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
45期
8955-8960
,共6页
陆华%苗宗宁%吴卫江%蒋云召%葛风%房文峰%徐杰%朱爱华%陈革%周建宏%陆爻忠%唐志放%王泳
陸華%苗宗寧%吳衛江%蔣雲召%葛風%房文峰%徐傑%硃愛華%陳革%週建宏%陸爻忠%唐誌放%王泳
륙화%묘종저%오위강%장운소%갈풍%방문봉%서걸%주애화%진혁%주건굉%륙효충%당지방%왕영
骨髓间充质干细胞%免疫调节%神经元样细胞%分化
骨髓間充質榦細胞%免疫調節%神經元樣細胞%分化
골수간충질간세포%면역조절%신경원양세포%분화
背景:骨髓间充质干细胞作为具有多向分化潜能的干细胞,属于未分化的前体干细胞,其表型分化尚不成熟,因此在同种异体移植后无排斥反应或反应较弱.但是在体外条件下通过化学和生物诱导物使骨髓间充质干细胞转化为神经元,影响骨髓间充质干细胞向神经细胞、胶质细胞系统定向分化的因素比较复杂.目的:观察人骨髓间充质干细胞的免疫调节作用,及向神经元样细胞诱导分化的能力.设计、时间及地点:对比观察,于2008-01/2009-03在无锡市第三人民医院细胞室完成.材料:骨髓来源于人髋关节手术时的松质骨碎片或髂骨游离移植.方法:首先分离和培养骨髓间充质干细胞,建立双向混合淋巴细胞反应体系,在双向混合淋巴细胞反应中,将来自2个志愿者的外周血单个核细胞(各1×10~5/孔)等比例加入96孔板中,根据不同效靶比E/T ratios(骨髓间充质干细胞/外周血单个核细胞)加入丝裂霉紊处理过的骨髓间充质干细胞.在骨髓间充质干细胞诱导分化为神经元样细胞实验中分别选择两种诱导培养基,方法1诱导:DMEM+体积分数为10%胎牛血清+1 μmol/L全反式维甲酸+20 μg/L 碱性成纤维细胞生长因子+20 μg/L表皮生长因子;方法2诱导:DMEM+2%二甲基亚砜+100 μmol/L 丁羟茴醚.主要观察指标:~3H掺入法测定淋巴细胞增殖率,观察骨髓间充质干细胞对淋巴细胞增殖的影响;通过条件培养基定向诱导,免疫荧光和免疫组织化学染色观察其分化为神经细胞的能力.结果:①骨髓间充质干细胞与混合淋巴细胞反应体系共同培养抑制了淋巴细胞增殖,其增殖抑制率与加入骨髓间充质干细胞的数量呈正比.②方法1诱导2 h后,光镜下可见骨髓间充质干细胞的细胞质向核收缩,呈典型核周体形态.3～5 h后多数细胞能形成神经元样细胞形态,但细胞数目无明显增加.3 d后大多数细胞转变为双极或多极神经元细胞样形态,部分细胞之间拉成网状.染色可见60%～70%神经元特异性烯醇化酶、45%～50%胶质纤维酸性蛋白阳性,巢蛋白阳性细胞下降为3.4%.方法2诱导2 h即可见细胞体积变小,形成双极或多极的细胞体,可持续诱导48 h,后大部分细胞浮起死亡.染色可见40%～50%神经元特异性烯醇化酶,35%～40%胶质纤维酸性蛋白阳性,巢蛋白阳性细胞在诱导2 h时一过性上升到63%,48 h时下降为1.6%.结论:骨髓间充质干细胞具有向神经细胞分化能力并且可以抑制混合淋巴细胞反应体系中淋巴细胞的增殖,对同种异体免疫反应具有负调节作用.
