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@#Abstract：Objective To investigate the adaptability and genetic stability of hepatitis A virus（HAV）SYX1 strain in human diploid cell MRC⁃5. Methods HAV SYX1 strain isolated from feces of patients with hepatitis A was continuously propagated in MRC⁃5 cells for 28 passages，of which the 1st ~ 26th passages were determined for antigen contents and virus titers，the 6th passage was observed for the morphology under microscope and detected for physicochemical properties，and the 13th ~ 15th passages were studied for virus proliferation dynamics to determine the peak yield of virus proliferation. Genomic RNA was extracted from the 8th，12th，18th，20th，22nd，25th，26th and 28th passages and sequenced to analyze the genetic stability. The main seed batch and working seed batch of HAV SYX1 strain were established and verified according to the requirements of Chinese Pharmacopoeia（VolumeⅢ，2020 edition）. Results The antigen content of HAV SYX1 was stable at 160 ~ 320 EU／mL and the titer was maintained at 7. 3 ~ 8. 3 lgCCID50／mL after the 8th passages in MRC 5 cells；Virus particles showed two types：hollow and solid，with a diameter of 27 ~ 32 nm，spherical，without envelope and protrusions on the surface，which tolerated low pH value and ether. The peak period of virus proliferation was 10 d with an antigen content of more than 160 EU／mL and a virus titer of more than 7. 0 lgCCID50／mL. HAV SYX1 was a subtype of HAV IB，and no mutation in the coding region of all structural proteins during passage was observed. The verification results of main seed batch and working seed batch of HAV all met the relevant requirements. Conclusion HAV SYX1 strain showed good adapt⁃ ability and genetic stability in MRC⁃5，which might be used for the development and production of inactivated hepatitis A vaccine.
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@#Abstract：Objective To improve the replication level of varicella⁃zoster virus（VZV）in human diploid cell line MRC⁃5 and increase the yield of VZV vaccine by reducing the expression of interferon（IFN）related genes via optimizing the cell line MRC⁃5. Methods Interferon receptor 1（IFNAR1）silenced MRC⁃5 cell line（MRC⁃5IFNAR1⁃）was constructed by CRISPR／Cas9 gene editing technology，which was determined for the relative expression of IFNAR1 mRNA，and for those of mRNA of IFN related genes IFNβ and OAS1 after VZV infection by qRT⁃PCR to evaluate the effect of gene silencing. Gene mutation sequences were further identified by sequencing of the silenced sites. The replication of VZV in MRC⁃5 and MRC⁃5IFNAR1⁃ cell lines was compared 168 h after VZV infection by using qRT⁃PCR and plaque formation unit（PFU）assay， to evaluate the effect of MRC⁃5IFNAR1⁃cell line on VZV replication. Results The growth status of MRC⁃5IFNAR1⁃ cell line wasconsistent with that of MRC ⁃ 5 cells，and the relative expression of IFNAR1 mRNA decreased by 73%；The relative expressions of IFNβ and OAS1 mRNA in MRC⁃5IFNAR1⁃ cell line were 61% and 90% lower than those in MRC⁃ 5 cells respectively after VZV infection；In addition，168 h after VZV infection，the level of DNA replication and the titer of VZV increased by 5. 7 folds and 4 folds respectively. Conclusion The successful establishment of MRC⁃5IFNAR1⁃ cell line may be a potential scheme to increase the yield of vaccines based on human diploid cells，and provided a reference for expanding production of VZV vaccine.
