OBJECTIVES: To reprogram human placental fibroblasts (HPFs) into chemically induced epithelioid-like cells (ciEP-Ls) using a combination of small-molecule compounds. METHODS: HPFs cultured under normoxic conditions were identified using immunofluorescence assay, PCR and chromosomal karyotyping. Under hypoxic conditions (37 ℃, 5% O₂), HPFs were cultured in a medium containing small-molecule compounds C6TSEDRVAJZ (CHIR99021, 616452, TTNPB, SAG, EPZ5676, DZNep, Ruxolitinib, VTP50469, Afuresertib, JNK-IN-8, and EZM0414), and the cell morphology was observed daily. The expression levels of epithelial cell markers in the induced cells were detected by immunofluorescence, Western blotting and PCR. Chromosomal karyotyping of the induced cells was performed and the induction efficiency was calculated. RESULTS: Before induction, HPFs showed positive expressions of fibroblast surface markers CD34 and vimentin and were negative for epithelial surface markers. PCR results showed high expressions of fibroblast-specific genes S100A4 and COL1A1 in HPFs with a normal human diploid karyotype. After one day of induction, the HPFs underwent morphological changes from a multinodular spindle shape to a round or polygonal shape, which was morphologically characteristic of ciEP-Ls. On day 4 of induction, the cells exhibited high expressions of the epithelial cell markers E-cadherin and Lin28A. RT-qPCR results also showed that the cells expressed the epithelial markers Smad3, GLi3, PAX8, WT1, KRT19, and KRT18 with significantly down-regulated expressions of all the fibroblast surface markers and a normal human diploid karyotype. The reprogramming efficiency of HPFs into ciEP-Ls ranged from (64.53±2.8)% to (68.10±3.6)%. CONCLUSIONS The small-molecule compound combination C6TSEDRVAJZ is capable of inducing HPFs into ciEP-Ls under hypoxic conditions with a high induction efficiency.
Humans ; Fibroblasts/drug effects* ; Pregnancy ; Female ; Cell Differentiation/drug effects* ; Pyrimidines/pharmacology* ; Placenta/cytology* ; Cells, Cultured ; Pyridines/pharmacology* ; Pyrazoles/pharmacology* ; Epithelial Cells/cytology*

Objective:To investigate the changes of respiratory function and lung volume after bariatric surgery using spiral CT three-dimensional imaging technology.Methods:Using the prospective study method, the medical records of 30 subjects undergoing sleeve gastrectomy (LSG) in the Eighth Department of General Surgery, the Second Hospital of Hebei Medical University from Jan. 2023 to Jun. 2024 were collected. Among them, 12 were males and 18 were females, aged from 20 to 45 years, with the average age of 31.1 years old. Chest CT scans were completed within 1 week before surgery and 6 months after surgery. The subject′s CT plain scan reconstructed thin-layer images of the mediastinal window were transferred to a GE workstation, and the lung volumes of both lungs and each lobe and the tracheal wall area of the five bronchial segments (RB1, RB4, RB10, LB1 + 2 and LB10) were calculated as a percentage of the airway cross-sectional area (WA%), and the posterior intercostal position corresponding to the diaphragm was recorded. Paired t-test and rank-sum test were used to compare preoperative and postoperative differences.Results:Compared with pre-operation, the subject′s body mass index decreased significantly 6 months after bariatric surgery [(42.22±7.31) kg/m 2vs (30.12±5.59) kg/m 2,t=7.31, P<0.001)]. Except for left lower lobe [(1.15±0.23) L vs (1.27±0.24) L, t=1.97, P=0.054] and right middle lobe [(0.57±0.16) L vs (0.83±0.16) L, t=1.38, P=0.172], the remaining lung parts were significantly larger after surgery than before surgery: right upper lobe [(0.80±0.17) L vs (0.94±0.19) L, t=2.79, P=0.007], right lower lobe [(1.08±0.14) L vs (1.22±0.19) L, t=3.23, P=0.002], left upper lobe [(1.12±0.20) L vs (1.24±0.23) L, t=2.26, P=0.014]. Overall, right lung volume [(2.44±0.33) L vs (2.79±0.41) L, t=3.62, P=0.001], left lung volume [(2.27±0.36) L vs (2.52±0.39) L, t=2.53, P=0.014] and total lung volume [(4.71±0.60) L vs (5.30±0.71) L, t=3.48, P=0.001] all increased significantly at 6 months after surgery compared with before surgery. All five segments of bronchus (WA%) were significantly reduced after surgery than before surgery: RB1: [(62.82±4.66) vs (66.85±3.99), t=3.60, P=0.001]; RB4: [(61.24±5.28) vs (64.31±5.51), t=2.20, P=0.031]; RB10: [(60.03±4.64) vs (62.97±5.73), t=2.18, P=0.033]; LB1+ 2: [(63.61±5.05) vs (67.90±4.30), t=3.54, P=0.001]; LB10: [(58.73±6.49) vs (62.01±5.06), t=2.17, P=0.034)]. The posterior intercostal position corresponding to the diaphragm dropped from an average of 7-8 intercostal spaces to 8-9 intercostal spaces, with a significant difference (rank mean 22.77 vs 38.23, Z=-3.67, P<0.001). Conclusion:Bariatric surgery can significantly reduce weight, reduce the pressure of chest and abdominal, improve lung compliance, reduce the internal pressure of the chest, lower the diaphragm, expand the lung volume and airway cross-sectional area, restore the original airway anatomy and respiratory physiology, so it can effectively improve the respiratory function and lung structure abnormalities caused by obesity.

Objective To investigate the factors influencing the development of extra-pulmonary tuberculosis(EPTB)in patients with viral hepatitis complicated by pulmonary tuberculosis(PTB).Methods A retrospective analysis was conducted on 427 patients with Hepatitis B Virus(HBV)and Hepatitis C Virus(HCV)infections complicated by PTB admitted to the tuberculosis department of Kunming Third People's Hospital from January 2015 to December 2020.Patients were divided into the EPTB complication group(n=72)and the non-EPTB complication group(n=355)based on the presence of EPTB.Clinical treatment data of patients were collected.Univariate and multivariate Logistic regression analyse were used to screen independent risk factors for EPTB as predictive factors.A nomogram prediction model was established for Extrapulmonary Tuberculosis(EPTB)complications in patients with viral hepatitis and Pulmonary Tuberculosis(PTB),evaluated using the Hosmer-Lemeshow test and ROC curve analysis.Results Among the 427 patients,292(68.3%)were male and 135(31.7%)were female,with 72 cases of EPTB,resulting in an incidence rate of 16.86%.In the EPTB group,there were 34 males(47.2%)and 38 females(52.8%).The types of EPTB included tuberculous pleuritis(21 cases,29%),tuberculous peritonitis(16 cases,22%),lymph node tuberculosis(13 cases,18%),tuberculous encephalitis(5 cases,6%),intestinal tuberculosis(6 cases,8%),bone tuberculosis(5 cases,6%),pelvic tuberculosis(3 cases,4%),and genitourinary tuberculosis(3 cases,4%).Multivariate logistic regression analysis showed that gender(OR=0.425,95%CI:0.250-0.722,P=0.02),low triglyceride(TG)levels(OR=0.837,95%CI:0.717-0.978,P=0.025),the tuberculosis-specific antigen A(ESAT-6)(OR=1.007,95%CI:1.003～1.011 were independent influencing factors for EPTB in patients with PTB complicated by HBV and HCV infections.The optimal cutoff value for the nomogram model is 0.192,with a sensitivity of 0.611,specificity of 0.710,Youden index of 0.741,positive likelihood ratio of 2.103,and negative likelihood ratio of 0.548.The Hosmer-Lemeshow test yielded χ2=2.631,P=0.955.ROC curve analysis showed an AUC of 0.693,95%CI:0.629 1～0.7574.Conclusion The prediction model based on gender,low TG levels and ESAT-6 can well predict the occurrence of EPTB to some extent,providing a reference for clinical treatment.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

