[Study on DNA methylation in HEB cells exposed to PM(2.5) by application of methylation chip technology].

Objective: To screen the differential methylation sites, genes and pathways of air pollution fine particles (PM(2.5)) on human bronchial epithelial (HBE) cells by methylation chip and bioinformation technology, so as to provide scientific basis for further study of the toxicological mechanism of PM(2.5) on HBE cells. Methods: In August 2020, HBE cells were infected with 10 µg/ml and 50 µg/ml PM(2.5) aqueous solution for 24 h, namely PM(2.5) 10 µg/ml exposure group (low dose group) and PM(2.5) 50 µg/ml exposure group (high dose group) ; uninfected HBE cells were used as control group. The DNA fragments were hybridized with the chip, the chip scanned and read the data, analyzed the data, screened the differential methylation sites, carried out GO analysis and KEGG analysis of the differential methylation sites, and analyzed the interaction relationship of the overall differential methylation sites by functional epigenetic modules (FEMs). Results: Compared with the control group, 127 differential methylation sites were screened in the low-dose group, including 89 genes, including 55 sites with increased methylation level and 72 sites with decreased methylation level. The differential methylation sites were mainly concentrated in the Body region and UTR region. Compared with the control group, 238 differential methylation sites were screened in the high-dose group, including 168 genes, of which 127 sites had increased methylation level and 111 sites had decreased methylation level. The differential heterotopic sites were mainly concentrated in the Body region and UTR region. Through FEMs analysis, 8 genes with the most interaction were screened, of which 6 genes had significant changes in methylation level. MALT1 gene related to apoptosis was found in the heterotopic site of methylation difference in low-dose group; PIK3CA and ARID1A genes related to carcinogenesis were found in the heterotopic sites of methylation difference in high-dose group; TNF genes related to tumor inhibition were found in the results of FEMs analysis. Conclusion: After PM(2.5) exposure to HBE cells, the DNA methylation level is significantly changed, and genes related to apoptosis and carcinogenesis are screened out, suggesting that the carcinogenic mutagenic effect of PM(2.5) may be related to DNA methylation.

[Clinical and molecular characteristics of Streptococcus pneumoniae-associated hemophagocytic lymphohistiocytosis in children].

Objective: To summarize the clinical features of Streptococcus pneumoniae-associated hemophagocytic syndrome (SP-HLH), and the serotypes and drug-resistant characteristics of the isolated strains. Methods: There were 15 children with SP-HLH admitted to the Pediatric Intensive Care Unit (PICU) of Beijing Children's Hospital, Capital Medical University from January 2013 to December 2020 were included in this study. Clinical data including children's general characteristics, clinical features, laboratory examinations, treatments, prognosis and the outcomes of follow-up by May 2021 were analyzed retrospectively. The serotypes and drug resistance of the isolated strains were identified. All children were divided into the clinical improvement group and the death group. Mann-Whitney U test, Fisher's exact test were used to compare the data of the two groups. Results: Among the 15 children with SP-HLH, 8 were males and 7 were females. The age of these children was 1.0 (1.0, 2.5) years. Regarding the primary infection, there were 9 cases of severe pneumonia, 3 cases of meningitis and 3 cases of blood stream infection. None of these children had received pneumoniae conjugate vaccine (PCV) and all of them were admitted to the PICU. Respiratory failure was observed in 10 patients, acute renal injury in 5, and hemolytic uremic syndrome in 3 patients. All children received glucocorticoids and high-dose intravenous immunogloblin (IVIG) in addition to anti-infective treatment. Eight of the children were cured while the other 7 died. The neutrophil count in the death group was lower than that in the clinical improvement group ((5.0 (1.7, 9.3) × 109 vs. 5.2 (3.4, 10.5) ×109/L, Z =-2.43, P<0.015), and the length of hospital stay and days of PICU stay in the death group were both shorter than those in the improvement group statistically (3 (1, 11) vs. 39 (34, 48) d, 2 (1, 4) vs. 19 (12, 31) d, Z=-3.25, -3.24, both P=0.001). Ten serotypes of Streptococcus pneumoniae were identified, including 4 strains of 19F, 3 of 19A, 1 of 23F, 1 of 15A and 1 of 14, among which 9 strains (9/10) were covered by PCV13. All strains were resistant to erythromycin yet sensitive to vancomycin and linezolid. Conclusions: SP-HLH is more common in children under the age of 3, with a high mortality rate. The death cases have lower neutrophil count and rapid disease progression. The comprehensive treatment is anti-infective combined with glucocorticoids and high-dose IVIG. The predominant serotypes are 19F and 19A and all isolated strains were susceptible to vancomycin and linezolid.

