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Objective To investigate the functions of Monocyte chemotactic protein-induced protein 1 (MCPIP1) in human breast cancer cell line MDA-MB-231.Methods MDA-MB-231 cells were transfected with GFP-tagged MCPIP1 by Tet-on inducing expression system.Endogenous MCPIP1 was knocked down by stable expressing shRNA.MTT assay was performed to measure the growth of MDA-MB-231 cells after overexpression or knockdown of MCPIP1.FACS method was used to analyze cell cycle in MDA-MB-231 cells.Real-time PCR was used to test the expression of cell cycle-related mRNAs expression and their half-lives.RNA-IP experiment was conducted to detect the mRNA directly enriched by MCPIP1.Luciferase assay was performed to determine whether the mRNA decay was mediated through 3′UTR.Results MCPIP1 overexpression significantly inhibited cell proliferation(P<0.05), while knockdown MCPIP1 promoted cell proliferation with statistical significances (P<0.05).MCPIP1 induced cell cycle arrest in MDA-MB-231 with statistical significance (P<0.01).MCPIP1 overexpression reduced the half-lives of cell cycle mRNAs (CDK2,CDK6,cyclin D1,cyclin E1,respectively) with significance (P<0.01).In addition, cell cycle-related mRNAs were able to be pulled down by GFP-MCPIP1 but not isotype IgG(P<0.05).Compared with control vector, MCPIP1 significant suppressed luciferase activities of all four 3′UTR reporters (P<0.05).Conclusions MCPIP1 functions as a tumor suppressor in human breast cancer cell line MDA-MB-231 through inducing G1 cell cycle arrest.
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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.
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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.
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Objective To screen the potent permeation enhancers used in transcutaneous immunization with inactivated highly pathogenic avian influenza vaccine. Methods Five different permeation enhancers, ethanol, propylene glycol, dimethyl sulfoxide, ratinoic acid, oleic acid, were used to treat the skin of BALB/c mice before transcutaneous immunization. Sera were collected before the flist transcutaneous immunization and every two weeks post immunization. The titers of influenza virus-specific humoral IgG and IgA were assayed in serum, lung and nasal lavages by ELISA. The titers of hemagglutination inhibition ( HAI), IFN-γand IL-4 produced by splenic lymphocytes were also detected. Except that, clinical symptom of the skin in different time points and skin pathological changes were observed. Results The serum IgG titers, HAI titers and the influenza virus-specific lgA and IgG in lung and nasal lavages in the groups of HA +CT + DMSO, HA + CT + RA and HA + CT + OA were significantly higher than those of HA and HA + CT groups( P ＜0.05). Moreover, the numbers of splenic lymphocytes producing IFN-γ and IL-4 were increased in the above three groups than those in control groups. In addition, no evident clinical symptoms were observed, but stratum corneum of the skin in different groups showed different changes. Conclusion DMSO,RA and OA are potent permeation enhancers in mouse model inoculated with inactivated high pathogenic avian influenza vaccine transcutaneously.