中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
5期
928-930
,共3页
张金池%蔡森林%吴佳文%吴明祥%郑国富%欧阳亮远
張金池%蔡森林%吳佳文%吳明祥%鄭國富%歐暘亮遠
장금지%채삼림%오가문%오명상%정국부%구양량원
碱性成纤维生长因子%骨髓间充质干细胞%基因转染%分化
堿性成纖維生長因子%骨髓間充質榦細胞%基因轉染%分化
감성성섬유생장인자%골수간충질간세포%기인전염%분화
Basic fibroblast growth factor%Mesenchymal stem cells%Gene transfection%Differentiation
目的 观察人碱性成纤维生长因子(bFGF)基因修饰大鼠骨髓间充质干细胞(BMSCs)在体外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SD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3组:阴性对照组(普通的BMSCs组)、空白对照组(不含bFGF基因但含有GFP的慢病毒载体转染组)、实验组(含bFGF目的基因的慢病毒载体转染组),3组同法培养[培养基10％胎牛血清、50 μg/L血管内皮生长因子(VEGF)的L-DMEM培养基].1周后各组上清液ELSIA法测定bFGF含量,4周后各组行细胞形态学观察,免疫细胞化学法检测细胞表面标记分子CD31、CD34及Ⅷ子的表达及Dil-AC-LDL吞噬实验等.结果 流式细胞仪显示分离、培养的第3代BMSCs表面抗原阳性表达率:CD11 b/c和CD34分别为(14.16±0.72)％和(1.29±0.46)％,而CD44和CD90分别为(97.87±0.84)％和(96.85±1.49)％.实验组bFGF分泌量(5.50 ±0.12) ng/L明显高于空白对照组(2.65 ±0.40) ng/L和阴性对照组(2.76±0.25) ng/L,实验组与空白对照组以及阴性对照组比较差异有统计学意义(P＜0.01)；3组大部分细胞形态变化不明显,呈长梭形、杆状、多角形.实验组CD31、CD34及Ⅷ因子阳性表达率分别为(22.73±5.42)％、(26.32±3.95)％和(30.34±6.62)％,而空白对照组和阴性对照组则为[(7.85±1.83)％、(8.93±1.37)％、(10.23 ±1.21)％[和[(7.57±1.21)％、(8.41±0.97)％、(9.97±0.52)％],实验组与空白对照组以及阴性对照组比较差异有统计学意义(P＜0.01)；诱导后实验组细胞能有效地吞噬Dil-AC-LDL,表达荧光的平均吸光度值,实验组(0.52±0.23)明显高于阴性对照组(0.05 ±0.03)和空白对照组(0.07 ±0.02),实验组与空白对照组以及阴性对照组比较差异有统计学意义(P＜0.01).结论 人bFGF基因修饰的BMSCs能分泌bFGF,在VEGF的诱导下该细胞能够更有效地向血管内皮细胞样细胞分化.
目的 觀察人堿性成纖維生長因子(bFGF)基因脩飾大鼠骨髓間充質榦細胞(BMSCs)在體外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SD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3組:陰性對照組(普通的BMSCs組)、空白對照組(不含bFGF基因但含有GFP的慢病毒載體轉染組)、實驗組(含bFGF目的基因的慢病毒載體轉染組),3組同法培養[培養基10％胎牛血清、50 μg/L血管內皮生長因子(VEGF)的L-DMEM培養基].1週後各組上清液ELSIA法測定bFGF含量,4週後各組行細胞形態學觀察,免疫細胞化學法檢測細胞錶麵標記分子CD31、CD34及Ⅷ子的錶達及Dil-AC-LDL吞噬實驗等.結果 流式細胞儀顯示分離、培養的第3代BMSCs錶麵抗原暘性錶達率:CD11 b/c和CD34分彆為(14.16±0.72)％和(1.29±0.46)％,而CD44和CD90分彆為(97.87±0.84)％和(96.85±1.49)％.實驗組bFGF分泌量(5.50 ±0.12) ng/L明顯高于空白對照組(2.65 ±0.40) ng/L和陰性對照組(2.76±0.25) ng/L,實驗組與空白對照組以及陰性對照組比較差異有統計學意義(P＜0.01)；3組大部分細胞形態變化不明顯,呈長梭形、桿狀、多角形.實驗組CD31、CD34及Ⅷ因子暘性錶達率分彆為(22.73±5.42)％、(26.32±3.95)％和(30.34±6.62)％,而空白對照組和陰性對照組則為[(7.85±1.83)％、(8.93±1.37)％、(10.23 ±1.21)％[和[(7.57±1.21)％、(8.41±0.97)％、(9.97±0.52)％],實驗組與空白對照組以及陰性對照組比較差異有統計學意義(P＜0.01)；誘導後實驗組細胞能有效地吞噬Dil-AC-LDL,錶達熒光的平均吸光度值,實驗組(0.52±0.23)明顯高于陰性對照組(0.05 ±0.03)和空白對照組(0.07 ±0.02),實驗組與空白對照組以及陰性對照組比較差異有統計學意義(P＜0.01).結論 人bFGF基因脩飾的BMSCs能分泌bFGF,在VEGF的誘導下該細胞能夠更有效地嚮血管內皮細胞樣細胞分化.
