中华创伤骨科杂志
中華創傷骨科雜誌
중화창상골과잡지
CHINESE JOURNAL OF ORTHOPAEDIC TRAUMA
2012年
10期
889-892
,共4页
苏万汉%陈滨%方锦涛%赵培冉
囌萬漢%陳濱%方錦濤%趙培冉
소만한%진빈%방금도%조배염
间质干细胞%血管紧张素Ⅱ%血管内皮生长因子A%基因表达
間質榦細胞%血管緊張素Ⅱ%血管內皮生長因子A%基因錶達
간질간세포%혈관긴장소Ⅱ%혈관내피생장인자A%기인표체
Mesenchymal stem cells%Angiotensin Ⅱ%Vascular endothelial growth factor A%Gene expression
目的 探讨血管紧张素Ⅱ对人骨髓基质干细胞( BMSCs)源性成骨细胞血管内皮生长因子(VEGF)基因表达及蛋白分泌的影响. 方法 分离、培养人BMSCs,并向成骨细胞方向诱导,用碱性磷酸酶(ALP)染色鉴定成骨细胞.取诱导14d的人BMSCs,分成5组(n=3),分别用0、50、100、150、200 nmol/L血管紧张素Ⅱ处理细胞,以确定最佳作用浓度.取诱导21d的人BMSCs,分成5组(n=6),分别用最佳作用浓度血管紧张素Ⅱ处理细胞0、6、12、24、48 h,采用实时定量逆转录-聚合酶链反应( RT-PCR)检测不同时间组VEGF mRNA的表达,采用酶联免疫吸附测定法(ELISA)检测不同时间组VEGF蛋白的表达. 结果 人BMSCs经诱导后ALP染色大部分呈阳性反应.0、50、100、150、200nmol/L血管紧张素Ⅱ处理的BMSCs VEGF mRNA表达平均分别为1.000±0.000、2.304±0.315、4.336±0.400、5.573±0.608、5.492±0.460,其中0、50 nmol/L浓度组分别与100、150、200 nmol/L浓度组比较差异均有统计学意义(P＜0.05).选取150 nmol/L为血管紧张素Ⅱ的最佳作用浓度.0、6、12、24、48 h组细胞VEGF mRNA表达平均分别为1.000±0.000、1.984±0.160、4.372±0.378、5.573±0.586、4.994±0.445,其中0、6h组分别与12、24、48 h组比较差异均有统计学意义(P＜0.05).0、6、12、24、48 h组细胞VEGF蛋白分泌量逐渐增加,且呈时间依赖性. 结论 用血管紧张素Ⅱ处理人BMSCs源性成骨细胞能够使VEGF mRNA表达量和蛋白分泌量快速增加.
目的 探討血管緊張素Ⅱ對人骨髓基質榦細胞( BMSCs)源性成骨細胞血管內皮生長因子(VEGF)基因錶達及蛋白分泌的影響. 方法 分離、培養人BMSCs,併嚮成骨細胞方嚮誘導,用堿性燐痠酶(ALP)染色鑒定成骨細胞.取誘導14d的人BMSCs,分成5組(n=3),分彆用0、50、100、150、200 nmol/L血管緊張素Ⅱ處理細胞,以確定最佳作用濃度.取誘導21d的人BMSCs,分成5組(n=6),分彆用最佳作用濃度血管緊張素Ⅱ處理細胞0、6、12、24、48 h,採用實時定量逆轉錄-聚閤酶鏈反應( RT-PCR)檢測不同時間組VEGF mRNA的錶達,採用酶聯免疫吸附測定法(ELISA)檢測不同時間組VEGF蛋白的錶達. 結果 人BMSCs經誘導後ALP染色大部分呈暘性反應.0、50、100、150、200nmol/L血管緊張素Ⅱ處理的BMSCs VEGF mRNA錶達平均分彆為1.000±0.000、2.304±0.315、4.336±0.400、5.573±0.608、5.492±0.460,其中0、50 nmol/L濃度組分彆與100、150、200 nmol/L濃度組比較差異均有統計學意義(P＜0.05).選取150 nmol/L為血管緊張素Ⅱ的最佳作用濃度.0、6、12、24、48 h組細胞VEGF mRNA錶達平均分彆為1.000±0.000、1.984±0.160、4.372±0.378、5.573±0.586、4.994±0.445,其中0、6h組分彆與12、24、48 h組比較差異均有統計學意義(P＜0.05).0、6、12、24、48 h組細胞VEGF蛋白分泌量逐漸增加,且呈時間依賴性. 結論 用血管緊張素Ⅱ處理人BMSCs源性成骨細胞能夠使VEGF mRNA錶達量和蛋白分泌量快速增加.
