中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2008年
38期
7582-7586
,共5页
林海泓%施海明%肖萍%朱军%罗心平
林海泓%施海明%肖萍%硃軍%囉心平
림해홍%시해명%초평%주군%라심평
骨髓间充质干细胞%抗血小板药物%分化%增殖%分泌
骨髓間充質榦細胞%抗血小闆藥物%分化%增殖%分泌
골수간충질간세포%항혈소판약물%분화%증식%분비
背景:有研究指出氯吡格雷、噻氯匹定可引起粒细胞减少,阿司匹林可抑制内皮祖细胞增殖.目前缺乏这些抗血小板药物会对移植入心肌的骨髓间充质干细胞产生何种作用的报道.目的:探讨氯吡格雷、噻氯匹定和阿司匹林对人骨髓间充质干细胞增殖及分泌功能的影响.设计、时间和地点:细胞学体外观察,于2006-03/12在上海市疾病预防控制中心毒理学实验室完成.材料:第2代人骨髓间充质干细胞由上海第九人民医院组织工程研究中心提供.氯吡格雷、噻氯匹定为杭州赛诺菲圣德拉堡民生制药有限公司产品,批号分别为国药准字J20040006和国药准字H19980186.阿司匹林由拜耳医药有限公司生产,批号为国药准字20050059.方法:骨髓间充质干细胞解冻后,加入含 100 U/mL青霉素、100 mg/L链霉素、10%胎牛血清的低糖DMEM体外扩增培养,传至第6代,分别用不同浓度的氯吡格雷、噻氯匹定、阿司匹林进行干预处理,并设立空白对照.主要观察指标:MTT比色法检测细胞增殖情况,流式细胞仪检测细胞表面抗原表达,ELISA法测定细胞培养液中血管内皮生长因子浓度.结果:反映骨髓间充质干细胞增殖变化的A570值在0.02,0.1,0.4,2,10,40 μmol/L氯吡格雷或噻氯匹定作用后均明显高于空白对照(P < 0.01);经60,600,2 000 μmol/L阿司匹林处理后则低于空白对照(P < 0.05).细胞表面抗原CD34、CD105、CD106阳性细胞百分率经3种抗血小板药物作用后与空白对照基本相似.与空白对照比较,经 0.02 μmol/L氯吡格雷及5,2 000μmol/L阿司匹林处理后血管内皮生长因子含量无明显变化(P > 0.05);40 μmol/L氯吡格雷或噻氯匹定作用后血管内皮生长因子含量均显著降低;0.02 μmol/L噻氯匹定作用后血管内皮生长因子含量显著升高(P < 0.01).结论:氯吡格雷和噻氯匹定对人骨髓间充质干细胞具有明显的促增殖作用,阿司匹林可抑制其增殖.高浓度氯吡格雷和噻氯匹定能够抑制人骨髓间充质干细胞分泌血管内皮生长因子,低浓度噻氯匹定可促进其分泌.此3种抗血小板药物对人骨髓间充质干细胞的进一步分化无明显影响.
揹景:有研究指齣氯吡格雷、噻氯匹定可引起粒細胞減少,阿司匹林可抑製內皮祖細胞增殖.目前缺乏這些抗血小闆藥物會對移植入心肌的骨髓間充質榦細胞產生何種作用的報道.目的:探討氯吡格雷、噻氯匹定和阿司匹林對人骨髓間充質榦細胞增殖及分泌功能的影響.設計、時間和地點:細胞學體外觀察,于2006-03/12在上海市疾病預防控製中心毒理學實驗室完成.材料:第2代人骨髓間充質榦細胞由上海第九人民醫院組織工程研究中心提供.氯吡格雷、噻氯匹定為杭州賽諾菲聖德拉堡民生製藥有限公司產品,批號分彆為國藥準字J20040006和國藥準字H19980186.阿司匹林由拜耳醫藥有限公司生產,批號為國藥準字20050059.方法:骨髓間充質榦細胞解凍後,加入含 100 U/mL青黴素、100 mg/L鏈黴素、10%胎牛血清的低糖DMEM體外擴增培養,傳至第6代,分彆用不同濃度的氯吡格雷、噻氯匹定、阿司匹林進行榦預處理,併設立空白對照.主要觀察指標:MTT比色法檢測細胞增殖情況,流式細胞儀檢測細胞錶麵抗原錶達,ELISA法測定細胞培養液中血管內皮生長因子濃度.結果:反映骨髓間充質榦細胞增殖變化的A570值在0.02,0.1,0.4,2,10,40 μmol/L氯吡格雷或噻氯匹定作用後均明顯高于空白對照(P < 0.01);經60,600,2 000 μmol/L阿司匹林處理後則低于空白對照(P < 0.05).細胞錶麵抗原CD34、CD105、CD106暘性細胞百分率經3種抗血小闆藥物作用後與空白對照基本相似.與空白對照比較,經 0.02 μmol/L氯吡格雷及5,2 000μmol/L阿司匹林處理後血管內皮生長因子含量無明顯變化(P > 0.05);40 μmol/L氯吡格雷或噻氯匹定作用後血管內皮生長因子含量均顯著降低;0.02 μmol/L噻氯匹定作用後血管內皮生長因子含量顯著升高(P < 0.01).結論:氯吡格雷和噻氯匹定對人骨髓間充質榦細胞具有明顯的促增殖作用,阿司匹林可抑製其增殖.高濃度氯吡格雷和噻氯匹定能夠抑製人骨髓間充質榦細胞分泌血管內皮生長因子,低濃度噻氯匹定可促進其分泌.此3種抗血小闆藥物對人骨髓間充質榦細胞的進一步分化無明顯影響.
