Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

The organization of the brain follows a topological hierarchy that changes dynamically during development. However, it remains unknown whether and how cognitive training administered over multiple years during development can modify this hierarchical topology. By measuring the brain and behavior of school children who had carried out abacus-based mental calculation (AMC) training for five years (starting from 7 years to 12 years old) in pre-training and post-training, we revealed the reshaping effect of long-term AMC intervention during development on the brain hierarchical topology. We observed the development-induced emergence of the default network, AMC training-promoted shifting, and regional changes in cortical gradients. Moreover, the training-induced gradient changes were located in visual and somatomotor areas in association with the visuospatial/motor-imagery strategy. We found that gradient-based features can predict the math ability within groups. Our findings provide novel insights into the dynamic nature of network recruitment impacted by long-term cognitive training during development.
Child ; Humans ; Cognitive Training ; Magnetic Resonance Imaging ; Brain ; Brain Mapping ; Motor Cortex

BACKGROUND:Endogenous neurogenesis and exogenous stem cell transplantation in the brain show great therapeutic potential for neurological diseases including ischemic stroke,repairing and replacing lost neurons,promoting synaptic remodeling,and inhibiting apoptosis.Traditional Chinese medicine and compound therapy for supplementing qi,activating blood circulation and inducing resuscitation for the treatment of neurological dysfunction after ischemia have certain advantages,targeting nerve repair through a variety of ways,including promoting endogenous neurogenesis and exogenous stem cell survival,proliferation,homing,and inducing neuronal differentiation. OBJECTIVE:To summarize the mechanism of traditional Chinese medicine and compound for supplementing qi,activating blood circulation and inducing resuscitation to promote nerve repair in the acute phase of ischemic stroke,in order to provide a reference for the research and treatment of new drugs in ischemic stroke. METHODS:The articles from CNKI and PubMed databases about traditional Chinese medicine and compound for supplementing qi,activating blood circulation and inducing resuscitation in promotion of nerve repair in the acute phase of ischemic stroke from 2010 to 2022 were searched,with"supplementing qi and activating blood circulation;inducing resuscitation;traditional Chinese medicine(TCM);compounds;ischemic stroke;nerve repair;stem cells"as Chinese and English search terms.After excluding old and duplicate views,the retrieved literature was analyzed and collated,and a total of 124 articles were included for analysis. RESULTS AND CONCLUSION:(1)The definition of stem cells,ischemic stroke and the nerve repair pathway in the acute phase of ischemic stroke were sorted out.(2)The mechanism of action of traditional Chinese medicine and compound for supplementing qi,activating blood circulation and inducing resuscitation to promote nerve repair in the acute phase of ischemic stroke was summarized,mainly including promoting stem cell proliferation,improving stem cell viability and survival rate,promoting nerve cell homing,inducing stem cell differentiation to neurons,inhibiting apoptosis of nerve cells,promoting axon regeneration,regulating angiogenesis and remodeling,improving the level of neurotrophic factors and repairing the integrity of the blood-brain barrier.(3)Through the existing research,the relevant factors and signaling pathways of traditional Chinese medicines and compounds for supplementing qi,activating blood circulation and inducing resuscitation to promote nerve repair in the acute phase of ischemic stroke were summarized,such as Nestin protein expression,DCX protein expression,brain-derived neurotrophic factor,vascular endothelial growth factor and Wnt/β-catenin signaling pathway,Notch signaling pathway,PI3k/Akt signaling pathway,BDNF/TrkB signaling pathway and ERK/MAPK signaling pathway.It provides a relevant reference for future research on ischemic stroke-specific drugs and new clinical treatment methods.

BACKGROUND:Bone marrow mesenchymal stem cells have been widely used to treat neurological diseases.However,due to limitations of the blood-brain barrier,low survival rate and differentiation rate of stem cells at damaged sites,the therapeutic effect is limited. OBJECTIVE:To investigate the effects of Shexiang Huangqi compound dripping pills on proliferation,migration and astrocyte differentiation of bone marrow mesenchymal stem cells. METHODS:Male SD rats were treated with Shexiang Huangqi compound dripping pills for 5 days after continuous gavage.Blood was collected from the abdominal aorta and serum was separated for later use.The effect of 5%,10%and 20%drug-containing serum on the proliferation of bone marrow mesenchymal stem cells was detected by CCK-8 assay.The effect of 10%drug-containing serum on lateral migration of bone marrow mesenchymal stem cells was observed by scratch test.Bone marrow mesenchymal stem cells were cultured in Transwell cells.The effects of 10%drug-containing serum on longitudinal migration of bone marrow mesenchymal stem cells were observed by crystal violet staining and DAPI nuclear staining.Differentiation of bone marrow mesenchymal stem cells into astrocytes was observed by inducing solution with 10%drug-containing serum or co-culture with astrocytes. RESULTS AND CONCLUSION:(1)10%and 20%drug-containing serum promoted cell proliferation more significantly on days 2 and 3,and there was no statistical difference between the two concentrations.(2)At 30 and 48 hours,bone marrow mesenchymal stem cell migration in 10%drug-containing serum group was significantly higher than that in the control group.(3)The number of bone marrow mesenchymal stem cells filtered through Transwell cells in 10%drug-containing serum group was higher than that in the control group.(4)10%drug-containing serum might promote the differentiation of bone marrow mesenchymal stem cells to astrocytes,but the differentiation effect was weak,and astrocytes might further promote the differentiation of bone marrow mesenchymal stem cells into astrocytes induced by drug-containing serum.(5)The results exhibited that the 10%drug-containing serum could promote the proliferation and migration of bone marrow mesenchymal stem cells in vitro.Co-culture with astrocytes may promote the differentiation of bone marrow mesenchymal stem cells towards astrocytes.

