The MRNA Expression Of Genes Related To Fatty Acid β-oxidation And Metabolism Of Cholesterol In Glial And Glioma Cells

Objective: Very long chain fatty acid (VLCFA) is toxic to cells, and oxidized into short chain fatty acid and acetyl-CoA in peroxisome. In patients with enzyme deficiency and rats of these genes knocked out, VLCFA accummulates in brain, the migration of neurons is affected, leading to demyelination and toxicity to central nervous system(CNS) and eventually resulting in brain malformation or maldevelopment of newborn. High-cholesterol is a risk factor of Alzheimer disease. But so far, there is little known about the metabolism of fatty acid and cholesterol in CNS, so it is important to research the expression of genes related to the metabolism of fatty acids and cholesterol in glial cells in brain.Fatty acidβ-oxidation presents in mitochondria and peroxisome. In mitochondria short- and middle- even long- fatty acids are oxidized and Carnitine Acetyl transferaseⅠ(CPT-1) is the rate-limiting enzyme of mitochondriaβ-oxidation. In peroxisome VLCFA are mainly oxidized and Acyl-CoA oxidase 1(Acox1), Acyl-CoA oxidase2(Acox2), Acyl-CoA oxidase3(Acox3), D-bifunctional protein(DBP), L-bifunctional protein(LBP), Thiolase A (THLA), Thiolase B(THLB) and sterol carrier protein x(SCPx) are involved in. It was reported that Acox1, DBP and THLA expresse in rat brain and DBP expresses in neurons. Glial cells are important group in CNS and play a role in assisting neurons.Glioma comes from glial cells. It is unclear which genes of peroxisomal fatty acidβ-oxidation express in glial cells and whether they does in glioma either. Peroxisome Proliferator Activated Receptorα(PPARα) regulates the metabolism of fatty acids in cells. It was known that PPARαexpresses in neurons. However it is unknown whether PPARαexpresses in glial cells too.In this experiment, we assay the mRNA expression of PPARα, CPT-1, CAT, Acox1, Acox2, Acox3, DBP, LBP, THLA, THLB and SCPx mRNA in primary cultured cortical neuroglia cells and glioma cells to investigate the metabolic mechanism of fatty acids in glial and glioma cells, and the functions of glial cells in CNS.Cholesterol is synthesized in glial cells in brain of adult rat and the key enzyme for this pathway is 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMGR). Cholesterol in glial cells binds with ATP binding cassette transporter A1(ABCA1) and is submitted to extracellulary apoliprotein and then is uptook by scavenger receptor class B typeⅠ(SR-BI) in neuron membrane. Cholesterol 24-hydroxylase (CYP46) in neurons catalyzes cholesterol to 24S-hydroxycholesterol which can be transported through the blood brain carrier to blood, then is converted to bile acid in liver. Liver X receptorα(LXRα) regulates the metabolism of cholesterol and expresses in brain. Our previous experimental data showed that LXRα, HMGR, CYP46, ABCA1 and SR-BI expressed in both neurons and glial cells. It is unclear whether these genes express in glioma cells too. So we measure the mRNA expression of LXRα, HMGR, CYP46, ABCA1 and SR-BI mRNA in primary cultured glial cells and glioma cells to research the metabolic mechanism of cholesterol in glioma and the association between lipid metabolism and glioma.Methods:1 The primary culture of cortical glial cell3-day old neonatal rat pups were decapitated and the intact brain was removed and transferred into DMEM in a 60 mm Petri dish. Cerebral cortices were dissected and cut into 1 mm3 and then digested in 0.125% trypsin in PBS without calcium or magnesium at 37°C for 30 min. After the tissues descended, abandon the supernatant, supplement the same volume of DMEM culture medium containing 10%FBS and 100u/ml benzylpenicillin and streptomycin to terminate digestion for 10 minutes, and then wash twice. Add the culture medium again, dissociate manually with a fire-bored Pasteur pipette untill the remaining undigested tissures disappeared. The cell suspension was plated at a concentration 1×106/ml on polyL-lysine-coated 6-well plates containing 1 mL of medium at 37°C and 5%CO2 in a humidified atmosphere. The culture medium was changed every 3 days afterwards with DMEM culture medium containing 10%FBS and 100u/ml benzylpenicillin and streptomycin.2 The culture of C6Glioma was cultured in conventional subculturing and the cells from the tenth to the fifteenth were used in our experiment.3 GroupsThere were glial cell group(Glia) and glioma group(C6).4 The identification of cortical glial cellThe cortical glial cell was identified by Immunocytochemical stain by using the antibody of glial fibrillary acidic protein(GFAP).5 Extraction of cell total RNACell total RNA was extracted by Trizol regent.6 The expression of mRNA assayThe relative mRNA content of genes in glial cell and glioma containing