严重烧伤大鼠血清对脂肪间质干细胞生物学特性的影响  

Effects of severely burned rat serum on biological characteristics of rat adipose-derived stem cells

作　　者：谢松涛[1] 贾文斌[1] 白晓智[1] 陶克[1] 韩夫[1] 方小兵[1] 杨龙龙[1] 蔡维霞[1] 汤朝武[1] 胡大海[1] 

机构地区：[1]第四军医大学附属西京医院全军烧伤中心,西安710032

出　　处：《中华损伤与修复杂志（电子版）》2013年第6期13-17,共5页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：国家自然科学基金(81372056)

摘　　要：目的探讨严重烧伤大鼠血清对体外培养的大鼠脂肪间充质干细胞(ADSCs)生物学特性的影响。方法取雄性SD大鼠双侧腹股沟脂肪组织,采用胶原酶消化法、分离纯化大鼠ADSCs,取第3代细胞,采用流式细胞仪检测细胞表面标志物,行成脂成骨诱导分化鉴定;ADSCs传代后按随机数字表法分为10%胎牛血清组(正常培养组)、10%正常大鼠血清组,10%烧伤大鼠血清组,采用噻唑蓝(MTT)法测定各组细胞的生长曲线。通过流式细胞仪检测各组细胞的生长周期、Real-time PCR法检测各组细胞血管内皮生长因子(VEGF)、凋亡相关因子Caspase-3的表达情况。结果分离培养的ADSCs传至第3代时形态规则,经流式细胞仪检测,所培养的ADSCs均表达CD29、CD90、CD105,阳性率分别为88.6%、99.7%、92.8%,而CD31、CD34、CD45阳性率分别为4.4%、4.7%、3.3%;经诱导培养后可向成骨成脂分化。MTT法检测结果显示:10%烧伤大鼠血清组细胞增殖较快,各时间点吸光度值(A值)明显高于10%胎牛血清组及10%正常大鼠血清组。流式细胞仪检测结果显示:培养1周后10%烧伤大鼠血清组、10%正常大鼠血清组、10%胎牛血清组ADSCs G1期细胞比例分别为:(72.20±5.13)%、(83.50±4.74)%、(90.20±6.37)%;S期细胞比例分别为:(21.40±2.84)%、(12.50±1.96)%和(8.60±1.31)%,10%烧伤大鼠血清组ADSCs G1期细胞比例显著低于10%正常大鼠血清组、10%胎牛血清组(t=2.39、4.86;P<0.05);10%烧伤大鼠血清组ADSCs S期细胞比例显著高于10%正常大鼠血清组、10%胎牛血清组(t=5.54,7.93;P<0.01)。Real-Time PCR检测结果显示:10%烧伤大鼠血清组VEGF的表达明显上调,Caspase-3表达显著下调与10%正常大鼠血清组、10%胎牛血清组相比差异均有统计学意义(P<0.05)。结论 10%烧伤大鼠血清可明显促进ADSCs的增殖、抑制细胞凋亡发生、促进ADSCs的分泌功能,严重烧伤后可望移植ADSCs用于细胞治疗。Objective To investigate the effects of severely burned rat serum on the biological characteristics of rat adipose-derived mesenchymal stem cells( ADSCs) in vitro. Methods ADSCs were isolated and cultured from Sprague Dawley( SD) rat bilateral inguinal adipose tissue by ollagenase digestion in vitro. ADSCs of passage 3 were detected surface markers with flow cytometry,and were induced into adipocytes and osteocytes by adipogenic differentiation medium and osteogenic differentiation medium in vitro. Cultured ADSCs were randomly divided into 3 groups,which were cultured with 10% fetal bovine serum( normal culture group),10% normal rat serum,and 10% burned rat serum. The cell proliferation activities,cell cycles and the vascular endothelial growth factor( VEGF),Caspase-3 mRNA levels of cultured ADSCs were detected by using Methyl Thiazolyl Tetrazolium( MTT) colorimetric assay,flow cytometry and real-time polymerase chain reaction( PCR) techniques in all groups,respectively. Results The third generation of cultured ADSCs proliferated well with fusiform shape. Flow cytometry results showed the rates of positive expression of CD29,CD90 and CD105 were 88. 6%,99. 7%,92. 8%,respectively. The positive rates of CD31,CD34,CD45 expression were 4. 4%,4. 7%,3. 3%,respectively. The induced cells were presented the phenotypic characteristics of the adipocytes and bone osteocytes with oil red O staining and alizarin red staining, respectively. The 10% burned rat serum could significantly enhance ADSCs proliferation. As compared with the other two groups,the absorbance( A) values of MTT in the 10% burned rat serum groups were significantly increased at each point. The 10% burned rat serum could significantly enhance ADSCs proliferation. As compared with the other two groups,the absorbance( A) values of MTT in the 10% burned rat serum groups were significantly increased at each point. After cultured 7 days with 10% burned rat serum,10% normal rat serum,and 10% fetal bovine serum,the flow cytometry results showed the percentage of G1

关 键 词：烧伤 脂肪间充质干细胞 血清 细胞治疗 生物学特性 

分 类 号：R644[医药卫生—外科学]