胰岛素干预后脂肪干细胞旁分泌对人血管内皮细胞的作用

Biological effects of paracrine from insulin stimulated adipose-derived stem cells （ADSC） on human vascular endothelial cells

目的了解胰岛素刺激后脂肪干细胞（ADSC）旁分泌因子的变化及其对人血管内皮细胞的作用。方法（1）分离培养人ADSC，按照随机数字表法将细胞分为胰岛素刺激组（含终浓度1×10 -7mol／L胰岛素的无血清DMEM培养液培养）和对照组（未加胰岛素的无血清DMEM培养液培养），每组6孔。3d后收集ADSC培养上清液（ADSC—CM），ELISA法测定血管内皮生长因子（VEGF）、肝细胞生长因子（HGF）表达量。（2）分离培养人脐静脉内皮细胞（HUVEC），将细胞按照随机数字表法分为4组，均采用内皮细胞专用培养液培养：胰岛素刺激ADSC—CM组，培养液中加入经胰岛素刺激后的ADSC—CM；无刺激ADSC—CM组，培养液中含无胰岛素刺激的ADSC—CM；胰岛素组，培养液中加入终浓度为1×10 -7 mol／L的胰岛素；空白对照组，培养液中不添加刺激因素。3d后噻唑蓝法测定HUVEC增殖情况，数据用吸光度值表示。（3）另取HUVEC同上分为4组，培养12h时，膜联蛋白V-异硫氰酸荧光素双染色法测定细胞凋亡情况。（4）另取HUVEC同上分为4组后，体外细胞划痕法测定划痕后12、24、36、48h时HUVEC迁移距离。对实验数据行t检验。结果（1）胰岛素刺激组VEGF、HGF含量分别为（643±64）、（930±68）pg／mL，显著高于对照组的（287±47）、（577±84）pg／mL（t值分别为18．869、18．475，P值均小于0．05）。（2）胰岛素刺激ADSC—CM组、无刺激ADSC—CM组吸光度值分别为0．847±0．042、0．798±0．022，均高于胰岛素组的0．665±0．028（t值分别为4．579、3．732，P值均小于0．01）及空白对照组的0．674±0．031（t值分别为3．761、4．073，P值均小于0．01）；胰岛素刺激ADSC—CM组吸光度值较无刺激ADSC—CM组明显增高（t=2．576，P〈0．05）。（3）胰岛素刺激ADSC-CM组、无刺激ADSC—CM组细胞凋亡率分别为（5．8±1．9）％、（9．0±2．0）％，均明显低于胰岛素组的（30．4±6．0）％（t值分别为12．891、10．417，P值均小于0．05）和空白对照组的（31．4±7．4）％（t值分别为11．474、9．783，P值均小于0．05）；无刺激ADSC—CM组细胞凋亡率高于胰岛素刺激ADSC—CM组（t=8．548，P〈0．05）。（4）划痕后36、48h时，胰岛素刺激ADSC—CM组、无刺激ADSC—CM组HUVEC迁移距离明显长于胰岛素组与空白对照组，且胰岛素刺激ADSC—CM组细胞迁移距离大于无刺激ADSC—CM组（t值分别为4．076、4．573，P值均小于0．05）。结论胰岛素干预后ADSC旁分泌能更有效促进人血管内皮细胞增殖、迁移并抑制其凋亡，有利于组织血管化。

Objective To study the biological effects of the paracrine from ADSC after being stimulated by insulin on vascular endothelial cells. Methods （ 1 ） ADSC was isolated from human adipose tissue and cultured in vitro. The third generation cells were collected and divided into insulin group （ I, cultured with serum-free DMEM containing 1 × 10 -7 mol/L insulin） and control group （C, cultured with serum-free DMEM） according to the random number table, with 6 slots in each group. Three days later, ADSC culture medium （ADSC-CM） was collected for determination of levels of vascular endothelial growth factor （VEGF） and hepatocyte growth fator （HGF） by ELISA. （2） Human umbilical vein endothelial cells （HUVEC） were cultured to the third generation, and they were cultured with special nutrient solution and divided into ADSC-CM with insulin stimulation group （AI） , ADSC-CM without insulin stimulation group （AC）, insulin group （I, with same concentration as above） , blank control group （BC） according to the random number table. Three days later, proliferation of HUVEC was determined with MTT method （with expression of absorbance value）. Another two samples of HUVEC were respectively divided into 4 groups as above for determination of apoptosis rate with Annexin V/FITC double-staining 12 hours after culture, and HUVEC migration with scratch adhesion test at post scratch hour （PSH） 12, 24, 36, 48. Data were processed with t test. Results （ 1 ） Compared with those in C group [ （ 287 ＋- 47 ）, （577 ＋- 84） pg/mL, respectively ], the secretion levels of VEGF and HGF in I group [ （643 ＋-64）, （930 ＋-68） pg/mL, respectively] were significantly increased （ with t value respectively 18. 869, 18. 475, P values all below 0.05 ）. （2） The absorbanee value of HUVEC in AI and AC groups was 0. 847 ＋- 0. 042, 0. 798 ＋- 0. 022, respectively, which were higher than that in I and BC groups [0.665＋-0.028 （withtvalue respectively 4. 579, 3. 732）, 0.674＋-0.031 （witht value respectively 3. 761, 4. 073 ） , P values all below 0.01 ] , and that in AI group was higher than that in AC group （ t =2.576, P 〈0.05）. The apoptosis rates of HUVEC in AI and AC groups [（5.8＋-1.9）%, （9.0 ＋-2.0） % , respectivelyl were obviously lower as compared with that in I and BC groups [ （30.4 ＋- 6.0）% （withtvalue respectively 12.891, 10.417）, （31.4＋-7.4）% （witht value respectively 11.474, 9. 783） , P values all below 0.05 ] , and that in AC group was higher than that in AI group （ t = 8. 548, P 〈 0.05 ）. The distance of migration of HUVEC in AI and AC groups were greater than that in I and BC groups at PSH 36, 48, and that in AI group was greater as compared with that in AC group （ with t value respectively 4. 076, 4. 573, P values all below 0.05 ）. Conclusions Paracrine from ADSC after being stimulated by insulin can promote proliferation and migration of HUVEC, and suppress its apoptosis, and it is beneficial for tissue vascularization.