A novel unsaturated polyphosphoester (UPPE) was devised in our previous research, which is a kind of promising scaffold for improving bone regeneration. However, the polymerization process of UPPE scaffolds was unfavorable, which may adversely affect the bioactivity of osteoinductive molecules added if necessary, such as recombinant human bone morphogenetic protein-2 (rhBMP2). The purpose of this study was to build a kind of optimal scaffold named UPPE-PLGA-rhBMP2 (UPB) and to investigate the bioactivity of rhBMP2 in this scaffold. Furthermore, the cytotoxicity and biocompatibility of UPB scaffold was assessed in vitro. A W1/O/W2 method was used to fabricate PLGA-rhBMP2 microspheres, and then the microspheres were added to UPPE for synthesizing UPB scaffold. The morphological characters of PLGA-rhBMP2 microspheres and UPB scaffolds were observed under the scanning electron microscopy and laser scanning confocal microscopy. The cumulative release of UPB scaffolds was detected by using ELISA. The cytotoxicity and biocompatibility of UPB scaffolds were evaluated through examining the adsorption and apoptosis of bone marrow stromal cells (bMSCs) seeded on the surface of UPB scaffolds. The bioactivity of rhBMP2 in UPB scaffolds was assessed through measuring the alkaline phosphates (ALP) activity in bMSCs seeded. The results showed that UPB scaffolds sequentially exhibited burst and sustained release of rhBMP2. The cytotoxicity was greatly reduced when the scaffolds were immersed in buffer solution for 2 h. bMSCs attached and grew on the surface of soaked UPB scaffolds, exerting well biocompatibility. The ALP activity of bMSCs seeded was significantly enhanced, indicating that the bioactivity of rhBMP2 remained and still took effect after the unfavorable polymerization process of scaffolds. It was concluded that UPB scaffolds have low cytotoxicity, good biocompatibility and preserve bioactivity of rhBMP2. UPB scaffolds are promising in improving bone regeneration.

A novel unsaturated polyphosphoester (UPPE) was devised in our previous research, which is a kind of promising scaffold for improving bone regeneration. However, the polymerization process of UPPE scaffolds was unfavorable, which may adversely affect the bioactivity of osteoinductive molecules added if necessary, such as recombinant human bone morphogenetic protein-2 (rhBMP2). The purpose of this study was to build a kind of optimal scaffold named UPPE-PLGA-rhBMP2 (UPB) and to investigate the bioactivity of rhBMP2 in this scaffold. Furthermore, the cytotoxicity and biocompatibility of UPB scaffold was assessed in vitro. A W1/O/W2 method was used to fabricate PLGA-rhBMP2 microspheres, and then the microspheres were added to UPPE for synthesizing UPB scaffold. The morphological characters of PLGA-rhBMP2 microspheres and UPB scaffolds were observed under the scanning electron microscopy and laser scanning confocal microscopy. The cumulative release of UPB scaffolds was detected by using ELISA. The cytotoxicity and biocompatibility of UPB scaffolds were evaluated through examining the adsorption and apoptosis of bone marrow stromal cells (bMSCs) seeded on the surface of UPB scaffolds. The bioactivity of rhBMP2 in UPB scaffolds was assessed through measuring the alkaline phosphates (ALP) activity in bMSCs seeded. The results showed that UPB scaffolds sequentially exhibited burst and sustained release of rhBMP2. The cytotoxicity was greatly reduced when the scaffolds were immersed in buffer solution for 2 h. bMSCs attached and grew on the surface of soaked UPB scaffolds, exerting well biocompatibility. The ALP activity of bMSCs seeded was significantly enhanced, indicating that the bioactivity of rhBMP2 remained and still took effect after the unfavorable polymerization process of scaffolds. It was concluded that UPB scaffolds have low cytotoxicity, good biocompatibility and preserve bioactivity of rhBMP2. UPB scaffolds are promising in improving bone regeneration.
Bone Morphogenetic Protein 2 ; chemistry ; pharmacology ; Bone Regeneration ; drug effects ; Humans ; Lactic Acid ; chemistry ; pharmacology ; Phosphatidylinositol Phosphates ; chemistry ; pharmacology ; Polyglycolic Acid ; chemistry ; pharmacology ; Tissue Scaffolds

