论文部分内容阅读
目的对兔不同部位的脂肪来源干细胞(adipose-derived stem cells,ADSCs)体外成肌诱导能力进行比较,观察部位来源是否与干细胞分化能力有关,以便为尿路组织工程种子细胞的提取选择较佳取材部位。方法从1只4月龄雄性新西兰大白兔附睾附近、腹膜后、颈背部无菌取出脂肪组织,Ⅰ型胶原酶酶解法提取ADSCs,体外培养并传代后差速贴壁培养进行纯化,用鼠源性兔CD44分子的单克隆抗体做免疫细胞化学染色行初步鉴定,然后取各部位第5代ADSCs行成脂、成骨、成肌多向诱导分化,采用油红O染色行成脂诱导鉴定,von Kossa染色行成骨诱导鉴定,RT-PCR法对诱导对照组(含10%FBS的L-DMEM培养基培养)及诱导组(成肌诱导培养基培养)细胞行肌肉早期标志物α-actin基因的检测。结果 3个部位脂肪均可获取贴壁的ADSCs;差速贴壁培养细胞呈铺路石样生长;兔CD44免疫细胞化学染色呈阳性。各部位第5代ADSCs行成脂诱导14d油红O染色可见红色脂滴;成骨诱导28d后von Kossa染色可见细胞外不同程度黑色钙沉积。RT-PCR检测示,成肌诱导42d后诱导对照组α-actin条带较弱,诱导组呈强阳性条带;各部位ADSCs成肌诱导42d的α-actin增长率分别为:附睾附近38.446%±4.852%,腹膜后28.622%±4.879%,颈背部35.471%±3.434%,腹膜后增长率与其余2个部位比较差异均有统计学意义(P<0.05),其余2个部位间比较差异无统计学意义(P>0.05)。结论兔不同部位ADSCs在体外培养时表现出不同的成肌分化能力,附睾附近和颈背部脂肪可作为下尿路组织工程种子干细胞来源的较佳取材部位。
OBJECTIVE: To compare the induction ability of adipose-derived stem cells (ADSCs) in vitro from different parts of rabbits and observe whether the site origin is related to the differentiation ability of stem cells so as to select the best material for the extraction of urinary tissue engineering seed cells Site. Methods Adipose tissue was removed aseptically from the proximal epididymis of a 4-month-old male New Zealand white rabbits. ADSCs were extracted by enzymatic digestion of type Ⅰ collagenase and purified by differential adherent culture in vitro. Rabbit CD44 molecule monoclonal antibody immunocytochemical staining line initial identification, and then take the 5th generation of each site ADSCs line adipogenic, osteogenic, myogenic multi-directional differentiation, using oil red O staining line of fat-induced identification, von Kossa staining. The expression of α-actin, an early marker of muscle, in control group (cultured in L-DMEM medium containing 10% FBS) and induced group (cultured in myoblast induction medium) were detected by RT- Detection of genes. Results Adherent adherent cells could be obtained from all three adipose tissues. Passaged adherent cells were found to be cobblestone-like. Rabbit CD44 immunocytochemical staining was positive. The fifth generation of ADSCs at each site were induced to adipose red lipid droplets by oil red O staining for 14 days. After von Kossa staining for 28 days after osteogenic induction, different levels of extracellular calcium deposition were observed. The results of RT-PCR showed that the α-actin band in the control group was weak after induction of myoblast induction for 42 days and the strong positive band was observed in the induction group. The growth rates of α-actin on the 42th day after induction of ADSCs were 38.446% ± 4.852%, 28.622% ± 4.879%, 35.471% ± 3.434%. The retroperitoneal growth rate was significantly different from the other two sites (P <0.05), the other two sites showed no difference Statistical significance (P> 0.05). Conclusions ADSCs in different parts of rabbit showed different myogenic differentiation ability in vitro. Fetal fat near the epididymis and dorsal neck could be used as a better source of tissue engineering stem cells derived from lower urinary tract.