中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
1期
55-57
,共3页
张金池%欧阳亮远%吴佳文%王敬瀚
張金池%歐暘亮遠%吳佳文%王敬瀚
장금지%구양량원%오가문%왕경한
糖尿病%骨髓间充质干细胞%诱导分化%内皮样细胞
糖尿病%骨髓間充質榦細胞%誘導分化%內皮樣細胞
당뇨병%골수간충질간세포%유도분화%내피양세포
Diabetic%Bone marrow mesenchymal cell%Induction differentiation%Endothelial-like cell
目的 观察链脲佐菌素(STZ)诱导的糖尿病大鼠骨髓间充质干细胞(BMSCs)的体外培养方法和向血管内皮样细胞分化的能力.方法 利用密度梯度离心分离和贴壁培养相结合的方法从STZ诱导的糖尿病大鼠骨髓中提纯单个核细胞,体外扩增3代,倒置显微镜观察细胞生长及形态,流式细胞仪检测BMSCs表面抗原表达.取扩增第3代的BMSCs,分为2组,诱导组加入诱导培养剂[含10％胎牛血清、10μg/L血管内皮生长因子(VEGF)、2μg/L碱性成纤维细胞生长因子(bFGF)、100 U/ml青霉素、100 mg/L链霉素的M199培养基]中诱导分化,对照组则不加任何细胞因子.2周后行细胞形态学观察,免疫细胞化学法检测VEGF受体(VEGFR)-2表达,流式细胞仪测定CD34表达量,紫外线分光光度法测定一氧化碳(NO)含量,电镜观测胞质WeibelPalade小体.结果 STZ腹腔注射可诱导糖尿病大鼠模型.流式细胞仪显示第3代BMSCs表达表面抗原:CD44和CD90阳性细胞表达率分别为(97.8±0.9)％和(96.8±1.4)％,而CD11 b/c和CD34阳性细胞表达率分别为(13.2±0.6)％和(1.2±0.5)％.诱导组大部分细胞形态变化不明显,呈长梭形、杆状、多角形,诱导组VEGFR-2、CD34阳性表达率分别为(97.1±1.0)％和(65.0±3.9)％,而对照组则为(7.0±1.0)％和(0.9±0.3)％,两组比较差异有统计学意义(P＜0.05)；诱导组胞外NO含量(94.14±3.25) μmol/L亦明显高于对照组(70.37±2.10) μmol/L(P ＜0.05)；电镜两组均未观察到胞质Weibel Palade小体.结论 经密度梯度离心分离和贴壁培养相结合的方法可从骨髓中提纯BMSCs.糖尿病鼠BMSCs可在体外诱导向血管内皮样细胞方向分化,BMSCs有望作为治疗糖尿病下肢缺血性疾病的种子细胞.
目的 觀察鏈脲佐菌素(STZ)誘導的糖尿病大鼠骨髓間充質榦細胞(BMSCs)的體外培養方法和嚮血管內皮樣細胞分化的能力.方法 利用密度梯度離心分離和貼壁培養相結閤的方法從STZ誘導的糖尿病大鼠骨髓中提純單箇覈細胞,體外擴增3代,倒置顯微鏡觀察細胞生長及形態,流式細胞儀檢測BMSCs錶麵抗原錶達.取擴增第3代的BMSCs,分為2組,誘導組加入誘導培養劑[含10％胎牛血清、10μg/L血管內皮生長因子(VEGF)、2μg/L堿性成纖維細胞生長因子(bFGF)、100 U/ml青黴素、100 mg/L鏈黴素的M199培養基]中誘導分化,對照組則不加任何細胞因子.2週後行細胞形態學觀察,免疫細胞化學法檢測VEGF受體(VEGFR)-2錶達,流式細胞儀測定CD34錶達量,紫外線分光光度法測定一氧化碳(NO)含量,電鏡觀測胞質WeibelPalade小體.結果 STZ腹腔註射可誘導糖尿病大鼠模型.流式細胞儀顯示第3代BMSCs錶達錶麵抗原:CD44和CD90暘性細胞錶達率分彆為(97.8±0.9)％和(96.8±1.4)％,而CD11 b/c和CD34暘性細胞錶達率分彆為(13.2±0.6)％和(1.2±0.5)％.誘導組大部分細胞形態變化不明顯,呈長梭形、桿狀、多角形,誘導組VEGFR-2、CD34暘性錶達率分彆為(97.1±1.0)％和(65.0±3.9)％,而對照組則為(7.0±1.0)％和(0.9±0.3)％,兩組比較差異有統計學意義(P＜0.05)；誘導組胞外NO含量(94.14±3.25) μmol/L亦明顯高于對照組(70.37±2.10) μmol/L(P ＜0.05)；電鏡兩組均未觀察到胞質Weibel Palade小體.結論 經密度梯度離心分離和貼壁培養相結閤的方法可從骨髓中提純BMSCs.糖尿病鼠BMSCs可在體外誘導嚮血管內皮樣細胞方嚮分化,BMSCs有望作為治療糖尿病下肢缺血性疾病的種子細胞.
