CAJ | 학술논문

背景:骨髓间充质干细胞取材方便,易于体外扩增,其体外培养方法及诱导分化仍需要改良.目的:对大鼠骨髓间充质干细胞体外培养方法及诱导剂配比进行改良,观察骨髓间充质干细胞向成骨细胞分化的潜能.设计:观察实验.单位:解放军广州军区广州总医院医学实验科.材料:实验于2004-07/2006-06在解放军广州军区总医院医学实验科干细胞组织工程实验室完成,选用20只成年SD大鼠,清洁级,雌雄不拘,体质量140～180 g,由解放军广州军区广州总医院动物实验中心提供.实验过程中对动物的处置符合动物伦理学标准.β-甘油磷酸纳,地塞米松,维生素C均为美国Sigma公司产品,羊抗大鼠骨钙素抗体购自美国DSL公司,S-P免疫组化试剂盒为福建迈新生物技术开发有限公司产品.方法:采用改良法进行细胞的培养及诱导细胞成骨.①骨髓间充质干细胞分离及培养:大鼠麻醉后处死分离双侧股骨、胫骨骨髓制备单细胞悬液接种于培养瓶中,培养后48及96 h半量更换培养液,以去除未贴壁的造血细胞,以后每3 d换液1次,进一步去除未贴壁的细胞,待细胞汇合约80%时,用0.25%胰蛋白酶消化,按1:2传代培养.将第2代骨髓间充质干细胞接种于6孔培养板和玻璃平皿中,48 h后吸去基础培养液.②骨髓间充质干细胞向成骨细胞诱导分化:应用含终浓度分别为10 mmol/L、10-7 mol/L、50 mg/L的地塞米松、β-甘油磷酸纳和维生素C的诱导分化培养液定向诱导传代细胞向成骨细胞分化.主要观察指标:①应用改良的培养方法及自行配比的培养液诱导分化后10 d采用钙钴法测定碱性磷酸酶活性.②培养后12 d采用免疫组织化学法检测骨钙素分泌情况.③诱导培养后2周采用Von kossa染色检测细胞矿化作用.结果:①碱性磷酸酶活性:经诱导后细胞碱性磷酸酶染色明显,胞质中刚性反应呈现灰黑色颗粒或块状沉淀,碱性磷酸酶活性部位呈棕黑色.②骨钙素分泌情况:细胞经诱导后骨钙素阳性较明显,胞核呈蓝色,胞浆呈棕色.③细胞矿化作用:细胞经诱导培养后呈复层生长,并出现不透明结节,Von kossa染色可见黑色的矿化结节颗粒,颗粒大小不均一,提示有矿化基质沉积.结论:改良法可成功培养及诱导大鼠骨髓间充质干细胞成骨. 揹景:骨髓間充質榦細胞取材方便,易于體外擴增,其體外培養方法及誘導分化仍需要改良.目的:對大鼠骨髓間充質榦細胞體外培養方法及誘導劑配比進行改良,觀察骨髓間充質榦細胞嚮成骨細胞分化的潛能.設計:觀察實驗.單位:解放軍廣州軍區廣州總醫院醫學實驗科.材料:實驗于2004-07/2006-06在解放軍廣州軍區總醫院醫學實驗科榦細胞組織工程實驗室完成,選用20隻成年SD大鼠,清潔級,雌雄不拘,體質量140～180 g,由解放軍廣州軍區廣州總醫院動物實驗中心提供.實驗過程中對動物的處置符閤動物倫理學標準.β-甘油燐痠納,地塞米鬆,維生素C均為美國Sigma公司產品,羊抗大鼠骨鈣素抗體購自美國DSL公司,S-P免疫組化試劑盒為福建邁新生物技術開髮有限公司產品.方法:採用改良法進行細胞的培養及誘導細胞成骨.①骨髓間充質榦細胞分離及培養:大鼠痳醉後處死分離雙側股骨、脛骨骨髓製備單細胞懸液接種于培養瓶中,培養後48及96 h半量更換培養液,以去除未貼壁的造血細胞,以後每3 d換液1次,進一步去除未貼壁的細胞,待細胞彙閤約80%時,用0.25%胰蛋白酶消化,按1:2傳代培養.將第2代骨髓間充質榦細胞接種于6孔培養闆和玻璃平皿中,48 h後吸去基礎培養液.②骨髓間充質榦細胞嚮成骨細胞誘導分化:應用含終濃度分彆為10 mmol/L、10-7 mol/L、50 mg/L的地塞米鬆、β-甘油燐痠納和維生素C的誘導分化培養液定嚮誘導傳代細胞嚮成骨細胞分化.主要觀察指標:①應用改良的培養方法及自行配比的培養液誘導分化後10 d採用鈣鈷法測定堿性燐痠酶活性.②培養後12 d採用免疫組織化學法檢測骨鈣素分泌情況.③誘導培養後2週採用Von kossa染色檢測細胞礦化作用.結果:①堿性燐痠酶活性:經誘導後細胞堿性燐痠酶染色明顯,胞質中剛性反應呈現灰黑色顆粒或塊狀沉澱,堿性燐痠酶活性部位呈棕黑色.②骨鈣素分泌情況:細胞經誘導後骨鈣素暘性較明顯,胞覈呈藍色,胞漿呈棕色.③細胞礦化作用:細胞經誘導培養後呈複層生長,併齣現不透明結節,Von kossa染色可見黑色的礦化結節顆粒,顆粒大小不均一,提示有礦化基質沉積.結論:改良法可成功培養及誘導大鼠骨髓間充質榦細胞成骨.
