BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.

Objective To explore psychological resilience, anxiety, depression, coping styles and social supports status among parents of preterm infants of ophthalmic clinic, and analyze their relationship. Methods A total of 217 parents of preterm infants at ophthalmic clinic of hospital were selected by convenience sampling method and investigated by self- designed general information questionnaire, Connor-Davidson Resilience Scale, Self-rating Anxiety Scale, Self-rating Depression Scale, Social Support Rating Scale, and Simplified Coping Style Questionnaire. Results The total score of psychological resilience was (67.48 ± 14.20) points. The average score of positive coping styles dimension was (1.98±0.50) points, and negative coping style dimension score was (1.19±0.55) points. The social support score was moderate with a total score of (42.75 ± 6.17) points, the anxiety and depression got a total score of (36.77 ± 8.17) points and (39.67 ± 9.02) points respectively. Psychological resilience was negatively correlated with anxiety and depression (r=-0.363--0.242, P<0.01), and was positively correlated with coping styles and the social support (r=0.141-0.312, P<0.05 or 0.01). Multi-factor linear regression analysis showed that depression and social support were the influence factors of psychological resilience(t=-4.376, 2.516, P<0.01 or 0.05). Conclusions The parents of preterm infants are at the poor psychological states. Coping styles and depression are the important factors which affect psychological resilience among parents of preterm infants. Nurses should assess the psychological status of the parents, provide targeted interventions to relieve stress, guide the parents use social support effectively, and improve their psychological state.

BACKGROUND:Stem cels can induce immune tolerance, prolong graft survival time and reduce rejection in organ transplantation, which have become a hot research. OBJECTIVE:To induce immune tolerance to alogenic kidney transplantation with amniotic fluid stem cels in recipient rats and to explore the mechanism underlying immune tolerance. METHODS: Amniotic fluid stem cels were isolated from Wistar rats. Two inbred male rat strains, Wistar rats and Sprague-Dawley rats, were selected as donors and recipients of kidney transplantation. The rat models of renal orthotopic transplantation were divided into the folowing four groups: a sham-operated group (n=10, Sprague-Dawley rats); an isograft group (n=10, Sprague-Dawley to Sprague-Dawley rats); a control group (n=10, Wistar to Sprague-Dawley rats, treated with 1 mL saline); and an experimental group (n=10, Wistar to Sprague-Dawley rats, treated with 1 mL of 3×106/L amniotic fluid stem cels). Serum levels of creatinine, urea nitrogen, interleukin-2, interferon-γ, parameters of oxidative stress were detected at 5 days after operation. Flow cytometry was employed to determine the percentage of CD4+ and CD8+ lymphocytes in the peripheral blood. Kidney transplants were observed pathologicaly. RESULTS AND CONCLUSION:Compared with the control group, the levels of creatinine, urea nitrogen, interleukin-2, interferon-γ, parameters of oxidative stress and proteinuria were lower in the experimental group (P < 0.05). Percentages of CD4+, CD8+ and CD4+/CD8+ ratio were also significantly lower in the experimental group than the control group. However, the rate of cretinemia clearance in the experimental group was significantly higher than that in the control group (P < 0.05). Furthermore, the degree of kidney injury in the experimental group was significantly lower than that in the control group. Our findings demonstrate that the amniotic fluid stem cel transplantation can induce immune tolerance, extenuate oxidative stress, attenuate pathological damage to the kidney transplant and preserve kidney function from acute rejection in rats undergoing kidney transplantation.