목적 관찰련뇨좌균소(STZ)유도적당뇨병대서골수간충질간세포(BMSCs)적체외배양방법화향혈관내피양세포분화적능력.방법 이용밀도제도리심분리화첩벽배양상결합적방법종STZ유도적당뇨병대서골수중제순단개핵세포,체외확증3대,도치현미경관찰세포생장급형태,류식세포의검측BMSCs표면항원표체.취확증제3대적BMSCs,분위2조,유도조가입유도배양제[함10％태우혈청、10μg/L혈관내피생장인자(VEGF)、2μg/L감성성섬유세포생장인자(bFGF)、100 U/ml청매소、100 mg/L련매소적M199배양기]중유도분화,대조조칙불가임하세포인자.2주후행세포형태학관찰,면역세포화학법검측VEGF수체(VEGFR)-2표체,류식세포의측정CD34표체량,자외선분광광도법측정일양화탄(NO)함량,전경관측포질WeibelPalade소체.결과 STZ복강주사가유도당뇨병대서모형.류식세포의현시제3대BMSCs표체표면항원:CD44화CD90양성세포표체솔분별위(97.8±0.9)％화(96.8±1.4)％,이CD11 b/c화CD34양성세포표체솔분별위(13.2±0.6)％화(1.2±0.5)％.유도조대부분세포형태변화불명현,정장사형、간상、다각형,유도조VEGFR-2、CD34양성표체솔분별위(97.1±1.0)％화(65.0±3.9)％,이대조조칙위(7.0±1.0)％화(0.9±0.3)％,량조비교차이유통계학의의(P＜0.05)；유도조포외NO함량(94.14±3.25) μmol/L역명현고우대조조(70.37±2.10) μmol/L(P ＜0.05)；전경량조균미관찰도포질Weibel Palade소체.결론 경밀도제도리심분리화첩벽배양상결합적방법가종골수중제순BMSCs.당뇨병서BMSCs가재체외유도향혈관내피양세포방향분화,BMSCs유망작위치료당뇨병하지결혈성질병적충자세포.
Objective To investigate the methods for separation and culture of bone marrow mesenchymal stem cells (BMSCs) in vitro in diabetic rats and the capability of differentiation into endotheliallike cells.Methods Rat diabetic model (DM) was induced by injection of stretopzin (STZ) in SD rats.After 12 weeks of DM duration,BMSCs were separated from bone marrow with density gradient centrifugation,wall sticking screening and amplified in vitro.The surface markers of BMSCs were evaluated with fluorescence-activated cell sorter (FACS).BMSCs of the third passage were obtained and divided into two groups.In the induction group,the cells were induced to differentiate in M199 medium containing 20％ fatal bovine serun ( FBS),vascular endothelial growth factor ( VEGF,10 μg/L) and basic fibroblast growth factor (bFGF,2 μg/L).No cytokine was added in the blank control group.Two weeks later,the cell morphology was observed under the phase contrast microscopy.The expression levels of VEGFR-2 were detected by using immunohistochemistry.EPC-specific marker CD34 was evaluated with FACS,and release of nitrous oxide (NO) from culture cells was also detected by NO detection reagent.Weibel-Palade (W-P) bodies in the cytoplasm were observed under the transmission electron microscopy.Results Rat diabetic model in SD rats that were injected with STZ intraperitoneally was established.Cultured BMSCs at the third passage were positive for CD44 and CD90 [ (97.8 ± 0.9 ) ％ and (96.8 ± 1.4) ％,respectively ],but negative for CD11 b/c and CD34 [ ( 13.2 ± 0.6 ) ％ and ( 1.2 ± 0.5 ) ％,respectively ].On the 14 day in the induction group,the cell morphology observed under the phase contrast microscopy was not distinctly different.Differentiation antigens VEGFR2 and CD34 in the induction group were higher [ (97.1 ± 1.0) ％,(65.0 ± 3.9) ％ ] than in the blank control group [ (7.0 ± 1.0 ) ％,( 0.9 ± 0.3 ) ％ ] ( P ＜ 0.05 ).The content of nitrous oxide in the induction group was also obviously increased as compared with the blank control group [ (94.14 ± 3.25 ) vs.( 70.37 ± 2.1 0) μmol/L,P ＜ 0.05 ).W-P bodies in the cytoplasm of the cultured cells were not found in the two groups.Conclusion BMSCs in diabetic rats can be isolated by density gradient centrifugation and adherent culture.VEGF and bFGF can induce differentiation of BMSCs in diabetic rats into endothelial-like cells in vitro.It is suggested that BMSCs may be used as a new type of seed cells for the diabetic lower limb ischemia disease.