Objective To investigate the regulating effects of Krüppel-like factor 6 (KLF6) on the apoptosis of human lens epithelial cells (HLECs) by activating transcription factor 4 (ATF4) pathway and explore the bio-molecular mechanism of KLF6/ATF4-induced HLECs apoptosis.Methods HLECs (HLE-B3) were cultured using high glucose DMEM medium.The eukaryotic expression plasmid pEGFP-C2-ATF4 was transfected into the cells by liposome 2000 in the ATF4-transfected group,and pEGFP-C2 was transfected in the empty plasmid group.Then the cells were exposed to 20 mJ/cm2 ultraviolet ray B (UVB) for 200 seconds,The morphological changes of the cells were observed by hematoxylin & eosin staining and Hoechst33258 fluorescein staining.Cultured cells were transfected using pEGFP-C2-KLF6 and pEGFP-C2 plasmid and pSilencer-KLF6 (siKLF6) and pSilencer plasmid,respectively,and the expression of ATF4 protein in the cells was detected by Western blot assay.Culture cells were divided into four groups.pEGFP-C2 and pSilencer plasmids were co-transfected into the cells in the empty plasmid group;pEGFP-C2-KLF6 and pSilencer empty plasmid were co-transfected into the cells of the KLF6 + pSilencer group;pEGFP-C2 empty plasmid and pSilencer-ATF4 were co-transfected in the cells of the siATF4 + pEGFP-C2 group;pEGFP-C2-KLF6 and pSilencer-ATF4 plasmids were co-transfected in the cells of the KLF6 + siATF4 group,and then the cells were exposed to UVB.The apoptosis of the cells were detected by ELISA assay.Results Cultured cells grew well in the normal control group with the uniform morphology and regular arrangement.The karyopyknosis,karyorrhexis and enlargement of intercellular space were found in the cells exposed to UVB.In the ATF4 transfected group,the number of cells was decreased.The relative expression level of the ATF4 protein in the cells was 0.99±0.06 and 0.13±0.02 in the UVB+ATF4 transfected group and UVB+pEGFP-C2 plasmid group,respectively,with a significant difference between them (t =23.13,P＜0.01).The relative expression levels of KLF6 and ATF4 proteins in the KLF6 transfected group were higher than those in the empty plasmid group,and the relative expression levels of KLF6 and ATF4 proteins in the siKLF6 group were significantly lower than those in the empty plasmid group (all at P＜0.01).ELISA assay showed that the apoptotic rate in the ATF4 transfected group was 1.37± 0.11,which was significantly higher than 0.31 ±0.11 in the normal control group (t =8.034,P =0.001);the apoptotic rate of the cells was increased in the KLF6+pSilencer group and decreased in the siATF4+pEGFP-C2 group in comparison with the empty plasmid group (P＜0.01,P=0.02).In addition,the apoptotic rate in the KLF6+ siATF4 group was remarkably lower than that in the KLF6 + pSilencer group (P＜ 0.01).Conclusions KLF6 promotes the apoptosis of HLECs induced by UVB radiation.Silence of ATF4 gene reduces the apoptotic rate of the cells.ATF4 is probably a target factor in the regulating oathwav of KLF6 to apoptosis.

Objective To investigate the effect of the overexpression of Krüppel-like factor 6 (KLF6)towards the apoptosis of human lens epithelial cells (HLECs) induced by ultraviolet B (UVB) radiation.Methods The eukaryotic expression plasmid pEGFP-C2-KLF6 which was successfully constructed were transfected into HLECs,followed by the detection of KLF6 level by using Western blot,and then companied by UVB stimulation.Cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay.The morphology of the cells was observed by using hematoxylin-eosin staining method.The cell damage was examined by Live/Dead staining.The apoptotic markers bax and bcl-2 were detected by Western blot.Quantitative apoptotic levels were measured with the apoptosis detection kit;the expression level of reactive oxygen species (ROS) was analyzed by DCFH-DA probe.Results The cell viability of the 0.5 μg transfection group and the 1.0 μg transfection group was significantly lower than that of the blank vector control group (both at P＜0.05).In high KLF6 expression group,the cells were sparse,long and narrow in size and shape,and the cytoplasm was concentrated.The cells in the normal control group were green living cells with stable morphology and even quantity.The number of red dead cells was increased significantly in the KLF6 highexpression group.After UVB irradiation,the apoptosis value,relative bax expression,bax/bcl-2 ratio and ROS expression of HLECs cells in the KLF6 high-expression group were all higher than those in the blank vector control group,with statistically significant differences between them (all at P＜0.05).Conclusions Overexpression of KLF6 can exacerbate apoptosis of HLECs caused by UVB,by regulating the expression of apoptosis-related proteins and promoting the accumulation of ROS in the endoplasmic reticulum.Down-regulation of KLF6 expression by biological tools may play a protective role on LECs to a certain extent.

