Objective To observe the effects of basic fibroblast growth factor (bFGF) on osteogenic differentiation abili?ty and cell proliferation of human gingival fibroblasts (HGFs), and to explore the role of bFGF on the process of osteogenic differencitiaion in vitro. Methods HGFs were cultured in vitro until the 3rd passage when they were divided into four groups:normal medium as group 1, normal medium with 10μg/L bFGF as group 2, osteogenic medium as group 3 and osteo?genic medium with 10μg/L bFGF as group 4. MTT assay was used to evaluate the proliferation of HGFs. Alkaline phospha?tase (ALP) staining and Alizarin red staining were applied to investigate osteogenic potential of HGFs under different culture conditions. Results bFGF at concentration of 10 μg/L could increase HGFs proliferation in both normal and osteogenic medium (P<0.01). HGFs could be induced towards osteogenic differentiation and form mineralized nodule in osteogenic me?dium. However, 10μg/L bFGF had no effects on ALP activity and mineralized nodule formation of HGFs during osteogenic differentiation. Conclusion bFGF could promote the proliferation of HGFs but show no effects on osteogenic differentiation of HGFs at concentration of 10μg/L.

Objective To prepare the sinomenine hydrochloride multivesicular liposomes with high entrapment efficiency and sustained release character.Methods Multiple emulsion method was used to prepare the sinomenine hydrochloride multivesicular liposomes.Uniform design was applied to optimize the formulation and pharmaceutical process.The shape,the particle size,and the release charcter of the liposome were evaluated.Results The sinomenine hydrochloride multivesicular liposomes prepared were spherical and the size of majority particles was in the range of 20—30 ?m and well distributed.The encapsulation efficiency was more than 80% and its in-vitro release profile accorded well with the Higuchi model with t1/2 up to 52.7 h.Conclusion The formulation and pharmaceutical process of the sinomenine hydrochloride multivesicular liposomes are stable and feasible with the high encapsulation efficiency and good sustained-release character.

OBJECTIVE To establish an in vitro test method and to evaluate the genotoxicity of chemicals using primary cultured mouse hepatocytes and the changes in phosphorylated histone H2AX(γH2AX)expression levels to provide a more reliable marker of the identification of genotoxicity. METHODS Hepatocytes were isolated from BALB/c mice by an improved two-step collagenase diges?tion method and then cultured in sandwich configuration. The primary cultured hepatocytes were treat?ed with various concentrations of four known genotoxic agents bleomycin(BLM),benzo(a)pyrene〔B (a)p〕,styrene and styrene-7,8-oxide(SO)within the range of 40 μmol · L-1 and two non-genotoxic agents azathioprine(Aza)and ciclosporin A(CsA)at different time points within 24 h. The cytotoxicity induced by these toxicants was assessed by CCK-8 assay. Then,the changes in γH2AX expression levels in treated cells were determined by flow cytometry. RESULTS The four genotoxic agents could be detected and two non-genotoxic agents could not be detected by this method. The γH2AX expression level was the highest when hepatocytes were exposed to BLM and SO for 3 h,or B(a)p and styrene for 6 h(P<0.01). The production of γH2AX was 25.67,18.36,12.43 and 14.25 for the four types of genotoxic agents,respectively,and was approximately 19,13,9 and 11 times that of the vehicle control group(P<0.01)at the optimum time point and concentration. There was a significant positive corre?lation between the indicated concentrations of genotoxic chemicals and γH2AX expression levels(P<0.01). In addition,the production ofγH2AX indicated no marked increase in two non-genotoxic agents such as Aza and CsA in comparison with the control group. CONCLUSION This test method can effec?tively distinguish genotoxic agents from non-genotoxic agents,and direct genotoxic agents from indirect genotoxic agents in the absence of S9. γH2AX might be a reliable marker for the identification of the potential genotoxicity of chemicals.