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@#To investigate whether rare ginsenosides could alleviate idiopathic pulmonary fibrosis (IPF), C57BL/6 mice were randomly divided into control group, bleomycin (BLM)-induced IPF group, rare ginsenoside Rk1 group, rare ginsenoside Rk3 group, rare ginsenoside Rh4 group and rare ginsenoside Rg5 group.All mice except those in the control group were given bleomycin injection.The IPF model was established by BLM for 28 days.The treatment group was given ginsenoside intragastrically at the same time.After the experiment, the lung tissues of mice were collected and the pathological changes of the mice lungs were observed.The content of hydroxyproline (HYP) in mouse lung tissue was measured.The expression of IPF-related genes in mouse lung tissues was detected.In in vitro experiments, Medical Research Council cell strain-5 (MRC-5) was used to induce IPF cell model using transforming growth factor-β1 (10 ng/mL).The effects of four saponins on the expression of IPF-related genes were analyzed by MTT assay, HYP content determination and RT-qPCR.All four rare ginsenosides could effectively alleviate the pathological process such as alveolar structure destruction caused by IPF, reduce the content of HYP, and down-regulate the expression of IPF-related genes, indicating that rare ginsenosides can effectively alleviate IPF.
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Aim To explore the role of transcription factor F0XM1 in collagen synthesis in MRC-5 cells in-duced by high glucose.Methods I ▪ The optimal time and concentration of the hyperglycemia model in MRC-5 cells were explored by CCK8: the time gradi¬ents: 6,12,24,48,72 h; the concentration gradients: 5.5,15 ,30,45 mmol • L 1 , and 30 mmol • L 1 man- nitol was used to be the hypertonic control group.(2) Hie effect of collagen synthesis in MRC-5 cells induced by high glucose was detected: the cells were divided into normal control group, hypertonic control group (30 mmol • L 1 mannitol) and high glucose (30 mmol • L 1 ) group.WB and qPCR assays were used to de¬tect the expression of conllagen synthesis factors ( Fn, COL 1, COL m, a-SMA, MMP9, TIMP1 ) and TGF-p signaling pathway factors (TGF-pi , p-Smad2/ Smad2).(3 The role of FOXMl in promoting collagen synthesis by high glucose was investigated: the cells were divided into normal control group, hypertonic control group (30 mmol • L 1 mannitol) , high glucose ( 30 mmol • L 1 ) group and high glucose (30 mmol • L 1 ) + thiostrepton ( 1 (xmol) group, and the expres¬sions of FOXM1 , collagen synthesis factors were detec¬ted by WB and qPCR assays.Results Mannitol had no significant effect on proliferation of MRC-5 cells, hut their proliferation activity was significantly lower than that of control group when MRC-5 cells were trea¬ted with 30 mmol • L 1 high glucose for 24 h; the ex¬pressions of COL 1 , COL IH , F0XM1 and other fac¬tors were promoted when MRC5 cells were treated with high glucose; the expression of F0XM1 was signifi¬cantly inhibited after the addition of thiostrepton, and the expressions of collagen synthesis factors also de-creased compared with high glucose group, and the a- bove differences were all statistically significant (P < 0.05).Conclusion FOXM1 is a factor related to the increase of collagen synthesis in MRC-5 cells induced by high glucose.
