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Objective To investigate the effects of simulated microgravity by RCCS on proliferation and cell cytoskeleton of human HaCaT keratinocyte. Methods The rotary cell culture system (RCCS) was used to simulate the microgravity environment, and human HaCaT keratinocytes were divided randomly(random number) into the simulated microgravity group (SMG) and normal gravity group (NG). HaCaT cells in the two groups were harvested respectively after 32, 36 and 42 h culture. The HaCaT cells proliferation and cycles were detected by flow cytometry, the concentration of hb-EGF in supernatant was detected by ELISA, and the cell cytoskeleton was observed after 42 hours' culture under laser confocal microscope with FITC-labeled technique. SPSS 23.0 statistical software was used for statistical analysis, and P <0.05 was considered statistically significant. Results The flow cytometry showed that the proportions of human HaCaT keratinocytes in G1 and G2/M phases were increased while the proportion of HaCaT cells in S stage was decreased significantly after 32, 36 and 42 h RCCSculture compared with those in the normal gravity group. The HaCaT cells in G1 stage were declined along with incubation time. ELISA results showed that the hb-EGF concentration in HaCaT supernatant under simulated microgravity culture for 24 and 36 h was lower than that in the normal control group (P<0.01). The laser confocal microscope revealed that the HaCaT fluorescence intensity was decreased,and there were disordered microfilaments, structural ambiguity, pseudopodia reduction and irregularshape among FITC-labeled HaCaT cells cultured 42 h in RSSC compared with the normal gravity group.Conclusions RCCS simulated microgravity environment could inhibit the cell cycle transformation and proliferation of human HaCaT keratinocyte, affect the keratinocyte-secreting function, and induce alterations of the cell cytoskeleton.
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Objective To investigate the effects of cerium oxide nanoparticles of different sizes on the number and constructions of immune cells in peripheral blood of mice after X-ray irradiation. Methods Mice were randomly divided into 4 groups according to body weight layer and the weight of each mouse was weighed. All mice were divided into 6 groups according to weight from high to low, and there were 4 mice in each group. Then 1 mouse was randomly taken from each group to form the control group. Model group, 5 nm and 25 nm cerium oxide nanoparticles groups were formed in turn. There were 6 mice in each group. The mice in model group and cerium oxide nanoparticles administration groups were irradiated once with 3 Gy of X-rays. The mice in cerium oxide nanoparticles groups began to be intraperitoneally administrated once a day with 10 μg 5 nm or 25 nm cerium oxide nanoparticles per kilogram body weight on the 4th day before irradiation and once every other 2 days after irradiation. The mice in the control group and model group were intraperitoneally administrated with 0.9 % saline. The mice were killed on the 10th days after irradiation. White cells count (WBC) and classification in peripheral blood were detected by using automatic globulimeter, and lymphocyte subsets were analyzed by using flow cytometry. Results Compared with the control group, the number of WBC, neutrophil granulocytes, monocytes, lymphocytes, total T lymphocytes, CD4+and CD8+T lymphocytes and the percentages in the model group were decreased (all P＜0.05), and percentages of the lymphocytes, B cells and NK cells and ratio of CD4 to CD8 were increased in model group (all P＜ 0.05). Compared with the model group, the above parameters except percentages of T lymphocytes, CD4+and CD8+T lymphocytes were improved in mice of 5 nm cerium oxide nanoparticle group (all P ＜0.05). Compared with the control group, the number of WBC and lymphocytes were decreased in the 5 nm cerium oxide nanoparticle group (P＜0.05), and there were no significances in other parameters compared with the control group (all P ＞0.05). Compared with the control group, the number of WBC and lymphocytes, the number and percentages of T lymphocytes, CD4+and CD8+T lymphocytes and the percentages were decreased (all P＜ 0.05), and percentage of NK cells and ratio of CD4 to CD8 were significantly increased in 25 nm cerium oxide nanoparticles group (all P＜ 0.05). The number of lymphocytes and CD8+T lymphocytes in 25 nm cerium oxide nanoparticles group was lower than that in 5 nm cerium oxide nanoparticles group (all P ＜ 0.05). Conclusions The effects of cerium oxide nanoparticles of different sizes on the immune cells of mice after X-ray irradiation are different, and 5 nm cerium oxide nanoparticle is superior to 25 nm cerium oxide nanoparticle.
