APP5肽模拟物P165对体外培养大鼠海马神经干细胞增殖和分化的影响

目的研究一种小分子多肽─APP5肽的模拟物P165对体外培养的大鼠胚胎海马神经干细胞（neuralstem cells,NSCs）增殖和分化的影响,以期能找到一种可代替神经营养因子的小分子物质,能够促进NSCs的增殖或分化,为将来的临床应用提供理论依据。方法（1）原代培养SD大鼠胚胎脑海马NSCs;（2）利用5-溴脱氧尿嘧啶核苷（BrdU）和神经元、星型胶质细胞、少突胶质细胞的特异性标记物微管相关蛋白2（MAP2）、胶质纤维酸性蛋白（GFAP）、2,3-环核苷酸-3磷酸二酯酶（CNPase）对培养的NSCs进行鉴定;（3）将培养的NSCs分为对照组、血清组、APP5肽反序列组和P165组,观察各组细胞形态的变化;（4）将培养的NSCs分为对照组、APP5肽反序列组和P165组,利用细胞计数,测定干细胞克隆形成率、干细胞克隆形成大小的方法分析P165对海马NSCs增殖的影响。结果（1）海马神经干细胞呈神经球聚集生长,BrdU染色阳性;加入血清后神经球周围有细胞呈放射状向四周生长,并带有突起。染色呈MAP2、GFAP或CNPase阳性;（2）海马NSCs加入P165及其反序列后细胞形态上与对照组相比没有明显改变;（3）与对照组相比,加P165后海马NSCs数量明显增加,克隆形成率和克隆形成的直径均有明显的增加,并有统计学差异。结论P165能够促进海马NSCs的增殖,但并不促进其分化。

Effect of P165 Peptide on Proliferation and Differentiation of Cultured Rat Hippocampal Neural Stem Cells

Objective To look for a small molecular neurotrophin, which can promote proliferation and/or differentiation of neural stem cells （NSCs）. Methods 1. Embryonic hippocampal NSCs were cultured in vitro. 2. The NSCs were identified by bromodeoxyuridine （BrdU）, antibodies of glial fibrillary acid protein （GFAP）, microtubule-associated protein 2 （MAP2） and 2,3- cyclic nucleotide 3-phosphodiesterase （CNPase）. 3. The cells were divided into four groups： control group, FBS-treated group, antisense sequence of APP 5-mer peptide-treated group, and P165 peptide-treated group. The morphology of NSCs in the above mentioned four groups was observed 4. Cell count, deteetion of clone information rate and diameter of clone were done to study the effect of P165 peptide on proliferation of NSCs. Results 1. The NSCs formed neurospheres and grew floating. They were BrdUpositive, GFAP-positive, MAP2-positive and CNPase-positive cells appeared after FBS were added into the medium. 2. The morphology of NSCs were not changed in P165 peptide-treated group and antisense sequence group compared with that of the control group. 3. The cell number increased in P165 peptide-treated group compared with that of the control group. There were no apparent differences between the control group and the antisense-sequence-treated group. 4. The clone formation rate and diameter of neurospheres increased in the P165 peptide-treated group. Conclusion P165 peptide can promote the proliferation of embryonic hippocamal NSCs in vitro. P165 peptide doesn＇t promote the differentiation of embryonic hippocamal NSCs in vitro.