Objective To improve the efficiency of induced differentiation of primitive neural epithelial cells derived from human induced pluripotent stem cells(hiPSCs-NECs)into functional midbrain dopaminergic progenitor cells(DAPs).Methods HiPSCs were cultured in mTeSRTM medium containing DMH1(10 μmol/L),SB431542(10 μmol/L),SHH(200 ng/mL),FGF8(100 ng/mL),purmorphamine(2 μmol/L),CHIR99021(3 μmol/L),and N2(1%)for 12 days to induce their differentiation into primitive neuroepithelial cells(NECs).The hiPSCs-NECs were digested with collagenase IV and then cultured in neurobasal medium supplemented with 1%N2,2%B27-A,BDNF(10 ng/mL),GDNF(10 ng/mL),AA,TGF-β,cAMP,and 1%GlutaMax in the presence of different concentrations of Rho kinase inhibitor Y27632,and the culture medium was changed the next day to remove Y27632.Continuous induction was performed until day 28 to obtain DAPs.Results Human iPSCs expressed the pluripotency markers OCT4,SOX2,Nanog,and SSEA1 and were positive for alkaline phosphatase staining.The hiPSCs-NECs were obtained on day 13 in the form of neural rosettes expressing neuroepithelial markers SOX2,nestin,and PAX6.In digested hiPSCs-NECs,the addition of 5 μmol/L Y27632 significantly promoted survival of the adherent cells,increased cell viability and the proportion of S-phase cells(P<0.01),and reduced the rate of apoptotic cells(P<0.05).On day 28 of induction,the obtained cells highly expressed the specific markers of DAPS(TH,FOXA2,NURR1,and Tuj1).Conclusion Treatment with Y27632(5 μmol/L)for 24 h significantly promotes the survival of human iPSCs-NECs during their differentiation into DPAs without affecting the cell differentiation,which indirectly enhances the efficiency of cell differentiation.

Objective:To discuss the effect of cell division cycle protein 20(CDC20)on the proliferation and cell cycle of endometrial carcinoma(EC)cells,and to clarify its mechanism.Methods:Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of CDC20 mRNA and protein in human endometrial stromal T-HESC cell and EC cells(KLE,RL95-2,ZJB-ENC1,and ECC-1 cells).The RL95-2 cells were selected for the subsequent experiments.CDC20 shRNA interference lentivirus was transfected into the RL95-2 cells and the cells were divided into control group,sh-NC group(infected with negative control lentivirus),sh-CDC20 group(infected with CDC20 shRNA interference lentivirus),sh-NC+SM04690 group(infected with negative control lentivirus followed by treatment with 64 nmol·L-1 Wnt/β-catenin signaling pathway inhibitor SM04690 for 48 h),and sh-CDC20+SM04690 group(infected with CDC20 shRNA interference lentivirus followed by treatment with 64 nmol·L-1 SM04690 for 48 h).RT-qPCR and Western blotting methods were used to detect the expression levels of CDC20 mRNA and proteins in the cells in various groups;CCK-8 method was used to detect the proliferation activities of the RL95-2 cells in various groups;BrdU assay was used to detect the percentages of BrdU positive cells in various groups;flow cytometry was used to detect the percentages of the cells at G2/M stage in various groups;Western blotting method was used to detect the expression levels of β-catenin,oncogene c-Myc,and cyclin D1 proteins in the cells in various groups.Results:Compared with T-HESC cells,the expression levels of CDC20 mRNA and protein in the KLE,RL95-2,ZJB-ENC1,and ECC-1 cells were significantly increased(P＜0.05),and the highest expression levels of CDC20 mRNA and protein were observed in RL95-2 cells.Compared with sh-NC group,the proliferation activities and percentages of the BrdU positive cells in sh-CDC20 group and sh-NC+SM04690 group were significantly decreased(P＜0.05),the percentages of the cells at G2/M phase were significantly increased(P＜0.05),and the expression levels of β-catenin,c-Myc,and cyclin D1 proteins were significantly decreased(P＜0.05).Compared with sh-CDC20 group,the proliferation activity and percentage of BrdU positive cells in sh-CDC20+SM04690 group were significantly decreased(P＜0.05),the percentage of the cells at G2/M phase was significantly increased(P＜0.05),and the expression levels of β-catenin,c-Myc,and cyclin D1 proteins in the cells were significantly decreased(P＜0.05).Conclusion:CDC20 is highly expressed in the EC cells.Silencing CDC20 may inhibit the cell proliferation by inducing G2/M phase arrest in the RL95-2 cells through the regulation of Wnt/β-catenin signal transduction.