배경:골수간충질간세포취재방편,역우체외확증,기체외배양방법급유도분화잉수요개량.목적:대대서골수간충질간세포체외배양방법급유도제배비진행개량,관찰골수간충질간세포향성골세포분화적잠능.설계:관찰실험.단위:해방군엄주군구엄주총의원의학실험과.재료:실험우2004-07/2006-06재해방군엄주군구총의원의학실험과간세포조직공정실험실완성,선용20지성년SD대서,청길급,자웅불구,체질량140～180 g,유해방군엄주군구엄주총의원동물실험중심제공.실험과정중대동물적처치부합동물윤리학표준.β-감유린산납,지새미송,유생소C균위미국Sigma공사산품,양항대서골개소항체구자미국DSL공사,S-P면역조화시제합위복건매신생물기술개발유한공사산품.방법:채용개량법진행세포적배양급유도세포성골.①골수간충질간세포분리급배양:대서마취후처사분리쌍측고골、경골골수제비단세포현액접충우배양병중,배양후48급96 h반량경환배양액,이거제미첩벽적조혈세포,이후매3 d환액1차,진일보거제미첩벽적세포,대세포회합약80%시,용0.25%이단백매소화,안1:2전대배양.장제2대골수간충질간세포접충우6공배양판화파리평명중,48 h후흡거기출배양액.②골수간충질간세포향성골세포유도분화:응용함종농도분별위10 mmol/L、10-7 mol/L、50 mg/L적지새미송、β-감유린산납화유생소C적유도분화배양액정향유도전대세포향성골세포분화.주요관찰지표:①응용개량적배양방법급자행배비적배양액유도분화후10 d채용개고법측정감성린산매활성.②배양후12 d채용면역조직화학법검측골개소분비정황.③유도배양후2주채용Von kossa염색검측세포광화작용.결과:①감성린산매활성:경유도후세포감성린산매염색명현,포질중강성반응정현회흑색과립혹괴상침정,감성린산매활성부위정종흑색.②골개소분비정황:세포경유도후골개소양성교명현,포핵정람색,포장정종색.③세포광화작용:세포경유도배양후정복층생장,병출현불투명결절,Von kossa염색가견흑색적광화결절과립,과립대소불균일,제시유광화기질침적.결론:개량법가성공배양급유도대서골수간충질간세포성골.
BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are in advantages of easy collection and amplification in vitro, but the culture and induction in vitro still need to be modified. OBJECTIVE: To investigate the differentiated potency of BMSCs into ostcoblasts by modified in vitro culture methods and inductor matching. DESIGN: Observational study. SETTING: Department of Medical Laboratory, General Hospital of Guangzhou Military Area Command of Chinese PLA. MATERIALS: This study was performed in the Stem Cell Tissue Engineering Laboratory, Department of Medical Laboratory, Genera Hospital of Guangzhou Military Area Command of Chinese PLA from July 2004 to June 2006. Twenty adult SD rats of clean grade, irrespective of gender, weighing 140-180 g were provided by Animal Experimental Center, General Hospital of Guangzhou Military Area Command of Chinese PLA. The experimental animals were disposed according to ethical criteria. Β-sodium glycerophosphate, dexamethasone, and vitamin C were provided by Sigma Company, USA; goat-anti-rat osteocalcin antibody by DSL Company, USA; S-P immunohistochemical kit by Maixin Biotechnology Developing Co., Ltd., Fujian. METHODS: Cells were cultured and induced into osteoblasts by modified methods. Separation and culture of BMSCs: By anesthesia, bone marrow of bilateral femur and tibia was separated to prepare single cell suspension; subsequently, the suspension was inoculated in culture bottle, and the culture media was semi-quantitatively changed after 48 and 96 hours in order to get rid of non-adherent hematopoietic cells. The liquid was changed every three days to further get rid of non-adherent cells. At 80% cell confluence, the medium was digested with 0.25% trypsin and cells were subcultured in the ratio of 1:2. MSCs in the second generation were inoculated in 6-well culture plate and glass fiat plate; after 48 hours, basic culture fluid was removed. Differentiation of BMSCs into osteoblasts: Subcultured BMSCs differentiated into osteoblasts induced by dexamethasone (10 mmol/L), β-sodium glycerophosphate (10-7 mol/L), and vitamin C (50 mg/L). MAIN OUTCOME MEASURES: Ten days after differentiation by modified culture methods and inductor matching, alkaline phosphatase activity was measured with Ca-Co technique. Twelve days after culture, osteocalcin secretion was detected with immunohistochemical method. Two weeks after culture, cell mineralization was detected with Von kossa staining. RESULTS: Alkaline phosphatase activity: Alkaline phosphatase staining of cells was apparent; gray-black particles or massive precipitations were observed in cytoplasm after positive reaction; regions expressing alkaline phosphatase activity were brown-black. Osteocalcin secretion: Osteocalcin was apparently positive; nucleus was blue; cytoplasm was brown. Cell mineralization: Induced cells grew layer by layer, and adiaphanous nodus was observed at the same time. Black mineralized nodus granules in various sizes were observed after Von kossa staining, and this suggested that mineralized apposition occurred. CONCLUSION: BMSCs may be successfully cultured and induced into osteoblasts by modified culture method.