BACKGROUND:Spleen has the functions of blood storage,hematopoiesis,and immunity.With the increase of age,the structural degeneration and functional decline of spleen lead to the impairment of immune system function,thus accelerating the aging process of the body.The treatment of spleen aging in tree shrews with highly active umbilical cord mesenchymal stem cells has not been reported. OBJECTIVE:To explore the intervention effect and mechanism of highly active umbilical cord mesenchymal stem cells on spleen aging in tree shrews. METHODS:Highly active umbilical cord mesenchymal stem cells were isolated,cultured,and obtained from the umbilical cord tissue of newborn tree shrews by caesarean section.The differentiation abilities of adipogenesis,osteogenesis,and chondrogenesis were detected by three-line differentiation kit.Cell cycle and surface markers were detected by flow cytometry.The second generation of highly active umbilical cord mesenchymal stem cells were transfected with Genechem Green Fluorescent Protein with infection complex values of 100,120,140,160,180,and 200,respectively,to screen the best transfection conditions.After transfection,the fourth generation of highly active umbilical cord mesenchymal stem cells was injected into the tail vein of tree shrews in the elderly treatment group.The young control group and the aged model group were not given special treatment.After 4 months of treatment,the spleen tissue was taken and the structure of the spleen was observed by hematoxylin-eosin staining.β-Galactosidase staining was used to detect the activity of aging-related galactosidase.Immunohistochemical staining was used to detect the expression levels of p21 and p53 proteins.Ki67 and PCNA immunofluorescence staining was used to detect cell proliferation activity.Immunofluorescence staining was used to detect the expression levels of spleen autophagy protein molecules Beclin 1 and APG5L/ATG5.Reactive oxygen species fluorescence staining was used to detect the content of reactive oxygen species in spleen tissue.CD3 immunofluorescence staining was used to detect the change of the proportion of total T lymphocytes.The secretion levels of interleukin 1β and transforming growth factor β1 in spleen were detected by enzyme linked immunosorbent assay.The distribution of highly active umbilical cord mesenchymal stem cells labeled with green fluorescent protein in spleen tissue was observed by DAPI double staining of nucleus. RESULTS AND CONCLUSION:(1)Highly active umbilical cord mesenchymal stem cells grew in a short spindle shape with fish-like growth,with a large proportion of G0/G1 phase,and had the potential to differentiate into adipogenesis,osteogenesis,and chondrogenesis.(2)Multiplicity of infection=140 and transfection for 72 hours were the best conditions for labeling tree shrews highly active umbilical cord mesenchymal stem cells with Genechem Green Fluorescent Protein.(3)Compared with the aged model group,in the aged treatment group,the spleen tissue cells of tree shrews were arranged closely,and the area of white pulp was increased(P<0.01);the boundary between red pulp and white pulp was clear;the proportion of germinal centers did not show statistically significant difference(P>0.05).The activity level of galactosidase related to spleen tissue aging was decreased(P<0.001),and the expression levels of aging protein molecules p21 and p53 were down-regulated(P<0.001).The expression levels of proliferation-related molecules Ki67 and PCNA were up-regulated(P<0.001,P<0.05);expression levels of autophagy-related molecules Beclin 1 and APG5L/ATG5 were up-regulated(P<0.001),and the content of reactive oxygen species decreased(P<0.001),and the proportion of CD3+T cells increased(P<0.05).The secretion level of interleukin 1β in the aging-related secretion phenotype decreased(P<0.001);no significant difference was found in transforming growth factor β1 level(P>0.05).Compared with the young control group,the above indexes were significantly different in the elderly treatment group(P<0.05).(4)Green fluorescent cells labeled with green fluorescent protein were observed in spleen tissue of tree shrews the elderly treatment group by frozen tissue section observation.The results show that intravenous infusion of highly active umbilical cord mesenchymal stem cells can migrate to spleen tissue,inhibit the production of reactive oxygen species,down-regulate the expression of aging-related proteins,induce autophagy,promote cell proliferation,reduce chronic inflammation,and then improve the structure and function of spleen tissue.

Objective To explore the role of regulation of nuclear pre-mRNA-domain-containing 1B(RPRD1B)in human ovarian cancer.Methods The expression level of RPRD1B in ovarian cancer was detected by RT-qPCR and Western blot experiments.Based on the CRISPR/Cas9 system and lentiviral system,the A2780 and SKOV3 cell lines with RPRD1B gene knockout and over-expression were respectively constructed,and their functions in ovarian cancer were verified by wound-healing assay,Transwell assay and mouse subcutaneous tumor formation model.Results RPRD1B was highly expressed in ovarian cancer cell lines(P＜0.001).Knockout of RPRD1B in-hibited the colony formation and proliferation ability of ovarian cancer cells(P＜0.001),as well as the cell migra-tion(P＜0.05)and invasion ability(P＜0.001).Meanwhile,knockout of RPRD1B inhibited the tumorigenesis ability of SKOV3 ovarian cancer cell lines in vivo(P＜0.001).Conclusions RPRD1B is highly expressed in hu-man ovarian cancer cell lines and promotes the growth of subcutaneous tumors in mice.

