利用共軛焦顯微鏡研究紫外線A對正常及葡萄糖六磷酸脫氫酶缺乏症人類纖維母細胞株造成程式凋亡之分子機制探討

Molecular Mechanism Underlines UV-A Irradiation Induced Apoptosis in Normal and Glucose-6-phosphate Dehydrogenase (G6PD) –deficient Human Skin Fibroblasts: A Confocal Microscopic Study

林旻萱(Min-Shuan Lin)

指導教授 ： 蔡麗玉

高雄醫學大學/健康科學院/生物醫學檢驗學研究所/碩士(2008年)

摘要

葡萄糖六磷酸去氫酶(簡稱G6PD)主要的功能是產生NADPH藉以維持細胞內氧化還原狀態的平衡。文獻報導在G6PD的缺乏會導致人類纖維母細胞的生長延遲以及加速老化。紫外線A (UVA) 對於正常纖維母細胞胞內成分的影響已經源自經由photodynamic action的活性氧(簡稱ROS) 之研究而建立了。然而，經由UVA照射而促成的細胞死亡的細部流程仍然有待釐清。另外，在人類纖維母細胞由於G6PD缺乏對於UVA調控之光化學的衝擊迄今仍未被探索。所以，本論文的研究主旨是擬利用共軛焦雷射掃瞄顯微鏡去探討與UVA調節之光化學ROS引起一系列損傷作用的機轉能在快速之下，目睹單細胞之變化情形。因此，我們利用了兩種對於UVA誘發效應有不同敏感性的G6PD正常及缺乏的纖維母細胞來做研究模式。首先，我們明確地以DCF-DA探針，證明了單細胞產生ROS之量是與UVA劑量(25至100 KJ/cm2) 之照射成正比。同時，我們亦以電腦軟體計算ROS產量分佈在不同胞器的情形。由此結果證實，G6PD缺乏之細胞產生粒線體ROS(mROS)之量比正常細胞要強度超越甚多。顯然這些結果UVA對G6PD缺乏細胞所造成mROS之較為敏感。接續下來，我們針對mROS對兩株在細胞下游損傷之過程做詳細的研究。首先，我們要證實兩株細胞經UVA誘發之mROS ，會造成胞內谷胱甘肽的耗損(GSH depletion)以及脂質過氧化作用(lipid peroxidation)。的確，我們發現mROS可以造成兩株細胞胞內谷胱甘肽之耗損以及脂質過氧化作用。此結果明顯證明粒線體胞內之GSH容易被耗損因為此胞器無法合成GSH。故當粒線體胞內之ROS增加，對GSH定會耗損，因其量在胞內的存量是固定的。同時，當我們比較兩株細胞GSH之耗損，G6PD缺乏細胞確實比正常細胞嚴重甚多，同樣地，G6PD缺乏細胞胞膜過氧化作用之嚴重度亦較正常細胞為甚。接續下來，我們更進一步的探討GSH之耗損以及胞膜過氧化作用是否會造成細胞質以及其他胞器，包括粒線體之鈣質的恒定作用(homeostasis)有影響。利用Fluo4(測胞質鈣質之探針)以及Rhod-2(測粒線體鈣質之探針)兩種探針，我們證實了，UVA(50 KJ/cm2)的確會造成胞質鈣之恆定作用改變(胞質鈣增加現象)。接著我們又觀察到粒線體鈣有過度堆積的現象(calcium overload)。同時，這個現象也造成粒線體之形態大大改變(由條狀變為圓形)。G6PD缺乏細胞對UVA造成鈣離子之堆積程度較正常細胞嚴重甚多，這是可以預期到的結果。最後，我們以TUNEL方法證實兩株細胞受UVA照射死亡是以程式凋亡(apoptosis)之型式。而G6PD缺乏細胞則對UVA照射程式凋亡之程度較正常細胞嚴重(相差兩倍之多)。總結之，G6PD缺乏(蠶豆症患者)之纖維母細胞對UVA之耐受性非常差，故本研究提供寶貴之資訊並証明蠶豆症患者必須嚴防陽光之曝曬，否則容易造成不可逆的皮膚老化。同時，本實驗並提出一項值得研究的課題，篩選抗UVA之天然物以防UVA照射傷害，是刻不容緩的事。本實驗室亦正努力從事這方面的工作中。

關鍵字

葡萄糖六磷酸去氫酶紫外線A

並列摘要

Glucose-6-phosphate dehydrogenase (G6PD) is involved in the generation of NADPH for the proper maintenance of the cellular redox balance. G6PD deficiency predispose human fibroblasts (HFF) to retarded growth and accelerated cellular senescence. In addition, human dermal fibroblasts are subjected to deeply penetrating UVA irradiation. The effects of UVA on cell components of normal fibroblasts have been documented to result from a photodynamic action producing reactive oxygen species (ROS) that can indirectly affect different cellular targets. However, the detailed cascades of events leading to the final demise of cell by UVA-irradiation remain elusive. In addition, the impact of G6PD deficiency on UVA-mediated photodynamic action of human fibroblasts has never been explored. The objective of this study is to explore the possible underlying mechanism(s) in relation to UVA-mediated ROS-evoked cascade of events using laser scanning confocal microscopic technique (LSCMT). The potential disparity in the susceptibility to UVA-induced photodynamic effect between the normal and G6PD-deficient human skin fibroblasts will also be investigated. Using normal human (HFF3) and G6PD-deficient (HFF1) as experimental cell models, we first demonstrate unequivocally the rapid production of ROS in a dose-dependent manner, as reflected by the green fluorescence intensity of DCF when these cells were exposed to various fluences of UVA irradiation (25 to 100 KJ/cm2). Furthermore, ROS production in various cellular compartments was much more extensive for HFF1 than HFF3 cells. Accumulation of mitochondrial ROS(mROS) was appeared much quicker for HFF1 than HFF3 cells. Subsequently, we showed that UVA irradiation could induce intracellular GSH depletion as well as lipid peroxidation in both types of cells dose-dependently.
Next, we looked at the possibility if UVA irradiation of both types of cells alter the mitochondrial calcium homeostasis. Using Rhod 2 as the probe, we could demonstrate that indeed UVA irradiation on both types of cells could result in mitochondrial calcium (mCa+2) overload. From the confocal image, we could vividly see the morphological changes of mitochondrion from rod-like shape to ball-like configuration in HFF1 cells indicating that the degrees of mCa2+ overload was much more extensive than HFF3 cells. Finally, we demonstrated that UVA irradiation-mediated cell death for both types of cells was apoptotic in nature. Approximately two-fold increase of TUNEL-positive cells was observed in HFF1 than HFF3 cells when exposed to a dose of UV irradiation (50 KJ/cm2). Collectively, our data indicate clearly that fibroblasts devoid of G6PD are imparted with weaker tolerance to UVA irradiation than normal fibroblast. It is suggested that persons with G6PD deficiency should avoid exposure to solar light in order to prevent severe oxidative damages to the skin.

並列關鍵字

G6PD ； UVA

參考文獻

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