목적 탐토혈관긴장소Ⅱ대인골수기질간세포( BMSCs)원성성골세포혈관내피생장인자(VEGF)기인표체급단백분비적영향. 방법 분리、배양인BMSCs,병향성골세포방향유도,용감성린산매(ALP)염색감정성골세포.취유도14d적인BMSCs,분성5조(n=3),분별용0、50、100、150、200 nmol/L혈관긴장소Ⅱ처리세포,이학정최가작용농도.취유도21d적인BMSCs,분성5조(n=6),분별용최가작용농도혈관긴장소Ⅱ처리세포0、6、12、24、48 h,채용실시정량역전록-취합매련반응( RT-PCR)검측불동시간조VEGF mRNA적표체,채용매련면역흡부측정법(ELISA)검측불동시간조VEGF단백적표체. 결과 인BMSCs경유도후ALP염색대부분정양성반응.0、50、100、150、200nmol/L혈관긴장소Ⅱ처리적BMSCs VEGF mRNA표체평균분별위1.000±0.000、2.304±0.315、4.336±0.400、5.573±0.608、5.492±0.460,기중0、50 nmol/L농도조분별여100、150、200 nmol/L농도조비교차이균유통계학의의(P＜0.05).선취150 nmol/L위혈관긴장소Ⅱ적최가작용농도.0、6、12、24、48 h조세포VEGF mRNA표체평균분별위1.000±0.000、1.984±0.160、4.372±0.378、5.573±0.586、4.994±0.445,기중0、6h조분별여12、24、48 h조비교차이균유통계학의의(P＜0.05).0、6、12、24、48 h조세포VEGF단백분비량축점증가,차정시간의뢰성. 결론 용혈관긴장소Ⅱ처리인BMSCs원성성골세포능구사VEGF mRNA표체량화단백분비량쾌속증가.
Objective To study the effect of Angiotensin Ⅱ (Ang Ⅱ) on the expression and protein secretion of vascular endothelial growth factor (VEGF) in human bone marrow stromal cells (hBMSCs) derived osteoblasts. Methods hBMSCs were isolated,cultured and induced to differentiate into osteoblasts.The cells were identified by alkaline phosphatase (ALP) staining.The hBMSCs induced for 14 days were divided into 5 groups ( n =3) and treated by Ang Ⅱ at concentrations of 50,100,150 and 200 nmol/L to determine the optimal one.The hBMSCs induced for21 days were divided into 5 groups ( n =6) and treated by Ang Ⅱ at the optimal concentration for 0,6, 12,24,48 hours.The level of mRNA VEGF was determined by SYBRGREN fluorescent quantitation and the level of VEGF protein in the medium was measured by ELISA.Results When the hBMSCs were cultured in the conditioned culture medium,most of the ALP staining was positive.The expressions of mRNA VEGF in the hBMSCs treated by Ang Ⅱ at concentrations of 0, 50, 100, 150 and 200 nmol/L were respectively 1.000±0.000,2.304±0.315, 4.336t0.400,5.573 ± 0.608 and 5.492 ± 0.460. There were significant differences regarding the expression levels respectively between the first 2 concentrations and the Iatter 3 concentrations ( P ＜ 0.05).The concentration of 150 nmol/L was used as the optimal one to treat Ang Ⅱ.The expressions of mRNA VEGF in the hBMSCs treated by Ang Ⅱ at the optimal concentration for 0,6,12,24,48 hours were respectively 1.000 ± 0.000,1.984 ± 0.160,4.372 ± 0.378,5.573 ± 0.586 and 4.994 ± 0.445.There were significant differences regarding the expression levels respectively between the first 2 time points and the latter 3 time points ( P ＜0.05).The protein secretions by VEGF induced by Ang Ⅱ at the optimal concentration for 0,6,12,24,48hours were increased in a time-dependent manner. Conclusion Incubation of hBMSCs derived osteoblasts with Ang Ⅱ may induce a rapid increase in both VEGF mRNA expression and protein synthesis.
