氯化钴预处理对大鼠脂肪干细胞成脂及增殖的影响

目的观察脂肪来源问充质干细胞（ADSCs）在经氯化钴（CoCl2）预处理的条件下，其增殖、细胞状态变化以及成脂肪化倾向。方法通过有限稀释法分离、培养SD大鼠ADSCs，取第3代ADSCs，分别向骨细胞、脂肪细胞定向诱导分化鉴定，利用流式细胞仪检测大鼠ADSCs细胞表面标志物CD29，CD34，CIM4，CIM5的表达。将ADSCs经不同浓度CoCl2干预，利用Westernblot检测缺氧诱导因子1d（HIF-101）的表达情况。同时，利用MT＇F实验来检测大鼠ADSCs在不同浓度的CoCl2下细胞增殖的改变；并且通过油红O定量检测大鼠ADSCs在经历缺氧后的成脂情况。结果第3代ADSCs在倒置显微镜下观察，大多呈梭形，经流式鉴定，大鼠ADSCs细胞表面标志物CD29、CD44表达率高，而CD34、CD45表达率低，体外诱导培养的大鼠ADSCs能够向成骨细胞和成脂细胞分化，具有较高的自我更新能力。在MTr实验中，400、200ixmol／LCoCl2组，ADSCs增值减弱；而100、50Ixmol／LCoCl2的实验组，缺氧造成的对细胞增殖和细胞毒性与空白对照组相比，没有变化（P〉0．05）。HIF-1d的表达随着培养基中的CoCl2的浓度而增加。在光镜下观察发现，随着CoCl2浓度的升高以及时间的延长，大鼠ADSCs的成脂倾向越发明显（P〈0．001）。结论ADSCs在处于体外适量的CoCl2干预情况时，其增殖并不会受到影响，并且呈显著的增强其成脂倾向。

Effect of cobalt chloride preprocessing on the proliferation and adipogenic differentiation of rat adipose- derived stem cells

Objective To observe the effect of cobalt chloride（ Cocl2 ） preprocessing on the proliferation and adipogenic differentiation of rat adipose-derived stem cells（ADSCs）. Methods Rat ADSCs were isolated from adipose tissue obtained from rat and were cultured, ADSCs at Passage 3 were cultured in adipogenic, os- teogenetic differentiation medium and underwent identification, flow cytometfic analysis for cell surface antigen CD29, CD34, CD44 and CD45 were performed. To confirm the effect of cobalt chloride preprocessing on adipo- genie differentiation of ADSCs, CoC12 , a HIF stabilizer was used to mimic the hypoxic condition. The expres- sion of HIF-lc~ were observed by Western blotting. Growth condition of the cells was observed by inverted mi- croscope. MTT method was used to observe cell proliferation activity at different concentrations after hypoxic culture. The effects of bypoxic condition to ADSCs adipogenic differentiation was detected by Oil Red O quanti- tative assay. Results Most ADSCs at Passage 3 were spindle-shaped under the inverted microscope. Flow cy- tometric analysis showed CD29 and CD44 were positive; CD 34 and CIM5 were negative. ADSCs were induced in vitro to osteoblasts and lipoblasts. MTT method revealed the Optical Density value of 100 and 50 μmol/L^-1 CoC12 and control group were no significant differences between each groups （ P 〉 0.05 ）. HIF-1 alpha protein was increased with the CoC12 concentration increasing. At 7th and 14th days after hypoxia culture, Oil Red Oquantitative assay showed hypoxia had a significant impact on ADSCs adipogenic capacity （ P〈 0. 001 ）. Con- clusion Cobalt chloride preprocessing condition will enhance the adipogenic differentiation ability of ADSCs and proliferation activity will not be affected.