Pericytes are a very important cellular constituent of the blood-brain barrier.They play a regulatory role in brain angiogenesis,endothelial cell tight junction formation,blood-brain barrier differentiation,microvascular dynamic motion and structural stability.Pericytes exhibit unique functional characteristics in some diseases,such as cerebrovascular disease,neurodegenerative disease,neuroimmune disease and traumatic brain injury.This article reviews the roles of pericytes in the blood-brain barrier.

Objective To explore the method of primary isolation, cultivation and identification of rat brain microvessel pericytes. Methods The brain tissue of 10 3 week-old Wistar rats was separated sterilely. The brain microvessel fragments were separated using two-step enzyme digestion and one-step gradient centrifugation and were seeded in 35-mm dishes for primary culture. The cell morphology was observed by phase contrast microscopy; the immunofluorescence assay was used to identify the associated antigns, such as the α-smooth muscle actin (α-SMA), neuron-glial antigen 2 (NG2), von Willebrand factor (vWF), and glial fibrillary acidic protein (GFAP). Methyl thiazolyl tetrazolium was used to determine the cell growth curve. Results Pericytes climbed out from the adherent brain microvascular fragments around,showing polygonal, and the cell fusion was 95％ after 12-14 days. Immunofluorescence staining revealed that the molecular markers of the pericytes α-SMA and NG2 related antigens showed double positive, while the vWF and GFAP related antigens showed double negative and the cultured cells were confirmed as brain microvascular pericytes. The growth rate of primary cells was slower. The passage cells entered into logarithmic growth phase after 36 to 60 hours and entered into plateau phase after 72 to 108 hours. Conclusions This method may successfully isolate rat brain microvascular pericytes with higher purity.

Objective To establish a stable in vitro model of blood-brain barrier (BBB) simulating in vivo state using the primary-cultured rat brain microvascular endothelial cells (BMVECs) and pericytes.Methods The primary rat BMVECs and pericytes were isolated,purified and cultured.The isolated cells were identified by immunocytochemical staining method.An in vitro model of BBB was constructed using Transwell inserts (pore size 0.4 μm) coculture.Its barrier function was evaluated by the 4-hour leakage test,tight junction protein identification,transendothelial resistance detection,and permeability test.The difference between the cocultured model and simple BMVEC model across the membrane resistance values,and the permeability difference of the small molecule sodium fluorescein (Na-F) were compared.Results Confluent BMVEC monolayers demonstrated a typical cobblestone appearance and the pericytes displayed irregular shape and overlapping growth.Immunodouble labeling technique identification showed that the pericytes positively expressed α-srmooth muscle actin (α-SMA) and neuron-glial antigen 2 (NG2); after the fusion of cocultured model endothelial cells,the surface leakage test became positive; immnocytochemical staining shows that a continuous and dense tight junction formed between the endothelial cells; compared to the BMVEC model,the transendothelial electrical resistance of the cocultured model increased significantly (190.762 ± 10.326 Ω/cm2 vs.96.503 ± 8.012 Ω/cm2; t=- 24.489,P ＜0.01),and the permeability decreased significantly (56.149％ ± 3.572％ of the single endothelial model; t =19.330,P ＜ 0.01 ).Conclusions The primary isolated rat BMVECs and pericytes cocultured the morphology,structure and barrier function of in vitro model have the basic characteristics of BBB,and they have provided a useful tool for the research of BBB.

Objective:To compare the function and action pathways of VEGFA, VEGFB and VEGFC in VEGF family of mouse eye.Methods:Using the BXD mouse gene data in Genenetwork database as template to compare and study the similarities and differences of VEGFA, VEGFB and VEGFC molecular pathways or potential functions in the whole genome expression spectrum of BXD recombinant mouse inbred line population, with multiple analytical methods and statistical strategies were used, such as gene expression level, target genes comparison, top genes comparison associated to target genes, expression Quantitative Trait Loci (eQTL).Results:Matrix comparison showed strong positive correlation between two probes of VEGFC ( r=0.732, P<0.01), weak correlation between VEGFA 1420909 and VEGFC 1440739, VEGFA 1451959 and VEGFC 1451803, VEGFC 1419417959 and VEGFC 1439766, VEGFC 1451803 and VEGFC 1439766 ( P<0.05); there was no correlation between VEGFA 1420909 and four other genes except VEGFC 1440739, VEGFA 1451959 and VEGFC 1440739, VEGFB 1451803 and VEGFA 1420909/VEGFC 1419417/VEGFC 1440739 ( P >0.05). In the comparative analysis of the relevant Top 50 genes of each VEGF gene, most of the genes in BXD mouse were not significantly correlated with VEGFA, VEGFB and VEGFC except for the weak association of individual related genes. The results of eQTL analysis showed that each probe of VEGF gene was located on different chromosomes. Conclusions:The expression levels and positive and negative correlations of VEGFA, VEGFB and VEGFC were different in the VEGF family of mouse eye, suggesting that these genes may play their role through different pathways.

