中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2011年
11期
673-677
,共5页
成纤维细胞%糖基化终产物,高级%半胱氨酸天冬氨酸蛋白酶%细胞凋亡
成纖維細胞%糖基化終產物,高級%半胱氨痠天鼕氨痠蛋白酶%細胞凋亡
성섬유세포%당기화종산물,고급%반광안산천동안산단백매%세포조망
Fibroblasts%Glycosylation end products,advanced%Caspases%Apoptosis
目的 观察晚期糖基化终产物(advanced glycation end products,AGE)对人牙龈成纤维细胞(human gingival fibroblasts,HGF)凋亡的诱导作用,同时通过检测胱天蛋白酶(caspase)-8,9,3酶活性改变及caspase抑制剂对凋亡作用的影响,探讨凋亡蛋白酶依赖性凋亡途径在AGE诱导HGF凋亡过程中的作用.方法 将AGE修饰的人血清白蛋白(AGE-human serum albumin,AGE-HAS)与HGF共培养(以不加AGE-HAS的HGF为空白对照组),利用酶标仪分别测定12、24h后caspase-8,9,3酶活性的变化；分别添加caspase-8,9,3及caspase-8 +caspase-9抑制剂后(分别为caspase-8,9,3及caspase-8+ caspase-9抑制剂组,不添加caspase抑制剂的为阳性对照组),检测24h后Hoechst33258染色及膜联蛋白V-碘化丙啶双染检测细胞凋亡率,酶标仪测定caspase-3酶活性的变化.结果 caspase酶活性检测结果表明:HGF与AGE-HAS共培养后,caspase-8,9,3的酶活性分别为0.1097±0.0051、0.0965±0.0051及0.1280±0.0103,共培养24h后酶活性分别为0.1558±0.0053、0.1308±0.0035及0.1954±0.0051,组间差异均有统计学意义(P＜0.05)；Hoechst33258染色结果显示caspase-9抑制剂组、caspase-8抑制剂组、caspase-8+ caspase-9抑制剂组、caspase-3抑制剂组阳性细胞数分别为(247.7±32.4)、(200.1±14.6)、(154.1±14.4)及(131.3±14.6)个,与阳性对照组[ (350.4±20.0)个]间差异均有统计学意义(P＜0.05)；膜联蛋白V-碘化丙啶双染结果显示,caspase-9抑制剂组、caspase-8抑制剂组、caspase-8+ caspase-9抑制剂组及caspase-3抑制剂组凋亡率分别为(38.87±3.31)％、( 25.57±2.20)％、(17.17±2.24)％和(14.73±2.48)％,与空白对照组及阳性对照组相比差异均有统计学意义(P＜0.05)；caspase-3酶活性在caspase-8、caspase-9及caspase-8+caspase-9抑制剂组分别为0.1274±0.0076、0.1465±0.0062、0.1044±0.0051,组间差异均有统计学意义(P＜0.05).结论 AGE-HAS主要通过激活caspase依赖性凋亡途径诱导HGF凋亡；其中死亡受体途径可能在凋亡过程中占主导地位.
目的 觀察晚期糖基化終產物(advanced glycation end products,AGE)對人牙齦成纖維細胞(human gingival fibroblasts,HGF)凋亡的誘導作用,同時通過檢測胱天蛋白酶(caspase)-8,9,3酶活性改變及caspase抑製劑對凋亡作用的影響,探討凋亡蛋白酶依賴性凋亡途徑在AGE誘導HGF凋亡過程中的作用.方法 將AGE脩飾的人血清白蛋白(AGE-human serum albumin,AGE-HAS)與HGF共培養(以不加AGE-HAS的HGF為空白對照組),利用酶標儀分彆測定12、24h後caspase-8,9,3酶活性的變化；分彆添加caspase-8,9,3及caspase-8 +caspase-9抑製劑後(分彆為caspase-8,9,3及caspase-8+ caspase-9抑製劑組,不添加caspase抑製劑的為暘性對照組),檢測24h後Hoechst33258染色及膜聯蛋白V-碘化丙啶雙染檢測細胞凋亡率,酶標儀測定caspase-3酶活性的變化.結果 caspase酶活性檢測結果錶明:HGF與AGE-HAS共培養後,caspase-8,9,3的酶活性分彆為0.1097±0.0051、0.0965±0.0051及0.1280±0.0103,共培養24h後酶活性分彆為0.1558±0.0053、0.1308±0.0035及0.1954±0.0051,組間差異均有統計學意義(P＜0.05)；Hoechst33258染色結果顯示caspase-9抑製劑組、caspase-8抑製劑組、caspase-8+ caspase-9抑製劑組、caspase-3抑製劑組暘性細胞數分彆為(247.7±32.4)、(200.1±14.6)、(154.1±14.4)及(131.3±14.6)箇,與暘性對照組[ (350.4±20.0)箇]間差異均有統計學意義(P＜0.05)；膜聯蛋白V-碘化丙啶雙染結果顯示,caspase-9抑製劑組、caspase-8抑製劑組、caspase-8+ caspase-9抑製劑組及caspase-3抑製劑組凋亡率分彆為(38.87±3.31)％、( 25.57±2.20)％、(17.17±2.24)％和(14.73±2.48)％,與空白對照組及暘性對照組相比差異均有統計學意義(P＜0.05)；caspase-3酶活性在caspase-8、caspase-9及caspase-8+caspase-9抑製劑組分彆為0.1274±0.0076、0.1465±0.0062、0.1044±0.0051,組間差異均有統計學意義(P＜0.05).結論 AGE-HAS主要通過激活caspase依賴性凋亡途徑誘導HGF凋亡；其中死亡受體途徑可能在凋亡過程中佔主導地位.
