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 observe the effect of different synovial cell secretions on chondrocytes after LPS-induced inflammation,and to explore the mechanism of two synovial cell secretions causing cartilage damage in the progres-sion of KOA disease.Methods Two kinds of synovial cells were co-cultured at 1∶4 and LPS-induced inflamma-tion.The supernatant and exocrine were extracted,and then the normal and LPS-induced inflammation were extrac-ted.The human cartilage tissue obtained during the operation was isolated and cultured into chondrocytes,which were divided into five groups:the first group was added with FLS secretion,the second group was added with nor-mal FLS secretion,the third group was added with secretion after co-culture of two kinds of synovial cells,the fourth group was added with inflammatory MLS secretion,and the fifth group was added with inflammatory FLS se-cretion.CCK-8 was used to detect the viability of chondrocytes in each group.TNF-α,IL-1β,IL-6 level in the su-pernatant of chondrocytes in each group was detected by ELISA.The protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in chondrocytes of each group was detected by Western blot method.Results CCK-8 showed that the activity of chondrocytes in the three groups of inflammatory secretions decreased compared with the secretions from normal synovial cells(P＜0.05);ELISA showed TNF-α,IL-1 β,IL-6 level in the supernatant of group Ⅲ,Ⅳ and V was higher than that of group Ⅰ and Ⅱ(P＜0.05),TNF-α,IL-1 β,IL-6 level in group Ⅲ was higher than that in group Ⅳ but lower than that in group Ⅴ(P＜0.05).Western blot showed the protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in chondrocytes of group Ⅲ,Ⅳ and Ⅴ was higher than that in group Ⅰ and Ⅱ(P＜0.05),the protein expression of TLR4,NF-κB,IkK,IκB,ADAMTS5 in group Ⅲ was higher than that in group Ⅳbut lower than that in group Ⅴ(P＜0.05).Conclusion Two kinds of synovial cell-derived secretions after LPS-induced inflammation can regulate cartilage TLRs/NF-κB signal pathway,causing cartilage inflammation.The in-flammatory effect of MLS secretion is stronger than that of FLS secretion,but the inflammatory effect of MLS secre-tion under two co-cultures is weaker than that of MLS secretion alone.

Objective: To investigate the effects of acupotomy on skeletal muscle fibrosis and collagen deposition in a rabbit knee osteoarthritis (KOA) model. Methods: Rabbits (n = 18) were randomly divided into control, KOA, and KOA + acupotomy (Apo) groups (n = 6). The rabbits in the KOA and Apo groups were modeled using the modified Videman's method for 6 weeks. After modeling, the Apo group was subjected to acupotomy once a week for 3 weeks on the vastus medialis, vastus lateralis, rectus femoris, biceps femoris, and anserine bursa tendons around the knee. The behavior of all animals was recorded, rectus femoris tissue was obtained, and histomorphological changes were observed using Masson staining and transmission electron microscopy. The expression of transforming growth factor-β1 (TGF-β1), Smad 3, Smad 7, fibrillar collagen types I (Col-I) and III (Col-III) was detected using Western blot and real-time polymerase chain reaction (RT-PCR). Results: Histological analysis revealed that acupotomy improved the microstructure and reduced the collagen volume fraction of rectus femoris, compared with the KOA group (P = .034). Acupotomy inhibited abnormal collagen deposition by modulating the expression of fibrosis-related proteins and mRNA, thus preventing skeletal muscle fibrosis. Western blot and RT-PCR analysis revealed that in the Apo group, Col-I, and Col-III protein levels were significantly lower than those in the KOA group (both P < .01), same as Col-I and Col-III mRNA levels (P = .0031; P = .0046). Compared with the KOA group, the protein levels of TGF-β1 and Smad 3 were significantly reduced (both P < .01), as were the mRNA levels of TGF-β1 and Smad 3 (P = .0007; P = .0011). Conversely, the levels of protein and mRNA of Smad 7 were significantly higher than that in the KOA group (P < .01; P = .0271). Conclusion Acupotomy could alleviate skeletal muscle fibrosis and delay KOA progress by inhibiting collagen deposition through the TGF-β/Smad pathway in the skeletal muscle of KOA rabbits.