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Objective:To investigate the effect of tetrandrine on transforming growth factor-β1（TGF-β1）stimulated MRC-5 cells. Method:Different concentrations of TGF-β1 (0, 2.5, 5, 10, 20, 40 μg·L-1) were applied to MRC-5 cells. Proliferation toxicity of TGF-β1 to MRC-5 was detected by cell counting kit-8 (CCK-8) method. Detection of alpha smooth muscle actin (α-SMA) and Vimentin's expression levels in MRC-5 by Western blot. Detection of changes of collagen I(Col-I) and fibronectin (FN)'s expression levels in MRC-5 supernatants by enzyme linked immunosorbent assay(ELISA) kit. And the appropriate concentration of TGF-β1 activated MRC-5 cells was screened. The appropriate concentration of TGF-β1 and different concentrations of Tet (0, 2.5, 5, 10, 20, 40 μmol·L-1) were applied to MRC-5 cells, and CCK-8 method was used to screen safe concentration again. Western blot was used to detect changes in α-SMA and Vimentin expression levels in MRC-5 cells, and ELISA method to detect changes in Col-I and FN in MRC-5 cell supernatant. Result:Compared with the blank group, 20，40 μg·L-1 of TGF-β1 had toxic effects on MRC-5 cells at 24 hours (P<0.05), and 10，20，40 μg·L-1 of TGF-β1 had toxic effects on MRC-5 cells at 48 h (P<0.05).When Tet is added for 24 h, the half inhibitory concentration (IC50) value was 14.07 μmol·L-1, and when cultured for 48 h, the IC50 value was 7.51 μmol·L-1. Compared with the blank group, the relative contents of α-SMA, FN and Col-I in the 5 μg·L-1 of TGF-β1 group were obviously increased (P<0.05), and the relative contents of Vimentin were significantly increased (P<0.01), and the relative contents of FN and Col-I, α-SMA and Vimentin in 10 μg·L-1 group were significantly increased (P<0.01). 10 μg·L-1 of TGF-β1 was co-cultured with Tet at different concentrations. Compared with the TGF-β1 group, the relative levels of α-SMA, Vimentin and FN in the 5 μmol·L-1 of Tet group were significantly reduced (P<0.01), and the relative levels of Col-I were obviously reduced (P<0.05). In the Tet 10 μmol·L-1 group, the relative contents of the α-SMA, Vimentin, FN and Col-I were significantly reduced (P<0.01). Conclusion:TGF-β1 can increase the levels of Col-I, FN and other extracellular matrices in MRC-5 cells, and Tet can effectively inhibit the occurrence of this change. It is suggested that Tet may inhibit secreting extracellular matrix of fibroblasts in the formation of pulmonary fibrosis.
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OBJECTIVE: To investigate the differentially expressed microRNAs(miRNAs) in human embryonic lung fibroblast MRC-5 cells stimulated by transforming growth factor-β1(TGF-β1) using microarray chip, and screen for key genes and signaling pathways of fibroblast trans-differentiation. METHODS: The miRNA expression gene chip dataset GSE43992 on TGF-β1 stimulated MRC-5 cells were downloaded from high-throughput Gene Expression Omnibus(GEO) database of National Center for Biotechnology Information of the United States. The R language Limma package was used to screen the differentially expressed miRNAs. Corresponding target genes were predicted by miRWalk database performed by Gene Ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analysis. The protein-protein interaction(PPI) network was constructed by the search tool for the Retrieval of Interacting Genes database. RESULTS: A total of five differentially expressed miRNAs were identified, including four up-regulated miRNAs and one down-regulated miRNA; and 42 corresponding differentially expressed target genes were predicted. GO analysis indicated that the target genes were significantly enriched in collagen catabolic process, extracellular matrix organization, membrane organization, collagen fibril organization, and cellular response to amino acid stimulus. The results of KEGG pathway analysis showed that the signaling pathways corresponding to miRNAs and target genes were mainly concentrated in 18 signaling pathways, that were mainly related to the age-ethnic signaling pathways and protein digestion and absorption miRNAs in tumors and diabetic complications. The core genes transfected into the myofibroblasts by the three fibroblasts screened by the PPI network were threonine kinase 1, estrogen receptor 1 and β-catenin. CONCLUSION: Five differentially expressed miRNAs, 42 target genes, 18 signaling pathways, and 3 core genes related to TGF-β1-induced MRC-5 cell trans-differentiation were screened. It can provide new reference for the treatment and research of many diseases including pneumoconiosis and pulmonary fibrosis.
