绿色荧光蛋白转基因小鼠脂肪干细胞治疗后肢缺血的实验研究

Study on the treatment for mouse hindlimb ischemia with green fluorescent protein transgenic mouse adipose-derived stem cells

目的观察绿色荧光蛋白(GFP)转基因小鼠来源的脂肪干细胞(ADSCs)治疗小鼠后肢缺血的效果及其自身所带荧光标记的有效性。方法取4周龄GFP转基因小鼠的脂肪组织,消化获取GFP来源的脂肪干细胞(GFP-ADSCs),并用流式鉴定其表面的干细胞抗原。建立C57BL/6小鼠左后肢缺血模型并随机分成两组(每组16只):一组后肢缺血的肌肉组织内注射P3代的GFPADSCs 1×106个/100μl,对照组于同样部位注射100μl PBS。1个月后利用苏木素-伊红(HE)、免疫组织化学(IHC)及免疫荧光(IF)染色行CD31染色观察缺血肌肉组织内血管新生情况。结果1从GFP转基因小鼠的脂肪组织中可以获得大量GFP-ADSCs并表达干细胞表面抗原CD90及CD105;2 GFP-ADSCs被成功地诱导成脂成骨;3 GFP-ADSCs组总残肢恢复率显著高于PBS组(P<0.05);4 1个月后GFP-ADSCs治疗组缺血的肌肉组织内IHC染色CD31可见较多的新生血管,其微血管密度数显著高于PBS组(P<0.05);IF染色显示GFP-ADSCs治疗组的新生血管表达内皮细胞的特异性标记CD31和GFP,而PBS组则未见GFP绿色荧光表达。结论从GFP转基因小鼠的脂肪组织中可以获取大量的ADSCs,其能够促进小鼠后肢缺血肌肉组织内的血管新生,自带的GFP可以示踪ADSCs在受体内的存活、迁移及分化。

Objective To evaluate the efficacy of the treatment for hindlimb ischemia with green fluorescent protein transgenic mouse adipose-derived stem cells(GFP-ADSCs)and the reliability of fluorescence.Methods GFP-ADSCs were isolated from the adipose tissues from 4 weeks old GFP mice by digestion, and the stem cell surface antigens were identified by flow cytometry. C57BL/6 left hindlimb ischemia models were established and then randomly divided into two groups(sixteen mice each group). 1×106/100μl of P3 GFP-ADSCs and 100μl PBS were respectively injected into the hindlimb ischemic muscle in the same place of treatment group and control group. One month later,the angiogenesis in muscles were detected by hematoxylin-eosin(HE),immunology and histology chemistry(IHC)and immunofluorescence(IF)staining with CD31. Results 1 A lot of GFP-ADSCs which expressed CD90 and CD105 could be isolated from GFP transgenic mice; 2 GFP-ADSCs were successfully induced into lipoblasts and osteoblasts; 3 Total residual limb recovery rate of GFP-ADSCs Group was obviously higher than that of PBS Group(P<0.05); 4 There were more new blood vessels among the muscle bundles in GFP-ADSCs Group by staining with CD31 after one month. Micro vessel density(MVD)of new vessels in GFPADSCs Group were obviously higher than that of PBS Group(P<0.05). IF staining indicated that the new vesselsof GFP-ADSCs group expressed CD31 and GFP,while green fluorescence could not be found in PBS group.Conclusion A lot of GFP-ADSCs can be isolated from GFP transgenic mice,and they can induce angiogenesis in muscles of hindlimb ischemia mice. GFP can monitor the survival,migration and differentiation of ADSCs.