PPARα, LXRα, CPT-1, CAT, Acox1, Acox2, Acox3, DBP, LBP, THLA, THLB, SCPx, HMGR, CYP46, ABCA1 and SR-BI were measured by RT-PCR using GAPDH as inner standard.Results:1 Morphology of cortical glial cellsCells observed with phase contrast microscope adhered mostly after plated for 12 hours, round or ellipse,some cells had small processes and distrubuted well. The third or forth day after plated, cells grew fastly and formed clusters. There are many karyokinesis around the cells. The volume and quantity of glial cells increased and the processes also grew in number while the dispersing neurons reduced. On the sixth or seventh day, astrocytes took on two types: polygon and fibriform. The clusters between cells started to fuse and emerged a carpet-like growth. Solitary glial cell had a comparatively large body and obvious nucleus, round or orbicular-ovate, locating one side of the body. As the incubation continuing, neurons almost disppeared. On the thirteenth or fourteenth day, oligodendrocytes start to increase on the surface layer with small actinoid processes extending from the body while on the bottom there are astrocytes fusing completely and spreading the whole plate.2 Immunocytochemical stainThe result of immunocytochemical stain by using the antibody of glial fibrillary acidic protein(GFAP) showed that the proportion of astrocytes was 85% and it is suit to our experiment.3 The relative expression of CAT mRNA in cellsThere was the expression of CAT mRNA in Glia and C6 groups; There was no statistical significance between Glia and C6 groups (2.406±0.318 vs 2.578±0.221, P>0.05). This result indicated that there are peroxisomes in glial and glioma cells.4 The relative expression of CPT-1 mRNA in cellsThere was the expression of CPT-1 mRNA in Glia and C6 groups; The relative expression of CPT-1 mRNA in C6 group (2.371±0.294) was significantly higher than that in Glia group (1.364±0.232, P<0.01). This result indicated thatβ-oxidation in mitochondria in glial cells was higher than that in glioma.5 The relative expression of Acox1, Acox2 and Acox3 mRNA in cellsThere was the expression of Acox1 and Acox2 mRNA but there was not the experssion of Acox3 mRNA in Glia group and there was the expression of Acox1 and Acox3 mRNA but there was not the expression of Acox2 mRNA in C6 group; The relative expression of Acox1 mRNA in C6 group (2.777±0.342) was significantly higher than that in Glia group (1.873±0.240, P<0.01) .6 The relative expression of DBP and LBP mRNA in cellsThere was the expression of DBP mRNA in Glia and C6 groups but there was not the expression of LBP mRNA in Glia and C6 groups; There was no statistical significance between Glia and C6 groups (0.919±0.128 vs 1.079±0.207, P>0.05).7 The relative expression of THLA, THLB and SCPx mRNA in cellsThere was the expression of THLA and SCPx mRNA but there was not the expression of THLB mRNA in Glia group and there was the expression of THLA and THLB mRNA but there was not the expression of SCPx mRNA in C6 group; There was no statistical significance of the expression of THLA mRNA between Glia and C6 groups (0.188±0.024 vs 0.187±0.031, P>0.05).8 The relative expression of PPARαmRNA in cellsThere was the expression of PPARαmRNA in Glia and C6 groups; The relative expression of PPARαmRNA in C6 group (1.064±0.121) was significantly higher than that in Glia group (0.463±0.091, P<0.01) .9 The relative expression of LXRαmRNA in cellsThere was the expression of LXRαmRNA in Glia and C6 groups; There was no statistical significance between Glia and C6 groups (0.734±0.147 vs 0.746±0.134, P>0.05).10 The relative expression of HMGR mRNA in cellsThere was the expression of HMGR mRNA in Glia and C6 groups; The relative expression of HMGR mRNA in C6 group (2.324±0.100) was significantly higher than that in Glia group (1.175±0.189, P<0.01) .11 The relative expression of CYP46 mRNA in cellsThere was the expression of CYP46 mRNA in Glia group but there was not the expression of CYP46 mRNA in C6 group.12 The relative expression of ABCA1and SR-BI mRNA in cellsThere was the expression of ABCA1and SR-BI mRNA in Glia and C6 groups; The relative expression of SR-BI mRNA in C6 group (1.778±0.214) was significantly higher than that in Glia group(1.107±0.185, P<0.01) ; There was no statistical significance of the expression of ABCA1 mRNA between Glia and C6 groups(1.397±0.270 vs 1.255±0.227, P>0.05).Conclusion:1 Genes involved in the peroxisomalβ-oxidation of unsaturated VLCFA and branched VLCFA in peroxisomes express in glial cells. It indicated that glial cells may play a role in detoxicating VLCFA, inactivating hormones and translocating cholesterol out in brain. 2 The synthesis of cholesterol and the oxidation of fatty acid in glioma are more than that in normal glial cells. It was not found that the mRNA expression of CYP46 and Acox2, SCPx involved in peroxisomalβ- oxidation occured.