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Objective To explore the effect of dose rate of X-rays on migration of non-small cell lung cancer (NSCLC) cells and provide the experimental basis for developing radiotherapy scheme. Methods Human NSCLC cell line A549 was cultured and irradiated with X-rays at dose of 6 Gy from a linear accelerator. The dose rates of 1, 2, 4 and 6 Gy/min were selected. Monolayer adherent cells were scratched and photographed at 0 hour and 24 hours under a microscope to measure the scratch width. Results After 24 hours, the scratch width of nonirradiated control cells was (640.7±8.1)μm. The scratch widths of cells were different when cells were irradiated with X-rays of various dose rates. Scratch widths were the largest in cells irradiated at dose rates of 1 Gy/min [(691.4±7.6)μm] and 6 Gy/min [(691.8±12.1)μm]. The scratch width was (666.2±1.3) μm of X-rays at 4 Gy/min, and there were significant differences compared with nonirradiated group (all P< 0.01), which suggested that inhibitory effect of X-rays at dose rates on A549 cell migration was obvious. However, the scratch width of cells irradiated at 2 Gy/min [(643.5 ±6.8) μm] had no difference compared with the control cells (t=-0.336, P=0.742). Conclusions The effect of X-rays irradiation on cell migration of human NSCLC cell line A549 is related with irradiated dose rate. The effect of different dose rates on cell migration is significantly different. Selecting appropriate dose rates for irradiation may help to improve the efficacy of radiotherapy.
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Objective To investigate the effects of simulated microgravity by rotary cell culture system (RCCS) on expression profiles of miRNA in mouse fibroblasts L929 cell line.Methods L929 cells were cultured in vitro and divided into simulated microgravity (SMG) group and normal gravity (NG) group according to the simple random method.Samples of two groups were collected on 7th day of culture and the total RNAs were extracted,labeled,and hybridized in sequence.Feature Extraction Software was used to collect the array images and get raw data,which were analyzed by Genespring Software.Differentially expressed miRNAs were identified and then validated by qRT-PCR.Target genes of differentially expressed miRNAs were predicted by the databases of Targetscan and microRNAorg,and the intersections of databases were identified as potential regulatory target genes.Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis were applied to determine the roles of these target genes.Relevant biological functions and/or signaling pathways of the regulated genes especially related with wound healing process were categorized based on their enrichments.Then integration predictions of the miRNA and mRNA expression profiles had been proposed to refine the functional miRNA-mRNA relationships.The miRNA-mRNA functional network and miRNA-mRNA control network were constructed.Results Four miRNA genes were up-regulated significantly including mmumiR-669j,-122-5p,-30a-3p,-6516-3p,among which mmu-miR-669j was up-regulated at 52.84 folds with the greatest significance (P ＜ 0.05).Seventeen miRNA genes were down-regulated significantly including mmu-miR-21a-3p,-miR-28a-5p,-218-5p,-210-3p,-miR-19a-3p,-miR-31-3p,and-miR-19b-3p,among which mmu-miR-28a-5p was down-regulated at 15.47 folds with the greatest significance (P ＜ 0.05).The qRT-PCR showed a high concordance with the microarray results (P ＜ 0.05).Target gene prediction and functional enrichment analysis suggested that a variety of biological processes and signaling pathways involved in wound repair were significantly enriched (P ＜ 0.05).Function network and regulation network of miRNA-mRNA covered all the differentially expressed miRNAs,which suggested that miR-21 a-3p and predicted target gene Smad3 might play an important role in wound healing under microgravity.Conclusions Simulated microgravity by RCCS can significantly affect the expression of stress-related miRNAs in mouse fibroblasts L929.The miRNA target gene prediction and functional enrichment analysis based on gene chip technology may provide theoretical basis for illustrating the mechanism and management of weightlessness stress injury.
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Objective To explore the effects of different dose rates of X-ray under the same dose on cell clonogenic formation in non-small-cell lung cancer cell line A549 in order to provide experimental basis for clinical radiotherapy plan. Methods The A549 cells were cultured at low density and irradiated with X-rays at dose of 4 Gy and selected dose rates of 1, 2, 4 and 6 Gy/min, respectively, from a linear accelerator. The 8th day after irradiation, the cells were fixed and stained with Giemsa solution, and colonies containing at least 50 cells were counted. The plating efficiency and surviving fraction were calculated. Results The clonogenic number in non-irradiated cells was 88.6±4.6. The numbers were significantly reduced in irradiated cells at dose rate 1, 2, 4 and 6 Gy/min (12.3±3.4, 9.0±0.8, 5.6±1.0, 11.5±1.7, respectively) than that in non-irradiated control cells (F=678.799, P<0.05). The plating efficiencies were decreased in irradiated cells, especially in 4 Gy/min irradiated cells, which was lower than that in any of the other three dose rate groups (P< 0.05). Conclusions Though at same radiation dose, cancer cells have different clonogenic formation efficiency when irradiation with X-ray at different dose rates. Thus, treatment with optimal dose rate may improve the radiotherapy efficacy.