[Effects of K-ras gene silence on the expression of oncogenes in HBE cells treated with PM(2.5)].

Objective: To explore the effects of K-ras gene on the expressions of oncogenes and cancer suppressor genes in human bronchial epithelial (HBE) cells which were exposed to PM(2.5). Methods: According to the mRNA sequence of K-ras gene provided by GenBank in September 2019, interference sequences were designed and synthesized, and the recombinant lentiviral vector was transfected into HBE cell to construct the K-ras gene-silenced cells. HBE cells and K-ras gene-silenced cells were exposed to 10 µg/ml, 50 µg/ml PM(2.5) suspension and 10 µmol/L Cr(6+). Real-time fluorescent quantitative PCR was used to detect the mRNA expression levels of c-myc, c-fos, N-ras, cyclin-D1, p16 and p53 genes, the expression levels of p53 and c-myc proteins were detected by Western blot. Results: In K-ras silenced cell group, K-ras mRNA expression level decreased (80.5%±3.6%) and K-ras protein level decreased (58.9%±4.7%) when compared with the control group (P<0.01) . Compared with the correspoding cell control group without exposure, the mRNA expression levels of c-myc, c-fos, N-ras and cyclin-D1 genes in HBE cell group exposed to different concentrations of PM(2.5), K-ras silenced cell group exposed to different concentrations of PM(2.5), HBE cell group exposed to 10 µmol/L Cr(6+) and K-ras silenced cell group exposed to 10 µmol/L Cr(6+) were increased, the mRNA expressions of p16 and p53 genes were decreased (P<0.01) . Compared with HBE cell group exposed to 10 µg/ml PM(2.5), the mRNA expressions of c-myc, c-fos and p16 genes in K-ras silenced cells exposed to 10 µg/ml PM(2.5) were decreased, and the p53 mRNA level was increased (P<0.01) . Compared with HBE cell group exposed to 50 µg/ml PM(2.5), the mRNA expression levels of c-fos, N-ras, cyclin-D1, p16 and p53 genes in K-ras silenced cell group exposed to 50 µg/ml PM(2.5) were decreased (P<0.01) . Compared with the HBE cell group without exposure, c-myc protein increased and p53 protein decreased in HBE cells exposed to 50 µg/ml PM(2.5) (P<0.05) . Compared with the K-ras silenced cell group without exposure, c-myc protein increased in K-ras silenced cells exposed to 50 µg/ml PM(2.5) (P<0.05) . Conclusion: PM(2.5) can increase the expression levels of oncogenes in HBE cells, and K-ras gene silencing can inhibit the expression levels of oncogenes in HBE cells treated with PM(2.5).

[Effect of p38MAPK gene silencing on expression of oncogenes and apoptotic genes induced by PM(2.5) in hepatocytes].

Objective: To study the effect of p38 mitogen-activated protein kinase (MAPK) gene silencing on expression of apoptotic genes and oncogenes in hepatocytes treated with PM(2.5). Methods: From June to September 2019, according to the p38MAPK gene mRNA sequence provided by GenBank, three interfering sequences were designed and synthesized, ligated into PLVX-shRNA2-puro after annealing, and the recombinant lentiviral vector was transfected into L02 hepatocytes. The p38MAPK silencing cells were identified by real-time fluorescent quantitative PCR and western blotting. The normal L02 cells and p38MAPK silencing cells were treated with 50 µg/mL PM(2.5) water soluble solution, 10 µmol/L positive control Cr(6+), and a blank control group was set up, the treatment time was 24 h. The mRNA levels of oncogenes (c-fos, c-myc, k-ras) , tumor suppressor gene (p53) and apoptotic genes (Caspase-3, Caspase-8, Caspase-9) were detected by real-time PCR. The protein levels of oncogenes and apoptotic genes were detected by Western blotting. Results: The expression levels of p38MAPK mRNA and protein in p38MAPK gene silencing cells were significantly lower than those in L02 hepatocytes (P<0.05) , and the p38MAPK gene silencing cell line was successfully constructed. Compared with the blank control group, the expression levels of the oncogenes c-fos, c-myc, k-ras and the apoptosis genes Caspase-3, Caspase-8 and Caspase-9 increased, the expression level of tumor suppressor gene p53 decreased in the L02 hepatocyte group treated with PM(2.5) water soluble matter, and the differences were statistically significant (P<0.05) . Compared with the L02 hepatocytes group treated with PM(2.5) water soluble matter, the expression levels of the oncogenes c-fos, c-myc, k-ras and apoptosis genes Caspase-3, Caspase-8 and Caspase-9 decreased, the expression level of tumor suppressor gene p53 increased in the p38MAPK gene silencing cells group treated with PM(2.5) water soluble matter, and the differences were statistically significant (P<0.05) . Conclusion: PM(2.5) has effects on the expression of oncogenes, tumor suppressor genes and apoptotic genes in L02 hepatocytes, while p38MAPK gene silencing can inhibit the effects of PM(2.5) on L02 hepatocytes.

