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
2009年
27期
5291-5295
,共5页
孙良%栾保华%李中华%王小霞%刘萌萌
孫良%欒保華%李中華%王小霞%劉萌萌
손량%란보화%리중화%왕소하%류맹맹
外周血间充质干细胞%培养基%α-MEM%L-DMEM%成骨诱导
外週血間充質榦細胞%培養基%α-MEM%L-DMEM%成骨誘導
외주혈간충질간세포%배양기%α-MEM%L-DMEM%성골유도
背景:研究证明外周血中存在间充质干细胞,但含量极少.目的:拟从兔外周血中提取间充质干细胞,并诱导其向成骨细胞分化.设计、时间及地点:细胞学体外观察,于2008-06/12在山东省立医院中心实验室完成.材料:新两兰大白兔6只,购自山东省农科院.α-MEM培养基、L-DMEM培养基为Hyclone公司产品.方法:每隔2 d从兔背部不同部位切去3 cm×3 cm的全层皮肤(保留背部肌层),所切皮肤做原何移植后包扎,每只兔连续创伤4次.分别于创伤前及末次创伤1周后从股静脉取外周血,采用密度梯度离心法获取间充质干细胞,设立2组,分别加入含体积分数为10%胎生血清的α-MEM培养基或L-DMEM培养基.细胞传至第2代以1×105cm2密度接种于12孔板,加入含成骨诱导剂的H-DMEM培养液进行成骨诱导.主要观察指标:创伤前后细胞形态观察,成纤维样细胞集落形成率,成骨诱导效果.结果:创伤前所获得的细胞首次换液后,未见有梭形细胞贴壁;创伤后所获得的细胞接种24 h即可见有短梭形及多角形细胞贴壁,五六天后出现细胞集落.与L-DMEM培养基组比较,α-MEM培养基组外周血成纤维样细胞集落形成率明显升高(P<0.05).外周血间充质干细胞成骨诱导后,呈梭形、三角形或多角形,有突起,可见两三个核仁和细胞分裂相,增殖速度缓慢,诱导7 d后碱性磷酸酶阳性率达80%以上,诱导21 d后茜素红染色形成钙结节.结论:皮肤损伤刺激后可成功从兔外周血中获取间充质干细胞,且具备成骨分化特性,α-MEM培养基较L-DMEM培养基更适合外周血间充质干细胞的体外生长.
揹景:研究證明外週血中存在間充質榦細胞,但含量極少.目的:擬從兔外週血中提取間充質榦細胞,併誘導其嚮成骨細胞分化.設計、時間及地點:細胞學體外觀察,于2008-06/12在山東省立醫院中心實驗室完成.材料:新兩蘭大白兔6隻,購自山東省農科院.α-MEM培養基、L-DMEM培養基為Hyclone公司產品.方法:每隔2 d從兔揹部不同部位切去3 cm×3 cm的全層皮膚(保留揹部肌層),所切皮膚做原何移植後包扎,每隻兔連續創傷4次.分彆于創傷前及末次創傷1週後從股靜脈取外週血,採用密度梯度離心法穫取間充質榦細胞,設立2組,分彆加入含體積分數為10%胎生血清的α-MEM培養基或L-DMEM培養基.細胞傳至第2代以1×105cm2密度接種于12孔闆,加入含成骨誘導劑的H-DMEM培養液進行成骨誘導.主要觀察指標:創傷前後細胞形態觀察,成纖維樣細胞集落形成率,成骨誘導效果.結果:創傷前所穫得的細胞首次換液後,未見有梭形細胞貼壁;創傷後所穫得的細胞接種24 h即可見有短梭形及多角形細胞貼壁,五六天後齣現細胞集落.與L-DMEM培養基組比較,α-MEM培養基組外週血成纖維樣細胞集落形成率明顯升高(P<0.05).外週血間充質榦細胞成骨誘導後,呈梭形、三角形或多角形,有突起,可見兩三箇覈仁和細胞分裂相,增殖速度緩慢,誘導7 d後堿性燐痠酶暘性率達80%以上,誘導21 d後茜素紅染色形成鈣結節.結論:皮膚損傷刺激後可成功從兔外週血中穫取間充質榦細胞,且具備成骨分化特性,α-MEM培養基較L-DMEM培養基更適閤外週血間充質榦細胞的體外生長.