Objective:To evaluate the effect of extracorporeal shock wave on the phosphoproteomics of the spinal cord in rats with diabetic neuralgia.Methods:Thirty-six healthy male SPF-grade Sprague-Dawley rats, aged 2 months, weighing 200-250 g, were divided into 3 groups ( n=12 each) using the random number table method: control group (group C), diabetic neuralgia group (group D), and extracorporeal shock wave + diabetic neuralgia group (group E). The rats were continuously fed a common diet in group C, while the rats were fed a high-sugar and high-fat diet for 8 weeks in D and E groups. Streptozotocin 35 mg/kg was intraperitoneally injected, and the successful induction of diabetic neuralgia was defined as the blood glucose >14.6 mmol/L and the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) ≤85% of baseline values. Group E received extracorporeal shock wave treatment after developing the model, with 1, 000 shocks per session at a frequency of 10 Hz and an energy of 1.0 bar, once per week for a total of 4 sessions. The MWT and TWL were measured before developing the model (T 0) and at 1, 2, 3 and 4 weeks after developing the model (T 1-T 4). After the last extracorporeal shock wave treatment, the rats were anesthetized and sacrificed, and lumbar spinal cord tissues were obtained for proteomic analysis and for detection of the expression of glial fibrillary acidic protein (GFAP), interleukin-1beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) (by immunohistochemistry). Results:Compared with group C, the MWT and TWL were significantly decreased at T 1-T 4 in D and E groups ( P<0.05). Compared with group D, the MWT and TWL were significantly increased at T 1-T 4 in group E ( P<0.05). The results of phosphoproteomics screening revealed 284 differentially phosphorylated proteins in D and C groups, 282 in E and C groups, and 303 in E and D groups ( P<0.05). The results of immunohistochemistry showed that the expression of GFAP, IL-1β and TNF-α was significantly up-regulated in group D compared with group C ( P<0.05); the expression of GFAP, IL-1β and TNF-α was significantly down-regulated in group E compared with group D ( P<0.05). Conclusions:The mechanism by which extracorporeal shock wave alleviates diabetic neuralgia is related to inhibition of astrocyte activation and excessive phosphorylation of mGluR5 in rats.

Objective:To retrospectively study the curative effect of Vbeam 595nm pulsed dye laser(PDL)in treating the mid-term infantile hemangiomas(IHs),and analyze and explore the possible influencing factors of that.Methods:A total of 219 IHs patients who underwent PDL treatment in Tangshan Maternal and Child Health Care Hospital from January 2020 to December 2022 were selected,and they received follow-up at least 6 months.The relationship between the treatment effect and the month age,location,thickness of hemangioma and the number of treatments was analyzed.Results:The median month age of the 219 pediatric children was 7 months,and the average month age of them was(4-10)months,and there was a total of 219 lesions.There were no statistically significant differences in the general data such as month age and the characteristics of lesion between different genders(P＞0.05).The overall effective rate of treatment was 92.7%,and the median number of treatments was 3 times,and the average number of treatments was(2-4)times.There were no statistically significant differences in the comparisons of gender,the number of treatments and the treatment outcome(P＞0.05).The curative effects of 6 pediatric children were poor,whose month age was 13(8,19)months.The curative effects of 10 pediatric children were general,whose month age was 13(8,14)months.The curative effects of 31 pediatric children were favorable,whose month age was 8(4,10)months.The curative effects of 172 pediatric children were excellent,whose month age was 6(4,9)months.The difference of month age of pediatric children among different curative effect was significant(Z=24.863,P＜0.001),and the pediatric children with better curative effect had smaller month age.The treatment outcome of pediatric children with hemangioma was correlated with the thickness of lesion,the area size of lesion and the number of treatment(r=-0.32,-0.46,-0.46,P＜0.05),respectively.The effective treatment mainly focused on the treatments of the 2nd-4th times.The adverse reaction caused by treatment was skin erythema,and the rate of the total residual after six months of the complication was 5.48%.Conclusion:The treatment of 595nm PDL combined with dynamic cooling device(DCD)can provide a safe and effective treatment for IHs.Early intervention for IHs can improve the curative effect,and reduce the course of treatment,and further reduce the possibility of corresponding complications.

