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 establish a methodological system for reprogramming rat embryonic fibroblasts(REF)into chemically induced neurons(ciNCs)via small molecule compounds to provide safe and effective donor cells for treatment of neurodegenerative diseases.Methods:Based on the method established by PEI Gang's research group to directly reprogram human fibroblasts into neurons,the induction medium and maturation medium was optimized by replacing the coating solution,mitigating oxidative stress injury,adding neurogenic protective factors,adjusting the concentration of trichothecenes,performing small-molecule removal experiments,and carrying out immunofluorescence and Western blotting on cells at different stages of induction to validate the effect of induction.Results:When the original protocol was used for induction,the cell survival rate was(34.24±2.77)%.After replacing the coating solution gelatin with matrigel,the cell survival rate increased to(45.41±4.27)%;after adding melatonin,the cell survival rate increased to(67.95±5.61)%and(23.43±1.42)%were transformed into neural-like cells;after adding the small molecule P7C3-A20,the cell survival rate was further increased to(76.27±1.41)%,and(39.72±4.75)%of the cells were transformed into neural-like cells.When the concentration of trichothecene was increased to 30 μmol/L,the proportion of neural-like cells reached(55.79±1.90)%;after the removal of SP600125,(86.96±2.15)%of the cells survived,and the rate of neural-like cell production increased to(63.43±1.60)%.With the optimized protocol,REF could be successfully induced into ciNC through the neural precursor cell stage,in which the neural precursor cells were able to highly express the neural precursor cell markers SRY-related HMG-box gene 2(Sox2)and paired box 6(Pax6)as well as neuron-specific marker tubulin 1(Tuj1),while the expression of fiber-associated protein vimentin was reduced.After two weeks of induction of neural precursor cells in a maturation medium,most cells displayed neuronal-like cell morphology.The induced ciNCs were able to highly express the mature neuronal surface markers Tuj1 and microtubule-associated protein 2(MAP2),while the expression of vimentin was reduced.Conclusion:The small molecule combinations optimized in this study can reprogram REF to ciNCs under normoxic conditions.

Objective:To investigate the characteristics, process, and prognosis of esophageal stricture after circumferential endoscopic submucosal dissection (ESD), and to preliminarily analyze the prevention and treatment effects of simple dilation, stent placement, mucosal transplantation, and glucocorticoid (hereinafter referred to as hormone) application in esophageal stricture.Methods:From August 2017 to March 2022, at the First Affiliated Hospital of Zhengzhou University, the clinical and follow-up data of 55 patients who underwent circumferential ESD for early esophageal cancer and precancerous lesions were retrospectively analyzed. According to the prevention and treatment methods for esophageal stricture, the patients were divided into two groups: simple dilation group (23 cases) and combined dilation group (32 cases). The combined dilation group was divided into mucosal transplantation subgroup (9 cases), stent placement subgroup (14 cases), hormone application subgroup (7 cases), and bleomycin subgroup (2 cases, excluded from comparative analysis due to limited cases). Overall prognosis of patients was observed. Treatment efficacy, prognosis, and adverse events were compared among the simple dilation group, mucosal transplantation subgroup, stent placement subgroup, and hormone application subgroup. Independent samples t-test, chi-square test, and Fisher′s exact test were used for statistical analysis. Results:Among the 55 patients, the follow-up time was (894.1±417.7) days. Refractory esophageal stricture (total dilation times ≥ 5) occurred in 33 patients (60.0%). Fifty-two patients (94.5%) achieved clinical remission of the stricture. The total number of dilations was 5.8±4.0, and the average dysphagia-free period was (52.3±37.1) days. The dysphagia-free period of mucosal transplantation subgroup was longer than that of the simple dilation group, stent placement subgroup, and hormone application subgroup ((114.5±50.0) days vs. (40.9±20.0), (39.7±10.0), and (40.9±25.5) days, respectively), and the differences were statistically significant ( t=4.82, 3.77 and 3.14, P<0.001, =0.011, =0.009). There were no statistically significant differences between the simple dilation group and the mucosal transplantation subgroup, stent placement subgroup, and hormone application subgroup in the total number of dilations (6.8±4.8 vs. 3.0±2.5, 5.8±2.2, and 5.7±5.0), stricture remission rate (95.7%, 22/23 vs. 8/9, 13/14, and 7/7), and incidence of adverse events (17.4%, 4/23 vs. 5/9, 5/14, and 2/7; all P>0.05). Conclusions:Esophageal stricture formed after circumferential ESD shows the characteristics of recurrence and intractability. The over all number of dilations is high, and the average dysphagia-free period is short. Most patients can achieve clinical remission of the stricture after multiple times of endoscopic dilation treatment. However mucosal transplantation, stent placement, and hormone application cannot well intervene the natural process of esophageal stricture.

