CAJ | 학술논문

目的探讨胰岛素对烫伤大鼠血清诱导大鼠骨骼肌成肌细胞株L6凋亡的调控作用及机制.方法体外培养L6细胞,按照随机数字表法分为对照组、烫伤血清组、胰岛素组和烫伤血清+胰岛素组.在上述4组细胞培养液中分别添加体积分数20％正常大鼠血清、体积分数20％烫伤大鼠血清、体积分数20％正常大鼠血清+100 nmol/L胰岛素、体积分数20％烫伤大鼠血清+100 nmol/L胰岛素,孵育48 h.行Hoechst 33258染色,于倒置荧光显微镜下观察细胞凋亡形态并计数凋亡细胞；行膜联蛋白V-异硫氰酸荧光素/碘化丙啶双标记染色,用流式细胞仪检测细胞凋亡率；蛋白质印迹法检测细胞磷酸化(p一)蛋白激酶B(Akt)、p-磷脂酰肌醇3激酶(PI3K)、Bax、Bcl-2、活化半胱氨酸天冬氨酸蛋白酶3( caspase-3)的蛋白表达量.对实验数据行组间或配对t检验.结果 (1)倒置荧光显微镜下可见,烫伤血清组凋亡细胞为每视野(59.6±3 9)个,显著多于对照组、胰岛素组、烫伤血清+胰岛素组[每视野(4 9±2.6)、(5 5±2 1)、(19.7±2 3)个,t值分别为28.53、29.86、21.53,P值均小于0.01].(2)流式细胞仪检测结果显示,烫伤血清组细胞凋亡率为(18.5±1.8)％,明显高于对照组、胰岛素组及烫伤血清+胰岛素组[(1.1±0 6)％、(1 5±0 3)％、(7.8±0.6)％,t值分别为22.41、22 83、13 92,P值均小于0 01].(3)蛋白质印迹检测显示,烫伤血清组Bax蛋白表达量(1.12±0.63)及活化caspase-3蛋白表达量(2.15±0.51)明显高于对照组(0.16±0 03、0.21±0.03,t值分别为3 80、10 69,P值均小于0 01),p-Akt蛋白表达量(0.20±0.03)明显低于对照组(0 42±0 07,t=-8.46,P＜0.01)；Bcl-2及p-PI3K蛋白表达量(0.19±0 03、0.17±0.03)与对照组(0.26±0 09、0 28±0.07)接近(t值分别为-2.73、-1.14,P值均大于0 05).与烫伤血清组比较,烫伤血清+胰岛素组Bax蛋白表达量(0.40±0.14)和活化caspase-3蛋白表达量(0.83±0.18)明显降低(t=-3.23,P ＜0.05;t =6.66,P ＜0.01),Bcl-2、p-Akt及p-PI3K蛋白表达量均升高(0.39±0.10、0 78±0 03、0.47±0.12,t值分别为4.07、18.71、5 05,P＜0.05或P＜0.01).结论烫伤大鼠血清可显著促进骨骼肌成肌细胞凋亡,胰岛素能通过PI3K/Akt信号途径抑制细胞凋亡. 目的探討胰島素對燙傷大鼠血清誘導大鼠骨骼肌成肌細胞株L6凋亡的調控作用及機製.方法體外培養L6細胞,按照隨機數字錶法分為對照組、燙傷血清組、胰島素組和燙傷血清+胰島素組.在上述4組細胞培養液中分彆添加體積分數20％正常大鼠血清、體積分數20％燙傷大鼠血清、體積分數20％正常大鼠血清+100 nmol/L胰島素、體積分數20％燙傷大鼠血清+100 nmol/L胰島素,孵育48 h.行Hoechst 33258染色,于倒置熒光顯微鏡下觀察細胞凋亡形態併計數凋亡細胞；行膜聯蛋白V-異硫氰痠熒光素/碘化丙啶雙標記染色,用流式細胞儀檢測細胞凋亡率；蛋白質印跡法檢測細胞燐痠化(p一)蛋白激酶B(Akt)、p-燐脂酰肌醇3激酶(PI3K)、Bax、Bcl-2、活化半胱氨痠天鼕氨痠蛋白酶3( caspase-3)的蛋白錶達量.對實驗數據行組間或配對t檢驗.結果 (1)倒置熒光顯微鏡下可見,燙傷血清組凋亡細胞為每視野(59.6±3 9)箇,顯著多于對照組、胰島素組、燙傷血清+胰島素組[每視野(4 9±2.6)、(5 5±2 1)、(19.7±2 3)箇,t值分彆為28.53、29.86、21.53,P值均小于0.01].