목적 관찰만기당기화종산물(advanced glycation end products,AGE)대인아간성섬유세포(human gingival fibroblasts,HGF)조망적유도작용,동시통과검측광천단백매(caspase)-8,9,3매활성개변급caspase억제제대조망작용적영향,탐토조망단백매의뢰성조망도경재AGE유도HGF조망과정중적작용.방법 장AGE수식적인혈청백단백(AGE-human serum albumin,AGE-HAS)여HGF공배양(이불가AGE-HAS적HGF위공백대조조),이용매표의분별측정12、24h후caspase-8,9,3매활성적변화；분별첨가caspase-8,9,3급caspase-8 +caspase-9억제제후(분별위caspase-8,9,3급caspase-8+ caspase-9억제제조,불첨가caspase억제제적위양성대조조),검측24h후Hoechst33258염색급막련단백V-전화병정쌍염검측세포조망솔,매표의측정caspase-3매활성적변화.결과 caspase매활성검측결과표명:HGF여AGE-HAS공배양후,caspase-8,9,3적매활성분별위0.1097±0.0051、0.0965±0.0051급0.1280±0.0103,공배양24h후매활성분별위0.1558±0.0053、0.1308±0.0035급0.1954±0.0051,조간차이균유통계학의의(P＜0.05)；Hoechst33258염색결과현시caspase-9억제제조、caspase-8억제제조、caspase-8+ caspase-9억제제조、caspase-3억제제조양성세포수분별위(247.7±32.4)、(200.1±14.6)、(154.1±14.4)급(131.3±14.6)개,여양성대조조[ (350.4±20.0)개]간차이균유통계학의의(P＜0.05)；막련단백V-전화병정쌍염결과현시,caspase-9억제제조、caspase-8억제제조、caspase-8+ caspase-9억제제조급caspase-3억제제조조망솔분별위(38.87±3.31)％、( 25.57±2.20)％、(17.17±2.24)％화(14.73±2.48)％,여공백대조조급양성대조조상비차이균유통계학의의(P＜0.05)；caspase-3매활성재caspase-8、caspase-9급caspase-8+caspase-9억제제조분별위0.1274±0.0076、0.1465±0.0062、0.1044±0.0051,조간차이균유통계학의의(P＜0.05).결론 AGE-HAS주요통과격활caspase의뢰성조망도경유도HGF조망；기중사망수체도경가능재조망과정중점주도지위.
Objective To investigate the effect of synthesized advanced glycation end products (AGE) on apoptosis of human gingival fibroblasts(HGF) and the possible role of caspase-dependent pathway in the process of AGE-induced apoptosis.Methods HGF were incubated with AGE-human serum albumin (AGE-HSA).The activity of caspase-8,caspase-9 and caspase-3 were detected by microplate reader after 12 and 24 hours.HGF were incubated with caspase inhibitors for 1 hour,and then incubated with AGE-HSA for 24 hours,HGF was first stained by Hoechst33258 and observed under inverted microscope,and then double stained by annexin V and propidine iodide(PI) and observed by flow cytometry(FCM).The activity of caspase-3 was determined by caspase-3 assay kit and observed by microplate reader.Results Caspases activity of caspase-8,-9,-3 was 0.1097 ±0.0051,0.0965 ±0.0051 and 0.1280 ±0.0103 after 12 h of incubation with AGE-HSA and HGF,respectively,and 0.1558 ±0.0053,0.1308 ±0.0035 and 0.1954 ±0.0051 after 24 h of incubation with AGE-HSA and HGF,respectively ( P ＜0.05).Positive cells number was 247.7 ±32.4,200.1 ± 14.6,154.1 ± 14.4 and 131.3 ± 14.6 in caspase inhibitor groups by Hoechst33258 staining,respectively.Apoptotic rate was ( 25.57 ± 2.20 ) ％,( 38.87 ± 3.31 ) ％,( 17.17 ± 2.24 ) ％ and ( 14.73 ±2.48)％ in caspase inhibitor groups by annexin V-PI staining,respectively.The difference between different groups was significant (P ＜ 0.05 ).Caspase-3 activity was reduced to 0.1274 ± 0.0076,0.1465 ± 0.0062,0.1044 ±0.0051 in caspase inhibitor groups,respectively.The difference between different groups was significant(P ＜ 0.05 ).Conclusions Apoptosis of HGF induced by AGE-HISA may be mainly through activation of caspase-dependant pathway in which cytoplasmic pathway may play a predominant role.
