Objective To isolate and culture neural crest cells(NCCs)from lung tissue of mice and to identify the characteristics of the cells in order to provide a new cell model for studying lung injury and injure repair.Methods The mT/mG dual-fluorescence reporter mice and Wnt1-Cre transgenic mice were hybridized,and mT/mG;Wnt1-Cre transgenic mice were screened to obtain enhanced green fluorescent protein(EGFP)permanently labeled NCCs.Cell suspension of mouse lung tissue was prepared by enzymolysis.EGFP+cells(namely NCCs)were har-vested by flow cytometry.Primary culture was performed with DMEM/F12 culture medium optimized in the labora-tory,NCCs was characterized by immunofluorescence microscopy.Then NCCs differentiation was directed by mouse bone marrow mesenchymal stem cells osteogenic induction.Results The mT/mG of EGFP permanently labeled NCCs was successfully obtained by hybridization and high-purity NCCs were isolated from Wnt1-Cre transgenic mice lung tissue.They can be cultured in vitro and with spindle morphology which was,similar to fibroblast adherent proliferation.NCCs expressed the neural crest stem cell marker Sox10 and induced to differentiate into osteoblasts.Conclusions NCCs isolated and cultured from lung tissue of mT/mG;Wnt1-Cre transgenic mice show stable prolif-eration and have the characteristics of neural crest stem cells,which may function as a potential cell model for re-search on lung tissue injury and the mechanism of repair.

Objective: To explore the changes in ribosomal DNA copy number in peripheral blood among patients with pneumoconiosis and its influencing factors, so as to provide insights into prevention and treatment of pneumoconiosis. Methods: Eighty-eight patients with pneumoconiosis who visited a designated hospital and 71 community residents with no history of pneumoconiosis or dust exposure were selected as the pneumoconiosis group and control group, and age, smoking history, drinking history and cumulative years of exposure to dust were collected through questionnaire surveys. The copy number of 45S rDNA and 5S rDNA was detected using real-time fluorescence quantitative PCR, and the differences between the two groups were compared. Factors affecting the copy number of 45S rDNA and 5S rDNA were identified by a multiple linear regression model. Results: The pneumoconiosis group had a median age of 56.00 (interquartile range, 15.25) and a mean cumulative dust exposure duration of (12.40±8.08) years, with 56.82% smoking and 62.50% drinking. The control group had a median age of 64.00 (interquartile range, 37.00) years, with 32.39% smoking and 26.76% drinking. The median copy number of 45S rDNA in the pneumoconiosis group was 1.29 (interquartile range, 0.59), which was lower than 2.10 (interquartile range, 1.88) in the control group; the median copy number of 5S rDNA in the pneumoconiosis group was 5.33 (interquartile range, 0.85), which was higher than 4.66 (1.34) in the control group (both P<0.05). Multiple linear regression analysis identified age (β=-0.034) and pneumoconiosis (β=-1.595) as factors affecting 45S rDNA copy number, age (β=-0.013) as a factor affecting 5S rDNA copy number, and age (β=0.018) as a factor affecting 5S rDNA copy number in the pneumoconiosis group (all P<0.05). Conclusions Compared with community residents with no history of pneumoconiosis or dust exposure, the copy number of 45S rDNA in peripheral blood among patients with pneumoconiosis is reduced and the copy number of 5S rDNA is increased.

Objective:To explore the relationship between gene mutation in the pre-C region of hepatitis B virus (HBV) and acute-on-chronic liver failure (ACLF) associated with hepatitis B.Methods:Fifty-eight patients with chronic hepatitis B (CHB) admitted to the General Hospital of Northern Theater Command of the Chinese People′s Liberation Army from May 2020 to May 2023 were selected as the CHB group, 51 patients with chronic hepatitis B liver cirrhosis (CHB-LC) were selected as the CHB-LC group, and 52 patients with hepatitis B-related acute-on-chronic liver failure were selected as the ACLF group. The clinicopathological data of the three groups were collected for retrospective analysis. Peripheral serum HBV DNA of the three groups was collected. The pre-C region genes of HBV in the three groups were amplified by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR), and then gene sequencing was performed. The variation at position 1896 of the pre-C region gene of HBV in the three groups was recorded. The general data, hepatitis B e antigen (HBeAg), HBV DNA quantification, triglycerides (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and gene mutation in the pre-C region of the three groups were observed and compared. Logistic regression analysis was performed on the related influencing factors of the onset of hepatitis B-related ACLF.Results:There were no statistically significant differences in gender, age, disease course, body mass index (BMI), TG, TC, and BUN among the three groups (all P>0.05), and there were statistically significant differences in HBeAg, HBV DNA quantification, ALT, and AST among the three groups (all P<0.05). The HBV DNA quantification, ALT, and AST in the ACLF group were higher than those in the CHB group and the CHB-LC group (all P<0.05). The mutation rate at position 1896 of the pre-C region gene of HBV in the ACLF group was higher than that in the CHB group and the CHB-LC group (all P<0.05); The results of logistic multivariate regression analysis showed that HBV DNA quantification, ALT, and gene mutation in the pre-C region were independent influencing factors for the onset of hepatitis B-related ACLF ( OR=1.042, 1.570, 1.413, P<0.05). Conclusions:The variation at position 1896 of the pre-C region gene of HBV is common in patients with HBV infection at different disease courses. The incidence of its variation shows a gradually increasing trend in CHB, CHB-LC, and ACLF. Elevated HBV DNA and ALT and gene mutation in the pre-C region of HBV are independent risk factors for the occurrence of hepatitis B-related ACLF. The progress of the disease in such patients requires clinical attention.

