CAJ | 학술논문

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GFP-ADSCs 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；②GFP-ADSCs被成功地诱导成脂成骨；③GFP-ADSCs组总残肢恢复率显著高于PBS组（P<0.05）；④1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。
목적：관찰록색형광단백（GFP）전기인소서래원적지방간세포（ADSCs）치료소서후지결혈적효과급기자신소대형광표기적유효성。방법취4주령GFP전기인소서적지방조직，소화획취GFP래원적지방간세포（GFP-ADSCs），병용류식감정기표면적간세포항원。건립C57BL/6소서좌후지결혈모형병수궤분성량조（매조16지）：일조후지결혈적기육조직내주사P3대적GFP-ADSCs 1×106개/100μl，대조조우동양부위주사100μl PBS。1개월후이용소목소-이홍（HE）、면역조직화학（IHC）급면역형광（IF）염색행CD31염색관찰결혈기육조직내혈관신생정황。결과①종GFP전기인소서적지방조직중가이획득대량GFP-ADSCs병표체간세포표면항원CD90급CD105；②GFP-ADSCs피성공지유도성지성골；③GFP-ADSCs조총잔지회복솔현저고우PBS조（P<0.05）；④1개월후 GFP-ADSCs치료조결혈적기육조직내IHC 염색CD31가견교다적신생혈관，기미혈관밀도수현저고우PBS조（P<0.05）；IF 염색현시GFP-ADSCs치료조적신생혈관표체내피세포적특이성표기CD31화GFP，이PBS조칙미견GFP록색형광표체。결론종GFP전기인소서적지방조직중가이획취대량적ADSCs，기능구촉진소서후지결혈기육조직내적혈관신생，자대적GFP가이시종ADSCs재수체내적존활、천이급분화。
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 GFP-ADSCs Group were obviously higher than that of PBS Group(P<0.05). IF staining indicated that the new vessels of 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.