Objective To investigate the pluripotency of human gingival fibroblasts (hGFs), and provide a novel cell source for tissue engineering. Methods With informed consent from volunteers, fresh and healthy gingiva were collected. The hGFs were obtained from the gingiva by tissue culture. The third passage of hGFs was cultured in osteogenic medium, chondrogenic medium and adipogenic medium. Cells without differentiation were taken as control. Cells were examined by al?kaline phosphatase (ALP) staining, Alizarin red staining, Alcian blue staining and oil red O staining for detecting of the abili?ty of differentiation pluripotency. Real-time polymerase chain reaction was applied to examine the expression of osteogenic marker genes ALP, runt-related transcript factor 2 (Runx2), chondrogenic marker aggrecan (AGR) and adipogenic marker peroxisome proliferator-activated receptor gamma 2 (PPARγ2). Results The hGFs cultured in osteogenic medium showed massive violet deposit at day 7 and calcium nodulus at day 28, meanwhile, the expressions of ALP and Runx2 were higher than those of control (P<0.01). In chondrogenic group cells were found blue deposit at day 14. In adipogenic group lipid-filled droplets stained with oil red O were found in cells at day 14. However, hGFs in control group had no any positive stain?ing. Furthermore, expressions of AGR and PPARγ2 were significantly higher than those of control (P<0.01). Conclusion Human gingival fibroblasts have the pluripotency of osteogenic, adipogenic and chondrogenic differentiation.

Objective To prepare sinomenine hydrochloride transfersomes and evaluate their qualities. MethodsThree different preparation methods including film dispersion, reverse phase evaporation, and ethanol injection methods were compared according to the encapsulation efficiency of transfersomes. Uniform design was applied to optimize the formulation and pharmaceutical process of reverse phase evaporation. The particle size, the appearance, the Z-potential, and the stability were also evaluated. ResultsThe transfersomes prepared by reverse-phase evaporation method possessed the highest encapsulation efficiency. The ideal combinations of preparation and formulation were: soya lecithin/sodium cholate was 200/30 mg/mg, chloroform/PBS was 5 mL/mL, pH of PBS was 6.5, added sinomenine hydrochloride was 10 mg. The transfersomes obtained were milky white translucent suspension, with a mean encapsulation efficiency of 62.2%. The shape of their particles was spherical or similar to spherical under microscope, which was smooth and disconglutinated with an average diameter of 96.4 nm, and a Z-potential of-35.93 mV. Aggregation or deposition was not observed after exposure under the temperature of 4 ℃ for 30 d. ConclusionThe preparation process of sinomenine hydrochloride transfersomes is feasible, the quality of obtained transfersomes is stable.It is expected to provide a new preparation for clinical use of sinomenine hydrochloride.

Objective:To explore the effects of laser irradiation parameters (irradiation frequency and single duration) on tear secretion, lens and retina.Methods:Thirty-six healthy guinea pigs were randomly divided into 6 groups with random number table method according to different frequency and single exposure duration of laser to the eye, namely, high frequency short time (HFST) group, high frequency long time (HFLT) group, medium frequency short time (MFST) group, medium frequency long time (MFLT) group, low frequency short time (LFST) group and low frequency long time (LFLT) group, 6 for each group.The right eyes were irradiated with 500 lx laser as experimental eyes, and the left eyes of the guinea pigs served as the control eyes.The high, medium and low irradiation frequencies were defined as 15 times, 10 times and 5 times, respectively, and the short and long period was defined as 30 seconds and 60 seconds each time, respectively.The right eyes were irradiated based on the grouping at a 10-minute interval.The tear secretion was detected by SchirmerⅠtest; lens opacity was assessed under the slit-lamp microscope; fundus photography was performed to evaluate the general morphology of retina; retinal function was evaluated by electroretinogram (ERG) record and the thickness of retinal outer nuclear layer was measured by histopathology examination.This study protocol was approved by the Medical Ethics Committee of Air Force Military Medical University (No.20181203), and the use and care of the experimental animals complied with the ARVO statement.Results:The tear secretion was 8.00(7.37, 9.00), 8.75(8.25, 9.00), 8.50(7.75, 9.50), 9.00(8.50, 9.50), 8.00(7.37, 8.75) and 8.25(7.75, 8.75) mm/5 min in the HFST group, HFLT group, MFST group, MFLT group, LFST group and LFLT group, respectively, without significant difference among the groups(χ 2=5.502, P=0.240); after laser irradiation, there were no statistically significant differences in tear secretion between the control eyes and laser-irradiated eyes in all the groups (all at P>0.05). The lenses were clear and the fundus was normal through the experimental duration in all the groups.The amplitude of ERG a-wave was significantly reduced in the HFST group in comparison with the LFST group (P<0.05), and there was no significant difference in the b-wave amplitude among the six groups (F=1.358, P=0.268). The ERG a-, b-wave amplitudes were not significantly different between the control eyes and laser-irradiated eyes in various groups (both at P>0.05). There was no significant difference in the thickness of the outer nuclear layer of retina among the HFST group, HFLT group, MFST group, MFLT group, LFST group and LFLT group (F=0.952, P=0.463). Conclusions:The 500 lx laser irradiation is safe to ocular surface and lens, but there are some injuries to retinal function, and the injury degree is related to laser irradiation frequency.