Objective: To establish a convenient non-invasive tracheal perfusion method for constructing a mouse model of silicosis-induced pulmonary fibrosis. Methods: The specific pathogen-free C57BL/6 mice were randomly divided into control group and model group, with 15 mice in each group. After anesthesia, a 22G arteriovenous indwelling needle was used to inset into the trachea through the mice's mouth. The model group mice were perfused with 0.1 mL of silica suspension with a mass concentration of 25 g/L, and the mice in the control group were perfused with an equal volume of 0.9% sodium chloride solution. On the 7th, 14th, and 30th day after modeling, the body weight of the mice was measured, and the lung tissue morphology and pathological changes were observed. The expression of α-smooth muscle actin (α-SMA) and collagen type 1 alpha 1 (COL1A1) protein in lung tissue of mice was detected by immunofluorescence on the 30th day after modeling. Results: There was no death of mice in the two groups during the experiment. There was no significant difference in body weight between the two groups (P>0.05). The lung tissues of the mice in the model group were pinkish-gray and uneven in color on the 7th and 14th days after dust exposure. On the 30th day after dust exposure, the lung tissue of the mice in the model group was gray and hard, and unevenly distributed silicon nodules were visible by the naked eyes. The histopathology results of lung tissue showed that compared with the mice in control group, the model group mice exhibited persistent aggravation of pulmonary inflammation, thickening of alveolar septum, infiltration of inflammatory cells gradually clustering into clumps, and an increasing number of fibrous foci.On the 30th day after dust exposure, the relative expression of α-SMA and COL1A1 proteins in the lung of the model group was higher than those in the control group (median： 72.59 vs 5.91, 35.62 vs 10.07, both P<0.05). Conclusion: The method of tracheal perfusion silica suspension of mice using 22G arteriovenous indwelling needle can successfully construct an animal model of silicosis fibrosis. This method is convenient, safe and effective, and is worth promoting.

Objective:To investigate the effect of protocatechuic acid on chronic neuropathic pain (NP) and its mechanism in rats.Methods:NP models were established in 32 SD rats by sciatic nerve ligation, and they were randomly divided into model group, low- and high-dose protocatechuic acid groups, and ibuprofen group ( n=8); on the 3 rd d of modeling, rats in the latter 3 groups were given 10 or 20 mg/kg protocatechuic acid solution via jugular vein injection or 20 mg/kg ibuprofen tablets by gavage, once a d for consecutive 21 d. A sham-operated group ( n=8) was set up; the sciatic nerve was dissociated but not ligated. The behavioral performance of rats in each group was continuously observed; on the 7 th, 14 th and 21 st d of administration, the mechanical pain threshold of both hind limbs of rats was measured by von-Frey filament stimulation and the thermal pain threshold was measured by BME-410A thermal pain stimulator. Then, rats were sacrificed. The serum levels of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISA. Cell apoptosis in the spinal cord tissues was observed by TUNEL. Western blotting was used to detect the expressions of nuclear factor-κB (NF-κB)/NOD-like receptor pyrin domain-related protein 3 (NLRP3) signaling pathway related proteins in the spinal cord tissues. Results:On the 7 th, 14 th, and 21 st d of administration, the thermal pain threshold and mechanical pain threshold in the model group were significantly decreased as compared with those in the sham-operated group ( P<0.05); as compared with those in the model group, those in the low- and high-dose protocatechuic acid groups and ibuprofen group were significantly increased ( P<0.05); as compared with those in the low-dose protocatechuic acid group, those in the high-dose protocatechuic acid group and ibuprofen group were significantly increased ( P<0.05); those in the ibuprofen group were significantly increased as compared with those in the high-dose protocatechuic acid group ( P<0.05). (2) On the 21 st d of administration, as compared with those in sham-operated group, the serum levels of TNF-α and IL-1β and number of apoptotic cells in the spinal cord tissues of the model group were significantly increased ( P<0.05); as compared with those in the model group, those in the low- and high-dose protocatechuic acid groups and ibuprofen group were significantly decreased ( P<0.05); as compared with those in the low-dose protocatechuic acid group, those in the high-dose protocatechuic acid group and ibuprofen group were significantly decreased ( P<0.05); those in the ibuprofen group were significantly decreased as compared with those in the high-dose protocatechuic acid group ( P<0.05). (3) On the 21 st d of administration, the protein expressions of phosphorylated (p)-NF-κb-65 (0.77±0.05), NLRP3 (1.03±0.08), apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1 and IL-1β in the spinal cord of rats in the model group were significantly increased as compared with those in the sham-operated group ( P<0.05); as compared with those in the model group, those in the low- and high-dose protocatechuic acid groups were significantly decreased (p-NF-κB-65: 0.49±0.03, 0.25±0.02; NLRP3: 0.81±0.06, 0.69±0.04; P<0.05); as compared with those in the low-dose protocatechuic acid group, those in the high-dose protocatechuic acid group were significantly decreased ( P<0.05). Conclusion:Protocatechuic acid may alleviate pain in chronic NP rats by downregulating NF-κB/NLRP3 signaling pathway transduction.