Objective:Induced pluripotent stem cells (iPSCs) cell lines were established using peripheral blood mononuclear cells (PBMCs) from a patient suffering from neonatal respiratory distress syndrome (NRDS) who carried Adenosine triphosphate-binding cassette transporter A3 ( ABCA3) compound heterozygous mutations. Methods:Cell experimental research.Peripheral venous blood was collected and PBMCs were isolated and cultured in vitro. PBMCs were transfected with non-integrated Sendai vector carrying reprogramming factors.The chromosome karyotypes of the established iPSCs were analyzed.Immunofluorescence and flow cytometry were used to detect pluripotency markers of stem cells and verify their differentiation potential.Sanger sequencing was performed to analyze gene mutations.In addition, short tandem repeat (STR) analysis was performed, polymerase chain reaction(PCR) and agarose gel electrophoresis were used to detect virus residual. Results:Karyotype analysis of established iPSCs cell lines showed normal diploid 46, XY karyotype.Immunofluorescence showed positive staining of stem cell pluripotency markers OCT4, SSEA4, Nanog and Sox2.Flow cytometry was used to detected stem cell pluripotency markers and showed expression of TRA-1-60, SSEA-4 and OCT4.After differentiation into all three germ layers, immunofluorescence was performed to detect ectoderm (Pax-6), mesoderm (Brachyury) and endoderm alpha-fetoprotein markers, and the results showed positive staining, which confirmed that the iPSCs had the potential to differentiate.Sanger sequencing showed c. 3997_3998del and c. 3137C>T compound heterozygous mutations.STR analysis showed they originate from PBMCs, and no Sendai virus residual was detected by PCR and agarose gel electrophoresis.Conclusions:In this study, PBMCs from patient carrying ABCA3 compound heterozygous mutations was used to establish iPSCs cell lines.The research lays a foundation for the study of pathogenesis, therapeutic drug screening and cell therapy of NRDS caused by ABCA3 gene mutations.

Objective:To summarize the clinical and prognostic features of children with opsoclonus-myoclonus-ataxia syndrome (OMAS).Methods:A total of 46 patients who met the diagnostic criteria of OMAS in the Department of Neurology, Beijing Children′s Hospital from June 2015 to June 2023 were retrospectively analyzed. Centralized online consultations or telephone visits were conducted between June and August 2023. The data of the children during hospitalization and follow-up were collected, including clinical manifestations, assistant examination, treatment and prognosis. According to the presence or absence of tumor, the patients were divided into two groups. The chi-square test or Mann-Whitney U test was used to compare the differences between the two groups. Univariate Logistic regression was used to analyze the factors related to OMAS recurrence and prognosis. Results:There were 46 patients, with 25 males and the onset age of 1.5 (1.2, 2.4) years. Twenty-six (57%) patients were diagnosed with neuroblastoma during the course of the disease, and no patients were categorized into the high-risk group. A total of 36 patients (78%) were followed up for≥6 months, and all of them were treated with first-line therapy with glucocorticoids, gammaglobulin and (or) adrenocorticotrophic hormone. Among the 36 patients, 9 patients (25%) were treated with second-line therapy for ≥3 months, including rituximab or cyclophosphamide, and 17 patients (47%) received chemotherapy related to neuroblastoma. At the follow-up time of 4.2 (2.2, 5.5) years, 10 patients (28%) had relapsed of OMAS. The Mitchell and Pike OMS rating scale score at the final follow-up was 0.5 (0, 2.0). Seven patients (19%) were mildly cognitively behind their peers and 6 patients (17%) were severely behind. Only 1 patient had tumor recurrence during follow-up. The history of vaccination or infection before onset was more common in the non-tumor group than in the tumor group (55%(11/20) vs. 23%(6/26), χ2=4.95, P=0.026). Myoclonus occurred more frequently in the non-tumor group (40%(8/20) vs. 4%(1/26), χ2=7.23, P=0.007) as the onset symptom. Univariate Logistic regression analysis showed that the tumor group had less recurrence ( OR=0.19 (0.04-0.93), P=0.041). The use of second-line therapy or chemotherapy within 6 months of the disease course had a better prognosis ( OR=11.64 (1.27-106.72), P=0.030). Conclusions:OMAS in children mostly starts in early childhood, and about half are combined with neuroblastoma. Neuroblastoma in combination with OMAS usually has a low risk classification and good prognosis. When comparing patients with OMAS with and without tumors, the latter have a more common infection or vaccination triggers, and myoclonus, as the onset symptom, is more common. Early addition of second-line therapy is associated with better prognosis in OMAS.