This article summarizes the definitions, influencing factors, and intervention measures of medication adherence in patients with ischemic stroke coexisting with atrial fibrillation. It serves as a reference for medical and nursing staff to timely and accurately identify patients with medication adherence issues, and to implement practical and effective intervention measures from multiple dimensions to enhance the medication adherence in patients with ischemic stroke coexisting with atrial fibrillation.

Objective: To explore the effect of Huangqi-Baoxin decoction combined with conventional western medicine on the cardiac function of the patients with diabetic cardiomyopathy (DCM). Methods: A total of 103 DCM patients who met the inclusion criteria were divided into the control group (51 cases) and the research group (52 cases) by random number table method. The control group was treated with conventional therapy, while the study group was treated with Huangqi-Baoxin decoction on the basis of the control group. Both groups were treated for 3 months. TCM syndromes were scored before and after treatment. The serum GSH-Px, SOD and CRP levels were measured by ELISA. Left ventricular ejection fraction (LVEF) and E/A ratio of early diastolic peak velocity to late diastolic peak velocity (E/A) were measured by echocardiography and then evaluate the clinical efficacy. Results: The total effective rate was 92.3% (48/52) in the study group and 76.5% (39/51) in the control group. There was significant difference between the two groups (χ²=4.922, P=0.027). After treatment, the scores of palpitation, shortness of breath, chest tightness and chest pain, fatigue, hypochondriac mass, spontaneous sweating, edema and total scores in the study group were significantly lower than those in the control group (t values were 10.603, 9.997, 10.546, 10.074, 5.211, 3.981, 6.438, 13.502, P<0.01). After treatment, LVEF (46.03% ± 1.70% vs. 42.33% ± 1.63%, t=11.271) and E/A (1.02 ± 0.17 vs. 0.90 ± 0.16, t=3.796) in the study group were significantly higher than those in the control group (P<0.01). After treatment, the serum GSH-Px and SOD levels in the study group were significantly higher than those in the control group (t values were 7.585, 6.816, P<0.01), and CRP level was significantly lower than the control group (t=10.854, P<0.01). Conclusions The Huangqi-Baoxin decoction combined with routine western medicine therapy can improve the clinical symptoms and cardiac function of DCM patients, reduce oxidative stress injury and inhibit inflammation.

BACKGROUND:Recent studies have found that long non-coding RNAs (lncRNAs) can regulate stem cel proliferation and differentiation. But it is unclear that how lncRNA NR_033474 regulate stem cel proliferation and cel cycle. OBJECTIVE:To investigate the effect of lncRNA NR_033474 on the proliferation and cel cycle regulation in C3H10T1/2 mesenchymal stem cel s after the NR_033474 overexpressed by lentivirus, and to study the possible regulation mechanism of NR_033474 on mesenchymal stem cel s. METHODS:LncRNA NR_033474 was cloned into a lentivirus vector. Lentivirus particles were infected into C3H10T1/2 cel s to upregulate the expression of NR_033474. The NR_033474 expression level was detected by real-time PCR. Compared with the empty lentivirus vector, the proliferation of C3H10T1/2 cel s which overexpressed NR_033474 was detected by cel counting assay and cel cycle was detected using flow cytometry. The expression of cel cycle-associated proteins such as CDK1, Cyclin B1, Cyclin D1 and P53 were detected by western blot assay. RESULTS AND CONCLUSION:Compared with the control group, lncRNA NR_033474 in C3H10T1/2 cel s which overexpressed NR_033474 was increased by about 100 times (P<0.01), and the proliferation of C3H10T1/2 cel s was significantly inhibited after NR_033474 overexpression by lentivirus (P<0.05). In addition, flow cytometry showed that C3H10T1/2 cel s overexpressing NR_033474 were arrested in G2/M phase compared to the control group. Western blot showed that the expression levels of CDK1 and Cyclin B1 were downregulated, while there were no changes in Cyclin D1 and P53 expression. To conclude, these findings suggest that the NR_033474 overexpression significantly inhibits the cel growth of C3H10T1/2 cel s, at least in part, through induction of cel cycle arrest at G2/M phase.

AIM:To explore the effects of transforming growth factor-α( TGF-α) in the monoclonal formation , proliferation, migration and adhesiveness of human endothelial progenitor cells ( EPCs).METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α(final concentrations of 1, 5, 10μg/L, respectively).At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10μg/L TGF-αplus 1∶1 000 EGFR-TKI) were set.The effects of TGF-αon monoclonal formation , proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment , thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays , respectively.The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor ( VEGF) were measured by Western blotting .RESULTS:Different concentrations of TGF-αall significantly induced the monoclonal formation , proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI.The results of Western blotting showed that TGF-αalso induced the expression of EGFR and VEGF with a cer-tain concentration effect ( P<0.01) .CONCLUSION:By combining with EGFR induced the expression of VEGF , TGF-αsignificantly promotes the related cell function of monoclonal formation , proliferation, migration, adhesiveness in EPCs.

