中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2013年
7期
496-500
,共5页
人参皂甙%紫外线%环丁烷嘧啶二聚体%角蛋白细胞
人參皂甙%紫外線%環丁烷嘧啶二聚體%角蛋白細胞
인삼조대%자외선%배정완밀정이취체%각단백세포
GINSENOSIDE%Ultraviolet rays%Cyclobutane pyrimidine dimers%Keratinocytes
目的 观察人参皂苷Rb1对中波紫外线(UVB)照射诱导小鼠表皮细胞、培养的HaCaT细胞产生与清除环丁烷嘧啶二聚体(CPD)的影响及对核苷酸切除修复蛋白XPC、ERCC1表达的影响.方法 42只去毛BALB/c小鼠分为4组:未处理组(6只),UVB组(12只),UVB+小剂量Rb1组(12只),UVB+大剂量Rb1组(12只).后2组照射前2h在背部按100 μl/cm2分别外用含0.5 g/L、2g/L人参皂苷Rb1的丙酮溶液,而前2组予以相应丙酮溶液.UVB剂量均为180 mJ/cm2,于照射后0.5、16 h分别处死半数小鼠,利用免疫组化法检测表皮CPD水平.培养的HaCaT细胞用含人参皂苷Rb1(5、20、50 mg/L)的培养基孵育4h,部分细胞于UVB照射(15、30 mJ/cm2)后0.5、12 h终止培养并提取基因组DNA,通过斑点杂交法检测CPD.其余细胞照射(30 mJ/cm2)后0、0.5、2、4、12h终止培养,提取细胞总蛋白,通过免疫印迹法分析XPC、ERCC1蛋白的表达.结果 UVB照射小鼠0.5 h后,各照光组表皮均产生大量CPD,但组间差异无统计学意义；在照射后16h,UVB+小剂量Rb1组、UVB+大剂量Rb1组表皮CPD分别为32.1±8.5、14.6±4.1,均较UVB组(67.3±11.2)显著减少,且大剂量组更为明显(P值均＜0.01).HaCaT细胞在UVB照射后0.5 h,各组CPD总量差异无统计学意义,但照射后12h,给药组CPD水平明显降低.UVB组HaCaT细胞XPC、ERCC1蛋白的表达均随着时间延长不断下降,在照射后即刻、0.5、2、4、12 h,XPC/GAPDH蛋白灰度比值分别为0.68±0.11、0.47±0.09(与照射后即刻比较,P＜0.05,下同)、0.45±0.08 (P＜0.05)、0.37±0.06(P＜0.01)、0.18±0.03(P＜0.01),ERCC 1/GAPDH分别为0.28±0.03、0.25±0.03 (P＞ 0.05)、0.21±0.02 (P＜0.05)、0.14±0.02 (P＜0.01)、0.11±0.01(P＜0.01);加入50 mg/L Rb1干预后,XPC、ERCC1蛋白的表达不断增加,XPC/GAPDH分别为0.56±0.07、0.48±0.14、0.68±0.15、0.97±0.20(P＜0.01)、0.79±0.12(P＜0.05),ERCC 1/GAPDH分别为0.27±0.04、0.24±0.04、0.29±0.05、0.35±0.05(P＜0.05)、0.39±0.05(P＜0.01).结论 人参皂苷Rb1对UVB诱导的CPD的产生无明显影响,但可显著加速其清除,这可能与其上调XPC、ERCC1蛋白的表达有关.
