CAJ | 학술논문

干细胞%骨髓干细胞%神经节苷脂%骨髓间充质干细胞%神经元样细胞%诱导分化%免疫组化%聚合酶链反应%激光扫描共聚焦显微镜%细胞内游离钙离子%生长相关蛋白 43%神经元特异性烯醇化酶%神经丝蛋白%神经巢蛋白%干细胞图片文章榦細胞%骨髓榦細胞%神經節苷脂%骨髓間充質榦細胞%神經元樣細胞%誘導分化%免疫組化%聚閤酶鏈反應%激光掃描共聚焦顯微鏡%細胞內遊離鈣離子%生長相關蛋白 43%神經元特異性烯醇化酶%神經絲蛋白%神經巢蛋白%榦細胞圖片文章
간세포%골수간세포%신경절감지%골수간충질간세포%신경원양세포%유도분화%면역조화%취합매련반응%격광소묘공취초현미경%세포내유리개리자%생장상관단백 43%신경원특이성희순화매%신경사단백%신경소단백%간세포도편문장

　　背景：单唾液酸四己糖神经节苷脂(Monosialotetrahexosylganglioside，GM1)在神经细胞的生长发育、分化、再生和细胞内外信息传递等多种生理过程中发挥重要作用。目的：探讨 GM1对骨髓间充质干细胞按照 Woodbury 经典诱导方案将其诱导分化为神经元样细胞前后基因方面和细胞内游离 Ca2+浓度的变化。方法：分离纯化 SD 大鼠骨髓间充质干细胞进行传代培养，传至第5代时，待细胞融汇成致密单层后，加入50 mmol/L 神经节苷脂设为 GM1组，预培养24 h 后按照 Woodbury 经典诱导方案进行诱导，设置对照组，采用免疫组化和 Realtime PCR 技术分别检测诱导前后生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白的蛋白和 mRNA 表达的变化，采用激光扫描共聚焦显微镜检测诱导前后细胞内游离钙离子浓度的变化。结果与结论：①加入 GM1组诱导分化后较对照组生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白表达水平增高(P <0.05)，GM1能够促骨髓间充质干细胞诱导分化为神经元样细胞。②更换诱导液后，两组细胞内荧光强度逐渐增加，到100 s 达高峰值，其后逐渐减弱，但20 min 时细胞荧光强度仍高于诱导前，加入 GM1组，细胞内游离 Ca2+浓度较对照组有所增加(P <0.05)。说明 GM1能够促进细胞内 Ca2+浓度增加，游离 Ca2+在诱导过程中可能有促进作用。③诱导后生长相关蛋白43、神经元特异性烯醇化酶、神经丝蛋白和神经巢蛋白基因表达改变不显著，说明 Woodbury 经典诱导方案可能为转录后水平即蛋白水平的调控。
　　배경：단타액산사기당신경절감지(Monosialotetrahexosylganglioside，GM1)재신경세포적생장발육、분화、재생화세포내외신식전체등다충생리과정중발휘중요작용。목적：탐토 GM1대골수간충질간세포안조 Woodbury 경전유도방안장기유도분화위신경원양세포전후기인방면화세포내유리 Ca2+농도적변화。방법：분리순화 SD 대서골수간충질간세포진행전대배양，전지제5대시，대세포융회성치밀단층후，가입50 mmol/L 신경절감지설위 GM1조，예배양24 h 후안조 Woodbury 경전유도방안진행유도，설치대조조，채용면역조화화 Realtime PCR 기술분별검측유도전후생장상관단백43、신경원특이성희순화매、신경사단백화신경소단백적단백화 mRNA 표체적변화，채용격광소묘공취초현미경검측유도전후세포내유리개리자농도적변화。결과여결론：①가입 GM1조유도분화후교대조조생장상관단백43、신경원특이성희순화매、신경사단백화신경소단백표체수평증고(P <0.05)，GM1능구촉골수간충질간세포유도분화위신경원양세포。②경환유도액후，량조세포내형광강도축점증가，도100 s 체고봉치，기후축점감약，단20 min 시세포형광강도잉고우유도전，가입 GM1조，세포내유리 Ca2+농도교대조조유소증가(P <0.05)。설명 GM1능구촉진세포내 Ca2+농도증가，유리 Ca2+재유도과정중가능유촉진작용。③유도후생장상관단백43、신경원특이성희순화매、신경사단백화신경소단백기인표체개변불현저，설명 Woodbury 경전유도방안가능위전록후수평즉단백수평적조공。
BACKGROUND: Monosialotetrahexosylganglioside plays an important role in a variety of physiological processes, such as the nerve cel growth and development, differentiation, regeneration and intracel ular and extracel ular information transmission. OBJECTIVE: To investigate the effects of monosialotetrahexosylganglioside on the changes of gene expression and calcium ion concentration in the course of inducing the mesenchymal stem cells into neuron-like cells with Woodbury’s method. METHODS: The mesenchymal stem cells from Sprague Dawley rats were cultured after isolated and purified, After 5 passages in culture, the cel integrated into a dense monolayer, and treated with 50 mmol/L monosialotetrahexosylganglioside for 24 hours as the monosialotetrahexosylganglioside group; then the mesenchymal stem cells were induced into neuron-like cells with the methods of Woodbury after pre-cultured for 24 hours, and set the control group. The protein and mRNA expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin were detected by immunocytochemistry and real-time PCR, respectively. The fluorescence intensity of intracel ular free calcium ion before and after inducing was detected by laser scan confocal microscope. RESULTS AND CONCLUSION: After induction, the expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin of the mesenchymal stem cells in the monosialotetrahexosylganglioside group were higher than those in the control group (P < 0.05), demonstrating that monosialotetrahexosylganglioside could promote the differentiation of mesenchymal stem cells into neuron-like cells. The fluorescence intensity in mesenchymal stem cells was increased gradual y in two groups after the medium was replaced by the induction medium, attained its peak value at 100 seconds and then decreased gradual y, but the fluorescence intensity was stil higher than that before the induction at 20 minutes. The fluorescence intensity of intracel ular free Ca2+ was increased significantly in the monosialotetrahexosylganglioside group when compared with the control group (P < 0.05), suggesting that monosialotetrahexosylganglioside could increase the concentration of intracel ular free Ca2+, and intracel ular free Ca2+ may be useful in the course of induction. The changes of protein expression levels of growth-associated protein 43, neuron-specific enolase, neurofilament and nestin were not significant after induction, indicating that Woodbury classic induced programme could regulate the post-transcriptive protein level.