Damaged PC12 cells in Transwell culture system for promoting the transdifferentiation of bone marrow stromal stem cells into neuron-like cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Drug treatment has unsatisfactory effect on Alzheimer disease, while many studies have indicated that the transplantation of bone marrow stromal cells (MSCs) is effective on Parkinson disease, cerebral ischemia, etc., but the mechanism is still unclear.OBJECTIVE: To imitate transplantation environment by co-culture of amyloid β1-40 (Aβ1-40) damaged PC12 cells and MSCs, observe the effect of bi-directional information feedback in the microenvironment on the transdifferentiation of MSCs to nerve cells, and observe its protective effect on the apoptosis of damaged PC12 cells.DESTGN A comparative observation.SETTING: Department of Neurology, China Medical University.MATERIALS SD rats of 2-3 weeks after birth either male or female were used. PC12 cell lines were purchased from the Institute of Cell Biology, Chinese Academy of Sciences; neuro-specific enolase (150, Boster, Wuhan);Methyl-thiazol-tetrazolium (MTT) 15 μL (terminal concentration of 0.5 g/L).METHODS: The experiment was carried out in the Experimental Center (provincial experimental animal center) of China Medical University from June to July in 2004. Bilateral femurs were aseptically removed from 1 SD rat, and MSCs were identified using CD44 antibody immunofiuorescently. PC12 was used to replace nerve cells. The PC12 cells were stimulated by Aβ1-40 then transferred by Transwell. There were 5 groups Group A normally cultured PC12 co-cultured with MSCs; Group B Aβ1-40 stimulated PC12 co-cultured with MSCs; Group C normal PC12 supernatant+MSCs; Group D damaged PC12 supernatant+MSCs; Group E MSCs cultured with common medium 1640.MAIN OUTCOME MEASURES:Routine immunohistochemical staining was performed. NSE positive cells were observed under inverted fluorescence microscope, 10 visual fields (200×) were randomly selected to count the positive cells. MTT metabolic rate was used to detect the proliferation of MSCs in each group. The differences of measurement data were compared using the one-way analysis of variance.fluorescent and bright fields, NSE positive cells appeared as red fluorescence, MSCs were bipolar, multipolar and cone shapes, and appeared as neuron-like forms with dendrite-like structure, and there were extensive connections among some neuron-like cells. The NSE positive rates was obviously higher in group B than groups A, C, D and E (P ＜ 0.01 ).in groups A, C, D and E (F=9.713, P＜ 0.01).