배경:유연구지출록필격뢰、새록필정가인기립세포감소,아사필림가억제내피조세포증식.목전결핍저사항혈소판약물회대이식입심기적골수간충질간세포산생하충작용적보도.목적:탐토록필격뢰、새록필정화아사필림대인골수간충질간세포증식급분비공능적영향.설계、시간화지점:세포학체외관찰,우2006-03/12재상해시질병예방공제중심독이학실험실완성.재료:제2대인골수간충질간세포유상해제구인민의원조직공정연구중심제공.록필격뢰、새록필정위항주새낙비골덕랍보민생제약유한공사산품,비호분별위국약준자J20040006화국약준자H19980186.아사필림유배이의약유한공사생산,비호위국약준자20050059.방법:골수간충질간세포해동후,가입함 100 U/mL청매소、100 mg/L련매소、10%태우혈청적저당DMEM체외확증배양,전지제6대,분별용불동농도적록필격뢰、새록필정、아사필림진행간예처리,병설립공백대조.주요관찰지표:MTT비색법검측세포증식정황,류식세포의검측세포표면항원표체,ELISA법측정세포배양액중혈관내피생장인자농도.결과:반영골수간충질간세포증식변화적A570치재0.02,0.1,0.4,2,10,40 μmol/L록필격뢰혹새록필정작용후균명현고우공백대조(P < 0.01);경60,600,2 000 μmol/L아사필림처리후칙저우공백대조(P < 0.05).세포표면항원CD34、CD105、CD106양성세포백분솔경3충항혈소판약물작용후여공백대조기본상사.여공백대조비교,경 0.02 μmol/L록필격뢰급5,2 000μmol/L아사필림처리후혈관내피생장인자함량무명현변화(P > 0.05);40 μmol/L록필격뢰혹새록필정작용후혈관내피생장인자함량균현저강저;0.02 μmol/L새록필정작용후혈관내피생장인자함량현저승고(P < 0.01).결론:록필격뢰화새록필정대인골수간충질간세포구유명현적촉증식작용,아사필림가억제기증식.고농도록필격뢰화새록필정능구억제인골수간충질간세포분비혈관내피생장인자,저농도새록필정가촉진기분비.차3충항혈소판약물대인골수간충질간세포적진일보분화무명현영향.
BACKGROUND: Results from clinical trials suggested that clopidogrel and ticlopidine had side effects of granulopenia, and aspirin could inhibit endothelial progenitor cell proliferation. There is no report of effects of these drugs on human bone marrow mesenchymal stem cells (hBMSCs) in stem cell transplantation. OBJECTIVE: To investigate the effects of antiplatelet drugs including clopidogrel, ticlopidine and aspirin on hBMSC proliferation and secretion. DESIGN, TIME AND SETTING: The cytology in vitro observation was performed at the Laboratory of Toxicology, Shanghai Municipal Center for Disease Control and Prevention from March to December 2006.MATERIALS: The second passage of hBMSCs was kindly donated from Shanghai Tissue Engineering Research & Development Center, Shanghai Ninth People's Hospital. Clopidogrel (Lot number J20040006) and ticlopidine (Lot number H19980186) were obtained from Hangzhou Sanofi-Synthelabo Minsheng Pharmaceutical CO., Ltd. Aspirin (Lot number 20050059) was obtained from Bayer Vital GmbH. METHODS: The standard culture medium consisted of DMEM-LG, 10% heat-inactivated FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. After being cultured in vitro expanded out to passage 6, hBMSCs were treated with antiplatelet drugs of different concentrations and compared with control group. MAIN OUTCOME MEASURES: Cell proliferation was assessed by 3- (4, 5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, level of vascular endothelial growth factor (VEGF) of culture medium was detected by enzyme-linked immunoadsordent assay (ELISA), and surface antigens of hBMSCs were analyzed by the flow cytometry. RESULTS: A570 values of hBMSCs treated by clopidogrel or ticlopidine of 0.02,0.1,0.4,2,10,40 μmol/L were higher than control group (P < 0.01), while A570 values of aspirin group of 60, 600, 2 000 μmol/L were lower than control group(P < 0.05). Antiplatelet drugs had no obvious effect on cell surface antigens(CD34, CD105, CD106)expressed by hBMSCs. Treated by high dose clopidogrel or ticlopidine (40 μmol/L), VEGF level from hMSCs was lower than that of control group(P < 0.01), but VEGF level of low dose (0.02 μmol/L) ticlopidine group was higher than control group(P < 0.01), and there was no significantly difference of VEGF level among low dose clopidogrel group (0.02 μmol/L), aspirin group (5, 2 000 μmol/L), and control group(P > 0.05). CONCLUSION: Clopidogrel and ticlopidine improve proliferation of hBMSCs, but aspirin inhibits proliferation of hBMSCs. High dose of clopidogrel and ticlopidine suppress VEGF secretion of hBMSCs, while low dose of ticlopidine promote it. Antiplatelet drugs have no obvious effect on hBMSCs differentiation.