揹景:骨髓間充質榦細胞作為具有多嚮分化潛能的榦細胞,屬于未分化的前體榦細胞,其錶型分化尚不成熟,因此在同種異體移植後無排斥反應或反應較弱.但是在體外條件下通過化學和生物誘導物使骨髓間充質榦細胞轉化為神經元,影響骨髓間充質榦細胞嚮神經細胞、膠質細胞繫統定嚮分化的因素比較複雜.目的:觀察人骨髓間充質榦細胞的免疫調節作用,及嚮神經元樣細胞誘導分化的能力.設計、時間及地點:對比觀察,于2008-01/2009-03在無錫市第三人民醫院細胞室完成.材料:骨髓來源于人髖關節手術時的鬆質骨碎片或髂骨遊離移植.方法:首先分離和培養骨髓間充質榦細胞,建立雙嚮混閤淋巴細胞反應體繫,在雙嚮混閤淋巴細胞反應中,將來自2箇誌願者的外週血單箇覈細胞(各1×10~5/孔)等比例加入96孔闆中,根據不同效靶比E/T ratios(骨髓間充質榦細胞/外週血單箇覈細胞)加入絲裂黴紊處理過的骨髓間充質榦細胞.在骨髓間充質榦細胞誘導分化為神經元樣細胞實驗中分彆選擇兩種誘導培養基,方法1誘導:DMEM+體積分數為10%胎牛血清+1 μmol/L全反式維甲痠+20 μg/L 堿性成纖維細胞生長因子+20 μg/L錶皮生長因子;方法2誘導:DMEM+2%二甲基亞砜+100 μmol/L 丁羥茴醚.主要觀察指標:~3H摻入法測定淋巴細胞增殖率,觀察骨髓間充質榦細胞對淋巴細胞增殖的影響;通過條件培養基定嚮誘導,免疫熒光和免疫組織化學染色觀察其分化為神經細胞的能力.結果:①骨髓間充質榦細胞與混閤淋巴細胞反應體繫共同培養抑製瞭淋巴細胞增殖,其增殖抑製率與加入骨髓間充質榦細胞的數量呈正比.②方法1誘導2 h後,光鏡下可見骨髓間充質榦細胞的細胞質嚮覈收縮,呈典型覈週體形態.3～5 h後多數細胞能形成神經元樣細胞形態,但細胞數目無明顯增加.3 d後大多數細胞轉變為雙極或多極神經元細胞樣形態,部分細胞之間拉成網狀.染色可見60%～70%神經元特異性烯醇化酶、45%～50%膠質纖維痠性蛋白暘性,巢蛋白暘性細胞下降為3.4%.方法2誘導2 h即可見細胞體積變小,形成雙極或多極的細胞體,可持續誘導48 h,後大部分細胞浮起死亡.染色可見40%～50%神經元特異性烯醇化酶,35%～40%膠質纖維痠性蛋白暘性,巢蛋白暘性細胞在誘導2 h時一過性上升到63%,48 h時下降為1.6%.結論:骨髓間充質榦細胞具有嚮神經細胞分化能力併且可以抑製混閤淋巴細胞反應體繫中淋巴細胞的增殖,對同種異體免疫反應具有負調節作用.
배경:골수간충질간세포작위구유다향분화잠능적간세포,속우미분화적전체간세포,기표형분화상불성숙,인차재동충이체이식후무배척반응혹반응교약.단시재체외조건하통과화학화생물유도물사골수간충질간세포전화위신경원,영향골수간충질간세포향신경세포、효질세포계통정향분화적인소비교복잡.목적:관찰인골수간충질간세포적면역조절작용,급향신경원양세포유도분화적능력.설계、시간급지점:대비관찰,우2008-01/2009-03재무석시제삼인민의원세포실완성.재료:골수래원우인관관절수술시적송질골쇄편혹가골유리이식.방법:수선분리화배양골수간충질간세포,건립쌍향혼합림파세포반응체계,재쌍향혼합림파세포반응중,장래자2개지원자적외주혈단개핵세포(각1×10~5/공)등비례가입96공판중,근거불동효파비E/T ratios(골수간충질간세포/외주혈단개핵세포)가입사렬매문처리과적골수간충질간세포.재골수간충질간세포유도분화위신경원양세포실험중분별선택량충유도배양기,방법1유도:DMEM+체적분수위10%태우혈청+1 μmol/L전반식유갑산+20 μg/L 감성성섬유세포생장인자+20 μg/L표피생장인자;방법2유도:DMEM+2%이갑기아풍+100 μmol/L 정간회미.주요관찰지표:~3H참입법측정림파세포증식솔,관찰골수간충질간세포대림파세포증식적영향;통과조건배양기정향유도,면역형광화면역조직화학염색관찰기분화위신경세포적능력.결과:①골수간충질간세포여혼합림파세포반응체계공동배양억제료림파세포증식,기증식억제솔여가입골수간충질간세포적수량정정비.②방법1유도2 h후,광경하가견골수간충질간세포적세포질향핵수축,정전형핵주체형태.3～5 h후다수세포능형성신경원양세포형태,단세포수목무명현증가.3 d후대다수세포전변위쌍겁혹다겁신경원세포양형태,부분세포지간랍성망상.염색가견60%～70%신경원특이성희순화매、45%～50%효질섬유산성단백양성,소단백양성세포하강위3.4%.방법2유도2 h즉가견세포체적변소,형성쌍겁혹다겁적세포체,가지속유도48 h,후대부분세포부기사망.염색가견40%～50%신경원특이성희순화매,35%～40%효질섬유산성단백양성,소단백양성세포재유도2 h시일과성상승도63%,48 h시하강위1.6%.결론:골수간충질간세포구유향신경세포분화능력병차가이억제혼합림파세포반응체계중림파세포적증식,대동충이체면역반응구유부조절작용.