目的 觀察人參皂苷Rb1對中波紫外線(UVB)照射誘導小鼠錶皮細胞、培養的HaCaT細胞產生與清除環丁烷嘧啶二聚體(CPD)的影響及對覈苷痠切除脩複蛋白XPC、ERCC1錶達的影響.方法 42隻去毛BALB/c小鼠分為4組:未處理組(6隻),UVB組(12隻),UVB+小劑量Rb1組(12隻),UVB+大劑量Rb1組(12隻).後2組照射前2h在揹部按100 μl/cm2分彆外用含0.5 g/L、2g/L人參皂苷Rb1的丙酮溶液,而前2組予以相應丙酮溶液.UVB劑量均為180 mJ/cm2,于照射後0.5、16 h分彆處死半數小鼠,利用免疫組化法檢測錶皮CPD水平.培養的HaCaT細胞用含人參皂苷Rb1(5、20、50 mg/L)的培養基孵育4h,部分細胞于UVB照射(15、30 mJ/cm2)後0.5、12 h終止培養併提取基因組DNA,通過斑點雜交法檢測CPD.其餘細胞照射(30 mJ/cm2)後0、0.5、2、4、12h終止培養,提取細胞總蛋白,通過免疫印跡法分析XPC、ERCC1蛋白的錶達.結果 UVB照射小鼠0.5 h後,各照光組錶皮均產生大量CPD,但組間差異無統計學意義；在照射後16h,UVB+小劑量Rb1組、UVB+大劑量Rb1組錶皮CPD分彆為32.1±8.5、14.6±4.1,均較UVB組(67.3±11.2)顯著減少,且大劑量組更為明顯(P值均＜0.01).HaCaT細胞在UVB照射後0.5 h,各組CPD總量差異無統計學意義,但照射後12h,給藥組CPD水平明顯降低.UVB組HaCaT細胞XPC、ERCC1蛋白的錶達均隨著時間延長不斷下降,在照射後即刻、0.5、2、4、12 h,XPC/GAPDH蛋白灰度比值分彆為0.68±0.11、0.47±0.09(與照射後即刻比較,P＜0.05,下同)、0.45±0.08 (P＜0.05)、0.37±0.06(P＜0.01)、0.18±0.03(P＜0.01),ERCC 1/GAPDH分彆為0.28±0.03、0.25±0.03 (P＞ 0.05)、0.21±0.02 (P＜0.05)、0.14±0.02 (P＜0.01)、0.11±0.01(P＜0.01);加入50 mg/L Rb1榦預後,XPC、ERCC1蛋白的錶達不斷增加,XPC/GAPDH分彆為0.56±0.07、0.48±0.14、0.68±0.15、0.97±0.20(P＜0.01)、0.79±0.12(P＜0.05),ERCC 1/GAPDH分彆為0.27±0.04、0.24±0.04、0.29±0.05、0.35±0.05(P＜0.05)、0.39±0.05(P＜0.01).結論 人參皂苷Rb1對UVB誘導的CPD的產生無明顯影響,但可顯著加速其清除,這可能與其上調XPC、ERCC1蛋白的錶達有關.
목적 관찰인삼조감Rb1대중파자외선(UVB)조사유도소서표피세포、배양적HaCaT세포산생여청제배정완밀정이취체(CPD)적영향급대핵감산절제수복단백XPC、ERCC1표체적영향.방법 42지거모BALB/c소서분위4조:미처리조(6지),UVB조(12지),UVB+소제량Rb1조(12지),UVB+대제량Rb1조(12지).후2조조사전2h재배부안100 μl/cm2분별외용함0.5 g/L、2g/L인삼조감Rb1적병동용액,이전2조여이상응병동용액.UVB제량균위180 mJ/cm2,우조사후0.5、16 h분별처사반수소서,이용면역조화법검측표피CPD수평.배양적HaCaT세포용함인삼조감Rb1(5、20、50 mg/L)적배양기부육4h,부분세포우UVB조사(15、30 mJ/cm2)후0.5、12 h종지배양병제취기인조DNA,통과반점잡교법검측CPD.기여세포조사(30 mJ/cm2)후0、0.5、2、4、12h종지배양,제취세포총단백,통과면역인적법분석XPC、ERCC1단백적표체.결과 UVB조사소서0.5 h후,각조광조표피균산생대량CPD,단조간차이무통계학의의；재조사후16h,UVB+소제량Rb1조、UVB+대제량Rb1조표피CPD분별위32.1±8.5、14.6±4.1,균교UVB조(67.3±11.2)현저감소,차대제량조경위명현(P치균＜0.01).HaCaT세포재UVB조사후0.5 h,각조CPD총량차이무통계학의의,단조사후12h,급약조CPD수평명현강저.UVB조HaCaT세포XPC、ERCC1단백적표체균수착시간연장불단하강,재조사후즉각、0.5、2、4、12 h,XPC/GAPDH단백회도비치분별위0.68±0.11、0.47±0.09(여조사후즉각비교,P＜0.05,하동)、0.45±0.08 (P＜0.05)、0.37±0.06(P＜0.01)、0.18±0.03(P＜0.01),ERCC 1/GAPDH분별위0.28±0.03、0.25±0.03 (P＞ 0.05)、0.21±0.02 (P＜0.05)、0.14±0.02 (P＜0.01)、0.11±0.01(P＜0.01);가입50 mg/L Rb1간예후,XPC、ERCC1단백적표체불단증가,XPC/GAPDH분별위0.56±0.07、0.48±0.14、0.68±0.15、0.97±0.20(P＜0.01)、0.79±0.12(P＜0.05),ERCC 1/GAPDH분별위0.27±0.04、0.24±0.04、0.29±0.05、0.35±0.05(P＜0.05)、0.39±0.05(P＜0.01).결론 인삼조감Rb1대UVB유도적CPD적산생무명현영향,단가현저가속기청제,저가능여기상조XPC、ERCC1단백적표체유관.
