Objective To explore the age-related biological properties of bone-derived mesenchymal stem cells(BMSCs)from mice of different age groups and their osteogenic differentiation induced by bone morphogenetic protein 2(BMP2).Methods Eight C57BL/6J mice were divided into a young group(4 weeks old,weighing 10～15 g,n=4)and an old group(12 months old,weighing 20～25 g,n=4),with half male and half female.MSCs were extracted from the whole bones of the 2 groups of mice.After the obtained cells were identified with flow cytometry for the surface markers,β-galactosidase staining was employed to compare the senescence level of BMSCs,MTT and EdU incorporation assays were conducted to compare the proliferation and self-renewal abilities of between the 2 groups.Western blotting was employed to analyze the expression of CyclinD1 and P21 in BMSCs.Then ALP staining,Alizarin Red staining and RT-qPCR were used to evaluate the osteogenic differentiation ability of the cells.RNA sequencing was performed to compare the differential gene expression in BMP2-induced BMSCs.Lastly,the sequencing results were re-confirmed by using flow cytometry.Results Flow cytometry showed that the sorted and cultured mouse BMSCs met the criteria for MSCs.The results of β-galactosidase staining indicated that the senescence level of BMSCs in the old group was significantly higher than that in the young group(P＜0.05).MTT and EdU doping experiments revealed that the cell viability and proliferation ability of BMSCs were significantly lower in the old group than the young group(P＜0.05).Western blotting displayed that the expression level of cell cycle protein CyclinD1 was lower,whereas that of cell cycle inhibitory factor P21 was significantly higher in the BMSCs from the old group than the cells from the young group(P＜0.05).ALP/Alizarin Red staining and RT-qPCR demonstrated that the BMSCs from the young group had stronger osteogenic differentiation capacity after BMP2 treatment when compared the cells of the old group(P＜0.05).RNA sequencing results displayed that the changing profile of CD51 expression was in opposite trends in the young and old BMSCs after BMP2 treatment.Finally,flow cytometry revealed that the percentage of CD51+cells within the CD45-cells was significantly higher in the young group than the old group.Conclusion The decrease in the percentage of CD51+cells among CD45-cells in aged BMSCs is closely associated with their decreased responsiveness to BMP2-induced osteogenic differentiation.

Objective:To investigate the analytical performance verification protocols and performance specifications of CD34+cell enumeration by flow cytometry for clinical laboratories.Methods:According to international guidelines and National Health Standard of China, we designed the performance verification protocols of CD34 +cell enumeration (including percent count and absolute count) by flow cytometry. Four quality assessment materials, three leukapheresis products and three samples of peripheral blood were selected to verify the precision, linearity, carryover, trueness and accuracy of FACSCanto Ⅱ measurement system, and the assessment criterion was set according to the detection technologies of clinical laboratories. Results:The CVs of intra-run precision of percent count and absolute count were 2.5% to 8.9% and 3.0% to 9.0%; the CVs of inter-run precision were 2.8% to 10.5% and 3.8% to 9.9%, respectively. The slopes of linearity regression equation of low range (3.6/μl to 123.6/μl) and high range (113.2/μl to 1196.3/μl) were 0.993 2 and 0.965 2, and R2 were 0.999 6 and 0.993 9, and the biases were -8.67% to 0.22%. The carryover of percent and absolute count were 0.07% and 0.00%. When percent count≤0.2% or absolute count≤20/μl, the absolute biases of trueness were in the range of ±0.006% or ±0.5/μl, and the absolute biases of accuracy were in the range of ±0.02% or ±0.9/μl; when percent count>0.2% or absolute count>20/μl, the relative biases of trueness were in the range of ±5.65%, and the relative biases of accuracy were in the range of ±8.19%. The verification results met the assessment criterion set in this study. Conclusions:The performance verification protocols and assessment criterion formulated in this study not only conform to the recommendations of domestic and foreign guidelines, but also conform to state of the detection technologies of native clinical laboratories, which can be taken as a reference of performance verification for clinical laboratories.