배경:연구증명외주혈중존재간충질간세포,단함량겁소.목적:의종토외주혈중제취간충질간세포,병유도기향성골세포분화.설계、시간급지점:세포학체외관찰,우2008-06/12재산동성립의원중심실험실완성.재료:신량란대백토6지,구자산동성농과원.α-MEM배양기、L-DMEM배양기위Hyclone공사산품.방법:매격2 d종토배부불동부위절거3 cm×3 cm적전층피부(보류배부기층),소절피부주원하이식후포찰,매지토련속창상4차.분별우창상전급말차창상1주후종고정맥취외주혈,채용밀도제도리심법획취간충질간세포,설립2조,분별가입함체적분수위10%태생혈청적α-MEM배양기혹L-DMEM배양기.세포전지제2대이1×105cm2밀도접충우12공판,가입함성골유도제적H-DMEM배양액진행성골유도.주요관찰지표:창상전후세포형태관찰,성섬유양세포집락형성솔,성골유도효과.결과:창상전소획득적세포수차환액후,미견유사형세포첩벽;창상후소획득적세포접충24 h즉가견유단사형급다각형세포첩벽,오륙천후출현세포집락.여L-DMEM배양기조비교,α-MEM배양기조외주혈성섬유양세포집락형성솔명현승고(P<0.05).외주혈간충질간세포성골유도후,정사형、삼각형혹다각형,유돌기,가견량삼개핵인화세포분렬상,증식속도완만,유도7 d후감성린산매양성솔체80%이상,유도21 d후천소홍염색형성개결절.결론:피부손상자격후가성공종토외주혈중획취간충질간세포,차구비성골분화특성,α-MEM배양기교L-DMEM배양기경괄합외주혈간충질간세포적체외생장.
BACKGROUND: A few mesenchymal stem cells (MSCs) can be found in peripheral blood.OBJECTIVE: To isolate rabbit peripheral blood MSCs and induce its differentiation into osteoblasts.DESIGN, TIME AND SE'I-I'ING: The cytological in vitro study was performed at the Central Laboratory of Shangdong Provincial Hospital from June to December 2008.MATERIALS: A total of 6 New Zealand rabbits were purchased from Shandong Academy of Agricultural Sciences. α-MEM and L-DiEM were bought from Hyclone.METHODS: Full-thickness skin (3 cm×3 cm) (dorsal muscular layer was left) was incised at various sites of rabbit back, every 3days. Incised skin was dressed following orthotopic transplantation. Each rabbit received four consecutive wounds. Peripheral blood was collected from femoral vein before injury and 1 week after injury. MSCs were harvested from peripheral blood by density gradient cantrifugation. MSCs were divided into 2 groups, which were respectively incubated in α-MEM supplemented with 10% fetal bovine serum and L-DiEM. Cells at passage 2 and 1 ×105/cm2 were incubated in a 12-well plate and induced with H-DiEM containing osteogenic inductor.MAIN OUTCOME MEASURES: The following parameters were measured: cell morphology before and after injury; colony forming efficiency of MSCs; outcome of osteogenic induction.RESULTS: Following primary medium change and before injury, obtained cells were normal. Twenty-four hours following incubation and after injury, MSCs were spindle or polygonal, and adhered to the wall. 5-6 days later, cell colonies appeared.Compared with the L-DiEM group, the number of primary culture colony formation in α-MEM group was significantly greater (P < 0.05). Peripheral blood MSCs were spindle, tdangle or polygonal, with the presence of processes, 2-3 nuclei and cell division phase, and slowly proliferated following osteogenic induction. At day 7 after differentiation of MSCs into osteoblasts, positive rate of alkaline phosphates was above 80%. At day 21, calcium deposition was detected by Alizarin Red S (positive staining).CONCLUSION: MSCs could be harvested from peripheral blood of wounded rabbits, with characteristics of osteogenic differentiation, α -MEM was more suitable than L-DiEM for peripheral blood MSCs to growin vitro.