목적 관찰인감성성섬유생장인자(bFGF)기인수식대서골수간충질간세포(BMSCs)재체외향혈관내피양세포분화적능력.방법 이용밀도제도리심분리화첩벽배양상결합적방법종SD대서골수중제순단개핵세포,체외확증3대,도치현미경관찰세포생장급형태,류식세포의검측BMSCs표면항원표체.취확증제3대적BMSCs,분위3조:음성대조조(보통적BMSCs조)、공백대조조(불함bFGF기인단함유GFP적만병독재체전염조)、실험조(함bFGF목적기인적만병독재체전염조),3조동법배양[배양기10％태우혈청、50 μg/L혈관내피생장인자(VEGF)적L-DMEM배양기].1주후각조상청액ELSIA법측정bFGF함량,4주후각조행세포형태학관찰,면역세포화학법검측세포표면표기분자CD31、CD34급Ⅷ자적표체급Dil-AC-LDL탄서실험등.결과 류식세포의현시분리、배양적제3대BMSCs표면항원양성표체솔:CD11 b/c화CD34분별위(14.16±0.72)％화(1.29±0.46)％,이CD44화CD90분별위(97.87±0.84)％화(96.85±1.49)％.실험조bFGF분비량(5.50 ±0.12) ng/L명현고우공백대조조(2.65 ±0.40) ng/L화음성대조조(2.76±0.25) ng/L,실험조여공백대조조이급음성대조조비교차이유통계학의의(P＜0.01)；3조대부분세포형태변화불명현,정장사형、간상、다각형.실험조CD31、CD34급Ⅷ인자양성표체솔분별위(22.73±5.42)％、(26.32±3.95)％화(30.34±6.62)％,이공백대조조화음성대조조칙위[(7.85±1.83)％、(8.93±1.37)％、(10.23 ±1.21)％[화[(7.57±1.21)％、(8.41±0.97)％、(9.97±0.52)％],실험조여공백대조조이급음성대조조비교차이유통계학의의(P＜0.01)；유도후실험조세포능유효지탄서Dil-AC-LDL,표체형광적평균흡광도치,실험조(0.52±0.23)명현고우음성대조조(0.05 ±0.03)화공백대조조(0.07 ±0.02),실험조여공백대조조이급음성대조조비교차이유통계학의의(P＜0.01).결론 인bFGF기인수식적BMSCs능분비bFGF,재VEGF적유도하해세포능구경유효지향혈관내피세포양세포분화.
Objective To investigate the capability of differentiation of human basic fibroblast growth factor (bFGF) gene-modified rat bone marrow mesenchvmal stem cells (BMSCs) into vascular endothelial-like cells in vitro.Methods BMSCs were separated from SD rat hone marrow with density gradient centrifugation,wall sticking screening and amplified in vitro.The surface antigens of BMSCs in 3rd generation were examined by fluorecene activated cell sorter (FACS).BMSCs of the third generati were obtained and divided into three groups:normal group (general BMSCs group),blank control group (the GFP lentiviral vector without bFGF gene was transfected into BMSCs),experimental group (the GFP lentiviral vector containing bFGF target gene was transfected into MBSCs).Three groups were were cultured in the same way.[10％ FBS,50 μg/L vascular endothelial growth factor (VEGF)-DMEM].One week later,the level of bFGF was measured by using enzyme-linked immunosorhent assay (ELISA) in the supernatant of each group.Four weeks later,the morphological changes were observed,the expression levels of CD31,CD34 and the factor Ⅷ were detected by using immunocytochemistry,and the phagocytosis phenomenon of Dil-AC-LDL was examined.Results BMSCs could be separated and amplified in vitro.Cultured BMSCs at the third passage were positive for CD44 and CD90 [(97.87 ± 0.84) ％ and (96.85 ± 1.49) ％,respectively],but negative for CD11h/c and CD34 [(14.16 ± 0.72) ％ and (1.29 ± 0.46) ％,respectively].The bFGF level in experimental group [(5.50 ± 0.12) ng/L] was higher than in blank control group [(2.65 ± 0.40) ng/L] and normal group [(2.76 ± 0.25) ng/L] (both P ＜ 0.01).There was no significant difference in the morphological changes of BMSCs among three groups.The positive expression rate of CD31,CD34 and the factor Ⅷ in experimental group [(22.73 ±5.42)％,(26.32 ±3.95)％ and (30.34±6.62)％] was higher than in blank control group [(7.85 ±1.83)％,(8.93 ±1.37)％ and (10.23±1.21)％] and normal group [(7.57 ±1.21)％,(8.41 ±0.97)％ and (9.97 ±0.52)％](all P ＜ 0.01).After induction,the cells in experimental group could swallow Dil-AC-LD.The expression of mean absorbance value of the fluorescence in experimental group (0.52 ± 0.23) was significantly higher than in blank control group (0.05 ± 0.03) and normal group (0.07 ± 0.02) (both P ＜ 0.01).Conclusion The human bFGF gene-modified rat BMSCs can secrete bFGF.The gene-modified cells in the induction of VEGF can more effectively differentiate into endothelial-like cells in vitro.