[Effect of c-myc gene silence on the expression of oncogenes and apoptotic genes in hepatocytes treated with PM(2).5].

Objective: To construct the c-myc gene silenced hepatocytes, study the effect of c-myc gene silence on expression of oncogenes and apoptosis genes in hepatocytes treated with PM2.5. Methods: According to the c-myc gene mRNA sequence provided by GenBank, three interfering sequences were designed and synthesized, the recombinant lentiviral vector was transfected into L02 hepatocytes. The real-time quantitative PCR and western blotting were used to identify the effect of c-myc gene silencing. L02 cells and c-myc gene silenced cells were used as experimental subjects. The normal L02 cells and c-myc silenced cells were treated with 50 µg/ml PM(2.5) water soluble solution, 10 µM positive control Cr(6+) and a blank control, the treatment period was 24 h. The mRNA levels of oncogenes (c-myc, c-fos, k-ras, p53) and apoptotic genes (Caspase-3, Caspase-8, Caspase-9) were detected by real-time PCR. The protein levels of oncogenes and apoptotic genes were detected by western blotting. Results: The mRNA level and protein level of c-myc decreased by 81% and 70% in c-myc silenced cells when compared with the normal L02 hepatocytes, the above results indicate that c-myc gene silenced cells were successfully constructed. After c-myc silenced cells were treated with PM2.5 water soluble solution, The mRNA levels of c-myc, c-fos, and k-ras decreased by 84.1%, 45.4%, and 54.6% (P<0.05) , p53 increased by 192.9% (P<0.05) , and the expression of Caspase-3, Caspase-8, and Caspase-9 decreased by 24.4%, 36.1%, 60.9% (P<0.05) . In the Cr(6+) positive control group, the expression of c-myc, c-fos, and k-ras decreased by 72.1%, 82.2%, and 54.0% (P<0.05) , p53 increased by 250.0% (P<0.05) , the expression of Caspase-3, Caspase-8, and Caspase-9 decreased by 34.6%, 36.0%, 68.9% (P<0.05) , respectively, when compared with the normal L02 hepatocytes (P<0.05) . Western blotting results showed that the protein levels of c-myc and c-fos increased, p53 decreased after PM(2.5) exposure; the protein levels of Caspase-3, Caspase-8, Caspase-9 increased after PM(2.5) exposure (P<0.05) . When in comparison with the c-myc silenced group, the protein levels of c-myc and c-fos decreased, p53 protein increased in PM(2).5 exposed group (P<0.05) . Conclusion: c-myc gene silenced cells were successfully constructed in this paper. PM(2.5) could promote the expression of oncogenes and apoptotic genes in L02 cells, and c-myc gene silencing can inhibit the expression of oncogenes and apoptotic genes after PM(2.5) treatment in L02 cells.

[Influence of low dose perfluorooctanoate acid exposure to the cell proliferation, migration and invasion of the human muscle rhabdomyosarcoma cell line].

