Objective To research the transfer of adenovirus human bone morphogenetic protein 4 (Ad-hBMP-4) to human degenerative lumbar intervertebral disc cells in vitro and analyze its effect on the proteoglycan, collagen type II, and Sox9 of intervertebral disc cells. Methods Identified Ad-hBMP-4 was amplified and detected. Degenerative lumbar intervertebral disc cells were aspirated from the degenerative lumbar intervertebral disc of patients with Modic III level disc protrusion (aged, 27-50 years). The expressing position of collagen type II was identified in the intervertebral disc cells through the laser confocal microscope. The intervertebral disc cells at passage 1 were transfected with Ad-hBMP-4 as experimental group. After 3 and 6 days of transfection, RT-PCR was used to detect the mRNA expressions of proteoglycan, collagen type II, and Sox9, and Western blot to detect the expressions of proteoglycan and collagen type II proteins. Non-transfected cells at passage 1 served as control group. Results The virus titer of Ad-hBMP-4 was 5 × 106 PFU/mL. No morphological changes in the cells after transfection by Ad-hBMP-4. Collagen type II mainly expressed in the cell cytoplasm. The mRNA expressions of the proteoglycan, collagen type II, and Sox9 in experimental group at 3 and 6 days after transfection were significantly higher than those in control group by RT-PCR (P lt; 0.05), and the expressions of proteoglycan and collagen type II proteins were significantly higher than those in contorl group by Western blot (P lt; 0.05). There were significant differences between 3 days and 6 days in experimental group (P lt; 0.05). Conclusion Ad-hBMP-4 could transfect human degenerative lumbar intervertebral cells with high efficiency and promote collagen type II, proteoglycan, and Sox9 expressions. hBMP-4 may play an important role in the repair process during early disc degeneration.
Objective To construct a replicationdefective recombinant adinovirus including the target gene human bone morphogenetic protein 4(fragment hBMP-4). Methods The hBMP-4 gene fragment was cut down from pCS2(+)/hBMP-4, cloned into the eukaryotic expressive vector pcDNA 3.1(+), then subcloned into pShuttle-CMV and transformed into the competent E. coli BJ5183/p by electroporation. The resulting recombinant plasmid pAdE/hBMP-4 was transformed into the packaging of thecell lines HEK293 to produce the replication-defective recombinant adenovirusescontaining the hBMP-4 gene. These replication-defective recombinant adinoviruses were transfected into HEK293 and HeLa cells. Then, total RNA and total protein were detected by RT-PCR and the Western-blot assay. Results The pAdE/hBMP-4 was confirmed by the restrictional endonuclease digestion. In HEK293 and HeLa cells, the specific transcription of the hBMP-4 gene was confirmed by RT-PCR, and the expression of the hBMP-4 protein was confirmed by theWestern-blot assay. Conclusion The replication-defective recombinant adinovirus expression vector containing the hBMP-4 gene can be constructed and expressed successfully, which has laid a foundation for the further research on the genetherapy of hBMP-4.
