Objective: To compare the changes in the concentration of relevant growth factors released from platelet-rich plasma (PRP) stored at -80℃ by cryopreservation and at 4℃ by refrigerated lyophilization over 2 years, aiming to provide a theoretical basis for prolonging PRP storage duration. Methods: PRP (n=15) was separated using a blood cell separator and stored under -80℃ cryopreservation (F-PRP group) and 4℃ refrigerated freeze-drying conditions (FD-PRP group). The contents of growth factors (PDGF-AA, PDGF-BB, EGF, TGF-β1, and VEGF) in both groups were measured by ELISA at 1, 3, 6, 9, 12 and 24 months. Results: PDGF-AA and VEGF maintained good stability in both groups for up to 24 months. PDGF-BB and TGF-β1 showed high stability in the first 12 months but their stability decreased gradually from 12th to 24th months. EGF demonstrated good stability in the first 6 months, and its stability gradually decreased from the 9th to 24th months. Comparing the F-PRP and FD-PRP groups, the concentrations of the five growth factors in the FD-PRP group were either not statistically different or higher than those in the F-PRP group at all time points. Specifically, the concentrations of EGF were significantly higher in the FD-PRP group at all time points. Conclusion: Both -80℃ freezing and 4℃ freeze-drying enable long-term preservation of PRP. Freeze-drying imposes less stringent storage requirements and facilitates growth factor compared to frozen storage.

Objective To investigate the correlation between different forms of serum vitamin D levels and liver fibrosis in patients with metabolic dysfunction-associated fatty liver disease (MAFLD). Methods Data from the National Health and Nutrition Examination Survey in 2021–2023 were analyzed. Logistic regression models were used to evaluate the relationship between serum total vitamin D, 25(OH)D₃ levels, and liver fibrosis in the MAFLD patients. Results A total of 2 628 patients were included. There were significant differences between MAFLD patients with liver fibrosis and those without fibrosis in age, smoking history, waist circumference, body mass index, high-density lipoprotein cholesterol, total cholesterol, fasting plasma glucose, hypertension history, vitamin D, and 25(OH)D₃ levels (P＜0.05). Logistic regression analysis revealed that compared to the low total serum vitamin D group (11.2-61.8 nmol/L), MAFLD patients with high total vitamin D levels (89.1 nmol/L＜vitamin D≤290 nmol/L) exhibited a 22% reduced risk of liver fibrosis (OR＝0.78, 95%CI 0.64-0.94, P＝0.015). Similarly, compared to the low 25(OH)D₃ group (4.1-57.0 nmol/L), those with high 25(OH)D₃ level [84.7 nmol/L＜25(OH)D₃≤288 nmol/L] showed a 23% lower risk of liver fibrosis (OR＝0.77, 95%CI 0.62-0.95, P＝0.021). After adjusting for covariates, high total vitamin D levels remained significantly associated with reduced liver fibrosis risk (OR＝0.63, 95%CI 0.42-0.94, P＝0.036). Conclusions Elevated serum total vitamin D and 25(OH)D₃ levels are protective factors against early liver fibrosis in MAFLD patients.

Objective:To deeply explore the decision-making experience of patients participating in clinical trials of neoadjuvant immunotherapy for lung cancer.Methods:Using the descriptive and qualitative research, 15 lung cancer patients who participated in clinical trials of neoadjuvant immunotherapy at the Cancer Hospital of the Chinese Academy of Medical Sciences were selected by purposive sampling from April 2021 to August 2022 for semi-structured in-depth interviews. Content analysis method was used for data analysis, summarization, and induction.Results:Three themes were extracted, namely decision-making information dilemma (insufficient or overloaded information, difficulty in understanding professional information, urgent need for decision-making information assistance), complex emotional experience (negative emotional experience, positive emotional experience), and hope for multi-party support (expecting psychological communication, hoping for family understanding, and longing for social recognition) .Conclusions:The decision-making experience of patients in clinical trials of neoadjuvant immunotherapy for lung cancer were summarized and described, which can help strengthen the understanding of the research team and medical and nursing staff on the live experience of such patients when making decisions, provide targeted decision support strategies, and promote good informed consent of patients.

