星形胶质细胞课件.ppt

1、Composition of Nervous TissueGlia are different from neurons:There are two main kinds in PNSSchwann cellThe Schwann cell making the myelin sheath(M)around the axon(A)was cut at the level of the nucleus(N).E-endoneurium.Scale=300 nm.(Rat,trigeminal nerve.)Schwann 细胞功能：There are four main kinds in CNS

2、Astrocytes(AS,星形胶质细胞）星形胶质细胞）Properties of ASFibrous astrocyteprotoplasmic astrocyte星形细胞的功能：星形细胞的功能：功能：功能：新的观点新的观点：葡萄糖葡萄糖乳糖酵解normaldisease储存糖原 1.2.参与脑内谷氨酸的代谢参与脑内谷氨酸的代谢Nature 411,261-268(2001)AChBP as anAChR decoy星形胶质细星形胶质细胞对突触的胞对突触的调节作用调节作用Astrocytes神经系统疾病的关系神经系统疾病的关系Oligodendrocytes (少突胶质细胞）少突胶质细胞）n

3、束间少突胶质细胞n血管周少突胶质细胞n神经元周少突胶质细胞n镀银染色发现突起很少；n通过特异性的GC单抗做免疫细胞化学标记，则可见少突细胞有很多突起，而且分支很多。根据在中枢神经系统中的位置和分布：功功 能能Ependymal cells (室管膜细胞）室管膜细胞）室管膜细胞分布于脑室和脊髓中央管腔面，形似单层立方或柱状上皮，细胞表面有纤毛和微绒毛 Ependymal cellsWho are the stem cells of the adult brain?astrocytes vs.ependymal cellsastrocytesEpendymal cellsJohansson et

4、al.,Identification of a neural stem cell in the adult mammalian central nervous system,Cell,96:25-34,1999.Doetsch et al.,Subventricular zone astrocytes are neural stem cells in the adult mammalian brain,Cell,97:703-16,1999.The subventricular zoneDr Jonas Frisen contends that neural stem cells are ce

5、lls that line ventriclesDr Arturo Alvarez-Buylla contends neural stem are astrocytes that lie one layer in from ventricle lining;Microglial cells (小胶质细胞）小胶质细胞）microgliaTypical features of neuronophagia,a cluster of microglia around a necrotic neuron,are shown(HE,high power).On the left side of the d

6、iagram,cells labeled B1,B2,and B3 represent chronically activated microglial cells.B3 is a markedly elongated cell,the nucleus in particular having a long,rod-shaped form.This rod cell is classically seen in general paresis(paretic neurosyphilis)but may also be seen in other chronic disease states o

7、f the brain.功功 能能胶质细胞培养http:/ 材料 仪器 设备步骤PREPARATION OF NEWBORN RATS FOR CULTURING步骤(细胞悬液的制备）Dissection:1.Remove brainstem,cerebellum and diencephalons.Immediately place in cold dissection buffer2.Peel off the meninges and transfer cortex to a tube containing cold dissection buffer.Immediately place

8、on ice3.Pour tissue into a dish and wash,using a syringe,with modified DMEM/F12 culture medium with 10%FBS,1%glutamine,and gentamicin antibiotic4 Mechanically digest tissue by mincing with a sterile razor bladeTransfer tissue back to a tube containing 5 ml 1X trypsin and 50uL DNAse.Incubate at 37C f

9、or 25 minutes.Remove only to swirl tube every 5 minutes5.Wash tissue with Glial Medium twice6.Dissociate tissue into a cell suspension by gently titrating through a 5mL pipette followed by a fire-polished Pasteur pipette.Transfer supernatant to a fresh tube during each titration.Do a total of three

10、titrations using both pipettes7.Dilute suspended cells in 10 mL of Glial Medium,and pass the solution through a 40 uM strainer8.Centrifuge cells at 1700 rpm for 5 minutes9.Remove supernatant,and resuspend pellet with 10 mL Glial Medium Seed 2 x 106 cells/T75 in 15 ml Glial medium10.Incubate the flasks at 37oC in 5%CO2 for 2-3 days without disruption.Change the medium in each flask every 2-3 days by aspirating and adding 15 mL fresh Glial Medium until confluency is achieved(after approximately 6-7 days)40 uM strainer步骤(细胞悬液的制备）步骤(纯化）步骤(鉴定）CD11b/DAPI 星形胶质细胞小胶质细胞少突胶质细胞Thanks！