中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2013年
32期
5741-5748
,共8页
干细胞%骨髓干细胞%骨髓间充质干细胞%神经元样细胞%甲钴胺%诱导%分化%神经巢蛋白%神经元特异性烯醇化酶%干细胞图片文章
榦細胞%骨髓榦細胞%骨髓間充質榦細胞%神經元樣細胞%甲鈷胺%誘導%分化%神經巢蛋白%神經元特異性烯醇化酶%榦細胞圖片文章
간세포%골수간세포%골수간충질간세포%신경원양세포%갑고알%유도%분화%신경소단백%신경원특이성희순화매%간세포도편문장
stem cel s%bone marrow-derived stem cel s%bone marrow mesenchymal stem cel s%neuron-like cel s%methylcobalamin%induction%differentiation%nestin%neuron-specific enolase%stem cel photographs-containing paper
背景：目前骨髓间充质干细胞移植到脊髓损伤动物体内后对脊髓损伤的恢复效果非常有限。甲钴胺是治疗神经系统疾病及损伤的常见药物，其对骨髓间充质干细胞的影响尚不清楚。目的：探讨甲钴胺体外定向诱导骨髓间充质干细胞向神经元样细胞分化的可行性，观察分化后的细胞增殖和生长情况。方法：取大鼠胫腓骨骨髓，采用密度梯度离心贴壁细胞培养法分离、培养骨髓间充质干细胞，取第四五代骨髓间充质干细胞，分别以25，50，100 mg/L的甲钴胺进行诱导分化24，48和72 h。倒置相差显微镜下连续观察细胞形态学变化，MTT法检测细胞活性，RT-PCR和Western blot法检测特异性标志物Nestin和NSE的表达。结果与结论：甲钴胺诱导骨髓间充质干细胞后大部分细胞变成神经元样细胞。与对照组比较，甲钴胺诱导后细胞活性无明显变化。不同剂量甲钴胺诱导48 h后，Nestin和NSE在mRNA和蛋白水平表达均上调，其中100 mg/L组表达上调最明显；100 mg/L 甲钴胺诱导24，48和72 h后，Nestin和NSE在mRNA和蛋白水平表达均上调，其中72 h表达上调最明显。说明甲钴胺可定向诱导骨髓间充质干细胞向神经元样细胞分化，100 mg/L为甲钴胺的较佳诱导浓度。
揹景：目前骨髓間充質榦細胞移植到脊髓損傷動物體內後對脊髓損傷的恢複效果非常有限。甲鈷胺是治療神經繫統疾病及損傷的常見藥物，其對骨髓間充質榦細胞的影響尚不清楚。目的：探討甲鈷胺體外定嚮誘導骨髓間充質榦細胞嚮神經元樣細胞分化的可行性，觀察分化後的細胞增殖和生長情況。方法：取大鼠脛腓骨骨髓，採用密度梯度離心貼壁細胞培養法分離、培養骨髓間充質榦細胞，取第四五代骨髓間充質榦細胞，分彆以25，50，100 mg/L的甲鈷胺進行誘導分化24，48和72 h。倒置相差顯微鏡下連續觀察細胞形態學變化，MTT法檢測細胞活性，RT-PCR和Western blot法檢測特異性標誌物Nestin和NSE的錶達。結果與結論：甲鈷胺誘導骨髓間充質榦細胞後大部分細胞變成神經元樣細胞。與對照組比較，甲鈷胺誘導後細胞活性無明顯變化。不同劑量甲鈷胺誘導48 h後，Nestin和NSE在mRNA和蛋白水平錶達均上調，其中100 mg/L組錶達上調最明顯；100 mg/L 甲鈷胺誘導24，48和72 h後，Nestin和NSE在mRNA和蛋白水平錶達均上調，其中72 h錶達上調最明顯。說明甲鈷胺可定嚮誘導骨髓間充質榦細胞嚮神經元樣細胞分化，100 mg/L為甲鈷胺的較佳誘導濃度。
배경：목전골수간충질간세포이식도척수손상동물체내후대척수손상적회복효과비상유한。갑고알시치료신경계통질병급손상적상견약물，기대골수간충질간세포적영향상불청초。목적：탐토갑고알체외정향유도골수간충질간세포향신경원양세포분화적가행성，관찰분화후적세포증식화생장정황。방법：취대서경비골골수，채용밀도제도리심첩벽세포배양법분리、배양골수간충질간세포，취제사오대골수간충질간세포，분별이25，50，100 mg/L적갑고알진행유도분화24，48화72 h。도치상차현미경하련속관찰세포형태학변화，MTT법검측세포활성，RT-PCR화Western blot법검측특이성표지물Nestin화NSE적표체。결과여결론：갑고알유도골수간충질간세포후대부분세포변성신경원양세포。여대조조비교，갑고알유도후세포활성무명현변화。불동제량갑고알유도48 h후，Nestin화NSE재mRNA화단백수평표체균상조，기중100 mg/L조표체상조최명현；100 mg/L 갑고알유도24，48화72 h후，Nestin화NSE재mRNA화단백수평표체균상조，기중72 h표체상조최명현。설명갑고알가정향유도골수간충질간세포향신경원양세포분화，100 mg/L위갑고알적교가유도농도。
BACKGROUND:Currently, transplantation of bone marrow mesenchymal stem cel s into the spinal cord is very limited to the recovery of animals fol owing spinal cord injury. Methylcobalamin is a common drug for the treatment of neurological diseases and injuries, but its effects on bone marrow mesenchymal stem cel s are unclear. OBJECTIVE:To study the feasibility of bone marrow mesenchymal stem cel s differentiating into neuron-like cel s induced by methylcobalamin in vitro and to observe the cel viability and proliferation of differentiated cel s. Methods:Rat bone marrow mesenchymal stem cel s were isolated, cultured and purified by density gradient centrifugation and adherent culture. The fourth to fifth generation of bone marrow mesenchymal stem cel s were treated for 24, 48 and 72 hours with different concentrations (25, 50 and 100 mg/L) of methylcobalamin. The morphological changes and cel growth were continuously observed under an inverted phase constract microscope. The viability of induced cel s was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The expressions of Nestin and neuron-specific enolase were identified by reverse transcription PCR and western blot. RESULTS AND CONCLUSION:Most of bone marrow mesenchymal stem cel s could differentiate into neuron-like cel s after induction with methylcobalamin. The expressions of Nestin and neuron-specific enolase were up-regulated after 48 hours of methylcobalamin treatment at different concentrations, especial y after treatment with 100 mg/L methylcobalamin. Similarly, the expressions of Nestin and neuron-specific enolase could be increased significantly after 100 mg/L methylcobalamin treatment for 24, 48 and 72 hours, especial y for 72 hours. It is indicated that methylcobalamin can induce bone marrow mesenchymal stem cel s differentiating into neuron-like cel s, and the optimal concentration of methylcobalamin is 100 mg/L.