BACKGROUND:Amyotrophic lateral sclerosis is a progressive neurodegenerative disease,which often leads to the death of neurons in the brain and spinal cord.The pathogenesis of amyotrophic lateral sclerosis is extremely complex,with high refractory rate and mortality rate.There are only two kinds of drugs for its treatment,so it is urgent to develop new treatment methods to improve the prognosis of patients. OBJECTIVE:To review the mechanism of Chinese medicine and mesenchymal stem cells regulating the immune response in the treatment of amyotrophic lateral sclerosis. METHODS:"Traditional Chinese medicine,medical stem cells,ALS,immune response"were Chinese and English search terms.Articles were retrieved from WanFang,CNKI,PubMed,Web of Science and other databases from 2010 to 2023.Finally,69 articles were included for review. RESULTS AND CONCLUSION:(1)The article summarizes in detail the five mechanisms of traditional Chinese medicine regulating the immune response in the treatment of amyotrophic lateral sclerosis:mainly including the promotion of expression of closed zone protein-1 and closed protein-5 by traditional Chinese medicine such as borneol and astragaloside IV to rebuild the integrity of the blood central nervous system barrier.Fufangteng Mixture can regulate the receptor molecules on the surface of the natural killer cells to inhibit their autotoxicity.The complement system factors such as Scutellaria barbata and patchouli can inhibit their abnormal activation.Tripterygium wilfordii and Uncaria rhynchophylla inhibit the activation of microglia by mediating the production of extracellular signal-regulated kinase 1/2 attenuated inducible nitric oxide synthase.Zuogui Pill and Trichosanthes kirilowii Root promote the expression of interleukin-10 and regulate T cells to improve the immune environment.(2)Through existing research,five mechanisms of mesenchymal stem cells regulating the immune response in the treatment of amyotrophic lateral sclerosis have been summarized,mainly including reducing the expression of aquaporin 4 and reducing endothelial nitric oxide synthase signal transduction to repair the integrity of the immune barrier;releasing indoleamine 2,3-dioxygenase,prostaglandin E2 and other factors to resist natural killer cell toxicity;secretion factor H interferes with the activity of invertase and inhibits abnormal activation of the complement system;regulating the CX3CL1/CX3CR1 system axis or secreting transforming growth factors β,which can change the phenotype of microglia and inhibit its activity by other ways;increasing the expression of interleukin-10 or activating the STATS phosphorylation pathway to restore T cell function.(3)At present,there are few studies on the combination of traditional Chinese medicine and mesenchymal stem cells in the treatment of amyotrophic lateral sclerosis.Relevant research reports have shown that Jiweiling Injection can promote stem cell proliferation and differentiation and that Buyang Huanwu decoction combined with bone marrow mesenchymal stem cells can significantly improve the integrity of the blood-brain barrier.In the future,further exploration is needed to explore the synergistic treatment effect of both on refractory amyotrophic lateral sclerosis.

Porcine circovirus type 2(PCV2)mainly damages the immune cells of pigs,causing lym-phocyte depletion and immune suppression.Interleukin(IL)-15 regulates immune functions wide-ly,and plays an important regulatory role in the survival,proliferation,differentiation and immune function of a variety of immune cells such as natural killer(NK),CD8+T cells and NKT cells.In this study,in order to determine the effect of PCV2 on IL-15 expression,4-week-old piglets(n=4)were infected with PCV2 and the negative control group(n=4)was set up.On day 7 post-infec-tion,the inguinal lymph nodes of the infected and control groups were collected,and porcine cyto-kine antibody microarray(QAP-CYT-1)was employed to quantify the expression of cytokines in the tissues,screen for differential cytokines,and GO and KEGG enrichment analysis for IL-15 were conducted.Real-time quantitative fluorescent PCR(qPCR)and ELISA were used to verify the level of IL-15 mRNA and protein,and those in porcine peripheral blood mononuclear cells(PBMC)and serum were simultaneously detected.Compared with the negative control group,the expression lev-el of IL-15 was significantly reduced in the infected group(P＜0.05);IL-15 was mainly involved in cytokine-cytokine receptor interactions,immune responses,cellular activation,and the regulation of JAK-STAT and TNF signaling pathways.The levels of IL-15 mRNA and protein in inguinal lymph nodes in the infected group were significantly lower than those in the control group(P＜0.05),which was consistent with the detection results of QAP-CYT-1.However,there was no significant difference in IL-15 mRNA and protein levels in PBMC and serum.These results indicate that PCV2 can inhibit IL-15 in the inguinal lymph node microenvironment of piglets.This study can provide important information for further revealing the immunosuppressive mechanism of PCV2.