{L-End}Objective To investigate the effect of motorcycle exhaust (ME) on the level of oxidative stress in different parts of respiratory tract epithelial cells. {L-End}Methods BEAS-2B and A549 cells in logarithmic growth phase were randomly divided into control group, low- and high-dose groups. The two kinds of cells growing on the membrane of Transwell inserts were treated with air-liquid interface (ALI) exposure technique for 60 minutes. The cells in the low- and high- dose groups were treated with diluted gas with the volume ratio of ME to clean air of 1∶20 and 1∶10, respectively, while the cells in the control group were treated with clean air. Cells were collected to detect their relative survival rate using CCK-8 method after exposure. And the levels of malondialdehyde, glutathione and the activity of superoxide dismutase (SOD) of the cells were detected using colorimetry. {L-End}Results The ME exposure dose affected the relative survival rate of cells (P<0.01), which showed a downward trend with the increasing ME exposure doses (all P<0.05). However, there was no significant difference in the main effect of cell types and the interaction effect of ME exposure dose and cell type (all P>0.05). There was a significant interaction between ME exposure dose and cell type in the level of glutathione and the activity of SOD (all P<0.01), and the level of malondialdehyde was a significant main effect of cell type (P<0.01). There was no significant difference in the glutathione level and SOD activity between the low-dose group and the control group (all P>0.05), while the glutathione level and SOD activity in high-dose group were higher than those in the control group and low-dose group in BEAS-2B cells (all P<0.05). The glutathione level decreased with increasing ME exposure dose in A549 cells (all P<0.05). Compared with the control group, the low-dose group had a significantly higher activity of SOD (P<0.05) in A549 cells. The SOD activity of A549 cells in high-dose group was lower than those in control group and low-dose group (all P<0.05). The level of malondialdehyde in A549 cells was higher than those in BEAS-2B cells(P<0.05). {L-End}Conclusion ME exposure can lead to changes in the production of oxidative stress biomarkers in respiratory tract epithelial cells. The oxidative stress response induced by ME exposure varies among respiratory tract epithelial cells from different regions.

