CAJ | 학술논문

目的了解钙离子对人永生化KC株HaCaT细胞整合素β1启动子活性、蛋白表达及细胞迁移的影响.方法 (1)体外培养HaCaT细胞(12孔板),按随机数字表法分为5组,每组18孔.分别用pGL3启动子(阳性对照组)、pGL3空载体(阴性对照组)及pGL3-1756 bp(全长启动子组)、pGL3-1442 bp(远端启动子组)、pGL3-261 bp(近端启动子组)报告质粒转染HaCaT细胞,转染分别在含0.00、0.03、0.09、0.30、0.80、1.20 mmol/L钙离子的无血清RPMI 1640培养液中进行(每组每种浓度3孔).24 h后用双荧光素酶报告基因检测系统检测荧光素酶相对活性.(2)取本室构建的稳定转染小干扰RNA-整合素β1载体(简称稳定转染)的HaCaT细胞,常规培养后按随机数字表法将细胞分为6部分,用前述6种浓度钙离子处理,每种浓度3个样本.采用蛋白质印迹法检测整合素β1蛋白表达水平.(3)于6孔板中培养正常HaCaT细胞和稳定转染的HaCaT细胞,每种细胞18孔.按常规进行划痕处理后,用含前述6种浓度钙离子的培养液(按随机数字表法划分.每种浓度3孔细胞)培养12 h.观察并检测细胞迁移率.(4)对各实验结果进行单因素方差分析和独立样本t检验.结果 (1)全长启动子组细胞在钙离子浓度由0.00 mmol/L升至0.09、0.30 mmol/L时,荧光素酶相对活性由0.16±0.09升至0.39±0.09、0.35±0.05(t值分别为3.143、3.140,P值均小于0.05);当钙离子浓度升至0.80、1.20 mmol/L时,荧光素酶活性下降.远端启动子组细胞荧光素酶相对活性随钙离子浓度变化的趋势与全长启动子组相似,但在0.09、0.30 mmol/L时,该酶活性分别为0.56±0.32和0.64±0.06,明显高于全长启动子组(t值分别为0.887、6.122,P值均小于0.05).各种浓度钙离子对近端启动子组荧光素酶相对活性无明显影响.(2)当钙离子浓度为0.00 mmol/L时,稳定转染的HaCaT细胞整合素β1蛋白表达量为0.53±0.10.当钙离子浓度为0.03、0.09、0.30、0.80、1.20 mmol/L时,整合素β1表达量分别为0.58±0.09、1.40±0.29、1.41±0.09、0.99±0.10、1.16±0.15,与0.00 mmol/L时比较均明显升高(t值分别为0.687、4.880、11.210、5.578、6.199,P值均小于0.05).(3)与钙离子浓度为0.00 mmol/L比较,当钙离子浓度为0.09、0.30 mmol/L时,正常HaCaT细胞迁移率明显增高;0.80、1.20 mmol/L时,迁移率明显降低.稳定转染的HaCaT细胞经各种浓度钙离子处理后,细胞迁移率无明显改变.结论整合素β1远端启动子是调节人表皮细胞整合素β1转录的主要区域,钙离子对整合素β1启动子活性、蛋白表达和细胞迁移具有调节作用. 目的瞭解鈣離子對人永生化KC株HaCaT細胞整閤素β1啟動子活性、蛋白錶達及細胞遷移的影響.方法 (1)體外培養HaCaT細胞(12孔闆),按隨機數字錶法分為5組,每組18孔.分彆用pGL3啟動子(暘性對照組)、pGL3空載體(陰性對照組)及pGL3-1756 bp(全長啟動子組)、pGL3-1442 bp(遠耑啟動子組)、pGL3-261 bp(近耑啟動子組)報告質粒轉染HaCaT細胞,轉染分彆在含0.00、0.03、0.09、0.30、0.80、1.20 mmol/L鈣離子的無血清RPMI 1640培養液中進行(每組每種濃度3孔).24 h後用雙熒光素酶報告基因檢測繫統檢測熒光素酶相對活性.(2)取本室構建的穩定轉染小榦擾RNA-整閤素β1載體(簡稱穩定轉染)的HaCaT細胞,常規培養後按隨機數字錶法將細胞分為6部分,用前述6種濃度鈣離子處理,每種濃度3箇樣本.採用蛋白質印跡法檢測整閤素β1蛋白錶達水平.(3)于6孔闆中培養正常HaCaT細胞和穩定轉染的HaCaT細胞,每種細胞18孔.按常規進行劃痕處理後,用含前述6種濃度鈣離子的培養液(按隨機數字錶法劃分.每種濃度3孔細胞)培養12 h.觀察併檢測細胞遷移率.(4)對各實驗結果進行單因素方差分析和獨立樣本t檢驗.結果 (1)全長啟動子組細胞在鈣離子濃度由0.00 mmol/L升至0.09、0.30 mmol/L時,熒光素酶相對活性由0.16±0.09升至0.39±0.09、0.35±0.05(t值分彆為3.143、3.140,P值均小于0.05);噹鈣離子濃度升至0.80、1.20 mmol/L時,熒光素酶活性下降.