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作者 標題 麻煩請生物或英文厲害的人點進來~跪求.....
時間 2014年03月26日 Wed. PM 05:25:58
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Extraction of total RNA and RT-PCR
Total RNA was isolated using the TRI reagent (Molecular Research Center, INC., Cincinnati, OH, USA) according to the manufacturer’s instructions.
總和 核糖核酸(RNA)和反轉錄•聚合酶鏈反應(RT-PCR)的萃取物
根據製造商家的說明用核酸蛋白質三相萃取試劑(TRI試劑)（分子研究中心，股份有限公司，辛辛那提，俄亥俄州，美國）把全部的RNA分離
Reverse transcription was performed using a First-Strand cDNA synthesis kit (Promega, Madison, WI, USA). Total RNA (1 μg) was incubated with oligo (dT)18 primer at 70oC for 5 min and cooled on ice for 5 minAfter addition of the reverse transcription (RT) premix, reac-tion ingredients were incubated at 37oC for 60 min.
使用第一鏈cDNA合成試劑盒(Promega公司, 麥迪森,WI,美國)進行進行反轉錄，全部的RNA（1微克）是培養於寡糖（DT）18引子在70℃進行5分鐘，然後在冰上冷卻5分鐘後加入反轉錄（RT）的預混合料，反應成份培養在37℃進行60分鐘
Reactions were terminated by raising the temperature to 70oC for 15 min.
經由提高溫度70℃進行15分鐘反應停止
The PCR reaction was conducted using i-Taq™ DNA poly¬merase (iNtRON Biotechnology, Korea) with the appropriate sense and antisense primers for TARC, MDC, and β-actin. The primer sequences were as follows: TARC primer sequence (F) 5’-ATG GCC CCA CTG AAG ATG CT-3’, (R) 5’-TGA ACA CCA ACG GTG GAG GT-3’ (351 bp); MDC primer sequence (F) 5’-GCA TGG CTC GCC TAC AGA CT-3’, (R) 5’-GCA GGG AGG GAG GCA GAG GA-3’ (497 bp); β-actin primer sequence (F) 5’-ATG GGT CAG AAG GAT TCC TAT G-3’, (R) 5’-CAG CTC GTA GCT CTT CTC CA-3’ (588 bp).
對TARC，MDC和β-肌動蛋白，使用i-Taq酶™ DNA聚合酶（內含子生物技術，韓國）進行聚合酶鏈鎖反應，與適當的效用和反義引物，引物順序如下：（F）5'-ATG GCC CCA CTG AAG ATG CT-3'，（R）5'-TGA CCA ACA ACG GTG GAG GT-3'（351個基點）; MDC引物順序（F）5'-GCA TGG CTCGCC TAC AGA CT-3'，（R）5'-GCA GGG AGG GAG GCA GAG GA-3'（497 bp）的;β-肌動蛋白的引物順序（F）5'-ATG GGT CAG AAG GAT TCC TAT G-3'，（R）5'-CAG CTC GTA GCT CTT CTC CA-3'（588個基點）。
PCR was performed using a C1000 instrument (Bio-Rad, Hercules, CA, USA). Thermal cycling conditions were set to 94oC for 30 sec, annealing at 55-60oC for 30 sec, and extending at 72oC for 2 min, repeated 30 to 35 times, and followed by incubation at 72oC for 10 min.
聚合酶鏈鎖反應用C1000儀器（Bio-Rad公司，赫拉克勒斯，CA，USA）執行，熱循環條件設定為94℃持續30秒，退火溫度為55-60℃，持續30秒，並在72℃，延長2分鐘，重複30至35次，然後藉由72℃10分鐘隨後培養
The reaction products were visualized by electrophoresis on a 1.2% agarose gel (Promega) and UV light illumination after staining with ethidium bromide.在1.2％瓊脂糖凝膠（Promega公司）和UV光照射之後反應物通過電泳顯現
The relative intensity was ana¬lyzed using Quantity One software, version 4.2.1 (Bio-Rad).