Objective To investigate the value of liver/muscle ratio (LMR) on susceptibility-weighted imaging (SWI) and serum markers in the diagnosis of the severity of chronic hepatitis B liver fibrosis after grouping based on alanine aminotransferase (ALT) level. Methods A retrospective analysis was performed for 255 patients with chronic hepatitis B who attended Affiliated Hospital of Yan'an University from October 2018 to September 2021, and the patients were divided into severe liver fibrosis group (SLF group) and non-severe liver fibrosis group (non-SLF group). The SLF group was defined as liver stiffness measurement (LSM) > 9.0 kPa and ALT level within the normal range or LSM > 12.0 kPa and ALT level greater than 1-5 times of the upper limit of normal. LMR was calculated by measuring the mean SWI value of the liver (SWI liver ) and the signal intensity of the erector spinae. The t -test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two group; the chi-square test was used for comparison of categorical data between two groups. The binary logistic regression analysis was used to investigate the influencing factors for SLF. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic performance of LMR and its combination with serum markers, and the DeLong test was used to compare the difference in the area under the ROC curve (AUC). Results Compared with the non-SLF group, the SLF group had significantly higher ALT ( Z =-3.569, P < 0.001), aspartate aminotransferase (AST) ( Z =-5.495, P < 0.001), hyaluronic acid (HA) ( Z =-6.746, P < 0.001), laminin (LN) ( Z =-5.459, P < 0.001), type Ⅳ collagen (Ⅳ-C)( Z =-8.470, P < 0.001), type Ⅲ procollagen (PCⅢ) ( Z =-6.326, P < 0.001), aspartate aminotransferase-to-platelet ratio index ( Z =-9.004, P < 0.001), and FIB-4 ( Z =-8.357, P < 0.001) and significantly lower prothrombin time activity (PTA) ( t =10.088, P < 0.001), platelet count ( t =9.163, P < 0.001), SWI liver ( t =2.347, P =0.02), and LMR×10 ( Z =-4.447, P < 0.001). PTA, HA, Ⅳ-C, and LMR×10 were independent influencing factors for SLF. LMR×10 had an AUC of 0.675 (95% confidence interval [ CI ]: 0.614-0.732) in the diagnosis of SLF, which was significantly higher than that of SWI liver (AUC=0.594, 95% CI : 0.531-0.655) ( Z =3.984, P < 0.001). PTA+HA+Ⅳ-C+LMR×10 and PTA+HA+Ⅳ-C had an AUC of 0.937 (95% CI : 0.896-0.966) and 0.905 (95% CI : 0.858-0.941), respectively, suggesting that PTA+HA+Ⅳ-C+LMR×10 had a better diagnostic performance than PTA+HA+Ⅳ-C ( Z =2.228, P =0.026). Conclusion LMR and serum markers can accurately distinguish SLF after grouping based on ALT level. LMR is a quantitative and objective imaging indicator and is better than SWI liver , and it can also improve the diagnostic performance of serum markers for SLF in clinical practice.

Objective:To summarize the clinical characteristics and efficacy of hemorrhage resulted from cortical venous infarction with seizure as the first symptom after craniotomy.Methods:Eleven patients with hemorrhage resulted from cortical venous infarction with seizure as the first symptom after craniotomy admitted to Neurosurgical Center, 988 th Hospital of PLA Joint Logistic Support Force from June 2011 to September 2019 were chosen in our study; primary diseases included meningioma in 7 patients, contusion and laceration of frontal lobe in 2, hypertensive cerebral hemorrhage in 1, and obsessive-compulsive disorder in 1 patient. Epilepsy was the first symptom after craniotomy. Clinical characteristics and efficacy of these patients were analyzed retrospectively; seizure control efficacy was evaluated by Engel grading. Results:First seizure occurred 4 h-7 d after craniotomy in these 11 patients, including 2 with focal sensory retention seizure, 3 with focal bilateral tonic-clonic seizure, and 6 with general tonic-clonic seizure. Follow-up cranial CT revealed hematoma in surgical region, adjacent cortex or subcortex in 9 patients (hematoma volume: 15-50 mL); emergency craniotomy (hematoma clearance) and decompressive craniectomy was performed in 5 patients; only emergency craniotomy (hematoma clearance) was performed in 3 patients; conservative treatment was performed in 1 patient. A small amount of diffuse bleeding with severe cerebral edema in the surgical region appeared in 2 patients, and the transient limb paralysis gradually recovered after 2 months of conservative treatment. Follow-up was performed for (4.5±1.7) years ([2.3-7.0] years). During the last follow-up, 4 patients were normal, 5 patients had mild to moderate hemiplegia, 1 had mild decreased vision in the right eye, and 1 had long-term coma. Epileptic control efficacy analysis indicated that 8 had Engel grading I and 3 grading II.Conclusion:Complete removal of hematoma and inactivated brain tissues can effectively control seizures and rebleeding in patients with hemorrhage resulted from cortical venous infarction.

