Biochemical properties of norepinephrine as a kind of neurotransmitter secreted by bone marrow-derived neural stem cells induced and differentiated in vitro

Jianrong Chen; Xiaodan Jiang; Ruxiang Xu; Peng Jin; Yuxi Zou; Lianshu Ding

摘要：BACKGROUND: It has been proved by many experimental studies from the aspects of morphology and immunocytochemistry in recent years that bone marrow stromal cells (BMSCs) can in vitro induce and differentiate into the cells possessing the properties of nerve cells. But the functions of BMSCs-derived neural stem cells(NSCs) and the differentiated neuron-like cells are still unclear.OBJECTIVE: To observe whether bone marrow-derived NSCs can secrete norepinephrine (NE) under the condition of in vitro culture, induce and differentiation, and analyze the biochemical properties of BMSCs-derived NSCs.DESIGN: A non-randomized and controlled experimental observation.SETTING: Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University.MATERIALS: This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5-month-old healthy New Zealand white rabbits.METHODS: This experiment was carried out in the Institute of Neuromedicine of Chinese PLA, Zhujiang Hospital, Southern Medical University. The bone marrow used in the experiment was collected from 1.5 month-old healthy New Zealand white rabbits. BMSCs of rabbits were isolated and performed in vitro culture, induce and differentiation with culture medium of NSCs and differentiation-inducing factor, then identified with immunocy-tochemical method. Experimental grouping: ①Negative control group: L-02 hepatic cell and RPMI1640 culture medium were used. ② Background culture group: Only culture medium of NSCs as culture solution was added into BMSCs to perform culture, and 0.1 volume fraction of imported fetal bovine serum was supplemented 72 hours later. ③Differentiation inducing factor group: After culture for 72 hours, retinoic acid and glial cell line-derived neurotrophic factors were added in the culture medium of BMSCs and NSCs as corresponding inducing factors. The level of NE in each group was detected on the day of culture and 5, 7, 14 and 20 days after culture with high performance liquid chromatography (HPLC). The procedure was conducted 3 times in each group. Standard working curve was made according to the corresponding relationship of NE concentration and peak area. The concentration of NE every 1 ×107 cells was calculated according to standard curve and cell counting.MATN OUTCOME MEASURES: The level of NE of cultured cells was detected with HPLC; immunocytochemistrical identification of Nestin and neuron specific nuclear protein was performed.RESULTS: ① On the 14th day after cell culture, BMSCs turned into agnus and round cells which presented Nestin-positive antigen, then changed into neuron-like cells with long processus and presented neuron specific nuclear protein -positive antigen at the 20th day following culture. ② The ratio of NE concentration and peak area has good linear relationship, and regression equation was Y=1.168 36+0.000 272 8X, r=0.998 4.Coefficient variation (CV) was ＜ 5% and the recovery rate was 92.39%(Y referred to concentration and X was peak area).③NE was well detached within 10 minutes under the condition of this experiment. ④ NE was detected in NSCs and their culture mediums, which were cultured for 7, 14 and 20 days respectively, but no NE in BMSCs, NSCs-free culture medium and L-02 hepatic cell which were as negative control under the HPLC examination. Analysis of variance showed that the level of NE gradually increased following the elongation of culture time (P ＜ 0.01). No significant difference in the level of NE existed at the same time between differentiation inducing factor group and basic culture group(P ＞ 0.05).CONCLUSION: BMSCs of rabbits can proliferate in vitro and express Nestin antigen; They can differentiate into neuron-like cells, express specific neucleoprotein of mature neurons, synthesize and secrete NE as a kind of neurotransmitter.