家兔脂肪基质干细胞的分离培养及多向分化诱导

Isolation,culture and multi-directional induced differentiation of rabbit adipose derived stromal stem cells

背景:脂肪基质干细胞在人体皮下脂肪中储备充足,体外能快速增殖,具有多向分化潜能,是目前组织工程种子细胞的研究热点。目的:体外分离培养家兔脂肪血管基质部分细胞,并验证其具有多分化潜能。方法:体外分离家兔脂肪血管基质部分细胞,在标准条件下培养,流式细胞仪检测第3代血管基质部分细胞表面标记物CD44,CD29,CD45,取第3代血管基质部分细胞进行成脂、成骨、成软骨诱导,油红O染色鉴定成脂诱导,碱性磷酸酶染色、茜素红染色、Vonkossa染色鉴定成骨诱导,Ⅱ型胶原免疫组织化学染色和Ⅱ型胶原mRNART-PCR检测鉴定成软骨诱导。结果与结论:原代血管基质部分细胞为短梭形和多角形,第3代血管基质部分细胞为长梭形,流式细胞仪检测提示CD44+,CD29+,CD45-,成脂诱导组,染色油红O阳性,成骨诱导组,碱性磷酸酶染色、Vonkossa染色、茜素红染色阳性,成软骨诱导组,诱导14dⅡ型胶原免疫组织化学染色阿利新蓝染色阳性,RT-PCR检测Ⅱ型胶原mRNA显示产物条带较未诱导前信号明显增强。提示体外分离培养的家兔血管基质部分细胞具有干细胞表型,具有向成骨、软骨、脂肪细胞分化的能力,初步鉴定为脂肪基质干细胞。

BACKGROUND：Subcutaneous fat of human body is a rich reservoir of adipose derived stromal stem cells（ADSCs）.ADSCs can proliferate rapidly when being cultured in vitro,and has the capacity of multi-directional differentiation.ADSCs attracted much attention in research of tissue engineered seed cells.OBJECTIVE：To isolate and culture stromal vascular fraction（SVF） cells from rabbit subcutaneous fat in vitro,and to testify whether it has multiple differentiation capacity.METHODS：SVF cells were isolated in vitro from rabbits,and cultured under standard condition.Cellular surface antigens CD44 CD45 and CD29 of passage 3 SVF cells were examined using flow cytometry.Passage 3 SVF cells were induced to differentiate into osteoblasts,chondrocytes,and lipocytes.Oil red staining was used to examine lipocyte induction.Alkaline phosphatase（ALP staining,alizarin red staining and von Kossa staining were used to examine osteoblast induction.Type Ⅱ collagen immunohistochemical staining and type Ⅱ collagen mRNA RT-PCR were used to examine chondrocyte differentiation.RESULTS AND CONCLUSION：Primary SVF cells were multi-angular or short spindle-shaped.Passage 3 SVF cells were long spindle-shaped.Flow cytometry showed CD44＋,CD29＋,CD45-.Oil red staining exhibited positive reaction in lipocyte induction group.ALP staining,alizarin red staining and Von kossa staining demonstrated positive reactions in osteoblast induction group.Type Ⅱ collagen immunohistochemical staining and alcian blue staining have suggested positive reactions at 14 days of chondrogenic induction group.RT-PCR of type Ⅱ collagen mRNA test showed that the product band had strong signal at 14 days of chondrogenic induction group compared with that before induction.Above mentioned results have indicated that SVF cells isolated from rabbit subcutaneous fat have identical surface makers of stem cells,and have the ability to differentiate into lipocytes,osteoblasts and chondrocytes in vitro by induction,and it could be concluded that the SVF cells were ADSCs.