遠耑啟動子組細胞熒光素酶相對活性隨鈣離子濃度變化的趨勢與全長啟動子組相似,但在0.09、0.30 mmol/L時,該酶活性分彆為0.56±0.32和0.64±0.06,明顯高于全長啟動子組(t值分彆為0.887、6.122,P值均小于0.05).各種濃度鈣離子對近耑啟動子組熒光素酶相對活性無明顯影響.(2)噹鈣離子濃度為0.00 mmol/L時,穩定轉染的HaCaT細胞整閤素β1蛋白錶達量為0.53±0.10.噹鈣離子濃度為0.03、0.09、0.30、0.80、1.20 mmol/L時,整閤素β1錶達量分彆為0.58±0.09、1.40±0.29、1.41±0.09、0.99±0.10、1.16±0.15,與0.00 mmol/L時比較均明顯升高(t值分彆為0.687、4.880、11.210、5.578、6.199,P值均小于0.05).(3)與鈣離子濃度為0.00 mmol/L比較,噹鈣離子濃度為0.09、0.30 mmol/L時,正常HaCaT細胞遷移率明顯增高;0.80、1.20 mmol/L時,遷移率明顯降低.穩定轉染的HaCaT細胞經各種濃度鈣離子處理後,細胞遷移率無明顯改變.結論整閤素β1遠耑啟動子是調節人錶皮細胞整閤素β1轉錄的主要區域,鈣離子對整閤素β1啟動子活性、蛋白錶達和細胞遷移具有調節作用.
목적 료해개리자대인영생화KC주HaCaT세포정합소β1계동자활성、단백표체급세포천이적영향.방법 (1)체외배양HaCaT세포(12공판),안수궤수자표법분위5조,매조18공.분별용pGL3계동자(양성대조조)、pGL3공재체(음성대조조)급pGL3-1756 bp(전장계동자조)、pGL3-1442 bp(원단계동자조)、pGL3-261 bp(근단계동자조)보고질립전염HaCaT세포,전염분별재함0.00、0.03、0.09、0.30、0.80、1.20 mmol/L개리자적무혈청RPMI 1640배양액중진행(매조매충농도3공).24 h후용쌍형광소매보고기인검측계통검측형광소매상대활성.(2)취본실구건적은정전염소간우RNA-정합소β1재체(간칭은정전염)적HaCaT세포,상규배양후안수궤수자표법장세포분위6부분,용전술6충농도개리자처리,매충농도3개양본.채용단백질인적법검측정합소β1단백표체수평.(3)우6공판중배양정상HaCaT세포화은정전염적HaCaT세포,매충세포18공.안상규진행화흔처리후,용함전술6충농도개리자적배양액(안수궤수자표법화분.매충농도3공세포)배양12 h.관찰병검측세포천이솔.(4)대각실험결과진행단인소방차분석화독립양본t검험.결과 (1)전장계동자조세포재개리자농도유0.00 mmol/L승지0.09、0.30 mmol/L시,형광소매상대활성유0.16±0.09승지0.39±0.09、0.35±0.05(t치분별위3.143、3.140,P치균소우0.05);당개리자농도승지0.80、1.20 mmol/L시,형광소매활성하강.원단계동자조세포형광소매상대활성수개리자농도변화적추세여전장계동자조상사,단재0.09、0.30 mmol/L시,해매활성분별위0.56±0.32화0.64±0.06,명현고우전장계동자조(t치분별위0.887、6.122,P치균소우0.05).각충농도개리자대근단계동자조형광소매상대활성무명현영향.(2)당개리자농도위0.00 mmol/L시,은정전염적HaCaT세포정합소β1단백표체량위0.53±0.10.당개리자농도위0.03、0.09、0.30、0.80、1.20 mmol/L시,정합소β1표체량분별위0.58±0.09、1.40±0.29、1.41±0.09、0.99±0.10、1.16±0.15,여0.00 mmol/L시비교균명현승고(t치분별위0.687、4.880、11.210、5.578、6.199,P치균소우0.05).(3)여개리자농도위0.00 mmol/L비교,당개리자농도위0.09、0.30 mmol/L시,정상HaCaT세포천이솔명현증고;0.80、1.20 mmol/L시,천이솔명현강저.은정전염적HaCaT세포경각충농도개리자처리후,세포천이솔무명현개변.결론 정합소β1원단계동자시조절인표피세포정합소β1전록적주요구역,개리자대정합소β1계동자활성、단백표체화세포천이구유조절작용.