BACKGROUND: A large number of experiments in vivo and in vitro have shown that mesenchymal stem cells may obviously inhibit the lymphocytes and other immunocytes.OBJECTIVE: To investigate the effect of human umbilical cord mesenchymal stem cell transplantation on the immune function and prognosis of patients suffering decompensated liver cirrhosisn due to hepatitis B.METHODS: 118 patients with decompensated cirrhosis due to hepatitis B were randomly divided into control group (n=59) and observation group (n=59). The two groups all received normal medical treatment, and in addition, the observation group also received human umbilical cord mesenchymal stem cell transplantation. (4.0-4.5)×108 stem cells were transplanted twice by intervention via proper hepatic artery (10 mL) and intravenous infusion (10 mL) within 1 week after admission. The levels of serum interleukin-6, tumor necrosis factor-α, interleukin-10, transforming growth factor-β and the percentage of lymphocyte subsets in the peripheral blood were determined in the two groups before and 1, 4 weeks after treatment. The model for end-stage liver disease (MELD) score and Child-Pugh score of 118 patients after treatment for 12 weeks were observed and recorded, and liver failure, complications and survival during follow-up period in the two groups were observed. RESULTS AND CONCLUSION: After treatment for 1 and 4 weeks, the levels of serum interleukin-6 and tumor necrosis factor-α in the observation group were significantly lower than those in the control group (P < 0.05 or P <0.001), but the levels of serum interleukin-10 and transforming growth factor-β in the observation group were significantly higher than those in the control group (P < 0.05 or P < 0.001). After treatment for 1 week, the percentagesof CD3+CD4+T cell and CD4+CD25+Treg cells in the observation group were significantly higher than those in the control group (P < 0.001), but the percentages of CD3+CD8+ T cells and CD3-CD19+ B cells were significantly lower than those in the control group (P < 0.05 or P < 0.001). After treatment for 4 weeks, the percentages of CD3+ T cell ,CD3+CD4+ T cells and CD4+CD25+ Treg cells in the observation group were significantly higher than those in the control group (P < 0.05 or P < 0.001), but the percentages of CD3+CD8+ T cell and CD3-CD19+ B cells were significantly lower than those in the control group (P < 0.05 or P < 0.001). After treatment for 12 weeks, the MELD and Child-Pugh scores in the observation group were significantly lower than those in the control group (P < 0.05).During the follow-up period, none of the cases in the observation group developed liver failure, but five cases in the control group did. In addition, the incidence of complications and cumulative mortality in the observation group were significantly lower than those in the control group (P < 0.05). These results show that the human umbilical cord mesenchymal stem cell transplantation may alleviate liver inflammation and improve liver function, and then may reduce the incidence of hepatic failure and mortality for patients with decompensated cirrhosis due to hepatitis B.

