脂肪分泌物对脂肪间充质干细胞的诱导研究

The Research on the Induction of Secretome by Adipose Tissue for Adipose-derived Stem Cells

目的研究脂肪分泌物对脂肪间充质干细胞(ADSCs)的诱导作用,探讨脂肪再生机制。方法利用组织块贴壁法和细胞流式技术分离、鉴定ADSCs,收集含有脂肪分泌物的培养基(CM)作为条件培养基并用来诱导ADSCs(CM诱导组),化学培养基诱导组作为阳性对照组,用普通培养基组作为空白对照组,通过细胞形态学观察脂滴的形成和Real-time PCR技术在基因水平上对其可靠性进行体外验证。用可溶性明胶海绵作为ADSCs的支架来研究脂肪分泌物在体内对ADSCs的诱导作用,并作HE切片观察。结果流式细胞术证实所培养的细胞为ADSCs。在体外实验中,CM诱导4d时,CM诱导组中的ADSCs胞浆内出现了明显的脂滴,Real-time PCR显示,与空白对照组比较,相关的成脂基因mRNA表达量也明显增加(P<0.05);体内实验HE染色结果可以观察到CM诱导组中有明显的血管化生成。结论脂肪组织分泌物中可能含有能诱导ADSCs分化形成成熟脂肪细胞以及血管化的目标因子。

Objective Study the role for adipose tissue-derived stem cells（ADSCs） of secretome by adipose tissue and hoped that makes some contributions for the fat regeneration.Methods Direct adherent culture methods and the flow cytometry（FCM） were used to isolate and identify ADSCs.Collected the medium that had contained secreted proteins as the conditional medium（CM） and was used to induce ADSCs,chemical medium group as the positive control and normal medium group as the negative control.The formation of lipid drops was observed through the cell morphology,and the reliability was verified through Real-time PCR of the mRNA level in vitro.To test the effect of CM on ADSCs in vivo,the absorbable gelatin sponge was used as a scaffold for ADSCs.Results FCM ensured that the cultured cells were ADSCs.Significant increased lipid drops and related mRNA levels were observed in induced ADSCs cultured for 4 days,which indicated that the CM could promote the ADSCs differentiate into adipocytes after cultured for 4 days in vitro.The secreted proteins could promote ADSCS differentiate into vascularization tissues in vivo.Conclusion The secreted proteome may contain some growth factors that can promote ADSCs to differentiate into adipocytes and vascularization,which may give new directions for the growth factors that could be applied in the repair of soft tissue.