使用維電泳分析軟體，版本4.2.1（Bio-Rad公司）進行了相對的強度分析
Real-time quantitative PCR was performed with a TaqMan® Universal Master Mix II (Applied Biosystems, Piscataway, NJ) with a StepOnePlus™ Real-Time PCR (Applied Biosys¬tems).
同時定量聚合酶鏈鎖反應進行了熒光定量通用預混液II（Applied Biosystems公司，新澤西州Piscataway）與StepOnePlus系統™ 擴增實時熒光定量PCR檢測系統（Applied Biosystems公司）。
Real-time PCR for the relative quantification of target gene copy numbers in relation to β-actin expression was con¬ducted using the following primers and probes: 5’-GTA CCA GAC ATC TGA GG-3’ (forward), 5’-ATT CTT CAC TCT CTT GTT GT-3’ (reverse), and 5’-Fam-TCC AGG GAT GCC ATC GTK TTT-BHQ-1-3’ (Taqman probe) for TARC; 5’-TGG ATC GCC TAC AGA CT-3’ (forward), 5’-GTA ATC ACG GCA GCA GA-3’ (reverse), and 5’-Fam-CTC GTC CTY CTT GCT GTG GCR-BHQ-1-3’ (Taqman probe) for MDC; 5’-CCA ACC GTG AAA AGA TG-3’ (forward), 5’-CGG AGT CCA TCA CAA TG-3’ (reverse), and 5’-Fam-ACC TTC AAC ACC CCA GCC A-BHQ-1-3’ (Taqman probe) for β-actin. Real-time PCR results were expressed using the StepOne™ software (Applied Bio¬systems) that measures amplification of the target and the endogenous control in experimental samples and in a refer¬ence sample.
即時聚合酶鏈鎖反應進行目標基因拷貝數相對於β-肌動蛋白表達的相對定量分析是濃度涵道使用下列引物和探，即時聚合酶鏈鎖反應進行目標基因拷貝數相對於相對定量，使用下面的引物和探針進行β-肌動蛋白的表現: 5'-GTA CCA GAC ATC TGA GG-3'（正向），5'-ATT CTT CAC TCT CTT GTT GT-3'（反向），和5'-FAM-TCC AGG GAT GCC ATC GTK TTT-BHQ-13'（熒光定量探針）用於TARC;5'-TGG GCC ATC TAC AGA CT-3'（正向），5'-GTA ATC ACG GCA GCA GA-3'（反向），和5'-FAM-CTC GTC CTY CTT GCT GTG GCR-BHQ-1-3'（熒光定量探針），用於MDC：5'-CCA ACC GTG AAA AGA TG-3'（正向），5'-CGG AGT CCA TCA CAA TG-3'（反向），和5'-FAM-ACC TTC AAC ACC CCA GCC A-BHQ-1-3'（熒光定量探針）用於β-肌動蛋白。即時聚合酶鏈鎖反應的結果使用StepOne™ 軟體（Applied Biosystems公司），其測量在實驗樣品和參考樣品中的目的和內對照物的擴增
Measurements were normalized using the en¬dogenous control.
測量標準是使用內對照物
SDS-PAGE and western blot analysis HaCaT cells were washed twice with ice-cold PBS and then disrupted in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonident P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM DTT, 1 mM phenylmethyl¬sulfonyl fluoride, 25 μg/ml leupeptin] on ice for 30 min.
十二烷基硫酸鈉聚丙烯醯胺凝膠電泳和免疫印跡分析的HaCaT細胞用冰冷的磷酸緩衝液洗滌兩次，然後在裂解緩衝液中分解[50 mM的三羥甲基氨基甲烷 - 鹽酸（pH值7.5），150受阻mM氯化鈉，1％Nonident P-40，2mM的乙二胺四乙酸，1毫乙二醇四乙酸，1mM的偏釩酸鈉，10mM的氟化鈉，1mM的二硫蘇糖醇，1mM苯甲磺酰氟，25微克/毫升亮肽素]冷卻30分鐘。
Cell lysates were centrifuged at 15,000 rpm for 15 min at 4oC and supernatants were used for western blotting.