(2)流式細胞儀檢測結果顯示,燙傷血清組細胞凋亡率為(18.5±1.8)％,明顯高于對照組、胰島素組及燙傷血清+胰島素組[(1.1±0 6)％、(1 5±0 3)％、(7.8±0.6)％,t值分彆為22.41、22 83、13 92,P值均小于0 01].(3)蛋白質印跡檢測顯示,燙傷血清組Bax蛋白錶達量(1.12±0.63)及活化caspase-3蛋白錶達量(2.15±0.51)明顯高于對照組(0.16±0 03、0.21±0.03,t值分彆為3 80、10 69,P值均小于0 01),p-Akt蛋白錶達量(0.20±0.03)明顯低于對照組(0 42±0 07,t=-8.46,P＜0.01)；Bcl-2及p-PI3K蛋白錶達量(0.19±0 03、0.17±0.03)與對照組(0.26±0 09、0 28±0.07)接近(t值分彆為-2.73、-1.14,P值均大于0 05).與燙傷血清組比較,燙傷血清+胰島素組Bax蛋白錶達量(0.40±0.14)和活化caspase-3蛋白錶達量(0.83±0.18)明顯降低(t=-3.23,P ＜0.05;t =6.66,P ＜0.01),Bcl-2、p-Akt及p-PI3K蛋白錶達量均升高(0.39±0.10、0 78±0 03、0.47±0.12,t值分彆為4.07、18.71、5 05,P＜0.05或P＜0.01).結論燙傷大鼠血清可顯著促進骨骼肌成肌細胞凋亡,胰島素能通過PI3K/Akt信號途徑抑製細胞凋亡.
목적 탐토이도소대탕상대서혈청유도대서골격기성기세포주L6조망적조공작용급궤제.방법 체외배양L6세포,안조수궤수자표법분위대조조、탕상혈청조、이도소조화탕상혈청+이도소조.재상술4조세포배양액중분별첨가체적분수20％정상대서혈청、체적분수20％탕상대서혈청、체적분수20％정상대서혈청+100 nmol/L이도소、체적분수20％탕상대서혈청+100 nmol/L이도소,부육48 h.행Hoechst 33258염색,우도치형광현미경하관찰세포조망형태병계수조망세포；행막련단백V-이류청산형광소/전화병정쌍표기염색,용류식세포의검측세포조망솔；단백질인적법검측세포린산화(p일)단백격매B(Akt)、p-린지선기순3격매(PI3K)、Bax、Bcl-2、활화반광안산천동안산단백매3( caspase-3)적단백표체량.대실험수거행조간혹배대t검험.결과 (1)도치형광현미경하가견,탕상혈청조조망세포위매시야(59.6±3 9)개,현저다우대조조、이도소조、탕상혈청+이도소조[매시야(4 9±2.6)、(5 5±2 1)、(19.7±2 3)개,t치분별위28.53、29.86、21.53,P치균소우0.01].(2)류식세포의검측결과현시,탕상혈청조세포조망솔위(18.5±1.8)％,명현고우대조조、이도소조급탕상혈청+이도소조[(1.1±0 6)％、(1 5±0 3)％、(7.8±0.6)％,t치분별위22.41、22 83、13 92,P치균소우0 01].(3)단백질인적검측현시,탕상혈청조Bax단백표체량(1.12±0.63)급활화caspase-3단백표체량(2.15±0.51)명현고우대조조(0.16±0 03、0.21±0.03,t치분별위3 80、10 69,P치균소우0 01),p-Akt단백표체량(0.20±0.03)명현저우대조조(0 42±0 07,t=-8.46,P＜0.01)；Bcl-2급p-PI3K단백표체량(0.19±0 03、0.17±0.03)여대조조(0.26±0 09、0 28±0.07)접근(t치분별위-2.73、-1.14,P치균대우0 05).여탕상혈청조비교,탕상혈청+이도소조Bax단백표체량(0.40±0.14)화활화caspase-3단백표체량(0.83±0.18)명현강저(t=-3.23,P ＜0.05;t =6.66,P ＜0.01),Bcl-2、p-Akt급p-PI3K단백표체량균승고(0.39±0.10、0 78±0 03、0.47±0.12,t치분별위4.07、18.71、5 05,P＜0.05혹P＜0.01).결론 탕상대서혈청가현저촉진골격기성기세포조망,이도소능통과PI3K/Akt신호도경억제세포조망.
