脂肪来源细胞体外增殖规律及定向诱导分化研究

脂肪组织由整形外科吸脂术获得(19例,31．5±5．8岁)。酶消化法分离抽吸物中细胞,体外扩增至第10代．测定细胞生长曲线、累计倍增数目,明确其体外生长规律和增殖能力；通过对表面抗原CD29、CD105、CD106、CD166、CD49d、CD34、CD31、3G5等的检测分析脂肪来源细胞的群体组成：分别向软骨、骨、脂肪定向诱导,进一步明确该细胞群体定向分化能力。实验表明,每300ml脂肪抽吸物平均可获得5×10~7个有核细胞,体外扩增10代,平均每代倍增数目为1．59±0．224．累计倍增数目为15．53。流式细胞学及免疫细胞化学检测显示,干细胞相关抗原CD29、CD105、CD106、CD166等表达率均＞60%,但与造血系相关的CD34、CD31表达率也分别达到7．3%、29．2%。ADC向软骨诱导可检测到Ⅱ型胶原表达；向成骨诱导可见矿化结节形成,并可检测到AKP、Osteonectin基因表达；向脂肪诱导可检测到PPARr2、GLU-4、Leptin基因表达,细胞内有脂滴形成。脂肪来源的细胞获得量大,体外增殖能力强,并含有具有多向分化潜能细胞,有可能作为组织构建的种子细胞。

IN VITRO STUDY ON PROLIFERATION AND MULTI-LINEAGE DIFFERENTIATION POTENTIAL OF ADIPOSE-DERIVED CELLS

Adipose were obtained from patients underwent liposuction treatment (total 19 female donors, 31.5 +/- 5.8 years old). Liposuction tissues were digested with type I collagenase, cells were isolated and cultured up to passage 10. To evaluate the proliferation potential of ADCs, growth curve and cumulative population doubling were achieved by cell counting. CD29,CD105, CD106, CD166, CD49d, CD34, CD31, 3G5 were analyzed by flow cytometry and immunocytochemistry to characterize the cell population. The multi-lineage potential of ADCs was testified by differentiating cells with osteogenic,chondrogenic and adipogenic inducer. A total of 5x10(7) nucleared cells could be obtained from 300ml liposuction tissues. After in vitro cultivation,cumulative population doubling number reached 15.53 at passage 10 (average 1.59 +/- 0.224 /passage). Flow cytometry and immunocytochemistry showed that ADCs expressed high level (>60%) of stem cell-related antigen (CD29, CD105, CD106, CD166), while cells expressed hematopoiesis-related antigen CD34 and CD31 around 7.3% and 29.2% respectively. Collagen II (both in mRNA and protein level) was detected in chondrogenic differentiation. The calcified nodules were observed by von Kossa and Alizarn Red staining and the expressions of AKP and Osteonectin were detected by RT-PCR in osteogenic differentiation. PPARr2, GLU-4, and Leptin genes were detected in adipogenic differentiation and intracellular lipid droplets could be observed by Oil Red staining. ADCs can be abundantly harvested and have high proliferative and multi-lineage differentiation potential.