Objective To investigate the effects of galectin-3(Gal3)on skin abscess development and activation of mast cells(MC)in mice infected with methicillin-resistant Staphylococcus aureus(MRSA).Methods Wild type mice and Gal3-knockout(Gal3-/-)mice,at 6～8 weeks of age,were divided into four groups:Wild type mice+PBS group,Wild type mice+MRSA group,Gal3-/-mice+PBS group,Gal3-/-mice+MRSA group,were subcutaneously injected with MRSA or the same volume of phosphate buffer saline,with five mice per group.The development and pathological changes of skin abscess were monitored and recorded.The bacterial load in skin tissues was compared,and the expression of associated cytokines,degranulation of MC,and the distribution of MC activation marker 5-hydroxytryptamine(5-HT)were detected.Results The skin of Wild type mice showed progressive abscesses after subcutaneous infection with MRSA,but the Gal3-/-mice showed smaller abscess areas.Compared to the Wild type mice+MRSA group,the Gal3-/-mice+MRSA group showed lower bacterial loading in the skin tissues(P＜0.01)and fewer infiltrating inflammatory cells with histopathological observation.The expression of cytokines,including IL-1β,TNF-α,IL-33,TGF-β,and IL-10,were significantly lower in Gal3-/-mice than Wild type mice(P＜0.05).The toluidine blue staining showed a large number of degranulated MCs in the skin tissues of the wild type mice+MRSA group,whereas only a few degranulated MCs were observed in the Gal3-/-mice+MRSA group.It was further found that the expression of 5-HT in Gal3-/-mice+MRSA group was significantly lower than that in wild-type mice+MRSA group with immunohistochemical staining.Conclusion Gal3 deficiency reduced the activation and degranulation of mouse skin MC after MRSA infection,resulting in changes to inflammatory responses and alleviating the severity of skin tissue abscesses.

Objective:To investigate the clinical infection status and trends in drug resistance of a rare pathogen Kluyveromyces marxianus ( Candida kefyr), and to provide experience for clinical diagnosis and treatment. Methods:Morphological and molecular biological identification tests and in vitro microdilution drug susceptibility test were conducted on a Kluyveromyces marxianus strain recently isolated from the midstream urine sample of a patient with urinary calculus in the Fungal Laboratory, Changhai Hospital. The ultrastructural damage of the strain caused by different antifungal drugs was observed by scanning electron microscopy. A retrospective analysis was conducted on clinical cases of Kluyveromyces marxianus infection in Changhai Hospital from 2009 to 2021. Results:The isolated strain formed smooth, soft, cheese-like yeast colonies on the Sabouraud′s agar medium, and ovoid or slender spores were observed under the microscope. Morphological analysis, mass spectrometry and sequencing analysis identified the strain as Kluyveromyces marxianus. The drug susceptibility test showed that minimum inhibitory concentrations (MICs) of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, flucytosine, caspofungin, and micafungin were 0.5, 0.5, 0.03, ≤ 0.03, 0.06, 0.5, ≤ 0.016, and 0.06 μg/ml, respectively. Under the scanning electron microscope, the strain was ovoid to slender before antifungal drug treatment, with a size of (3.0 - 6.5) μm × (5.5 - 11.0) μm; after 24-hour treatment with antifungal drugs at the dose of 1 μg/ml, cell membrane shrinkage was more obvious under the treatment with posaconazole, which exhibited a stronger destructive effect on the strain compared with amphotericin B and voriconazole. From 2009 to 2021, 8 cases of Kluyveromyces marxianus infection were collected, including 6 males and 2 females; the Kluyveromyces marxianus strains were isolated from ascites in 3 cases, bronchoalveolar lavage fluid in 1 case, sputum in 2 cases, and midstream urine samples in 2 cases. Conclusion:For suspected Kluyveromyces marxianus infection, it is crucial to determine the pathogenic species as early as possible using various identification methods, and to collect strain as well as evaluate drug susceptibility, which will be beneficial for targeted clinical treatment.