Objective To estimate the effect of ginsenoside Rb1 on the production and clearance of cyclobutane pyrimidine dimer (CPD) as well as on the expression of two nucleotide excision repair-associated proteins,xeroderma pigmentosum group C (XPC) and excision repair cross-complementing group 1 (ERCC1),by ultraviolet B (UVB)-irradiated murine epidermal cells and human HaCaT keratinocytes.Methods Totally,42 BALB/c mice were shaved on the back and divided into four groups: untreated group (n =6),UVB group irradiated with UVB only (n =12),low-dose and high-dose Rb1 group (both n =12) treated with Rb1 of 0.5 g/L and 2g/L (100 μl/cm2) respectively two hours before UVB irradiation.The dose of UVB in the animal experiment was 180 mJ/cm2.Half of the mice in each group were killed at 0.5 and 16 hours respectively after the irradiation,then,the back skin was resected and subjected to the determination of CPD levels in the epidermis by immunohistochemical SP method.Some cultured HaCaT cells were divided into several groups to be treated with different concentrations (5,20,50 mg/L) of Rb1 before or after different doses (15 and 30 mnJ/cm2) of UVB irradiation,and cells were collected at 0.5 and 12 hours after the irradiation.Subsequently,genomic DNA was extracted and CPD was detected by dot blot hybridization.Some HaCaT cells were cultured with or without the presence of Rb1 (50 mg/L) and irradiated with UVB (30 mJ/cm2),then,the cells were collected immediately or at 0.5,2,4 and 12 hours after the irradiation,and total protein was extracted and subjected to immunoblot analysis for the quantification of XPC and ERCC1 proteins.Results There was a high level of CPD in the epidermis of mice at 0.5 hour after the irradiation,with no significant differences between these groups (P ＞ 0.05).The number of CPD-positive cells per high power field (× 400) in the murine epidermis at 16 hours was statistically lower in the low-and high-dose Rb1 group than in the UVB group (32.1 ± 8.5 and 14.6 ± 4.1 vs.67.3 ± 11.2,both P ＜0.01).The CPD level in HaCaT cells was similar between these groups at 0.5 hour after UVB irradiation,but was markedly decreased at 12 hours in Rb1-treated groups.After UVB irradiation,the protein expressions of XPC and ERCC1 decreased with time in untreated HaCaT cells but increased with time in Rb1 (50 mg/L)-treated HaCaT cells.In detail,the XPC/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein ratio in untreated HaCaT cells was 0.68 ± 0.11 immediately after the irradiation,significantly higher than that at 0.5 hour (0.47 ± 0.09,P＜0.05),2 hours (0.45 ± 0.08,P＜0.05),4 hours (0.37 ± 0.06,P＜0.01),and 12 hours (0.18 ± 0.03,P ＜0.01),and that in Rb1-treated HaCaT cells was 0.56 ± 0.07 immediately after the irradiation,compared to 0.48 ± 0.14 at 0.5 hour (P＞ 0.05),0.68 ± 0.15 at 2 hours (P＞ 0.05),0.97 ± 0.20 at 4 hours (P＜0.01),and 0.79 ± 0.12 at 12 hours (P ＜0.05).The ERCC1/GAPDH protein ratio in untreated HaCaT cells was 0.28 ± 0.03 immediately after the irradiation,higher than that at 0.5 hour (0.25 ± 0.03,P ＞ 0.05),2 hours (0.21 ± 0.02,P＜0.05),4 hours (0.14 ± 0.02,P＜0.01) and 12 hours (0.11 ± 0.01,P＜0.01),and that in Rb1-treated HaCaT cells was 0.27 ± 0.04 immediately after the irradiation,compared to 0.24 ± 0.04 at 0.5 hour (P＞ 0.05),0.29 ± 0.05 at 2 hours (P＞ 0.05),0.35 ± 0.05 at 4 hours (P＜0.05),0.39 ± 0.05 at 12 hours (P ＜0.01).Conclusions Ginsenoside Rb1 shows no obvious effect on the UVB-induced production of CPD,but markedly accelerates the clearance of CPD,which may be partly associated with the upregulation of XPC and ERCC1 protein expression.