Objective: To clinically validate the feasibility and accuracy of cine images acquired through the multitasking method, with no electrocardiogram gating and free-breathing, in measuring left ventricular (LV) function indices by comparing them with those acquired through the balanced steady-state free precession (bSSFP) method, with multiple breath-holds and electrocardiogram gating. Materials and Methods: Forty-three healthy volunteers (female:male, 30:13; mean age, 23.1 ± 2.3 years) and 36 patients requiring an assessment of LV function for various clinical indications (female:male, 22:14; 57.8 ± 11.3 years) were enrolled in this prospective study. Each participant underwent cardiac magnetic resonance imaging (MRI) using the multiple breath-hold bSSFP method and free-breathing multitasking method. LV function parameters were measured for both MRI methods. Image quality was assessed through subjective image quality scores (1 to 5) and calculation of the contrast-to-noise ratio (CNR) between the myocardium and blood pool. Differences between the two MRI methods were analyzed using the Bland–Altman plot, paired t-test, or Wilcoxon signed-rank test, as appropriate. Results: LV ejection fraction (LVEF) was not significantly different between the two MRI methods (P = 0.222 in healthy volunteers and P = 0.343 in patients). LV end-diastolic mass was slightly overestimated with multitasking in both healthy volunteers (multitasking vs. bSSFP, 60.5 ± 10.7 g vs. 58.0 ± 10.4 g, respectively; P < 0.001) and patients (69.4 ± 18.1 g vs. 66.8 ± 18.0 g, respectively; P = 0.003). Acceptable and comparable image quality was achieved for both MRI methods (multitasking vs. bSSFP, 4.5 ± 0.7 vs. 4.6 ± 0.6, respectively; P = 0.203). The CNR between the myocardium and blood pool showed no significant differences between the two MRI methods (18.89 ± 6.65 vs. 18.19 ± 5.83, respectively; P = 0.480). Conclusion Multitasking-derived cine images obtained without electrocardiogram gating and breath-holding achieved similar image quality and accurate quantification of LVEF in healthy volunteers and patients.

Orthodontic tooth movement elicits alveolar bone remodeling and orofacial pain that is manifested by tooth mechanical hyperalgesia. Nerve growth factor (NGF) is upregulated in periodontium and may modulate tooth mechanical hyperalgesia. The objectives were to examine the role of NGF in tooth mechanical hyperalgesia and to elucidate the underlying mechanisms. Tooth mechanical hyperalgesia was induced by ligating closed coil springs between incisors and molars in Sprague-Dawley rats. Retrograde labeling was performed by periodontal administration of fluor-conjugated NGF and the detection of fluorescence in trigeminal ganglia (TG). Lentivirus vectors carrying NGF shRNA were employed to knockdown the expression of NGF in TG. The administration of agonists, antagonists, and virus vectors into TG and periodontium was conducted. Tooth mechanical hyperalgesia was examined through the threshold of biting withdrawal. Our results revealed that tooth movement elicited tooth mechanical hyperalgesia that could be alleviated by NGF neutralizing antibody and that NGF was upregulated in periodontium (mainly in periodontal fibroblasts) and TG. Retrograde labeling revealed that periodontal NGF was retrogradely transported to TG after day 1. Acid-sensing ion channel 3 (ASIC3) and NGF were co-expressed in trigeminal neurons and the percentage of co-expression was significantly higher following tooth movement. The administration of NGF and NGF neutralizing antibody into TG could upregulate and downregulate the expression of ASIC3 in TG, respectively. NGF aggravated tooth mechanical hyperalgesia that could be alleviated by ASIC3 antagonist (APETx2). Moreover, NGF neutralizing antibody mitigated tooth mechanical hyperalgesia that could be recapitulated by ASIC3 agonist (GMQ). NGF-based gene therapy abolished tooth mechanical hyperalgesia and downregulated ASIC3 expression. Taken together, in response to force stimuli, periodontal fibroblasts upregulated the expressions of NGF that was retrogradely transported to TG, where NGF elicited tooth mechanical hyperalgesia through upregulating ASIC3. NGF-based gene therapy is a viable method in alleviating tooth-movement-induced mechanical hyperalgesia.

Objective:To summarize the morphological characteristics and clinical experience of pulmonary artery sling(PAS) combined with anomalous bronchial branching, and provide new considerations for surgically treating PAS.Methods:A retrospective review of surgical experience of all patients with anomalous bronchial branching from January 2012 to December 2018 at Shanghai Children's Medical Center was conducted. There are 4 types according to the morphology of anomalous bronchial branching: tracheal bronchus, bronchial trifurcation, bridging bronchus, tracheal bronchus combined with bronchial trifurcation. The proportion of patients with anomalous bronchial branching combined with PAS was counted. The rate of tracheoplasty for four types was determined. We measured the interbronchial and subcarinal/subpseudocarinal angles before and after tracheoplasty.Results:140 patients were included in our study. The tracheal diameter of 11(21%, 11/53) patients with tracheal bronchus and 4(9%, 4/44) patients with bronchial trifurcation was normal. Among these patients, 125 patients had congenital tracheal stenosis(CTS). 115 patients underwent tracheoplasty. The rate of tracheoplasty of bridging bronchus was higher than tracheal bronchus(100% vs. 60%, P=0.000). One-stage surgery was performed in 76(61%) patients who had PAS. The interbronchial and subcarinal/subpseudocarinal angles were significantly reduced postoperatively( P=0.000). Conclusion:It is difficult to explain merely with vascular compression in patients with PAS with CTS and anomalous bronchial branching. One-stage slide tracheoplasty is currently the optimal option.