Objective:To investigate the influence of nonylphenol (NP) on cytoactive and the expression of G protein-coupled estrogen receptor 30 (GPR30) in human colon cancer SW480 cells.Methods:The experimental study was conducted. The human colon cancer SW480 cells were cultured in vitro. The influence of NP on proliferation, cell cycle, apoptosis and the expression of GPR30 in human colon cancer SW480 cells were analyzed by cell proliferation, cell cycle detection, cell apoptosis and gene expression and protein expression experiments. Cell grouping: SW480 cells cultured with medium were set as the control group, cultured with medium+1×10 ?8 mol/L estradiol were set as the estradiol group, cultured with medium+1×10 ?8 mol/L NP were set as the NP group, cultured with medium+1×10 ?8 mol/L NP+1×10 ?7 mol/L GPR30 specific antagonist G15 were set as the NP+G15 group, respectively. Observation indicators: (1) proliferation index of human colon cancer SW480 cells in the 4 groups; (2) cycle proportion of human colon cancer SW480 cells in the 4 groups; (3) apoptosis index of human colon cancer SW480 cells in the 4 groups; (4) GPR30 messenger RNA(mRNA) expression of human colon cancer SW480 cells in the 4 groups; (5) GPR30 protein expression of human colon cancer SW480 cells in the 4 groups. Measurement data with normal distribution were represented as Mean± SD and one way ANOVA was used for comparison between groups. The least significant difference method was used to test the pairwise comparison. Results:(1) Proliferation index of human colon cancer SW480 cells in the 4 groups. Results of the cell proliferation experiments showed that the proliferation indexes of human colon cancer SW480 cells in the control group, the estradiol group, the NP group and the NP+G15 group were 100.00±0.00, 89.19±4.86, 148.96±6.04 and 120.40±3.39, respectively, showing a significant difference among the 4 groups ( F=21.45, P<0.05). There was a significant difference between the control group and the NP group ( P<0.05), and there was no significant difference between the control group and the estradiol group, between the control group and the NP+G15 group ( P>0.05). (2) Cycle proportion of human colon cancer SW480 cells in the 4 groups. Results of the cell cycle detection experiments showed that the proportions of human colon cancer SW480 cells in the S phase of the cell cycles in the control group, the estradiol group, the NP group and the NP+G15 group were 39.96%±2.02%, 36.67%±0.62%, 43.85%±1.02% and 38.29%±1.42%, respectively, showing a significant difference among the 4 groups ( F=10.08, P<0.05). There were significant differences between the control group and the estradiol group, between the control group and the NP group ( P<0.05), and there was no significant difference between the control group and the NP+G15 group ( P>0.05). (3) Apoptosis index of human colon cancer SW480 cells in the 4 groups. Results of the cell apoptosis experiments showed that the apoptosis indexes of human colon cancer SW480 cells in the control group, the estradiol group, the NP group and the NP+G15 group were 1.67±0.18, 4.80±0.31, 0.75±0.11 and 2.20±0.19, respectively, showing a significant difference among the 4 groups ( F=136.79, P<0.05). There were significant differences between the control group and the estradiol group, between the control group and the NP group ( P<0.05), and there was no significant difference between the control group and the NP+G15 group ( P>0.05). (4) GPR30 mRNA expression of human colon cancer SW480 cells in the 4 groups. Results of quantitative real-time polymerase chain reaction detection showed that the relative expression rates of GPR30 mRNA in human colon cancer SW480 cells of the control group, the estradiol group, the NP group and the NP+G15 group were 1.00±0.00, 0.86±0.05, 1.89±0.27 and 0.64±0.12, respectively, showing a significant difference among the 4 groups ( F=26.61, P<0.05). There were significant differences between the control group and the NP group, between the control group and the NP+G15 group ( P<0.05), and there was no significant difference between the control group and the estradiol group ( P>0.05). (5) GPR30 protein expression human colon cancer SW480 cells in the 4 groups. Results of Western blot detection showed that the relative expression rates of GPR30 protein in human colon cancer SW480 cells of the control group, the estradiol group, the NP group and the NP+G15 group were 1.83±0.16, 1.68±0.15, 3.10±0.30 and 1.26±0.11, respectively, showing a significant difference among the 4 groups ( F=34.05, P<0.05). There were significant differences between the control group and the NP group, between the control group and the NP+G15 group ( P<0.05), and there was no significant difference between the control group and the estradiol group ( P>0.05). Conclusion:Low dose of NP can increase the proliferation index and the proportion of cells in the S phase of the cell cycles, decrease the apoptosis index, and promote the mRNA and protein expression of GPR30 in human colon cancer SW480 cells.

OBJECTIVETo evaluate the association between the two single nucleotide polymorphisms located in catechol-O-methyltransferase (COMT) gene and type 2 diabetes mellitus (T2DM)in Han population in Guangdong province.

METHODSTwo tagSNPs (rs4646312 and rs4680) were picked out from COMT gene. Using the SNPscan(TM) Kit, SNP genotyping was then performed, in two cohorts, including 595 cases and 725 controls. Finally, Chi-square test, logistic regression model and other methods were employed for statistical analysis.

RESULTSThe frequencies of TT, CT and CC of rs4646312 appeared to be 304(51.1%), 234(39.3%)and 57 (8.6%) in cases, 323 (44.6%), 319 (44.0%) and 83(11.4%)in controls, respectively. The frequencies of GG,GA and AA of rs4680 were 311(52.4%), 236 (39.8%) and 46(7.8%)in cases, 417(57.7%), 265 (36.6%) and 41 (5.7%) in controls, respectively.

RESULTSshowed that SNP rs4646312 was significantly associated with T2DM both in allelic association analysis (P = 0.020,OR = 1.26, 95%CI:1.04-1.53)and in recessive model (P = 0.022, OR = 1.35, 95% CI:1.05-1.74)after adjustment for sex,BMI and TG. The association between rs4680 and T2DM was not significant, but BMI was remarkably different among the three genotypes of rs4680 after controlling for other factors.

CONCLUSIONSNP rs4646312 of COMT gene was associated with the increased risk of T2DM in Han population in Guangdong province. However, rs4680 was not significantly associated with T2DM.

Adult ; Aged ; Alleles ; Case-Control Studies ; Catechol O-Methyltransferase ; genetics ; Diabetes Mellitus, Type 2 ; etiology ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide