胰岛素对烫伤血清诱导骨骼肌成肌细胞凋亡的调控作用

Modulatory effect of insulin on scalded rat serum-induced apoptosis of skeletal myoblast

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＋100nmol／L胰岛素、体积分数20％烫伤大鼠血清＋100nmol／L胰岛素，孵育48h。行Hoechst33258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-）蛋白激酶B（Akt）、p-磷脂酰肌醇3激酶（P13K）、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）倒置荧光照微镜下可见，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硅示，烫伤血清组细胞凋亡率为（18．5±1．8）％，明显高于对照组、腆岛素组及烫伤血清＋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easpase-3蛋白表达量（2．15±0．51）明照高于对照组（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蛋白发达量（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easpase-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。

Objective To study the modulatory effect of insulin on apoptosis of skeletal myoblast （1.6 cells） by serum of scahled ral and its mechanism. Methods 1,6 eells cuhured with DMEM medium containing 10% FBS were divided into control （C, added with 20% normal rat serum ） , serum from rat with seahl injury （S, added with 20% serum from seahted rat） , insulin （1, added with 20% normal rat serum and 100 nmol/L insulin） , and serum of scalded rat ＋ insulin （SI, added with 20% serum of seahted rat ＋ 100 nmol/L insulin） groups according to the random number table. After being cultured for 48 hours, upoptosis 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 phnsphorylated （p-） Akt, p-PI3K, Bax, Bcl-2, and active easpase-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 visual 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 ofp-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 easpase-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.