ObjectiveTo systematically review the correlation between the T538C polymorphism in bone morphogenetic protein 4 (BMP-4) and the risk of non-syndromic cleft lip with or without cleft palate (NSCL/P). MethodsWe electronically searched databases including PubMed, The Cochrane Library, EMbase, CBM, CNKI, VIP, and WanFang Data from inception to November 2014, to collect case-control studies of the correlation between the T538C polymorphism in BMP-4 and the risk of NSCL/P. Two reviewers independently screened literature according to the inclusion and exclusion criteria, extracted data and assessed the risk of bias of included studies. Then, meta-analysis was performed using RevMan 5.2 software. ResultsA total of 6 case-control studies involving 926 cases and 1 110 controls were included. The results of meta-analysis showed that:there was no significant association between the T538C polymorphism in BMP-4 gene and the risk of NSCL/P (C vs. T:OR=1.14, 95% CI 0.78 to 1.66; CC vs. TT:OR=0.75, 95% CI 0.50 to 1.11; CC vs. TT:OR=1.53, 95% CI 0.69 to 3.37; CC vs. CT+TT:OR=1.80, 95% CI 0.96 to 3.38; CC+CT vs. TT:OR=0.90, 95% CI 0.57 to 1.43). Subgroup analysis based on ethnicity showed that, the T538C polymorphism in BMP-4 gene was associated with increased risk of NSCL/P in Asian population (C vs. T:OR=1.54, 95% CI 1.26 to 1.87; CC vs. TT:OR=2.91, 95% CI 1.88 to 4.52; CC vs. CT+TT:OR=2.99, 95% CI 1.99 to 4.49), but decreased risk of NSCL/P in Latin populations (C vs. T:OR=0.69, 95% CI 0.50 to 0.96; CT vs. TT:OR=0.52, 95% CI 0.40 to 0.68; CC+CT vs. TT:OR=0.52, 95% CI 0.35 to 0.78). ConclusionAvailable evidence suggests that the T538C polymorphism in BMP-4 gene may be associated with increased risk of NSCL/P in Asians and decreased risk of NSCL/P in Latinas. Due to limited quality and quantity of the included studies, more high quality studies are needed to verify the above conclusion.
Objective To study the effects of connective tissue growth factor (CTGF) on retinal Müller cells based on transcriptome analysis of RNA-seq technology.MethodsRetinal Müller cells were divided into the control group and the CTGF treatment group which was continuously cultured with 10 ng/ml of CTGF for 24 h. The influence of CTGF on the migration of Müller cells were tested by scratching experiments. The RNA transcriptome analysis was applied to complete transcriptome sequencing, differentially expressed genes and functional enrichment analysis of the two groups of cells. HiSeq sequencing technology was used to sequence the whole transcriptome of the two groups of cells to obtain biological big data, and analyze the differentially expressed miRNAs on this basis. The functions and signal pathways of differential miRNAs were analyzed through gene annotation (GO) functional significance enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significant enrichment analysis. Based on transcriptome data, genes with differential expression multiples in the top ten between the two groups were screened out, and the expression of bone morphogenetic protein 4 (BMP4) gene was verified by real time fluorescence quantification PCR (qRT-PCR), immunofluorescence and Western blot.ResultsAfter CTGF stimulation of Müller cells, cell viability and mobility which compared with the control group were significantly increased, with statistically significant differences (t=3.453, P＜0.05). The differential gene expression profile of CTGF induced Müller cells was obtained by RNA transcriptome analysis. Comparing the sequencing results of the two groups, it was found that 325 differentially expressed genes included 152 up-regulated genes and 173 down-regulated genes. The results of GO functional significance enrichment analysis showed that the functions of differential miRNA were mainly divided into three categories: biological processes, cellular components, and molecular functions. These differentially expressed genes were involved in signaling between nervous systems, adhesion between cells, and the interaction between cytokines and their receptors. These differentially expressed genes were involved in different metabolic pathways and biological processes such as tissue inflammation and fibrosis. BMP4 gene was seected for verification through immunofluorescence, qRT-PCR and western blot. The results showed that the expression of BMP4 was significantly higher than that in the control group, and the difference was statistically significant (t=39.490, 10.110, 5.470; P=0.004, 0.001, 0.006).ConclusionCTGF promotes cell proliferation and migration by up-regulating the expression of BMP4 in Müller cells, leading to tissue fibrosis and inducing inflammation.
Objective To explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs). MethodsA experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by real-time quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups. ResultsThe ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant (F=53.40, 50.30; P＜0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant (F=54.35, 52.84, 84.35; P＜0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant (F=53.66, 83.54; P＜0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant (F=24.87, 53.84; P＜0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P＜0.001; BMP4 group: F=56.87, 59.35, 58.35; P＜0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group (F=48.32, 56.33, 55.01; P＞0.05). ConclusionBMP4 induces the proliferation and migration of hRMECs through glycolysis.
