间充质干细胞上清提取物对糖尿病皮肤溃疡大鼠模型溃疡组织中肿瘤坏死因子-α及凋亡蛋白caspase-3的作用研究

Effect of supernatant of mesenchymal stem cells on tumor necrosis factor-α and apoptosis protein caspase-3 in diabetic skin ulcer rat model

目的探讨脐带间充质干细胞(UCMSCs)上清提取物对糖尿病皮肤溃疡(DSU)大鼠模型溃疡组织中肿瘤坏死因子-α(TNF-α)及凋亡蛋白半胱氨酸天冬氨酸蛋白酶3(caspase-3)的影响.方法将45只Sprague-Dawley大鼠按随机数字表法分为急性损伤伤口组(对照组)、DSU+生理盐水干预组和DSU+UCMSCs上清提取物干预治疗组,每组15只.大鼠经尾静脉注射四氧嘧啶并给予高脂饲料喂养制作糖尿病模型,再在此基础上构建DSU模型.对照组大鼠建立糖尿病模型后只划伤口不注入金黄色葡萄球菌,皮下注射100μl生理盐水.另外两组大鼠构建DSU模型成功后,DSU+生理盐水干预组大鼠尾静脉注射100μl生理盐水,溃疡处滴加100μl生理盐水;DSU+UCMSCs上清提取物干预治疗组将UCMSCs上清冻干粉溶于200μl生理盐水中,尾静脉注射100μl,同时溃疡处滴加100μl.干预治疗5 d后,采用放射免疫法检测大鼠血清TNF-α水平,实时荧光定量PCR和蛋白质印迹法检测大鼠溃疡处组织中TNF-α和caspase-3的表达.结果 DSU+生理盐水干预组大鼠血清TNF-α水平[(35.9±3.7)μg/L]明显高于对照组[(11.4±4.9)μg/L]和DSU+UCMSCs上清提取物干预治疗组[(14.7±6.6)μg/L],差异均具有统计学意义(均P<0.05).DSU+UCMSCs上清提取物干预治疗组大鼠溃疡组织中TNF-α和caspase-3的mRNA及蛋白表达明显低于DSU+生理盐水干预组(均P<0.05);而对照组TNF-α和caspase-3的mRNA及蛋白表达与DSU+UCMSCs上清提取物干预治疗组比较,差异均无统计学意义(均P>0.05).结论 UCMSCs上清液提取物干预治疗可有效下调DSU大鼠溃疡组织中TNF-α和caspase-3的表达,起到抗炎、抗凋亡的作用.

Objective To investigate the supernatant of umbilical cord-derived mesenchymal stem cells (UCMSCs) on tumor necrosis factor-α(TNF-α) and apoptosis protein caspase-3 in diabetic rats model with skin ulcer. Methods 45 Sprague-Dawley rats were randomly divided into control group (acute wounds group), phosphate buffered saline (PBS) group and UCMSCs supernatant group. The diabetic rat model was constructed by injecting with alloxan by tail vein and feeding with high-fat diet. Diabetic skin ulcer (DSU) rat model was constructed by scratching a wound and infusing suspension of Staphylococcus aureus. In the control group, the diabetic rats (n=15) were scratched to form a wound and treated by tail vein injection of 100μl PBS. In the PBS group, DSU rats (n=15) were treated by tail vein injection of 100μl PBS, and then 100μl PBS was dropped at the ulcer site. In the UCMSCs supernatant group, freeze-dried powder of UCMSCs supernatant was dissolved in 200μl PBS, 100μl of which was injected into the tail vein of DSU rats (n=15), and other 100μl was dropped at the ulcer site. After 5 days of the treatments, the levels of serum TNF-αwere detected by radioimmunoassay method, and the expression of TNF-αand caspase-3 in the ulcer tissues of rats was detected by polymerase chain reaction and Western Blot. Results The levels of TNF-αin the PBS group [(35.9±3.7)μg/L] were significantly higher than that of the control group [(11.4±4.9)μg/L] and the UCMSCs group [(14.7±6.6)μg/L](all P<0.05). The levels of mRNA and protein expression of TNF-αand caspase-3 in the UCMSCs group were significantly lower than those of the PBS group (all P<0.05), and have no significant differences with respect to those of the control group (all P>0.05). Conclusions UCMSCs supernatant treatments can effectively down-regulate the expression of TNF-αand caspase-3 in ulcer tissue of DSU rats, and play an anti-inflammatory and anti-apoptotic effect.