Objective To retrospectively explore the diagnosis and treatment of severe immune thrombocytopenia(SIT)induced by programmed death-1(PD-1)monoclonal antibody.Methods Three patients with SIT caused by PD-1 monoclonal antibody treatment at Putuo District People's Hospital,Zhoushan City,Zhejiang Province from March 2020 to October 2022 were selected,and the diagnosis and treatment process and disease outcome were analyzed.Results All three patients were diagnosed with malignant tumors after postoperative pathological examination,and SIT was induced after PD-1 monoclonal antibody treatment,of which one patient died,and two patients improved after the treatment.Conclusion PD-1 induced SIT often occurs after 12 weeks,and clinical diagnosis can be confirmed by combining patients'medication history,platelet antibodies,and bone marrow examination.In terms of treatment,combination therapy such as ordered reduction of hormones,thrombopoietin,and platelet receptor agonists can promote the recovery of the patient.In addition,if necessary,CD20 monoclonal antibody therapy can be applied to antagonize platelet antibodies.

OBJECTIVE: To present on a prenatally diagnosed case with complex structural rearrangements of chromosome 8. METHODS: Chromosome karyotyping, chromosomal microarray analysis (CMA) and fluorescence in situ hybridization (FISH) were carried out for a fetus with increased nuchal thickness. RESULTS: The karyotype of the amniotic fluid sample showed extra materials on 8p. FISH revealed a centromeric signal at the terminal of 8p with absence of telomeric signal. CMA revealed partial deletion of 8p23.3 [(208049_2256732)×1], partial duplication of 8p23.3p23.2 [(2259519_3016818)×3], and partial duplication of 8q [8q11.1q12.2(45951900_60989083)×3]. CONCLUSION The complex structural rearrangements of chromosome 8 in this case has differed from the commonly seen inv dup del(8p).
Female ; Pregnancy ; Humans ; Chromosomes, Human, Pair 8/genetics* ; In Situ Hybridization, Fluorescence ; Gene Rearrangement ; Prenatal Diagnosis ; Centromere

Objectives:To compare the effects of manual and mechanical chest compression on patients receiving extracorporeal cardiopulmonary resuscitation (ECPR).Methods:Patients who underwent extracorporeal cardiopulmonary resuscitation admitted to Jinhua Municipal Central Hospital, Hangzhou First People's Hospital and the First Hospital of Jiaxing from September 2014 to July 2022 were enrolled in the study. The patients were divided into the manual group and mechanical group according to the compression method, and the clinical data of the two groups were compared. To explore the effects of the two compression method on the ECPR implementation, proportion of return of spontaneous circulation (ROSC) and hospital survival.Results:A total of 108 patients who underwent ECPR were included in the study, 50 patients in the manual group and 58 patients in the mechanical group. There were no significant differences in sex, age, laboratory tests before ECPR, ROSC proportion (90.0% vs. 86.2%) and survival (34.0% vs. 39.7%) between the two groups (all P>0.05). The puncture time in the mechanical group was shorter than that in the manual group [12 (9,15) min vs. 13 (11,16) min, P<0.05]. Conclusions:Compared with manual compression, mechanical compression during ECPR neither increase the probability of ROSC nor reduce in-hospital mortality in patients with cardiac arrest. However, mechanical compression may help to shorten the puncture time.

Objective:To provide reference for the development of a more intelligent and systematic nursing clinical decision support system based on the concept of precision nursing and data sharing, the nursing plan module of clinical decision support system.Methods:An evidence-based knowledge base was constructed based on the nursing process and the standardized nursing terminologies; the nursing plan module was designed according to clinical needs, and the logical reasoning rules were formulated from the generation, sequencing and stopping of nursing problems, objectives, measures and activities, and finally the nursing plan module of clinical decision support system was formed.Results:The nursing plan module of clinical decision support system included the basic information of patients, positive evaluation items and weight values, nursing problems, objectives, measures and activities, etc. the module could automatically deduce the nursing plan according to the patient's individual characteristic index (positive evaluation item), and sort the nursing problems and corresponding measures and activities according to the generation time, weight value and correlation degree. It could automatically distinguish nursing problems, goals, measures and the time of activity stop, and realize intelligent decision-making.Conclusion:The interface of nursing plan module of this system is clear and logical reasoning rules are rigorous. It breaks through the bottleneck of nursing decision-making based on personal professional knowledge and experience in clinical situation for a long time, which can ensure the homogeneity of nursing plan and improve the correctness of decision-making.