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Objective To study the effect of osgentide (OST) on proliferation of mouse preosteoblast MC3T3-E1 under simulated microgravity ( SMG ) .Methods Under normal conditions , cell proliferation was evaluated by MTT assay to screen an OST compound of an effective concentration after MC 3T3-E1 cells were treated with series OSTs .Furthermore, cell proliferation and cell cycle distribution of MC 3T3-E1 cells were analyzed after treatment with 1 nmol/L OST5 by MTT assay and by flow cytometry ( FCM) scanning under SMG .Results Under normal conditions , 1 nmol/L OST5 was able to significantly promote the proliferation of MC3T3-E1 cells (P<0.01).Under SMG, proliferation of MC3T3-E1 cells was significantly inhibited and more cells entered G 1 than under normal conditions (CN).The proportion of S phase of MC3T3-E1 cells after treatment with 1 nmol/L OST5 ( OST-SMG) for 3 d was higher than that of untreated MC 3T3-E1 cells under SMG,suggesting that OST5 could promote DNA synthesis ( P<0.05 ) .Conclusion OST5 facilitates the proliferation of MC3T3-E1 cells under SMG, which provides a basis for the use of OST5 in the prevention and treatment of bone loss relat-ed to microgravity .
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Objective To analyze the combination characteristics between Tir-IBD( intimin binding domain) and its ligand intimin or mutant intiminN916Y of EHEC O157 ∶H7.Methods The gene of TirIBD (tir-ibd) from EHEC O157 ∶H7 chromosome was cloned into pMD18-T vector.Thereafter,the amplified gene was cloned into prokaryotic expression plasmid pET-21 a (+).The recombinant pasmid pET-21 a( +)-tir-ibd was transformed into E.coli BL21 (DE3).After inducement,the protein Tir-IBD was successfully expressed and analyzed with SDS-PAGE and Western blot.It was purified by affinity chromatography and ionexchange chromatography and was coupled on the Ni-NTA chip of BIACore 3000.Then the ligand intimin and mutant intiminN916Y were flow through the chip and their combination characteristics were detected.Results In the present study,the gene of Tir-IBD(tir-ibd) was successfully cloned into pET-21a(+).The results of SDS-PAGE and Western blot assay showed that the protein was successfully expressed,which accounts for 15％ of total expression products,and its molecular weight was about 10×103.The purity was above 95％ after purification.After coupled on the Ni-NTA chip of BIACore 3000,their combination characteristics with ligand intimin and mutant intiminN916Y were successfully detected.The equilibrium binding constants Ka was obtained by fitting the data with the BIACore evaluation program ( Version 4.1 ).The result showed that the combination characteristics between Tir-IBD and intimin have some difference compared with that of mutant intiminN916Y and the difference is temperature dependent.Conclusion Tir-IBD of EHEC O157 ∶H7 was successfully constructed and purified.The method to analyze the combination characteristics between Tir-IBD and its ligand intimin or mutant was established.The combination characteristics between Tir-IBD and intimin or mutant intiminN916Y have some temperature dependent difference and the mutated amino acids residue is crucial for their receptor-ligand binding.
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AIM: To prepare Nano-Liposome encapsulated MAGE3/HSP70(NL M3H) and study its character and antitumor immunity in mouse. METHODS: NL M3H was prepared by the thin film-dispersion ultrasonic. The shape and size of NL M3H were detected by electron microscope. The encapsulation rate, drug-carrying capacity, stability and the releasing character were tested by Sephedex-G100 gel filtration. The mouse was immunized by NL M3H, and the antitumor immunity was detected by ELISPOT and LDH release assay. RESULTS: The mean size of NL M3H was lower than 100 nm. The encapsulation rate was 38％.The drug content was 0.038 g/L. NL M3H has good stability after stored in 4℃ for 6 months. The releasing profile showed that 74 percent of proteins was released during the first 24 hours in saline. The results of ELISPOT and LDH release assay showed that NL M3H generated tumor specific cytotoxic T lymphocyte(CTL)to damage tumor cel1. CONCLUSION: NL M3H has novel characters, it can generate specific CTL to kill tumor cell, and can be used as new kind of vaccine agsinst tumor.