Objective:To analyze the molecular characteristics of Echovirus 11 (Echo11) strains isolated in Xiangyang, Hubei Province from 2016 to 2017 based on the sequences of VP1 gene.Methods:Rectal and throat swab specimens were collected from children with hand, foot and mouth disease (HFMD) in Xiangyang from 2016 to 2017. Echo11 strains were detected by real-time reverse transcriptase PCR (RT-PCR) and isolated after cultured in human rhabdosarcoma (RD) cells. The VP1 regions of Echo11 strains isolated from RD cells and the whole genomes of three representative Echo11 strains were amplified by conventional RT-PCR and the sequences were analyzed. DNAStar7.0 (MegAlign) and MEGA6.0 (Data) were used to analyze the homology and mutation sites in nucleotide and amino acid sequences. Neighbor-joining method was used to construct phylogenetic trees. Recombination analysis was performed with SimPlot software (BootScanning).Results:A total of 11 Echo11 strains were isolated from 3 494 HFMD cases, accounting for 0.31%. They were highly homologous in the VP1 gene. These strains shared 98.4%-100.0% homology in nucleotide sequences and 98.3%-100.0% homology in amino acid sequences. The homology between the 11 Echo11 strains and the prototype strain (Echo11/Gregory, X80059) was 73.9%-74.8% in nucleotide sequences and 87.7%-88.7% in amino acid sequences. All of the Echo11 strains circulating in Xiangyang were classified into lineage D, having a similarity to the strains circulating in some regions of mainland China since 2013. In multiple regions of the genome, the Echo11 strains isolated in Xiangyang were highly similar to the Henan Echo1 strains in 2010 and the Hubei Echo6 strains in 2015, suggesting there was recombination within the genome of Echo11 strains in Xiangyang.Conclusions:The Echo11 strains circulating in Xiangyang from 2016 to 2017 belonged to lineage D and were recombinant strains.

Objective:To investigate the imaging and electrophysiological characteristics, surgical methods and efficacies of preschoolers with refractory temporal lobe epilepsy.Methods:A retrospective data study was conducted on 27 patients with refractory temporal lobe epilepsy accepted surgical treatment in our hospital from June 2014 to January 2019. By combined with clinical manifestations, preoperative epileptogenic zones were evaluated by imaging data, such as MR imaging, MR spectroscopy and positron emission tomography-CT, and interictal and ictal video-electroencephalogram (VEEG) data. During the surgery, cortical electroencephalography (ECoG) and deep electrode monitoring were used to monitor and locate the abnormal discharge areas to guide the surgical excision of epileptic zone. Engel grading was used to evaluate the efficacy after surgery.Results:All children had typical clinical manifestations of temporal lobe epilepsy; abnormal signal images were found in one side of the temporal lobe and the hippocampus in MR imaging; epileptic discharges were originated from one side frontotemporal region in interictal and ictal VEEG. Obviously persistent or paroxysmal spike waves, spike waves and slow spikes and spike composite waves were intraoperatively discovered by ECoG and depth electrode electroencephalography in the temporal lobe. All patients accepted standard anterior temporal lobectomy+lesion resection+peripheral abnormal discharge resection of the temporal lobe cortex; partial insular lobe was excised and frontal cortex was performed low power thermal coagulation in two patients. Follow-up was performed for 6 months; Engel grading I was reported in 22 patients (81.5%), Engel grading II in 3 patients (11.1%), and Engel grading III in 2 patients (7.4%).Conclusion:Early surgery and moderate extension of resection under intraoperative ECoG and deep electrode monitoring are the key factors to improve the surgical efficacy of preschoolers with refractory temporal lobe epilepsy.

Objective: New infectious diseases have become a global problem that seriously threatens human life and health and social development, which greatly increases the demand of CT examination for outpatients with fever. Through the discussion of the examination room design of the special CT for epidemic prevention, it can provide reference for medical institutions and reduce cross infection. Methods: Based on the requirements of CT equipment installation and environment, combined with the special requirements of epidemic prevention in fever clinic, the paper analyzed the location of examination room, room layout, site construction method, air purification and disinfection, intelligent image aided diagnosis of special CT for epidemic prevention, and put forward the design scheme. Results: Through the detailed analysis of the key points of engineering technology and the requirements of infection prevention and control of CT examination room, the design scheme of the examination room was given. Conclusions The establishment of special CT for epidemic prevention can meet the needs of clinical examination and effectively reduce cross infection. The design scheme given in this paper has certain reference value and can provide effective help for medical institution.