ObjectiveTo observe the effect of bone morphogenetic protein 4 (BMP4) on the proliferation and migration of human retinal microvascular endothelial cells (hRMEC) under oxidative stress. MethodsThe hRMEC cultured in vitro were divided into control group, 4-hydroxynonenal (HNE) treatment group (4-HNE group), 4-HNE+BMP4 group (BMP4 group). Cell culture medium of 4-HNE treatment group was added with 10 μmmol/L 4-HNE; cell culture of BMP4 group was cultured with 10 μmmol/L 4-HNE, and after stimulation for 6 h, 100 ng/ml recombinant human BMP4 was added. The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method. The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test. The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group, the fibronectin (FN) of BMP4 group, laminin (Laminin), α-smooth muscle contractile protein (α-SMA), and collagen type Ⅰ (Collagen Ⅰ), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) were detected by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group. The control group and 4-HNE group were compared by t test. ResultsCompared with the control group, cell viability (t=12.73, 16.26, P=0.000 2, <0.000 1), cell migration rate (t=28.17, 37.48, P<0.000 1, <0.000 1) in 4-HNE group and BMP4 group were significantly increased, and the difference was statistically significant; the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased, and the difference was statistically significant (t=16.36, 69.35, P=0.000 1, <0.000 1). The qRT-PCR test results showed that compared with the control group, the relative expression of VEGF, FN, Laminin, α-SMA, Collagen Ⅰ, and CTGF mRNA in the cells of the BMP4 group was significantly increased, and the difference was statistically significant (t=10.61, 17.00, 14.85, 7.78, 12.02, 10.61, P=0.0004, <0.000 1, 0.000 1, 0.001 5, 0.000 1, 0.000 4). ConclusionBMP4 can induce the proliferation and migration of hRMEC; it can also regulate the expression of angiogenesis factors and fibrosis-related factors in hRMEC.
ObjectiveTo investigate the effects of targeted regulation of SMAD9 expression by bone morphogenetic protein 4 (BMP4) on Müller cell migration, reactive oxygen species (ROS) generation and vascular endothelial growth factor (VEGF) expression. MethodsMüller cells cultured in vitro were divided into normal control group, BMP4 group, BMP4+ no-load plasmid group (BMP4+NC group) and BMP4+SMAD9 small interference plasmid group (BMP4+siSMAD9). Cells in BMP4 group, BMP4+NC group and BMP4+siSMAD9 group were induced by adding 100 ng/ml BMP4 into cell medium for 24 h. Subsequently, BMP4+NC group was transfected with empty plasmid. BMP4+siSMAD9 group was transfected with SMAD9 small interference plasmid for 48 h. The effect of BMP4 on Müller cell migration was determined by cell scratch test. The effect of BMP4 on the production of ROS in Müller cells was detected by flow cytometry. Western blots and real-time quantitative fluorescence polymerase chain reaction (qPCR) were used to detect the relative mRNA expression levels of glutamine synthetase (GS) and glial fibrinoacidic protein (GFAP) in Müller cells. VEGF expression in Müller cells was detected by immunofluorescence. One-way analysis of variance was used to compare groups. ResultsThe results of cell scratch test showed that the cell mobility of BMP4+siSMAD9 group was significantly lower than that of BMP4 and BMP4+NC group, and the difference was statistically significant (F=68.319, P＜0.001). Flow cytomethods showed that the level of ROS in BMP4+siSMAD9 group was significantly lower than that in BMP4 and BMP4+NC group, and the difference was statistically significant (F=52.158, P＜0.001). Western blot and qPCR results showed that the protein levels of GS and GFAP (F=42.715, 36.618) and mRNA relative expression levels (F=45.164, 43.165) in BMP4+siSMAD9 group were significantly lower than those in BMP4 and BMP4+NC group. The difference was statistically significant (P＜0.01). The results of immunofluorescence detection showed that the intracellular VEGF fluorescence intensity in BMP4 group and BMP4+NC group was significantly higher than that in BMP4+siSMAD9 group, and the difference was statistically significant (F=46.384, P＜0.05). ConclusionTargeted regulation of SMAD9 expression by BMP4 can up-regulate VEGF expression and promote the migration and ROS production of Müller cells.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.