Objective To study the inhibitory effect of fluorouracil combined with DDP on the growth of human osteosarcoma cell line MG-63,and to explore its influence on the expressions of transient receptor potential vanilloid 5 (TRPV5 )and transient receptor potential vanilloid 6 (TRPV6 )proteins.Methods The MG-63 cells were cultured by the density of 5 × 104 mL-1 .Fluorouracil group,DDP group,fluorouracil+ DDP group and control group containing 10% FBS were set up.The inhibitory rates of growth of MG-63 cells at different time were detected by CCK-8 assay.The apoptosis of MG-63 cells after treated with different drugs was determined by Hoechst staining Kit.The immunocytochemical staining was used to treatent to detect the expressions of TRPV5 and TRPV6 before and after treatment.Results Fluorouracil and DDP both inhibited the growth of MG-63 cells in a time-and dose- dependent manner.There were a lot of black particles in the MG-63 cells and the cells were smaller,aging or death when they were exposed to fluorouracil or DDP.Compared with 24 h group,the inhibitory rates of proliferation of MG-63 cells after treated with the sigle drug of fluorouracil or DDP for 48 and 72 h were increased significantly (P <0.05).Compared with control group,the apoptotic rates of MG-63 cells in fluorouracil group and DDP group 24,48,and 72 h after treatment were increased (P < 0.01)in a time-dependent manner. The expression levels of TRPV5 and TRPV6 in MG-63 cells 72 h after treatment of fluorouracil and DDP were decreased significantly compared with before treatment (P < 0.05 ). Conclusion Fluorouracil, DDP and fluorouracil combined with DDP could significantly inhibit the proliferation of MG-63 cells,induce the apoptosis, and decrease the expression levels of TRPV5 and TRPV6.

BACKGROUND:Recent studies have found that stem cellpluripotency and differentiation is regulated by many long non-coding RNAs (LncRNAs). The expression and effect of LncRNA AK089560 during differentiation of stem cells is unclear. OBJECTIVE:To investigate the expression of LncRNA AK089560 in mesenchymal stem cells C3H10T1/2 undergoing osteogenic and adipogenic differentiation. METHODS:Osteogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by recombinant human bone morphogenetic protein-2 and evaluated using alkaline phosphatase staining. The adipogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by three factors (dexamethasone, indomethacin and insulin) and evaluated by oil red O staining. The dynamical expression of LncRNA AK089560 was detected by qRT-PCR assay. The AK089560 secondary structure was predicted using RNAfold software. The relationship between AK089560 and neighboring protein-coding genes was analyzed using UCSC genome browser and visualized by fancyGENE online software. RESULTS AND CONCLUSION:Over 70%of C3H10T1/2 cells were positive for alkaline phosphatase after osteogenic induction and more than 80%of the cells positive for oil red O staining after adipogenic induction. qRT-PCR results showed that the expression of LncRNA AK089560 at days 2, 4, 6 of both osteogenic and adipogenic differentiation was significantly decreased compared with the control group (P<0.05). Bioinformatics analysis showed that there was a stem-loop structure for AK089560 and sense overlap relationship between AK089560 and protein-encoding gene Sema3a. These findings indicate that LncRNA AK089560 expression is reduced during osteogenic differentiation and adipogenic differentiation, showing that AK089560 may be involved in regulating the multi-directional differentiation of stem cells.

Objective To assess the clinical diagnostic value of left atrial function index (LAFI) for heart failure with normal left ventricular ejection fraction (HFNEF).Methods One hundred and ten patients with HFNEF were divided into 3 groups by New York Heart Association (NYHA) cardiac functional grading:NYHA Ⅱ grade group (43 cases),NYHA Ⅲ grade group (40 cases) and NYHA Ⅳ grade group (27 cases),and another 33 healthy subjects were selected as control group.The 4 groups were examined by ultrasoundcardiogram,LAFI and E/E' were calculated,and the plasma brain natriuretic peptide (BNP)levels were determined.The relation between LAFI and NYHA cardiac functional grading and the correlation between LAFI and plasma BNP,E/E' were observed.Results The worse heart function,the higher plasma BNP and E/E',and the lower LAFI,and there were statistical differences [control group:(40.9 ± 26.1) ng/L,6.4 ± 2.0,0.73 ± 0.10,NYHA Ⅱ grade group:(230.1 ± 85.9) ng/L,10.0 ± 3.7,0.46 ± 0.30,NYHA Ⅲ grade group:(398.6 ±98.7) ng/L,16.9 ±4.0,0.26 ± 0.30,NYHA Ⅳ grade group:(680.3 ± 146.6) ng/L,23.8 ± 3.9,0.17 ± 0.20,P ＜ 0.05].The correlation analysis results showed that LAFI and plasma BNP,E/E' were negatively correlated in patients with HFNEF (r =-0.868,-0.873,P ＜ 0.01).Conclusions In HFNEF patients,there is significant correlation between LAFI and left ventricular diastolic dysfunction.LAFI can well diagnose diastolic heart failure early and evaluate the diastolic function of the left ventricle.