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Aim To examine the role and uderlying mechanisms of Lin28 /let-7d axis in the proliferation of lung fibrobalsts and fibroblasts-into-myfibroblasts tran-sition,and provide novel strategy for the treatment of idiopathic pulmonary fibrosis (IPF).Methods We induced experimental lung fibrosis in mice by intratra-cheally injection of bleomycin (BLM).Ang Ⅱ and TGF-β1 were used to induce fibrogenesis in cultured MRC-5 cells;qRT-PCR and Western blot were applied to determine the changes of Lin28B,collagen 1 α1 and collagen 3α1 ;MTT assay,Edu satining and immun-ofluoresence were used to examine the cell viability, proliferation and fibroblasts-into-myofibroblasts transi-tion in MRC-5 cells.Results Lin28B was increased in the lung of mice with experimental lung fibrosis and in MRC-5 cells treated with AngⅡ or TGF-β1 .Moreo-ver,Lin28B enhanced collagen deposition via inhibi-ting expression of let-7d,which maybe contribute to the progression of IPF.In addition,further studies showed that Lin28B promoted proliferation and fibro-blasts-into-myofibroblasts in MRC-5 cells.Conclusion Lin28B /let-7d axis contributes to fibrogenesis via promotes fibroblasts-into-myofibroblasts transition, which may provide novel approaches for lung fibrosis treatment.
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Objective To optimize the culture conditions of MRC -5 human diploid cell.Methods To compare the growth status of MRC -5 cells,three kinds of culture medium with T25 bottles and Spinner cultivation system Cytodex1 micro carrier were used.Morphology,cell counting,growth curve,glucose -lactic acid value were observed and detected daily for screening a kind of suitable medium.Cell proliferation was compared with different levels of the bovine serum.Results There were no significant differences among the three kinds of culture medium.There were significant differences among MEM((43.25 ±0.60)×104 cells/mL,(12.98 ±1.27)×105 cells /mL),M199 ((35.40 ±1.41 )×104 cells/mL, (10.76 ±1.31)×105 cells /mL)and DMEM/F12 ((36.75 ±1.59)×104 cells/mL,(11.22 ±1.42)×105 cells /mL)(P<0.01).The cell proliferation of MEM cultures was 5.17 and 6.49 times better than those of M199 and DMEM/F12 cultures.Imported fetal bovine serum cell proliferation ((4.55 ±0.51)×105 cells /mL)was better than the other three bovine serum ((4.12 ±1.03,3.59 ±0.48,3.53 ±0.52)×105 cells /mL).Conclusion Tree kinds of culture medium can be used to culture MRC -5 human diploid cell.The MEMculture is better.Imported fetal bovine serum is better than other kinds of serum.
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ObjectiveStromal cell-derived factor -1 (SDF-1 ) is closely related to the biological characteristics of breast cancer. We aimed to explore whether estrogen affected breast cancer by SDF-1. MethodsThe breast cancer cell line MCF-7 and MRC5 were chosen, and divided into three groups: the control group, the estrogen group and the estrogen + estrogen receptor blocker group. Each group was cultured with different physiological concentrations of 17-β estrogen at certain time, and the same alcohol concentration of 17-β estradiol at different time points, and then the enzyme-linked immunosorbent assay (ELISA) was used to measure the concentration of SDF-1 in culture medium, and the semi-quantitative reverse transcriptionpolymerase chain reaction (RT -PCR) was used to detect the expression of SDF-1 mRNA in each group.ResultsSDF-1 can be detected in the culture medium of both MCF-7 and MRC5 cell lines. All different concentrations of 17-β estradiol may increase the secretion of SDF-1 in MCF-7 cells. When adding 17-β estradiol to the concentration of 107mol/L, the secretion of SDF-1 reached the peak in 2 hours, which was 6 times and 2.7 times that of control group ( P ＜ 0.01 ). The effect could be ehminated by pure estrogen receptor ICI182,780. In addition, the mRNA expression of SDF-1 was consistent with the SDF-1 protein levels-l07 mol/L group. The expression of SDF-1 mRNA was higher than both that of the control group and the blocking group in 2 hours (P ＜ 0.05 ). ConclusionsIn some breast cancer cell lines, physiological concentrations of estrogen can increase the secretion of SDF- 1, and this effect is mainly achieved through the estrogen receptor. Estrogen can influence the biological characteristics of breast cancer by SDF-1.