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 investigate the effect of intraoperative protection of the supraclavicular nerve on the healing of clavicular 1/3 mid-shaft fracture.Methods:A retrospective study was conducted to analyze the 83 patients who had been treated for clavicular 1/3 mid-shaft fractures at Department of Orthopaedics, The First Central Hospital of Baoding from June 2021 to March 2022. There were 57 males and 26 females with an age of (48.1±12.8) years. The patients were divided into 2 groups according to whether the supraclavicular nerve was protected or not during operation. There were 39 cases in the observation group (the supraclavicular nerve was protected during operation) and 44 cases in the control group (the supraclavicular nerve was not protected during operation). The incision length, operation time, intraoperative blood loss, hospital stay, visual analogue scale (VAS) pain scores before operation and 3, 6 and 12 months after operation, fracture healing at 3, 6 and 12 months after operation, and postoperative complications were recorded and compared between the 2 groups. Additionally, the number of microvessels in the middle clavicle was recorded and compared between the affected and healthy sides in 8 patients in the observation group at 6 weeks after operation.Results:There was no significant difference in the preoperative general data between the 2 groups, indicating comparability ( P>0.05). The operation time for the observation group [(72.2±5.4) min] was significantly longer than that for the control group [(61.1±4.7) min]. The VAS scores at postoperative 3 and 6 months for the observation group [2.7 (2.4, 3.1) and 2.1 (1.9, 2.6) points] were significantly lower than those for the control group [3.5 (3.2, 3.8) and 2.7 (2.4, 2.9) points] ( P<0.05). The fracture healing rates at postoperative 3 and 6 months in the observation group [97.4% (38/39) and 100% (39/39)] were significantly higher than those in the control group [81.8% (36/44) and 86.4% (38/44)] ( P<0.05), but there was no significant difference in the fracture healing between the 2 groups at 12 months after operation ( P>0.05). At 6 weeks after operation, the number of microvessels in the middle clavicle was respectively 85.3±0.7 and 87.1±0.8 in the 8 patients in the observation group, showing no significant difference ( P>0.05). After operation, delayed incision healing occurred in 3 cases in the observation group and in 4 cases in the control group, and abnormal sensation of the skin around the incision occurred in 9 cases in the observation group and in 26 cases in the control group, showing a significant difference between the 2 groups ( P<0.05). Conclusion:Intraoperative protection of the supra-clavicular nerve is beneficial for reduction of early postoperative pain and improvement of early fracture healing, and may have a positive effect on the postoperative reconstruction of microvascular network.

OBJECTIVES: To investigate the role of mitochondrial autophagy disorder caused by deletion of E3 ubiquitin ligase Parkin in neuroinflammation in a mouse model of MPTP-induced Parkinson's disease (PD). METHODS: Wild-type (WT) male C57BL/6 mice and Parkin^-/- mice were given intraperitoneal injections with MPTP or PBS for 5 consecutive days, and the changes in motor behaviors of the mice were observed using open field test. The effects of Parkin deletion on PD development and neuroinflammation were evaluated using immunofluorescence and Western blotting. The changes of the PINK 1/Parkin signaling pathway in the midbrain substantia nigra of the mice were examined to explore the molecular mechanism of Parkin-mediated regulation of mitochondrial autophagy and its effect on neuroinflammation in PD mice. RESULTS: Compared with their WT counterparts, the Parkin^-/- mice with MPTP injections exhibited significant impairment of motor function with decreased TH⁺ neurons, increased α-synuclein (α-syn) accumulation, and increased numbers of GFAP⁺ and I-ba1⁺ cells in the midbrain substantia nigra. Parkin deletion obviously affected PINK1/Parkin-mediated mitochondrial autophagy to result in significantly increased mtDNA and upregulated expressions of STING and NLRP3 inflammatosomes in the midbrain substantia nigra of MPTP-treated transgenic mice. CONCLUSIONS Parkin deletion causes mitochondrial autophagy disorder to accelerate PD progression and exacerbates neuroinflammation in mice by affecting the PINK1/Parkin signaling pathway, suggesting the important role of Parkin in early pathogenesis of PD.
Animals ; Ubiquitin-Protein Ligases/genetics* ; Mice ; Mice, Inbred C57BL ; Male ; Parkinson Disease/genetics* ; Protein Kinases/genetics* ; Mitochondria/metabolism* ; Disease Models, Animal ; Autophagy ; Signal Transduction ; Neuroinflammatory Diseases/metabolism* ; Mice, Knockout ; alpha-Synuclein/metabolism* ; Substantia Nigra/metabolism* ; Mitophagy ; Disease Progression

