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背景:药物治疗阿尔茨海默病难以达到满意效果,而多项研究表明,骨髓间质干细胞移植治疗帕金森病、脑缺血等有效,但其治疗机制尚不十分清楚。目的:采用淀粉样β1-40蛋白损伤的PC12细胞与骨髓间质干细胞共育,模拟移植环境,观察共育微环境中细胞间双向信息反馈对骨髓间质干细胞向神经细胞转分化的作用及对抗损伤的PC12凋亡的保护作用。设计:对比观察实验。单位:中国医科大学神经内科。材料:选用出生两三周的SD大鼠,雌雄不拘。PC12细胞株购自中国科学院细胞生物研究所。神经元特异性烯醇化酶(1∶50,博士德,武汉);MTT15μL(终浓度0.5g/L)。方法:实验于2004-06/07在中国医科大学实验中心完成。取1只SD大鼠双侧股骨骨髓,并培养骨髓间质干细胞,以CD44抗体用免疫荧光鉴定骨髓间质干细胞。PC12在实验中作为神经细胞的替代细胞。淀粉样β1-40蛋白刺激PC12后,用转移筛网转移PC12。实验分5组,A组:正常培养的PC12+骨髓间质干细胞共育;B组:淀粉样β1-40蛋白刺激的PC12+MSCs共育;C组:正常PC12的培养上清+骨髓间质干细胞;D组:受损PC12上清+骨髓间质干细胞;E组:普通1640培养的骨髓间质干细胞。主要观察指标:按常规进行细胞的免疫化学染色,采用荧光倒置显微镜观察神经元特异性烯醇化酶阳性细胞,随机选取10个200倍的视野,计数阳性细胞数。用四甲基偶氮唑盐代谢率(四唑盐比色试验)检测各组骨髓间质干细胞增殖情况。结果:①骨髓间质干细胞的生长:以荧光和明视野在同一视野下观察,骨髓间质干细胞生长良好,CD44免疫荧光鉴定,见95%以上细胞呈阳性表达。②神经元特异性烯醇化酶阳性细胞:以荧光和明视野在同一视野下观察,神经元特异性烯醇化酶阳性的骨髓间质干细胞呈现红色荧光,骨髓间质干细胞形态为双极形、多极形和锥形,出现类似神经元样细胞的形态,似树突样突起结构,个别神经元样细胞之间有广泛的联系。B组神经元特异性烯醇化酶细胞阳性率明显高于其他组(F=34.82,P<0.01)。③骨髓间质干细胞增殖情况:四甲基偶氮唑盐代谢率以B组最低,与其他比较,差异有显著性意义(F=9.713,P<0.01)。结论:淀粉样β1-40蛋白损伤PC12与骨髓间质干细胞非接触共培养的微环境细胞最有利于骨髓间质干细胞向神经元样分化,而非增殖方向发展。
Background: Drug treatment of Alzheimer's disease is difficult to achieve satisfactory results, and a number of studies have shown that bone marrow mesenchymal stem cell transplantation for the treatment of Parkinson's disease, cerebral ischemia and other effective, but its mechanism of treatment is not yet clear. OBJECTIVE: To co-culture PC12 cells damaged by amyloid β1-40 protein with bone marrow mesenchymal stem cells to simulate the transplantation environment and observe the effect of bidirectional feedback between cells on the transdifferentiation of bone marrow mesenchymal stem cells Protection of Injured PC12 Cells from Apoptosis. Design: Contrast observation experiment. Unit: Department of Neurology, China Medical University. MATERIALS: SD rats, born two to three weeks, were chosen, both male and female. PC12 cells purchased from Institute of Cell Biology, Chinese Academy of Sciences. Neuron-specific enolase (1:50, Bursty, Wuhan); MTT 15 μL (final concentration 0.5 g / L). Methods: The experiment was performed at the Experimental Center of China Medical University from June 2004 to July 07. One SD rat bilateral femur bone marrow was obtained and bone marrow mesenchymal stem cells were cultured. CD44 antibody was used to identify bone marrow mesenchymal stem cells by immunofluorescence. PC12 was used as a substitute for nerve cells in experiments. After amyloid-β1-40 protein stimulates PC12, PC12 is transferred using a transfer screen. Group A: normal cultured PC12 + mesenchymal stem cells co-education; Group B: amyloid-β1-40 protein-stimulated PC12 + MSCs co-education; Group C: normal PC12 culture supernatant + bone marrow mesenchymal stem cells ; Group D: injured PC12 supernatant + mesenchymal stem cells; Group E: ordinary 1640 cultured bone marrow mesenchymal stem cells. MAIN OUTCOME MEASURES: Immunocytochemical staining of cells was performed routinely. Neuron-specific enolase-positive cells were observed under a fluorescence inverted microscope. Ten 200-fold visual fields were randomly selected and the number of positive cells was counted. Metabolism rate (tetrazolium colorimetric assay) was used to detect the proliferation of bone marrow mesenchymal stem cells in each group. Results: ①The growth of bone marrow mesenchymal stem cells (BMSCs) was observed under the same field of vision with fluorescence and bright field. BMSCs grew well. CD44 immunofluorescence showed that more than 95% of cells were positive. ② neuron-specific enolase-positive cells: fluorescence and bright field in the same field of view, neuron-specific enolase-positive bone marrow stromal cells showed red fluorescence, bone marrow stromal cells in the form of bipolar, and more Poles and cones, similar to the appearance of neuron-like cells, like dendritic-like protuberance structure, there are a wide range of individual neuron-like cells. The positive rate of neuron-specific enolase cells in group B was significantly higher than that in other groups (F = 34.82, P <0.01). (3) Bone marrow mesenchymal stem cell proliferation: The MTB metabolism rate was the lowest in group B, which was significantly different from other groups (F = 9.713, P <0.01). CONCLUSION: Amyloid-β1-40 protein can damage MSCs differentiate into neurons rather than proliferate in the microenvironment of non-contact co-culture of PC12 and bone marrow mesenchymal stem cells.