A triple-transgenic (tyrosine hydroxylase/dopamine decarboxylase/GTP cyclohydrolase 1, TH/DDC/GCH1) bone marrow mesenchymal stem cell line (BMSCs) capable of stably synthesizing dopamine (DA) transmitters were established to provide experimental evidence for the clinical treatment of Parkinson's disease (PD) by using this cell line. The DA-BMSCs cell line that could stably synthesize and secrete DA transmitters was established by using the triple transgenic recombinant lentivirus. The triple transgenes (TH/DDC/GCH1) expression in DA-BMSCs was detected using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Moreover, the secretion of DA was tested by enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). Chromosome G-banding analysis was used to detect the genetic stability of DA-BMSCs. Subsequently, the DA-BMSCs were stereotactically transplanted into the right medial forebrain bundle (MFB) of Parkinson's rat models to detect their survival and differentiation in the intracerebral microenvironment of PD rats. Apomorphine (APO)-induced rotation test was used to detect the improvement of motor dysfunction in PD rat models with cell transplantation. The TH, DDC and GCH1 were expressed stably and efficiently in the DA-BMSCs cell line, but not expressed in the normal rat BMSCs. The concentration of DA in the cell culture supernatant of the triple transgenic group (DA-BMSCs) and the LV-TH group was extremely significantly higher than that of the standard BMSCs control group (P < 0.000 1). After passage, DA-BMSCs stably produced DA. Karyotype G-banding analysis showed that the vast majority of DA-BMSCs maintained normal diploid karyotypes (94.5%). Moreover, after 4 weeks of transplantation into the brain of PD rats, DA-BMSCs significantly improved the movement disorder of PD rat models, survived in a large amount in the brain microenvironment, differentiated into TH-positive and GFAP-positive cells, and upregulated the DA level in the injured area of the brain. The triple-transgenic DA-BMSCs cell line that stably produced DA, survived in large numbers, and differentiated in the rat brain was successfully established, laying a foundation for the treatment of PD using engineered culture and transplantation of DA-BMSCs.
Rats ; Animals ; Dopamine ; Parkinson Disease/metabolism* ; Mesenchymal Stem Cells/metabolism* ; Cell Line ; Brain/metabolism* ; Cell Differentiation ; Mesenchymal Stem Cell Transplantation

Objective:To explore the relationship between psychological distress and intimacy in stroke patients and the mediating effect of different dyadic coping style, to provide theoretical guidance for improving the intimate relationship of stroke patients.Methods:A convenient sampling method was used to select 203 patients with stroke who visited the General Hospital of Ningxia Medical University, the People's Hospital of Ningxia Hui Autonomous Region and the Department of Neurology of Yinchuan First People's Hospital from December 2020 to July 2021 as the survey objects. The general information questionnaire, Kessler Psychological Distress Scale, Quality of Relationship Index and Dyadic Coping Inventory were used to investigate patients.Results:The scores of psychological distress, intimate relationship, positive dyadic coping and negative dyadic coping were 19.33 ± 7.46, 32.75 ± 6.79, 86.25 ± 13.22, and 16.46 ± 4.08, respectively. Correlation analysis showed that psychological distress was negatively correlated with intimate relationship and positive dyadic coping ( r=-0.195, -0.204, both P<0.01), psychological distress was positively correlated with negative dyadic coping ( r=0.229, P<0.01). The mediating effect of positive dyadic coping and negative dyadic coping on psychological distress and intimate relationship was 35.87% and 53.26%, respectively. Conclusions:The psychological distress indirectly affects the intimate relationship of stroke patients through positive dyadic coping and negative dyadic coping. Health care workers can guide patients to actively cope with the disease and encourage couples to strengthen communication, so as to enhance the intimacy of couples and promote their physical and mental health development.