Objective:To summarize the clinical and genetic characteristics of mental retardation disorder (MRD) with TRIO gene variant in children.Methods:Case series study. The data of 9 children with TRIO gene variants were collected retrospectively from August 2019 to March 2024 in Department of Neurology, Beijing Children′s Hospital, Capital Medical University and Department of Pediatrics, the Third Affiliated Hospital of Zhengzhou University. The data included gender, age, intellectual and motor development, appearance, seizures, neuroimaging and genetic results. The clinical features and genotype-phenotype correlations were summarized.Results:Of the 9 children, 6 boys and 3 girls, 4 MRD63 children presented with moderate to severe developmental delays accompanied by macrocephaly; 5 MRD44 children had mild to moderate developmental delays with microcephaly. A total of 5 children had dysmorphic facial features (flat occiput, thick eyebrows, unibrow, large ears, short fingers, pale skin, yellow hair, and strabismus), 2 children experienced seizures (1 child with myoclonic seizure and 1 with absence seizure), 4 children had feeding difficulties, 1 child had congenital cataracts, 1 child had congenital heart disease, 1 child had recurrent infections, and 1 child had tiger-striped changes in the fundus examination. TRIO gene variants carried by the 9 children were all de novo, involving 8 variant sites, including 7 missense variants and 1 frameshift variant, c.3232C>T/p.R1078W (2 cases), c.3920A>G/p.Y1307C, c.4112A>T/p.H1371L, c.4283G>T/p.R1428L, c.4394A>G/p.N1465S, c.6041T>C/p.I2014T, c.6821G>A/p.R2274H, c.7027delC/p.Q2343Sfs*70. Among them, 2 sites are located in the Spectrin domain, 4 sites are in the GEFD1 domain, 2 sites are in the GEFD2 domain, and 1 site (frameshift variant) is in the PH2-SH3 domain. The individual with frameshift variant exhibit absence seizures, mild developmental delay, and the mildest phenotype. The child with myoclonic seizures was treated with valproic acid and levetiracetam for seizure control, while the child with absence epilepsy was treated with valproic acid and lamotrigine for seizure control. All 9 children underwent regular rehabilitation exercises, making slow progress.Conclusions:TRIO gene related MRD is characterized by varying degrees of developmental delay, and often accompanied by macrocephaly or microcephaly, dysmorphic facial features, and with or without seizures. The main variant types are missense variants, which are mostly concentrated in the Spectran domain and GEFD domain. p. R1078W may be a relative hotspot variant. The phenotype caused by the frameshift variant is relatively milder.

Objective:To investigate whether hsa_circ_0054595(circRTN4)is involved in glomerulosclerosis of lupus nephri-tis(LN)by regulating monocyte-derived TNF-α.Methods:RT-qPCR and immunofluorescence were used to detect the expression of TNF-α in monocytes of LN patients,and fluorescence in situ hybridization(FISH)was used to detect the expression of circRTN4.THP1 cells were transfected with circRTN4-siRNA and negative control NC-siRNA,respectively.RT-qPCR and Western blot were used to detect the expression of TNF-α in THP1cell,and ELISA was used to detect the expression of TNF-α in THP1 culture superna-tant.THP1 cells were transfected with mircoRNA mimics,and the expression of TNF-α was detected by Western blot.The direct bind-ing of miR-486-3p to TNF-α and circRTN4 were verified by reversion experiment and dual luciferase reporter gene experiment.MRL/lpr mice were injected with circRTN4 recombinant adeno-associated virus via tail vein,and the expression of circRTN4 in peripheral blood mononuclear cells of the mice was detected by RT-qPCR.ELISA was used to detect the expression of serum TNF-α.HE staining and PAS staining were used to observe the pathological changes,and immunofluorescence was used to detect the expression of FN.Results:The expressions of TNF-α and circRTN4 were increased in monocytes of LN patients(P<0.05).The expression of TNF-α in THP1 was significantly increased in the LN group(P<0.01).Knockdown of circRTN4 inhibited the expression and secretion of TNF-α(P<0.05),and this effect was achieved by binding to miR-486-3p(P<0.01).In vivo,knockdown of circRTN4 in peripheral blood monocytes of MRL/lpr mice reduced the expression of TNF-α in serum(P<0.05),and improved glomerular cell proliferation and FN deposition.Conclusion:Highly expressed circRTN4 in monocytes promotes the expression of TNF-α by binding to miR-486-3p,and participates in the occurrence and development of glomerulosclerosis in LN.

Parkinson disease is a common neurodegenerative disease.At present,the main therapeutic drugs have problems such as adverse reactions,lack of targeting,and low bioavailability.The development of new drugs is an important direction for research related to Parkinson disease.TCM flavonoids have various pharmacological activities and are widely used in anti-tumor and cardiovascular disease treatment,but there is relatively little research on their intervention in neurological diseases.This article elaborated on the mechanism of intervention of TCM flavonoids in Parkinson disease and summarized treatment strategies from six categories:flavonoids,flavonols,isoflavones,flavonoid glycosides,dihydroflavonols,and others,with the purpose to provide reference for the research and clinical application of TCM flavonoids.