Objective:To screen the characteristic gut microbiota and fecal metabolites related to the efficacy of oxaliplatin-based neoadjuvant chemotherapy in patients with colorectal cancer liver metastasis, to analyze the relationship between gut microbiota and fecal metabolites, and to evaluate the predictive value of relevant markers for the efficacy of neoadjuvant chemotherapy in patients with colorectal cancer liver metastasis.Methods:This is a case-control study, 34 patients with colorectal cancer liver metastasis who were treated in Qilu Hospital of Shandong University from October 2021 to July 2022 were selected as the research objects, and were divided into chemotherapy effective group (20 cases) and chemotherapy ineffective group (14 cases) according to the efficacy evaluation criteria. Logistic regression was used to construct a prediction model to screen the microbiota and metabolic markers capable of predicting the effect of chemotherapy, and the receiver operating characteristic (ROC) curve and survival analysis curve were plotted to evaluate the predictive effect of related microbiota and metabolites on the efficacy of neoadjuvant chemotherapy.Results:There was no significant difference in the α and β diversity of gut microbiota between the patients in the chemotherapy effective group and in the ineffective group (all P>0.05). In terms of species, the relative abundance of 5 species was up-regulated and 10 species were down-regulated in the chemotherapy-effective group compared with the chemotherapy-ineffective group, and the difference was statistically significant (all P<0.05), among which Prevotella salivae could effectively predict the chemotherapy effect (AUC=0.750, P=0.007), with a sensitivity of 80.0% and a specificity of 71.4%. The overall survival of patients with high abundance (17 cases) was lower than that of patients with low abundance (17 cases) ( χ 2=5.239, P=0.022). In terms of metabolites, 20 metabolites were up-regulated and 4 metabolites were down-regulated in the chemotherapy-effective group compared with the chemotherapy-ineffective group, and the difference was statistically significant (all P<0.05), among which threonine and prostaglandin F2α-1-ethanolamide could distinguish between patients who responded to chemotherapy and those who did not respond to chemotherapy (AUC=0.743, 0.707, all P<0.05), and the overall survival of patients with high levels of relative abundance (17 cases) was higher than that of patients with low levels (17 cases) ( χ 2=4.748, 5.407, all P<0.05). The Logistic regression model of Prevotella salivae and prostaglandin F2α-1-ethanolamide was obtained through screening analysis, and the ROC curve results showed that the model had a good predictive value (AUC=0.836, sensitivity: 90.0%, specificity: 78.6%), and the overall survival of patients with high predict probability (17 cases) predicted by the model was higher than that of patients with low predict probability (17 cases) ( χ 2=9.260, P=0.002). Conclusion:Prevotella salivae and prostaglandin F2α-1-ethanolamide can be used as predictive biomarkers of neoadjuvant chemotherapy for colorectal cancer liver metastasis, and the model has good clinical reference value for prognosis assessment of patients in this cohort.

Objective To design a pH-sensitive nintedanib liposomes(Nb-Lips)which targeted the acidic microenvironment of pulmonary fibrosis.The entrapment efficiency(EE%)was optimized by the formulation process.Methods Nintedanib liposomes were prepared by membrane hydration method,and the formulation of nintedanib liposomes were optimized by single factor experiments and response surface method(RSM).The particle size,polymer dispersity index(PDI),Zeta potential and encapsulation rate was investigated by dynamic light scattering technique and microcolumn centrifugation method.The release behavior of nintedanib liposomes in artificial lung fluid with pH 7.4 and artificial lung fluid with pH 5.3 was investigated by dialysis method.Nintedanib liposomes were atomized with a compressed air atomizer and its atomization stability and aerodynamic particle size were investigated.Results The particle size of nintedanib liposomes was(100.651±7.315)nm,the PDI was(0.328±0.026),the zeta potential was(21.633±2.004)mV,and the encapsulation rate was higher than 80%.Compared with nintedanib solution at pH 7.4,the total release of nintedanib liposomes in pH 5.3 artificial lung solution was 60.78%higher,and the release of nintedanib liposomes in pH 5.3 artificial lung solution was 48h delayed,which was much higher than that of nintedanib solution.The data reveals no significant differences in particle size,potential and PDI before and after atomization of nintedanib liposomes,and the encapsulation rate decreased 4.25%.The fine particle fraction of the atomized droplets was 37.49%.Conclusion The response surface method can effectively improve the encapsulation rate of nintedanib liposomes,and successfully prepare nintedanib liposomes that are sensitive to acidic environment,and can be inhaled and released slowly.

Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 μ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.