Objective: To investigate the effects and mechanisms of Xuebijing injection (XBJ) on Chimeric antigen receptor-T (CAR-T) cell function and its therapeutic potential against CAR-T therapy-associated cytokine storms (CRS). Methods: Anti-CD19 CAR-T cells were established based on FMC63 antibodies. Different doses of XBJ (1 and 10 mg/mL) were added to the culture system. Untreated anti-CD19 CAR-T cells served as negative controls. After 48-h co-culture, the effects of XBJ on CAR-T cell function were assessed. Carboxyfluorescein diacetate succinimidyl ester staining was used to assess the effect of XBJ on CAR-T cell proliferation. Flow cytometry, luciferase reporter gene assays, and real time cellular analysis were employed to evaluate the effects of XBJ on CAR-T cell cytotoxicity in vitro. RNA-sequencing was performed to analyze the effects of XBJ on CAR-T cell gene expression. Network pharmacology predicted potential XBJ therapeutic targets for CRS, which were verified in a THP-1 macrophage inflammation model. Results: XBJ enhanced both the proliferation and tumor killing capacities of CAR-T cells. Transcriptome analysis showed that XBJ treatment affects multiple genes and pathways in CAR-T cells, with differential gene enrichment in multiple cell proliferation and growth factor pathways. Potential targets for CRS control by XBJ were predicted using network pharmacology, and the inhibitory effect of XBJ on the expression of relevant genes was verified using a macrophage model. Conclusion The results of this study indicate that XBJ can enhance the killing effect of CAR-T cells on tumor cells and that the mechanism is related to the regulation of T cell proliferation and activation. Moreover, XBJ inhibited excessive inflammation associated with CAR-T therapy. However, the current findings remain to be further validated through in vivo experiments.

Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.

LZ-8 protein, isolated from a well known Chinese traditional medicinal fungus Ganoderma lucidum, is the first member of fungal immunomodulatory protein, members of which have been isolated from a variety of medicinal and edible mushrooms in the last two decades. The protein plays a multifunctional and important role in modulating immune system. In this report, in order to get LZ-8 protein crystals, the LZ-8 gene was expressed and purified by affinity chromatography, gel filtration chromatography and anion exchange chromatography subsequently. The protein was then crystallized using the hanging-drop vapour-diffusion method. The LZ-8 crystals were obtained and the phase information was calculated by X-ray diffraction. The resolution of LZ-8 crystals is 3.2A. This study will provide an insight into the structure of fungal immunomodulatory proteins.
Animals ; Crystallography ; Fungal Proteins ; biosynthesis ; genetics ; immunology ; Ganoderma ; genetics ; metabolism ; Genetic Vectors ; genetics ; Immunologic Factors ; biosynthesis ; genetics ; Mice ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes.

METHODSFive kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned, labeled as probes for Southern and Northern blot analysis and DNA sequencing.

RESULTSSample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40% (42.2% - 49.4%). There were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% (primer 8) to 68% (primer 2). Band B is a single copy fragment, and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005. Further analysis revealed that it might be part of a cis-regulatory element of a new tumor suppressor gene, containing a promoter of cis-action "CACA" box, an enhancer of "GATA" family and a start codon.

CONCLUSIONSIt was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.

Base Sequence ; Blotting, Southern ; Colonic Neoplasms ; genetics ; pathology ; Humans ; Liver Neoplasms ; genetics ; pathology ; Lung Neoplasms ; genetics ; pathology ; Molecular Sequence Data ; Neoplasms ; genetics ; pathology ; Polymorphism, Restriction Fragment Length ; Random Amplified Polymorphic DNA Technique ; Sequence Analysis, DNA ; Stomach Neoplasms ; genetics ; pathology

PD-L1 is a member of the B7 protein family, most of whose members so far were identified as dimers in a solution and crystalline state, either complexed or uncomplexed with their ligand(s). The binding of PD-L1 with its receptor PD-1 (CD279) delivers an inhibitory signal regulating the T cell function. Simultaneously with the Garboczi group, we successfully solved another structure of human PD-L1 (hPD-L1). Our protein crystallized in the space group of C222(1) with two hPD-L1 molecules per asymmetric unit. After comparison of reported B7 structures, we have found some intrinsic factors involved in the interaction of these two molecules. Based on these results, we tend to believe this uncomplexed hPD-L1 structure demonstrated its potential dimeric state in solution, although it could just be an evolutionary relic, too weak to be detected under present technology, or still a functional unit deserved our attentions.
Antigens, CD ; chemistry ; immunology ; B7-H1 Antigen ; Crystallography, X-Ray ; Evolution, Molecular ; Humans ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Secondary ; T-Lymphocytes ; chemistry ; immunology

