探討乳酸對人類皮膚角質細胞株(HaCaT)誘發細胞凋亡及細胞週期停滯的影響

Effects of Lactic Acid on the Induction of Apoptosis and Cell Cycle Arrest in Human Immortalized Keratinocyte Cell Line (HaCaT)

黃芯蘭(Hsin-Lien Huang)

指導教授 ： 楊仁宏 ； 鍾景光

中山醫學大學/醫學院/醫學研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00098

摘要

Alpha-hydroxyacids (AHAs) 成分近年來很廣泛的被使用於皮膚保養產品以及醫學美容上。然而，長期使用該成份的安全性尚未獲得解答。最近，化妝品專題研究指出含有AHAs 成分產品會造成使用者對陽光產生光敏感的現象以及皮膚刺激性。故在本篇研究中，我們想去調查乳酸是否會對人類皮膚角質細胞(HaCaT)產生細胞毒性作用。實驗結果指出，乳酸對人類皮膚角質細胞(HaCaT)產生細胞毒性作用其機轉為經由細胞週期的停滯及誘導細胞凋亡。結果指出，乳酸不僅是呈現劑量－依賴性的模式來抑制HaCaT 細胞增生，同時也引起細胞週期停滯在G1/S 期以及誘導細胞產生凋亡。我們也觀察到細胞型態上的改變，並利用DAPI 染色、彗星試驗偵測DNA 受損情
形，以及凝膠電泳偵測是否有產生細胞凋亡。流式細胞儀結果指
出，乳酸是經由粒線體膜電位下降，進而使粒線體釋放出AIF、Endo
G 及cytochrome c 到細胞質中。此外，乳酸也可能經由增加Bax
的表現及抑制Bcl-2及Bcl-xL的表現並活化了caspase 9 及caspase 3隨後啟動了caspase-依賴性及caspase-非依賴性的凋亡路徑。乳酸促使粒線體膜電位下降及活性氧的增加可能和細胞質鈣離子增加有
關。西方墨點法的結果指出，乳酸會增加Chk2、P21、P27 蛋白的
表現，以及降低cyclin E-CDK2、cyclin A-CDK2 複合體的表現而造
成細胞週期停滯在G1/S 期。此外，乳酸也能經由活化其他死亡接受
器活化caspase 8 並使Bid 活化。因此，乳酸可經由粒線體路徑及活化死亡接受器路經來誘發角質細胞的凋亡。

關鍵字

乳酸 ； 細胞凋亡 ； 細胞週期

並列摘要

Alpha-hydroxyacids (AHAs) have been widely used in skin care products and cosmetic dermatology in recent years. However, there is concern about its safety for long-term use. Recently, it has been found that AHA-containing products may induce photosensitivity and skin irritation .
In this study, we investigated the cytotoxic effect of lactic acid on human keratinocyte cell line(HaCaT). Our data suggested that lactic acid is cytotoxic to HaCaT cells via the mechanisms of cell cycle arrest and
induction of apoptosis. The results indicated that lactic acid not only inhibited proliferation of HaCaT cells in dose-dependent , but also induced cell cycle arrest at G1/S phase and apoptosis on HaCaT cells. We observed cell morphological changes, and also used DAPI stain, comet
assay, and agarose gel electrophoresis to investigate DNA damage. The results of flow cytometer showed lactic acid induced HaCaT cell apoptosis through mitochondrial membrane potential (MMP) decrease and AIF, Endo G and cytochrome c release from mitochondria to the cytosol. In addition, lactic acid increased the level of Bax and inhibited the level of Bcl-2, Bcl-xL and activate caspase 9 and caspase 3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathway. We also found that the decrease of MMP and the increase of reactive oxygen species (ROS) may be related to the increase of cytoplasmic calcium level. Western blotting results presented lactic acid increase the levels of Chk2, P21, P27 and decreased the levels of cyclin E-CDK2, cyclin A-CDK2 complexes that cause cell cycle arrest at
G1/S phase. Lactic acid also increased the levels of caspase 8 maybe other death receptor expression and activate Bid protein. Therefore, the lactic acid could induce apoptosis through mitochondrial pathway and death receptor pathway in human kerationocyte cell line (HaCaT).

並列關鍵字

lactic acid ； apoptosis ； cell cycle

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