Objective To evaluate the 15 years changing trends of prevalence of high risk HPV (HR?HPV) infection and the risks of cervical cancer and precancerous lesions (CIN2+) among a Chinese rural population. Methods The screening cohort with 1 997 women aged 35 to 45 years old was built in 1999 in Xiangyuan County, Shanxi province ( SPOCCS?I) and followed up by cytology and HR?HPV testing in the years of 2005, 2010, and 2014. The changes of HR?HPV prevalence and the risks of cervical precancerous lesions with CIN2+ as the endpoints were analyzed during the past 15 years. Results The detection rates of HPV infection and CIN2+ were 15.7%?22.3% and 1.1%?4.3% for the baseline visit and the other 3 follow?ups, respectively. The cumulative risk of CIN2+ in HR?HPV positive women at baseline was significantly higher than HR?HPV negative women ( P<0.01) during the 15?year follow?up. The risk of CIN2+ in the four?times HPV positive group was 40. 0%, while the group with four?times negative HPV results was 0.6% (Adjusted RR = 55.0, 95% CI: 11.3 to 268.4). Conclusions The prevalence of HR?HPV infection and CIN2+ lesions were high in Xiangyuan county during the 15 years. HR?HPV positivity elevated the risk of CIN2+ compared to women whose HR?HPV test was negative. The risks of CIN2+incidence in 6 years were low among women with negative HR?HPV test. The risk of CIN2+ increased with the numbers of HPV infection events. The screening interval could be extended to 5?6 years.

Objective To evaluate the 15 years changing trends of prevalence of high risk HPV (HR?HPV) infection and the risks of cervical cancer and precancerous lesions (CIN2+) among a Chinese rural population. Methods The screening cohort with 1 997 women aged 35 to 45 years old was built in 1999 in Xiangyuan County, Shanxi province ( SPOCCS?I) and followed up by cytology and HR?HPV testing in the years of 2005, 2010, and 2014. The changes of HR?HPV prevalence and the risks of cervical precancerous lesions with CIN2+ as the endpoints were analyzed during the past 15 years. Results The detection rates of HPV infection and CIN2+ were 15.7%?22.3% and 1.1%?4.3% for the baseline visit and the other 3 follow?ups, respectively. The cumulative risk of CIN2+ in HR?HPV positive women at baseline was significantly higher than HR?HPV negative women ( P<0.01) during the 15?year follow?up. The risk of CIN2+ in the four?times HPV positive group was 40. 0%, while the group with four?times negative HPV results was 0.6% (Adjusted RR = 55.0, 95% CI: 11.3 to 268.4). Conclusions The prevalence of HR?HPV infection and CIN2+ lesions were high in Xiangyuan county during the 15 years. HR?HPV positivity elevated the risk of CIN2+ compared to women whose HR?HPV test was negative. The risks of CIN2+incidence in 6 years were low among women with negative HR?HPV test. The risk of CIN2+ increased with the numbers of HPV infection events. The screening interval could be extended to 5?6 years.

Objective To construct and identify the siRNA eukaryotic expression vector targeting gene CXC chemo-kine receptor-4 and explore its role in invasion process of breast cancer cells in vitro. Methods Two siRNAs were designed and synthesized according to the coding sequence of CXCR4 gene and cloned into eukaryotic expression plasmid pGE-1-U6/kna. The constructed CXCR4-siRNA expression vector was transfected into MDA-MB-231 cells by liposome. Western blot was used to evaluate the suppression of CXCR4 expression in different groups. The inva-sion and migration of MDA-MB-231 cells were evaluated by cell invasion assay in vitro. Results Enzyme digestion and DNA sequencing confirmed that the CXCR4-siRNA expression vector was successfully constructed. After trans-fection,the CXCR4-siRNA obviously suppressed the expression of CXCR4 compared with control groups and the ability of cell migration was decreased markedly. Conclusion CXCR4-siRNA expression vector can effectively sup-press CXCR4 expression in the breast cancer cells and decrease potential of cell invasion, which may provide a no-vel strategy for gene therapy of breast cancer metastasis.

Objective To construct and identify the siRNA eukaryotic expression vector targeting gene CXC chemokine receptor-4 and explore its role in invasion process of breast cancer cells in vitro.Methods Two siRNAs were designed and synthesized according to the coding sequence of CXCR4 gene and cloned into eukaryotic expression plasmid pGE-1-U6/kna.The constructed CXCR4-siRNA expression vector was transfected into MDA-MB-231 cells by liposome.Western blot was used to evaluate the suppression of CXCR4 expression in different groups.The invasion and migration of MDA-MB-231 cells were evaluated by cell invasion assay in vitro.Results Enzyme digestion and DNA sequencing confirmed that the CXCR4-siRNA expression vector was successfully constructed.After transfection,the CXCR4-siRNA obviously suppressed the expression of CXCR4 compared with control groups and the ability of cell migration was decreased markedly.Conclusion CXCR4-siRNA expression vector can effectively suppress CXCR4 expression in the breast cancer cells and decrease potential of cell invasion,which may provide a novel strategy for gene therapy of breast cancer metastasis.