A Study On Mechanism Of Lithium Regulating Proliferation Of Hypoxic NSCs

Objective To culture and identify NSCs isolated from neonatal SD rat, and establish model of hypoxic NSCs and investigate whether lithium regulates the proliferation of hypoxic NSCs through PI3K/Akt signaling pathway and provide strongly experimental and theoretical evidence for clinical treatment of HIBD.Methods1. Isolation, cultivation and identification of NSCs: Removeing of the neck of neonatal rats within 1 day after birth, 75% alcohol immersion disinfection. Hippocampal tissue was isolated. The meninges and blood vessels were removed carefully and cut it.Hippocampal tissue was digested by 0.25%trypsin and centrifuged. According to the density of 5×105/ml of NSCs were cultured in 25 ml culture medium with serum-free medium in an incubator with a humidified atmosphere containing 5% CO2 at 37 °C.The medium was changed every 2-3 days. The primary cultured NSCs were passaged every 5-7days. The differentiation of NSCs was induced with serum medium. The function of proliferation and differentiation of NSCs was detected by immunohistochemistry.2. Establishing the model of hypoxic NSCs: The sugar-free medium using DMEM sugar-free medium similar to the basal medium. Hypoxic environment with the mixed gas include 5%CO2 and 95%N2. The morphological changes of the cells were observed by trypan blue staining, and the activity of the cells was detected by CCK-8 to determine the success of the modeling on 30 minutes, 1 hour and 2 hours.3. The regulation mechanism of lithium for hypoxic NSCs: The passage of NSCs were divided into normal control group, hypoxic group, normal saline intervention group,1m M, 3m M, 5m M lithium chloride intervention group. The NSCs of normal control group was cultured in serum-free medium.The other groups was cultured in DMEM sugar-free medium and a mix of CO2 with a volume fraction of 5% and N2 with a volume fraction of 95% for 1 hour. After hypoxia, NSCs were observed morphology and counted the number of the cells and cultured in serum-free medium. The culture medium of normal saline intervention group was immediately added normal saline,and lithium chloride intervention group was immediately added different concentration of lithium chloride after hypoxia for 3 days. NSCs expressing Akt /P-Akt,GSK-3b/P-GSK-3b,Caspase-9/P- Caspase-9 markers were detected by immunohistochemistry.Results1. Isolation, cultivation and identification of NSCs of neonatal SD rat: Primary cultured1-2 days, were growing in singles,pairs or three, and high refraction, clearly boundary and bright circular. Primary cultured 3 days, showed spherical cell, different size.Primary cultured 5-7 days, showed closely spherical shape increases, clearly boundary,yellowly center, refractive index significantly decreases and without neurite outgrowth.The passage cell morphology was the same as the primary cells. NSCs were identified by Nestin and NSCs were confirmed in proliferative condition by Brd U.NSCs were cultured in serum medium for 3 days, showed fusiform or triangle shape,and weaved into the fleecy networks. Positive NSE expressed as neurons and positive GFAP expressed as astroeytes.2. Modelling of hypoxic NSCs in vitro: The generation of cultured NSCs were cultured into the sugar-free DMEM medium and a mix of CO2 with a volume fraction of 5% and N2 with a volume fraction of 95% for an hour. After hypoxia, the amount of NSCs was decreasing and the characteristics of the NSCs was irregular in shape, low refraction, swollen perikarya or even membranolysis. The number of dead cells of NSCs in hypoxic group was increasing significantly(P<0.05). The activeness of NSCs in hypoxic group was decreasing significantly(P<0.05).3. The mechanism of lithium regulating proliferation of hypoxic NSCs: The red fluorescence of 3m M lithium chloride group was the strongest than the hypoxic group,normal saline group and 1m M lithium chloride group. Therefore, 3m M lithium chloride group mainly expressed P-Akt, P-GSK-3β, P-Caspase-9 markers. Compared with 3m M lithium chloride group, the red fluorescence of 5m M lithium chloride group was decreased gradually, the green fluorescence was increased gradually. Therefore, 5m M lithium chloride group mainly expressed Akt, GSK-3β,Caspase-9 markers. Compared with hypoxic group、normal saline intervention group,the number of P-Caspase-9/PGSK-3β/P-Akt expressed by hypoxic NSCs in 1m M、3m M lithium chloride group was increasing significantly(P<0.05);Compared with 3m M lithium chloride group,the number of P-Caspase-9/P-GSK-3β/P-Akt expressed by hypoxic NSCs in 5m M lithium chloride group was decreasing significantly(P<0.05).Conclusions1. Homemade box can establish the model of hypoxic NSCs.2. 1m M and 3m M lithium chloride can promote the proliferation of hypoxic NSCs,However, the ability of 3m M lithium chloride to promote the proliferation of hypoxic NSCs is stronger than 1m M lithium chloride. 5m M lithium chloride inhibits the proliferation of hypoxic NSCs.3. Akt, Caspase-9 and GSK-3b expressed by hypoxic NSCs are gradually decreased while P-Akt, P-Caspase-9 and P-GSK-3 b are gradually increased when 1m M and 3m M lithium chloride promote the proliferation of hypoxic NSCs.4.Akt, Caspase-9 and GSK-3? expressed by hypoxic NSCs are gradually increased while P-Akt, P-Caspase-9 and P-GSK-3? are gradually decreased when 5m M lithium chloride inhibits the proliferation of hypoxic NSCs.5. Lithium chloride increasing the activity of GSK-3? and Caspase-9 is related to its dosage.6. Lithium chloride regulate the proliferation of hypoxic NSCs through the PI3K/Akt signaling pathway.