Objective To observe the effect ofpolypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.Methods Experimental study.Müller cells were cultured and divided into groups according to the project design,plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro,then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay.The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit.Meanwhile,2',7'-diehlorofluorescin diaeetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.Results The morphology of cells in normal group was full and the cytoplasm staining was uniform.In N+AGEs group and Vec+AGEs group,cell volume decreased,cytoplasm was dense and concentrated,and eosinophilic staining was enhanced.The cell morphology of PSF+AGEs group was still full,with uniform cytoplasm staining and uniform nucleus staining.The viability of N+AGEs group,Vec+AGEs group and PSF+AGEs group were 0.42±0.11,0.35±0.12 and 0.68±0.12.The apoptosis values were 1.08 ± 0.16,0.96± 0.20 and 0.44± 0.08.The intracellular ROS levels were 28 833.67± 3 550.06,28 356.67±4 854.81,186 163.00±382.54.Compared with N+AGEs group and Vec+AGEs group,the cell viability of PSF+AGEs group was significantly improved (F=20.65,P=0.000),ce11 apoptosis value (F=43.43,P=0.000) and intracellular ROS level (F=1 8.86,P=0.000).Conclusion PSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müiller cells.

Objective To observe the effect ofpolypyramidine tract binding protein-associated splicing factor (PSF) on hydrogen peroxide (H2O2) induced apoptosis of retinal pigment epithelial (RPE) cells in vitro.Methods RPE cells were cultured and divided into a normal group,normal+H2O2 group,Vec+H2O2 group,PSF+H2O2 group according to the experimental design.Overexpression of PSF in RPE cells were achieved by pEGFP-PSF plasmid transient transfection into RPE cells,then RPE cells were exposed to H2O2.The morphological changes were observed by hematoxylin-eosin (HE) staining and Live/Dead staining while the survival rate of cells was detected by MTT assay.The effect of PSF on H2O2-induced RPE apoptosis was analyzed by Cell Death Detection ELISA kit.Meanwhile,intracellular reactive oxygen species (ROS) level was detected by using DCFH-DA method.Results Overexpression of PSF could effectively alleviate the morphological changes induced by H2O2 stimulation shown by HE staining,and effectively reduce dead cells number shown by Live/Dead staining.After H2O2 stimulation,the survival rate,apoptosis rate and ROS production level in PSF overexpression group were 0.68± 0.12,0.44± 0.08 and 18 616± 3 382.54 respectively,showing significant difference in comparison with the vector plasmid group and normal group (P＜0.05).Conclusion PSF overexpression plays a protective role in H2O2-induced apoptosis by inhibiting the production of ROS in RPE cells.

Objective To investigate the main pharmacological basis and mechanism of action of Guhuaisi Kangfu Pill(GHSKF)in the treatment of steroid-induced osteonecrosis of the femoral head(SONFH).Methods The active constituents and targets of GHSKF were screened by using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and other databases.The speculative targets of SONFH were screened out based on GeneCards and Online Mendelian Inheritance in Man(OMIM)databases.The gene modules and hub genes of SONFH were identified using a weighted gene co-expression network analysis(WGCNA).The intersection of the two targets and the result of WGCNA was taken to obtain the potential targets of GHSKF for the treatment of SONFH.The key active constituents were screened with the"active constituent-target"network,which was constructed by the Cytoscape software.Then,the STRING database was used to construct the protein interaction network to screen the key targets.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of key targets was performed,and the relationship between key active constituent,key targets and key signaling pathways was explored.Finally,the molecular docking between key active constituents and key targets was verified.In addition,the SONFH rat model was used for experimental verification.Results A total of 146 compounds and the corresponding 346 targets were identified based on the TCMSP database.A total of 4 187 targets of SONFH were obtained based on GeneCards and OMIM databases.In addition,twelve gene modules and 2 556 hub genes of SONFH were screened out based on WGCNA.Quercetin,luteolin and kaempferol were key active ingredients for the treatment of SONFH.Various signaling pathways such as PI3K/AKT were involved.Molecular docking showed the key active ingredients had good binding activity with the key targets.The results of animal experiments demonstrated that GHSKF could improve bone biological alterations by up-regulating AKT1,PI3K,RUNX2,and down-regulating the expression of Caspase-3 and IL-6(P＜0.01),which verified some results of the network pharmacology prediction.Conclusion We analyzed the potential mechanism of action of GHSKF for the treatment of SONFH using network pharmacology and animal experiments,which may provide a reference for further research on its pharmacological basis and targets.

Linear chromatin is compacted into eukaryotic nucleus through a complex and multi-layered architecture. Consequently, chromatin conformation in a local or long-distance manner is strongly correlated with gene expression. Chromosome conformation capture (3C) technology, together with its variants like 4C/5C/Hi-C, has been well developed to study chromatin looping and whole genome structure. In this review, we introduce new technologies including chromosome capture combined with immunoprecipitation, nuclei acid-based hybridization, single cell and genome sequencing, as well as their application.
Cell Nucleus ; Chromatin/genetics* ; Chromosomes/genetics* ; Genetic Techniques ; Genome/genetics*