Objective To study the modulatory effect of insulin on apoptosis of skeletal myoblast (L6 cells) by serum of scalded rat and its mechanism.Methods L6 cells cultured with DMEM medium containing 10％ FBS were divided into control ( C,added with 20％ normal rat serum),serum from rat with scald injury (S,added with 20％ serum from scalded rat),insulin (I,added with 20％ normal rat serum and 100 nmol/L insulin),and serum of scalded rat + insulin (SI,added with 20％ serum of scalded rat + 100 nmol/L insulin) groups according to the random number table.After being cultured for 48 hours,apoptosis was observed with Hoechst 33258 staining and its number counted,annexin V -FITC/PI double-labeling method was used to assess apoptosis rate,the protein levels of phosphorylated (p-) Akt,p-PI3K,Bax,Bc1-2,and active caspase-3 were determined by Western blotting.Data were processed with grouped or paired t test.Results ( 1 ) The amount of apoptosis with typical morphological change in S group [ (59.6 ± 3.9) per visual7 field ] was more than that in C,I,and SI groups [ (4.9 ± 2.6),( 5.5 ± 2.1 ),( 19.7 ± 2.3 ) per visual field,with t value respectively 28.53,29.86,21.53,P values all below 0.01 ].(2) Apoptotic rate in S group was (18.5 ± 1.8)％,which was markedly higher than that in C,I,and SI groups [ ( 1.1 ± 0.6) ％,( 1.5 ± 0.3 ) ％,( 7.8 ± 0.6) ％,with t value respectively 22.41,22.83,13.92,P values all below 0.01 ].(3) Compared with those in C group,the protein levels of Bax and active caspase-3 in S group were up-regulated (1.12 ± 0.63 vs.0.16 ± 0.03,2.15 ± 0.51 vs.0.21 ± 0.03,with t value respectively 3.80,10.69,P values all below 0.01 ),the protein level of p-Akt was lowered (0.20 ±0.03 vs.0.42 ±0.07,t =-8.46,P ＜0.01 ),and the protein levels of p-PI3K and Bcl-2 showed no statistical difference (0.19 ± 0.03 vs.0.26 ± 0.09,0.17 ± 0.03 vs.0.28 ± 0.07,with t value respectively - 2.73,- 1.14,P values all above 0.05 ).The protein levels of Bax (0.40 ± 0.14 ) and active caspase-3 (0.83 ±0.18) in SI group were lowered ( t =-3.23,P ＜0.05;t =6.66,P ＜0.01) and the protein levels of p-Akt,Bcl-2,and p-PI3K in SI group were elevated (0.39 ±0.10,0.78 ±0.03,0.47 ±0.12,with t value respectively 4.07,18.71,5.05,P ＜ 0.05 or P ＜ 0.01 ) as compared with those in S group.Conclusions Serum from scalded rat can induce apoptosis in skeletal myoblast,and the effect can be inhibited by insulin through PI3K/Akt signal pathway.