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@#Objective To reconstruct tissue-engineered 3D bronchial model using human bronchial epithelial cells and human embryo lung fibroblast as seeding cells, and liquid collagen mixed Matrigel as scaffold. Methods Human bronchial epithelial cells and human embryo lung fibroblast were mixed with liquid collagen supplementing with matrigel and casted in 12-wells plate to reconstruct cells-collagen sheet. Macroscopic observation, phase-contrast microscopy observation, routine HE staining and immunohistochemistry staining(CK ets) were employed to assess the engineered 3D model. Results We reconstructed engineered 3D bronchial model successfully in vitro by tissue engineering techniques and exerted static stretch onto the collagen sheet. From Macroscopic observation, we gained contracted well sheet. We also observed network structure in phase-contrast microscopy meanwhile the viability of cells was fine. HE staining showed the formation of 3D network structure. The immunohistochemistry staining of CK and Vimentin were positive.Conclusion We reconstructed engineered 3D bronchial model successfully in vitro and seeding cells could implement polarity growing in the scaffold materials then gained the network structure.
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BACKGROUND: For the diagnosis of varicella-zoster virus (VZV) infection, virus culture has been considered the reference method, but it gives delayed results and needs cell culture facilities. Shell vial culture is more rapid, but it also takes 2 days or more and needs cell culture. Immunofluorescent (IF) method has known to be rapid and sensitive. We compared the tube culture, shell vial culture, and direct IF method to find the most efficient diagnostic method. In addition, the MRC-5 cells were compared with A549 cells for the recovery of VZV in culture. METHODS: A total of 48 specimens were obtained from skin vesicles of patients with clinical herpes zoster. The vesicle smears were stained with FITC-conjugated monoclonal antibody. The vesicle aspirates obtained in 2 mL of viral transport media were inoculated into shell vials and tubes containing MRC-5 and A549 cell monolayers. After 48 h of incubation at 36degrees C the shell vials were stained with VZV-specific monoclonal antibody. The tubes were stained with the same antibody after 3 weeks or when the monolayer showed cytopathic effect. RESULTS: The positive rates of direct IF, shell vial culture, and tube culture were 67.5%, 87.5%, and 72.5% respectively. The positive rate of direct IF was increased to 96.4% when inadequate specimens for the direct IF were excluded. The MRC-5 and A549 cells showed no significant difference in the isolation rates of VZV in both shell vial and tube culture. CONCLUSIONS: Direct IF is the most rapid and practical method for the laboratory confirmation of VZV infection when the swab specimen is adequately obtained. The MRC-5 cells are recommended for the tube culture and A549 cells for the shell vial culture.
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Humanos , Técnicas de Cultivo de Célula , Diagnóstico , Herpes Zóster , Herpesvirus Humano 3 , PielRESUMEN
To investigate fragile sites induced by aphidicolin which is a specific inhibitor of eukaryotic DNA polymerase a which is primarily associated with chromosomal DNA replication in human lymphocytes, HaCat cells (human keratinocytes) and MRC-5 cells (human embryonic lung fibroblast), we cultured each cells in RPMI 1640 with 10% fetal calf serum and 2% PHA. Treatment of the cells with aphidicolin was generally carried out for the last 24 hours of culturing. The drug was dissolved in DMSO and used at final concentrations of 0.05~0.15 mg/ml, corresponding to a maximum DMSO concentration of 0.028%. Karyotypes of each cells were performed by routine method, and 50 metaphases were scored for each culture for analysis of breakage rate. Experimental cells treated with APC showed a dose dependent sensitivity and the amounts of chromosome breakage induced by APC are the highest in concentration of 0.15 mg/ml. The frequency of fragile sites on each cells appeared in MRC-5 cells, lymphocytes and HaCat cells in order. The common fragile sites on all experiments was 16q23, and the common fragile sites on embryonic cells was 1p31. It can be concluded that gene or nucleic acid which is located on 16q23 is the most important factor to induce chromosomal breakage with sensitivity to aphidicolin and 1p31 is important site to induce chromosomal breakage in embryonal cells.