Objective: This study aimed to explore the effect of perfluorooctanoate acid (PFOA) on the proliferation, migration and invasion of the human muscle rhabdomyosarcoma RD cell line and its related mechanisms. Methods: RD cells were cultured and exposed to PFOA of different concentrations with 6-72 hours. The cell viability was assessed by cell counting kit-8 (CCK-8) assay. Wound healing and transwell filter assay were used to evaluated the migration and invasion ability of the RD cells respectively. The cell cycles were detected by Flow cytometry. Quantitative real-time PCR and Western blot were used to quantify the mRNA and protein expression difference of related genes, respectively. Results: CCK-8 assay showed that, after treated the RD cell with different dose of PFOA for 72 h, low dose PFOA (1,10,50, 100 µmol/L) promotes the proliferation of RD cells while high dose PFOA (250, 500 mol/L) inhibits the proliferation (P<0.001). Flow cytometry showed that compared with the control group, there was no significant difference in G0/G1 phase, while cells in S phase deceased and G2/M phase cells increased after treated with PFOA (50 µmol/L) for 72 h. The relative proportions of S and G2/M were significantly different between the two groups (P<0.01). The results of qPCR showed that the mRNA relative expression of CDK2 of the control group and the PFOA (50 µmol/L) group were 0.97±0.07 and 2.64±0.11 respectively, and there was a significant difference (t=12.60, P<0.001); The mRNA relative expression of cyclin E2 of the control group and the PFOA (50 µmol/L) group were 1.33±0.17 and 3.35±0.22 respectively, and there was a significant difference (t=7.42, P<0.001); The results of Western blot showed that the protein relative expression of CDK2 of the control group and the PFOA (50 µmol/L) group were 0.35±0.01 and 0.84±0.03 respectively, and there was a significant difference (t=14.60, P<0.001); The protein relative expression of cyclin E2 of the control group and the PFOA (50 µmol/L) group were 0.67±0.04 and 0.86±0.01 respectively, and there was a significant difference (t=4.88, P<0.01); There was no significant difference in the mRNA and protein expression of p21 and p53 between the PFOA and control group (P>0.05). The wound healing rate of the PFOA (50 µmol/L) group was faster than that of the control group, and the relative migration area of the PFOA group was larger accordingly (P<0.001). After PFOA (50 µmol/L) treated, the number of the cell through the membranes was much more than the control group (t=54.40, P<0.001), which means PFOA significantly stimulated the invasion ability of the RD cells. The results of qPCR showed that the mRNA relative expression of vimentin of the control group and the PFOA (50 µmol/L) group were 0.71±0.03 and 2.53±0.16 respectively, and there was a significant difference (t=11.00, P<0.001); The mRNA relative expression of MMP2 of the control group and the PFOA (50 µmol/L) group were 1.09±0.04 and 10.73±1.20 respectively, and there was a significant difference (t=8.04, P<0.001). The results of Western blot showed that the protein relative expression of vimentin of the control group and the PFOA (50 µmol/L) group were 0.55±0.06 and 0.81±0.01 respectively, and there was a significant difference (t=4.50, P<0.05). The protein relative expression of cyclin E2 of the control group and the PFOA (50 µmol/L) group were 0.64±0.04 and 1.03±0.13 respectively, and there was a significant difference (t=2.94, P<0.05). Conclusions: Low dose PFOA (50 µmol/L) exposure promotes cell proliferation, migration and invasion in the human muscle rhabdomyosarcoma cell line through inducing the expressions of MMP2, vimentin and cell cycle related genes.

[Application advances of three-dimensional bioprinting in burn and plastic surgery field].

Three-dimensional bioprinting is one of the latest and fastest growing technologies in the medical field. It has been implemented to print part of the transplantable tissues and organs, such as skin, ear, and bone. This paper introduces the application status, challenges, and application prospect of three-dimensional bioprinting in burn and plastic surgery field.

[Clinical analysis of 21 cases with short fetal femur in the third trimester].

Objective: To analyze the clinical features and to explore the etiology of short fetal femur during the third trimester. Methods: From January 2010 to June 2016, 21 singleton pregnancies with short fetal femur detected by ultrasonography during the third trimester were referred to the Chinese PLA General Hospital. Clinical data were collected, karyotype or single nucleotide polymorphism microarray was carried out to detect chromosomal abnormalities, and FGFR3 c.1138G>A mutation detection was carried out to detect achondroplasia (ACH) via invasive procedure, respectively. The deviation of femur length from the mean value of the gestational age in ultrasonography was expressed as the Z-score. The difference between ACH and isolated short femur (ISF, in the absence of associated structure abnormality or genetic abnormality) was then explored. Results: In the 21 fetuses, 11 had abnormal genetic test results(52%, 11/21), including 9 cases of ACH, 1 case of Ellis-van Creveld Syndrome and 1 case of Pallister-Killian syndrome. In the 10 ISF fetuses (48%, 10/21), 3 cases were fetal growth restriction, 1 was normal small for gestational age infant and 6 cases were unexplained. The median Z-scores for 9 cases of ACH and 10 cases of ISF in the third trimester were -5.04, -3.20, respectively. The short femur in ACH was more severe than in ISF (P=0.005) in the third trimester. Conclusions: The etiology of short fetal femur is complicated, including skeletal dysplasia, chromosomal abnormality, fetal growth restriction, as well as normal variants during fetal development. Genetic test should be considered during the antenatal consultation.

[Recovery effect of cardiomyopeptidin fractions and fraction addition on cardiac muscle cells in rats damaged by adriamycin].

OBJECTIVE: To investigate the recovery effect of cardiomyopeptidin fractions and fraction addition on the cardiac muscle cells in rats damaged by adriamycin. METHODS: Observing the activity of the succinic dehydrogenase which is at mitochondrion in the cells damaged by adriamycin with MTT. RESULTS: Five fractions have all promoted the activating effect of the enzyme, the action of PI being higher than the others. Fraction addition has also promoted the activating effect of the enzyme, but without additive effect. CONCLUSION: The recovery effect of cardimyopeptidin depends on the interplay among the fractions.