Objective:To analyze the relationship between quantitative parameters of dual-energy CT and lymph node metastasis in patients with early non-small cell lung cancer (NSCLC).Methods:A total of 120 patients with NSCLC (NSCLC group) admitted to Dongtai People′s Hospital from April 2021 to April 2023 were selected retrospectively, and another 120 patients with pneumonia nodules (pneumonia nodules group) during the same period were selected. All patients were given dual-energy CT examination. CT quantitative parameters including standardized iodine concentration in venous phase (NICVP), standardized iodine concentration in arterial phase (NICAP) and standardized iodine concentration (ICD) were compared between the two groups. Patients in the NSCLC group were divided into the metastatic group (40 cases) and the non-metastatic group (80 cases) according to the presence or non-presence of lymph node metastasis. Quantitative CT parameters of the two groups were compared, CT signs of the two groups were compared. The receiver operating characteristics (ROC) curve was drawn, and the diagnostic efficacy of quantitative CT parameters on lymph node metastasis in NSCLC patients was analyzed.Results:The NICVP, NICAP and ICD in the NSCLC group were higher than those in the pneumonia nodules group: 0.23 ± 0.04 vs. 0.17 ± 0.02, 0.58 ± 0.16 vs. 0.42 ± 0.10, 0.36 ± 0.08 vs. 0.24 ± 0.03, there were statistical differences ( P<0.01). The NICVP, NICAP and ICD in the metastatic group were higher than those in non-metastatic group: 0.25 ± 0.03 vs. 0.18 ± 0.03, 0.61 ± 0.24 vs. 0.51 ± 0.13, 0.37 ± 0.10 vs. 0.26 ± 0.04, there were statistical differences ( P<0.01). The pleural depression sign, burr sign, mediastinum lymph node short diameter between metastasis group and non-metastatic group had statistical differences ( P<0.05). The results of ROC curve analysis showed that the area under the curve of the combined detection of NICVP, NICAP and ICD for the diagnosis of lymph node metastasis in NSCLC patients was 0.852 (95% CI 0.822 - 0.957), the sensitivity was 94.96%, the specificity was 90.12%, and were higher than those of single detection ( P<0.05). Conclusions:Quantitative dual-energy CT detection can improve the diagnostic sensitivity and specificity of lymph node metastasis in NSCLC patients, and provide CT signs to assist clinical diagnosis of lymph node metastasis.

Objective To investigate the clinical comprehensive value of finerenone in the treatment of diabetic nephropathy(DN),and to provide evidence-based medicine evidence for clinical drug decision.Methods PubMed,Web of Science,Embase,Cochrane Library,WanFang Data,CNKI and health technology assessment(HTA)official website were systematically searched to collect the systematic review/Meta-analysis and pharmacoeconomic evaluation on finerenone in treatment of DN from the inception to November 31,2023.The method of rapid HTA was used to evaluate the effectiveness,safety and economic evaluation.The innovation,suitability and accessibility of finerenone were analyzed by relevant data from drug instructions,professional websites such as the National Medical Products Administration(NMPA)and Center for Drug Evaluation,NMPA.Results In terms of effectiveness,finerenone significantly reduced the risk of the renal composite events and composite cardiovascular outcomes in DN compared with placebo and traditional mineralocorticoid receptor antagonist(MRA).In terms of safety,the incidence of adverse reactions and acute kidney injury of finerenone was similar to that of placebo and traditional MRA,but the incidence of hyperkalemia was higher than that of placebo.In terms of economy,two foreign HTA reports showed that finerenone was more economical than standard treatment.In terms of innovation,finerenone was the world's first approved non-steroidal,selective MRA innovative drug for the treatment of type 2 DN,making its efficacy and adverse reactions more advantageous.In terms of suitability,finerenone should only be taken once a day,which had good suitability in pharmaceutical properties and clinical use.In terms of accessibility,the domestic price of finerenone was lower than the international price,and it was included in the medical insurance,and the market coverage was high,it had a good affordability and availability.Conclusion Finerenone has good effectiveness and safety in the treatment of DN,but attention should be paid to the risk of hyperkalemia,and its economy requires further economic research in China.As the world's first approved non-steroidal,selective MRA innovative drug,finerenone has better innovation,suitability and accessibility.