細胞溶解物在4℃下以15,000 rpm離心15分鐘和取上清液用於西方墨點法
The total protein concentration of each sample was quantified by the Bio-Rad assay method (Bio-Rad). Extracts containing 30 μg of protein were loaded next to a prestained protein-mass lad¬der (Bio-Rad) on a NuPAGE 4-12% bis-Tris gel (Invitrogen, Carlsbad, CA, USA).
每個樣品的總蛋白質濃度藉由Bio-Rad測定法（Bio-Rad公司）進行定量，含有30微克蛋白萃取液，裝入在旁邊的一個預染蛋白質量途徑（Bio-Rad公司）上的NuPAGE4-12％的Bis-Tris凝膠（Invitrogen公司，卡爾斯巴德，加利福尼亞州，美國）
The proteins were electroblotted onto a polyvinylidene difluoride (PVDF) membrane using an iBlot gel transfer device (Invitrogen).
使用IBLOT凝膠轉印裝置(Invitrogen公司)，將蛋白質電吸到一個聚偏氟乙烯(PVDF)薄膜上
The membrane was blocked with blocking buffer (5% skim milk in TTBS) for 1 hr at room temperature (RT), followed by incubation with primary anti¬bodies (1:1,000) overnight at 4oC. All antibodies were diluted in 1% BSA in TTBS buffer.
在室溫（RT），用膜塞住封閉緩衝液（5％脫脂奶粉的TTBS中）1小時，接著培養主要抗體（1:1,000）持續整夜在4℃，所有抗體稀釋於TTBS緩衝液的1％BSA。After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-primary Ab host IgG diluted 1:5,000 for 1 hr at RT.
清洗後，將膜，與以山葵過氧化酶結合的免疫球蛋白(以1:5000稀釋)一同於室溫下培養1小時
After washing again, the result was visualized with a western blot detection system (iNtRON Biotechnology, Korea) according to the manufacturer’s instructions.
在次洗滌後，其顯示結果是根據製造商的說明用西方墨點法檢測系統（內含子生物技術，韓國）。
Fig. 1. Effects of various flavonoids contained within immature Citrus unshiu on inflammatory chemokines production and cell viability in IFN-γ- and TNF-α-stimulated HaCaT human keratinocytes.
圖1在IFN-γ- 及 TNF-α-刺激HaCaT人類角質形成細胞內，由未成熟的溫州蜜柑內不同的黃酮類化合物對於發炎趨化因子的產生及細胞存活率之影響
(B)HaCaT cells (3.0× 105 cells/ml) were pre-incubated with unsupplemented culture medium for 18 hr. They were then stimulated with IFN-γ (10 ng/ml) and TNF-α (10 ng/ml) for 24 hr in the presence of flavonoids (25, 50 μM). TARC and MDC productions were determined from the culture supernatants by ELISA assay, and cell viability was analyzed by MTT assay. The measurements were made in triplicate. Error bars indicate mean ± S.D.
(B) HaCaT細胞（3.0× 105細胞/毫升）預溫育與未補充培養基中持續18小時，然後將它們在黃酮類（25，50微米）的存在下刺激的IFN-γ（10納克/毫升）和TNF-α（10毫微克/毫升）處理24小時。TARC和MDC產品測定是從培養上清液由ELISA測定，細胞生存力由MTT進行分析，該測定法分成三份，誤差棒表示平均值±標準偏差
HPLC analysis
HPLC分析
HPLC analysis was done to identify the existence of quercetagetin and quercetin in the EtOH extract of immature C. unshiu, used in our previous study (Kang et al., 2011). This assay was conducted by Jeju Technopark (http://bio.jejutp.or.kr/english/, Korea).
HPLC分析是為了確定在未成熟C.蜜柑的乙醇萃取物中槲皮萬壽菊素和槲皮素的存在，在我們以前的研究（Kang等人，2011）中使用，此測定法是由濟州科（http://bio.jejutp.or.kr/engli
shStatistical analysis/，韓國）進行。Quantity One version 4.2.1 and Image-Pro plus version 4.5 software were used to transform images into numerical values. Student’s t-test and two-way analysis of variance were used to determine the statistical significance of differences between experimental and control group values. Data represent the mean ± standard deviation. Null hypotheses of no difference were rejected if p-values were less than .05.