BACKGROUND:As a kind of undifferentiated precursor cells,the phenotypic differentiation of bone marrow mesenchymal stem cells (BMSCs) remains immaturity,thus it presents weak rejection following transplantation.However,the in vitro directional differentiation of BMSCs into neuronal cells is easy affected by various factors.OBJECTIVE:To observe the immunomodulatory effect and the potential of BMSCs differentiate into neuronal-like cells.DESIGN,TIME AND SETTING:A contrast observation was conducted at the Department of Cytology,Third People's Hospital of Wuxi from January 2008 to March 2009.MATERIALS:Bone marrow was harvested from chips of cancellous or ilium bone dudng hip joint surgery.METHODS:Firstly,the BMSCs were separated and cultured to establish mixed lymphocyte reaction (MLR) system.Secondly,2 samples of peripheral blood mononuclear cells (1×10~5/well) were added into 96-well plate,and then BMSCs treated by mitomycin were added according to different ratios (BMSCs/peripheral blood monouclear cells).At the end,the cells were cultured as follows:Method 1:DMEM+10% fetal calf serum+1 μmol/L RA +20 μg/L basic fibroblast growth factor+20 μg/L epidermal growth factor.Method 2:DMEM+2% dimethyl sulfoxide +100 μmol/L butylated hydroxyanisole.MAIN OUTCOME MEASURES:The growth rate of lymphocyte was measured by ~3H-Thymidine,and the effect of BMSCs on lymphocyte proliferation was observed.Additionally,the differentiation potential of BMSCs into neuronel cells was determined by immunofluorescenca and immunohistochemistrical staining.RESULTS:①The BMSCs inhibited lymphocyte proliferation in MLR system and the influence on proliferation of lymphocyte was direct related to ratio of BMSCs.②Under a light microscope,cytoplasm of BMScs were shrinkd,which presented typical perikaryon morphology at hour 2 after culture with method 1.The majority of BMSCs were formed neuronal-like cells without number changes at hours 3-5,which turned to be dipolar or multipolar neuronal shapes at day 3.There were 60%-70% neuronspecific enolase,45%-50% glial fibrillary acidic protein were positive expressed.However,the positive rate of nidogen was decreased 3.4%.Cells cultured with method 2 became smaller after 2 hours,formed dipolar or multipolar body cells,and most of cells were died after 48 hours.The 40%-50% neuronspecific enolase,35%-40% glial fibrillary acidic protein was found to be positive.The positive rate of nidogen was temporary increased to 63% at hour 2 after culture；however,it was decreased to 1.6% after 48 hours.CONCLUSION:BMSCs can differentiate into neuronal-like cells,as well as inhibit lymphocyte proliferation in MLR system,which possess down regulation effect on alloimmunity-reaction.
