the condition of the telomere amplification and verifyingthe telomere length dynamic of the passage cell: We explored and improved the condition of real-time PCR, then established the standard curve of the telomere and the controlled single copy gene; and we also verified that the telomere length of the cell shortens with each cell passage.3. The effect of the UV irradiation on the fibroblast:3.1 Cells were irradiated by smaller dose (500 and 1000mJ/cm2) of UV and passage on. The DNA of the cell was extracted and then real-time PCR were performed. The result shows that the telomere length of the UV-treated group was shorter than the untreated cells but not significantly in statistics.3.2 The same passage cells were treated by several dose of UV and made a compare of the telomere length between the un-treated and treated group. We found that the telomere length of the high dose (10000mJ/cm2) of UV group were significant shorter than the un-treated group (p<0.05).This experiment has amplified the telomere successfully by the method of real-time PCR and verified that the telomere shortens with the cell passage; from the study we also showed that UV irradiation can shorten the telomere, especially the high one, which may present a clue to cell photoaging and help to understand the intrinsic mechanism of the skin photoaging.