維電泳分析軟體（4.2.1版本）和影像分析軟體版本4.5版本被用於圖像轉換為數值, 學生 t 測試和雙向方差分析用於確定實驗組和對照組的值之間的差異的統計意義，數據代表平均值±標準偏差，如果p值均小於0.05，沒有差異的零假設不被成立
Fig. 2. Effect of quercetagetin on TARC and MDC expressions in HaCaT human keratinocytes. HaCaT cells (5.0× 105 cells/ml) were pre-incubated for 18 hr in unsupplemented culture medium.
圖2. 在HaCaT人類角質形成細胞裡表示槲皮萬壽菊素《Quercetagetin》對TARC和MDC之效用，
HaCaT細胞（5.0 × 105個細胞/毫升）在未補充培養基中預培養18小時，
(A) TARC and MDC productions were measured in the culture supernatant of the cells stimulated with IFN-γ (10 ng/ml) and TNF-α (10 ng/ml) for 24 hr in the presence of quercetagetin (12.5, 25, 50 μM) by an ELISA method. The measurements of TARC and MDC were done in triplicate. Error bars indicate ± S.D. **p<0.01, ***p<0.001.
（A）TARC和MDC的產物，由酶聯免疫吸附法分別測定在培養上清液的細胞刺激與IFN-γ（10毫微克/毫升）和TNF-α（10毫微克/毫升），槲皮萬壽菊素《Quercetagetin》（12.5，25，50微米）持續存在24小時，TARC和MDC的測量進行三重複，誤差棒表示±標準偏差** P<0.01，*** P <0.001。
(B)Cells were pre-treated with quercetagetin at the indicated concentrations for 2 hr (12.5, 25, 50 μM). Cells were then stimulated with IFN-γ (10 ng/ml) and TNF-α (10 ng/ml) in the presence of quercetagetin for 18 hr. The expression levels of TARC, MDC, and β-actin mRNA were examined by real-time RT-PCR in triplicate.
槲皮萬壽菊素對細胞進行預先處理在指定的濃度持續2小時（12.5，25，50μM），然後細胞刺激IFN-γ（10納克/毫升）和TNF-α（10納克/毫升）在槲皮萬壽菊素的存在下持續18小時，由即時逆轉錄聚合酶鏈式反應進行三重複，對TARC，MDC和β-肌動蛋白mRNA的表現水平進行了檢查，
(C)HaCaT cells (3.0× 105 cells/ml) were pre-incubated with unsupplemented culture medium for 18 hr and then they were stimulated with IFN-γ (10 ng/ml) and TNF-α (10 ng/ml) for 24 hr in the presence of quercetagetin (12.5, 25, 50 μM). Cell viability was analyzed by the MTT assay. The measurements were made in triplicate. Error bars indicate mean ± S.D
(C)HaCaT細胞（3.0× 105個細胞/毫升）在未補充培養基中預培養18小時，然後將他們放在有槲皮萬壽菊素的存在下持續24小時，刺激的IFN-γ（10納克/毫升）和TNF-α（10納克/毫升）
RESULTS結果
Effects of various flavonoids abundantly contained in immature C. unshiu on TARC and MDC production in HaCaT human keratinocytes.
在HaCaT人類角質形成細胞裡，各種黃酮類化合物含有大量未成熟C.蜜柑，對TARC和MDC之影響
We reported recently that the EtOH extract of immature Citrus unshiu inhibits IFN-γ and TNF-α-induced inflammatory chemokines, TARC and MDC, in HaCaT keratinocytes.
我們最近的報導，從未成熟的溫州蜜柑用乙醇萃取物，抑制IFN-γ和TNF-α誘導的炎症趨化因子，TARC和MDC，在HaCaT細胞。
麻煩幫我看看哪裡不OK的謝謝大家
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※ 作者: flower113 時間: 2014-03-26 17:25:58
※ 看板: learner 文章推薦值: 0 目前人氣: 0 累積人氣: 448
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