Objective: The aim of this study was to investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells. Methods: 16HBE cells were exposed to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mmol/L chloroacetic acid for 24 h in vitro. The cytotoxicity induced by chloroacetic acid was assessed by CCK-8 and LDH assays. Cell apoptosis was detected by Annexin V-FITC and PI staining. The protein expression levels of phosphorylation of p38, ERK1/2 and JNK were determined by western blotting. 16HBE cells were pretreated with MAPK signaling pathway specific inhibitors including SB203580, U0126 and SP600125 for 1 h, and these cells were subsequently treated with 2.5 mmol/L chloroacetic acid for 24 h. The expressions of p-p38, p-ERK1/2 and p-JNK as well as the changes of cell viability and apoptosis were measured after pretreated with inhibitors for 1 h. Results: The cell viability by CCK-8 and LDH methods gradually reduced in a dose-dependent manner when chloroacetic acid concentrations elevated (P<0.05) , and their correlation coefficients were -0.902 and -0.825, respectively. The detection efficiency of CCK-8 assay significantly increased compared with LDH assay (P<0.05) . The cell apoptosis rates, which were (17.2±4.0) %, (24.6± 4.2) %, (39.3 ± 5.7) % in 1.5, 2.0, 2.5 mmol/L chloroacetic acid-treated groups, were higher than that of the control group[ (5.6 ± 3.0) %] (P<0.05) . There was a time-or dose-dependent change in the protein expressions of p-p38, p-ERK1/2 and p-JNK. Compared with the control, the levels of p-p38 had 2.1 and 2.6-fold increases in 16 and 24 h treated groups (P<0.01) , while the levels of p-ERK1/2 distinctly decreased by 37% and 52% (P<0.01) . In comparison with the control group, the expressions of p-p38 had 1.9 and 2.6-fold increases in 1.5 and 2.5 mmol/L treatment groups (P<0.01) , whereas the expressions of p-ERK1/2 significantly decreased by 40% and 50% (P<0.01) . No significant change was observed in p-JNK protein expression between the chloroacetic acid-treated and control groups. In comparison with the vehicle control and the exposed group, p-p38, p-ERK1/2, p-JNK protein expressions significantly declined in the inhibitor controls and inhibitor groups. Compared with the controls, the cell survival rates had significant reductions of 28%, 18%, 36% and 26% respectively in chloroacetic acid treated group, SB203580 group, U0126 group and SP600125 group, and the apoptosis rates in the abovementioned groups were 7, 4, 8 and 7 times. Compared with chloroacetic acid-treated group, the cell viability increased by 14% in SB203580 group and decreased by 11% in U0126 group, and the cell apoptosis rates decreased by 36% in SB203580 group and increased by 18% in U0126 group (P<0.05) . But no significant changes were observed in cell viability and apoptosis between SP600125 and chloroacetic acid-treated group. Conclusion Chloroacetic acid might activate p38 MAPK signaling pathway and inhibit ERK1/2 MAPK signaling pathway. The signaling pathways of p38 and ERK1/2 MAPK are involved in 16HBE cell apoptosis induced by chloroacetic acid, but JNK is not involved in chloroacetic acid-induced 16HBE cell apoptosis.

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median (P₂₅-P₇₅) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group (P<0.001). DNA methylation levels of P16, RASSF1A and MGMT were 2.04±0.41, 2.19 (1.94-2.51), 2.22 (1.94-2.46)%5mC in exposure group, and 2.19±0.40, 2.41 (2.11-2.67), 2.44 (2.15-2.91)%5mC in control group. DNA methylation levels were lower in exposure group (P values were 0.005, 0.002 and 0.001, respectively). Spearman correlation analysis showed that DNA methylation levels of P16, RASSF1A, and MGMT were negative associated with urinary εdA level (r values were -0.155, -0.137, and -0.198, respectively, P<0.05). No significant correlation was observed between the εdC level and any measured DNA methylation levels (P>0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95%CI) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95%CI) was -0.094 (-0.179--0.008), P=0.032). Conclusion DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A and MGMT.

Objective:To investigate the characteristics, contents and effects of outpatients′ telephone counseling during COVID-19 pandemic, and to promote the development of outpatient service.Methods:Frequency Retrospective analysis was made on contents of such consultations ranging from February to April 2020, to learn their medical consultation, epidemic consultation, outpatient registration and non-attendance consultation, inspection consultation and other 5 aspects.Results:72 525 cases of outpatient telephone consultations were received, and 98.48 percent of the problems had been solved. The evaluation of outpatient satisfaction was as high as 4.60 points. The quantity of consultation at 8-11 am and 14-17 pm on weekdays was the largest. The highest proportion of information content was about COVID-19.Conclusions:Telephone consultation during COVID-19 can not only effectively reduce outpatient visits, but also effectively diverse patient flow and prove conducive to extension services of outpatients in the future.