JP2014163749A5 - - Google Patents

Description

（１）HaCaT細胞培養および過酸化水素（H₂O₂）処理による酸化ストレスの負荷ならびに siRNA処理によるＤＪ−１遺伝子のノックダウン操作
HaCaT細胞をPBS(-)にて洗浄後、トリプシン処理により細胞を剥離し、1200rpmで3min遠心し、細胞を得た。培養は10%FBS含有DMEM培地を用い、5000cells/cm²の密度でフラスコに播種し、2〜3日置きにサブコンフルエントの状態で継代した。
HaCaT細胞を10%FBS含有DMEM培地にて12ウェルプレートに2.1×10⁵cells / well播種し24時間培養後、siRNA（終濃度3nM、Hs_PARK7_6 FlexiTube siRNA, SI02662107, Qiagen社製）、トランスフェクション試薬（2μl/well、HiPerFect Transfection Reagent, 301707, Qiagen社製）を前記培養培地と混合し、室温に10分放置した。ついでこの混合液を、100μl/well添加した。未処理の細胞をコントロールとした。
また、ノックダウン操作のネガティブコントロールとして、AllStars Negative Control siRNA (Qiagen社製)を用いて同様に操作した。
48時間培養後に、H₂O₂ (Wako製)を0、250、500、750、1000μM添加し、24時間後に培養上清を回収し、細胞をPBS(-)で洗浄後、Cell lysis buffer (50mM Tris-HCl , 1mM Na₃VO₄ , 0.4% NP-40 , 120mM NaCl)を100μl/well添加し、細胞溶解液を得た。 (1) Load of oxidative stress by HaCaT cell culture and hydrogen peroxide (H ₂ O ₂ ) treatment and knockdown operation of DJ-1 gene by siRNA treatment
After washing HaCaT cells with PBS (−), the cells were detached by trypsin treatment and centrifuged at 1200 rpm for 3 min to obtain cells. The culture was carried out using a DMEM medium containing 10% FBS, seeded in a flask at a density of 5000 cells / cm ² , and subcultured every ² to 3 days.
HaCaT cells were seeded in DMEM medium containing 10% FBS in a 12-well plate at 2.1 × 10 ⁵ cells / well, cultured for 24 hours, siRNA (final concentration 3 nM, Hs_PARK7_6 FlexiTube siRNA, SI02662107, manufactured by Qiagen), transfection reagent ( 2 μl / well, HiPerFect Transfection Reagent, 301707, manufactured by Qiagen) was mixed with the culture medium and allowed to stand at room temperature for 10 minutes. Then, 100 μl / wel l of this mixed solution was added. Untreated cells served as controls.
In addition, the same operation was performed using AllStars Negative Control siRNA (manufactured by Qiagen) as a negative control for the knockdown operation.
After culturing for 48 hours, H ₂ O ₂ (manufactured by Wako) was added at 0, 250, 500, 750, and 1000 μM. After 24 hours, the culture supernatant was collected, and the cells were washed with PBS (−), then the Cell lysis buffer ( 50 mM Tris-HCl, 1 mM Na ₃ VO ₄ , 0.4% NP-40, 120 mM NaCl) was added at 100 μl / well to obtain a cell lysate.

＜角層中ＤＪ−１測定＞
ガラスビーズとT-PERバッファー（Thermo scientific社）500μlの入ったチューブに角層を採取した角層チェッカーを入れ、25分ボルテックスミキサーにて振とうし、角層タンパクを抽出した。各サンプルのタンパク量はBCA protein Assay Kit (Thermo Scientific社)で測定した。測定には角層サンプルを10μlに reagentA: reagentB=50:1で混和した液200μlを加え、60℃30分でインキュベーションしたのち、562nmの吸光度で測定した。同時にウシ血清アルブミン（BSA）で検量線を作成した。この検量線と吸光度の値からタンパク量を算出した。
角層抽出液中に含まれるＤＪ−１量はHuman Park7/ＤＪ−１ DuoSet (R&D systems社)を用いて定量した。 <DJ-1 measurement in stratum corneum>
The stratum corneum checker which collected the stratum corneum was put into a tube containing glass beads and 500 μl of T-PER buffer (Thermo scientific), and shaken with a vortex mixer for 25 minutes to extract stratum corneum protein. The amount of protein in each sample was measured with BCA protein Assay Kit (Thermo Scientific). For the measurement, 200 μl of a stratum corneum sample mixed with 10 μl of reagent A : reagent B = 50: 1 was added, incubated at 60 ° C. for 30 minutes, and then measured by absorbance at 562 nm. At the same time, a calibration curve was prepared with bovine serum albumin (BSA). The protein amount was calculated from the calibration curve and the absorbance value.
The amount of DJ-1 contained in the stratum corneum extract was quantified using Human Park7 / DJ-1 DuoSet (R & D systems).