Objective To summarize the experience and practical value of living donor kidney harvesting in Bama miniature pigs with six gene modified. Methods The left kidney of Bama miniature pigs with six gene modified was obtained by living donor kidney harvesting technique. First, the ureter was occluded, and then the inferior vena cava and abdominal aorta were freed. During the harvesting process, the ureter, renal vein and renal artery were exposed and freed in sequence. The vascular forceps were used at the abdominal aorta and inferior vena cava, and the renal artery and vein were immediately perfused with 4℃ renal preservation solution, and stored in ice normal saline for subsequent transplantation. Simultaneously, the donor abdominal aorta and inferior vena cava gap were sutured. The operation time, blood loss, warm and cold ischemia time, postoperative complications and the survival of donors and recipients were recorded. Results The left kidney of the genetically modified pig was successfully harvested. Intraoperative bleeding was 5 mL, warm ischemia time was 45 s, and cold ischemia time was 2.5 h. Neither donor nor recipient pig received blood transfusion, and urinary function of the kidney transplanted into the recipient was recovered. The donor survived for more than 8 months after the left kidney was resected. Conclusions Living donor kidney harvesting is safe and reliable in genetically modified pigs. Branch blood vessels could be processed during kidney harvesting, which shortens the process of kidney repair and the time of cold ischemia. Living donor kidney harvesting contributes to subsequent survival of donors and other scientific researches.

BACKGROUND:Tissue engineering has brought new hope to the clinical challenge of liver failure,and the preparation of plant-derived decellularized fiber scaffolds holds significant importance in liver tissue engineering. OBJECTIVE:To prepare apple tissue decellularized scaffold material by using fresh apple slices and a solution of sodium dodecyl sulfate,and assess its biocompatibility. METHODS:Fresh apples were subjected to decellularization using phosphate buffer saline and sodium dodecyl sulfate solution,separately.Afterwards,the decellularized apple tissues and apple decellularized scaffold materials were decontaminated with phosphate buffer saline.Subsequently,scanning electron microscopy was used to assess the effectiveness of decellularization of the apple materials.Adipose-derived mesenchymal stem cells were extracted from the inguinal fat BALB/C of mice,and their expression of stem cell-related markers(CD45,CD34,CD73,CD90,and CD105)was identified through flow cytometry.The cells were then divided into a scaffold-free control group and a scaffold group.Equal amounts of adipose-derived mesenchymal stem cells were seeded onto both groups.The biocompatibility of the decellularized scaffold with adipose-derived mesenchymal stem cells was evaluated using CCK-8 assay,hematoxylin-eosin staining,and phalloidine staining.Cell adhesion and growth on the scaffold were observed under light microscopy and scanning electron microscopy.Furthermore,the scaffold was subdivided into the non-induced group and the hepatogenic-induced group.Adipose-derived mesenchymal stem cells were cultured on the decellularized apple scaffold,and they were cultured for 14 days in regular culture medium or hepatogenic induction medium for comparison.Immunofluorescent staining using liver cell markers,including albumin,cytokeratin 18,and CYP1A1,was performed.Enzyme-linked immunosorbent assay was used to detect the secretion of alpha fetoprotein and albumin.Additionally,scanning electron microscopy was employed to observe the morphology of the induced cells on the scaffold,verifying the expression of liver cell-related genes on the decellularized scaffold material.Finally,the cobalt-60 irradiated and sterilized decellularized apple scaffolds were transplanted onto the surface of mouse liver and the degradation of the scaffold was observed by gross observation and hematoxylin-eosin staining after 28 days. RESULTS AND CONCLUSION:(1)The scanning electron microscopy results revealed that the decellularized apple scaffold material retained a porous structure of approximately 100 μm in size,with no residual cells observed.(2)Through flow cytometry analysis,the cultured cells were identified as adipose-derived mesenchymal stem cells.(3)CCK-8 assay results demonstrated that the prepared decellularized apple tissue scaffold material exhibited no cytotoxicity.Hematoxylin-eosin staining and phalloidine staining showed that adipose-derived mesenchymal stem cells were capable of adhering and proliferating on the decellularized apple tissue scaffold.(4)The results obtained from immunofluorescence staining and enzyme-linked immunosorbent assay revealed that adipose-derived mesenchymal stem cells cultured on the decellularized apple scaffolds exhibited elevated expression of liver-specific proteins,including albumin,alpha-fetoprotein,cytokeratin 18,and CYP1A1.These results suggested that they were induced differentiation into hepatocyte-like cells possessing functional characteristics of liver cells.(5)The decellularized apple scaffold implanted at 7 days has integrated with the liver,with partial degradation of the scaffold observed.By 28 days,the decellularized apple scaffold has completely degraded and has been replaced by newly-formed tissue.(6)The results indicate that the decellularized scaffold material derived from apple tissue demonstrates favorable biocompatibility,promoting the proliferation,adhesion,and hepatic differentiation of adipose-derived mesenchymal stem cells.