Objective To study the effect of calcium on the activity and protein expression of integrin P, promoter in human immortal keratinocyte colony HaCaT cell and cell migration. Methods (1) HaCaT cells were cultured in vitro (12-slot plate) and divided into 5 groups according to the random number table, with 18 slots in each group: reporter plasmid pGL3 promoter (positive control group, PC) , pGL3 empty vector (negative control group, NC) , pGL3-1756 bp (total length promoter group, TL) , pGL3-1442 bp (distal promoter group, D) , and pGL3-261 bp (proximal promoter group, P) was respectively used to transfect HaCaT cells in non-serum RPMI 1640 culture medium with 0. 00, 0. 03, 0. 09, 0. 30, 0. 80, or 1. 20 mmol/L calcium (3 slots in each group with each concentration). Luciferase activity was detected with dual-luciferase reporter assay system 24 hours after transfection. (2) HaCaT cells steadily transfected with small interfering RNA-integrin β1 vector (steadily transfected in brief) constructed in our laboratory were normally cultured and divided into 6 parts according to the random number table. And then they were treated with former 6 different concentrations of calcium, with 3 samples for each concentration. Expression level of integrin β1 protein was determined with Western blot. (3) Normal and steadily transfected HaCaT cells were cultured in 6-slot plate, 18 slots for each kind of cells. They were cultured with former 6 kinds of calcium culture media (divided according to the random number table, with 3 slots of cells for each concentration) for 12 hours after scratch test. Cell migration rate was observed and determined. (4) Data were processed with one-way analysis of variance and independent samples t test. Results (1) The luciferase activity of cells in TL group increased from 0. 16 ± 0. 09 to 0. 39 ± 0. 09 and 0. 35 ± 0. 05 (with t value respectively 3. 143, 3. 140, P values all below 0.05) as calcium concentration increasing from 0.00 mmol/L to 0.09 and 0. 30 mmol/L, and it decreased as calcium concentration increased to 0. 80 and 1. 20 mmol/L. The change pattern of luciferase activity of cells along with calcium concentration in D group was similar to that in TL group, but its activity (0.56 ±0.32, 0.64 ±0.06) at the concentration of 0.09, 0.30 mmol/L was respectively higher than that in TL group (with t value respectively 0. 887, 6. 122, P values all below 0. 05). There was no obvious influence of calcium in either concentration on the luciferase activity of cells in P group. (2) The expression amount of integrin β1 of steadily transfected HaCaT cells cultured with 0.03, 0.09, 0.30, 0.80, 1.20 mmol/L calcium (0.58±0.09, 1.40±0.29, 1.41 ±0.09, 0.99 ±0.10, 1.16± 0. 15) were all increased as compared with that cultured with 0.00 mmol/L calcium (0.53 ±0. 10, with t value respectively 0.687, 4.880, 11.210, 5.578, 6. 199, P values all below 0.05). (3) Migration speed of normal HaCaT cells cultured with 0.09, 0. 30 mmol/L calcium increased obviously as compared with that cultured with 0. 00 mmol/L calcium, and it slowed down when cultured with 0. 80, 1.20 mmol/L calcium. There was no obvious difference of migration rate among steadily transfected HaCaT cells treated with different concentration of calcium. Conclusions Distal promoter region of integrin β1 plays a vital role in regulating integrin β1 transcription in human epidermal cells. And calcium regulates activity, protein expression of integrin β1 promoter and cell migration.