Objective:To investigate the pathogen distribution and influencing factors for pulmonary infection after radical resection of esophageal cancer.Methods:The retrospective case-control study was conducted. The clinical data of 555 patients who underwent radical resection of esophageal cancer in Heping Hospital Affiliated to Changzhi Medical College from January 2021 to December 2022 were collected. There were 344 males and 211 females, aged (64±8)years. Obser-vation indicators: (1) incidence of postoperative pulmonary infection and pathogen distribution; (2) analysis of influencing factors for postoperative pulmonary infection. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the t test. Count data were described as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test. The comparisons of ordinal data were analyzed using the nonparametric test. Univariate analysis was performed using the corresponding statistical methods based on data type. Multivariate analysis was performed using the Logistic stepwise regre-ssion model advance method. Results:(1) Incidence of postoperative pulmonary infection and pathogen distribution. Among 555 patients, 91 cases had postoperative pulmonary infection, with the incidence as 16.40%(91/555). In 91 patients with postoperative pulmonary infection, 59 strains of bacteria were isolated and cultured. There were 53 strains of gram-negative bacteria, 3 strains of gram-positive bacteria and 3 strains of fungi, including 20 multidrug-resistant bacteria. Among the 53 strains of gram-negative bacteria, there were 18 strains of Acinetobacter baumannii (12 strains were multidrug resistant), 18 strains of Klebsiella pneumoniae (3 strains were multidrug resistant), 6 strains of Pseudomonas aeruginosa (2 strains were multidrug resistant), 2 strains of Escherichia coli, 2 strains of Enterobacter cloacae, and 2 strains of Haemophilus influenzae (2 strains of Escherichia coli and 1 strain of Enterobacter cloacae were multidrug resistant strains), 1 strain of Serratia marcescens, 1 strain of Citrobacter keri, 1 strain of Corynebacterium striatum, 1 strain of Proteus mirabilis and 1 strain of Klebsiella acidogenes. Among the 3 strains of Gram-positive bacteria, there were 2 strains of Staphylococcus aureus and 1 strain of Streptococcus pneumoniae. All the three strains of fungi were Candida albicans. Among the 18 strains of Acinetobacter baumannii, there were 12, 12, 11, 9, 8, 6 and 5 strains resistant to imipenem, ceftriaxone, ceftazidme, cefoperazone or sulbactam, ciprofloxacin, amicacin and levofloxacin, respectively. The above indexes of 18 strains of Klebsiella pneumoniae were 0, 1, 1, 1, 2, 0 and 2, respectively. (2) Analysis of influencing factors for postoperative pulmonary infection. Results of multivariate analysis showed that tumor pathological staging as stage Ⅱ and Ⅲ, duration of preoperative hospital stay ≥6 days, operation time ≥240 minutes, mode of operation as thoracotomy, type of antibiotics used in peri-operative period ≥3, and postoperative antibiotic use time ≥5 days were independent risk factors for postoperative pulmonary infection ( P<0.05). Conclusions:The main pathogenic bacteria of pulmonary infection after radical resection of esophageal cancer are Acinetobacter baumannii and Klebsiella pneumoniae. Tumor pathological staging as stage Ⅱ and Ⅲ, duration of preoperative hospital stay ≥6 days, operation time ≥240 minutes, mode of operation as thoracotomy, type of antibiotics used in perioperative period ≥3, and postoperative antibiotic use time ≥5 days are independent risk factors for pulmonary infection in patients with esophageal cancer after radical surgery.

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

Osteochondral lesion of talus (OLT) is a foot and ankle disease characterized by ankle pain, which may impact the joint function and life quality. If managed improperly, it may lead to a further ankle arthritis, severely compromising the prognosis. The therapeutic effect of conservative treatment for OLT is still uncertain. Surgery is still the main treatment modality for OLT with various techniques. However, the optimized surgical technique is still inconclusive, furthermore, regeneration and repair of cartilage after debridement is also a great challenge for the treatment of OLT. Platelet-rich plasma (PRP) with good repair effect on cartilage injury is gradually applied in the treatment of OLT. However, there still lacks the unified understanding of the technique and specification of PRP for the treatment of OLT. Therefore, National Orthopedics Center of Shanghai Sixth People′s Hospital allied Foot Ankle Basic Research & Orthopedics Group, Chinese Association of Orthopedic Surgeons; Foot and Ankle Committee of Chinese Association of Sports Medicine Physicians; and Foot and Ankle Group of Orthopedic Specialized Branch of Shanghai Medical Association to organize related experts to formulate the Expert consensus on platelet- rich plasma treatment for osteochondral lesion of talus ( version2023). Fifteen recommendations were put forward upon PRP preparation, indications, contraindications and treatment methods of PRP for OLT, so as to standardize the PRP treatment for OLT.