Objective To investigate the change in interleukin-33 (IL-33) in the peripheral blood of hepatocellular carcinoma (HCC) patients and the role and potential mechanism of IL-33 in regulating CD8 + T cell function in HCC patients. Methods A total of 44 HCC patients who attended Shaanxi Provincial People's Hospital from April 2019 to January 2020 and 20 healthy controls were enrolled. Peripheral blood was collected, and plasma and peripheral blood mononucleated cells (PBMCs) were isolated; ELISA was used to measure the plasma levels of IL-33 and its receptor ST2, and quantitative real-time PCR was used to measure the relative mRNA expression levels of IL-33 and ST2 in PBMCs. CD8 + T cells were purified and stimulated with recombinant IL-33; CCK-8 assay was used to assess cell proliferation, enzyme-linked immunospot assay was used to measure the secretion of perforin and granzyme B, and flow cytometry was used to measure the expression of PD-1, LAG-3, and CTLA-4; changes in cell proliferation, secretion of cytotoxic molecules, and immune checkpoint molecules after IL-33 stimulation were compared. CD8 + T cells were co-cultured with HepG2 cells; the expression of lactate dehydrogenase was measured to calculate the proportion of dead HepG2 cells induced by CD8 + T cells, and the change in the killing function of CD8 + T cells after IL-33 stimulation was compared. The t -test or the paired t -test was used for comparison of continuous data between two groups, and a Pearson correlation analysis was performed. Results Compared with the control group, the HCC group had significantly lower plasma level of IL-33 (269.80±63.08 pg/ml vs 339.50±64.43 pg/ml, t =4.072, P < 0.001) and relative mRNA expression level of IL-33 in PBMCs (1.07±0.14 vs 2.45±0.87, t =10.250, P < 0.001). There were no significant differences in the plasma level of ST2 and the relative mRNA expression level of ST2 in PBMCs between the HCC group and the control group ( P > 0.05). The proportion of CD8 + T cells was not correlated with the plasma level of IL-33 or ST2 (both P > 0.05). Compared with the control group, the HCC group had significantly lower levels of perforin and granzyme B (both P < 0.05) and a significantly higher proportion of CD8 + T cells with positive PD-1, LAG-3, and CTLA-4 ( P < 0.05). Stimulation with recombinant IL-33 did not affect the proliferation of CD8 + T cells or the expression of immune checkpoint molecules ( P > 0.05), but it promoted the secretion of perforin and granzyme B ( P < 0.05). Compared with the control group, the HCC group had a significant reduction in the killing activity of CD8 + T cells ( P < 0.05), and stimulation with recombinant IL-33 enhanced the killing function of CD8 + T cells, which was mainly reflected in the increases in the proportion of dead HepG2 cells ( P < 0.05) and the secretion of IFNγ and TNFα ( P < 0.05). Conclusion There is a reduction in the plasma level of IL-33 in HCC patients. IL-33 can enhance the killing activity of CD8 + T cells by promoting the secretion of perforin and granzyme B, which provides a new target for the treatment of HCC.

Objective:To investigate the safety and efficacy of sacral neuromodulation(SNM)therapy for the treatment of lower urinary tract dysfunction(LUTD)in elderly patients.Methods:Clinical data of 91 elderly patients with LUTD from multiple medical institutions who received SNM during the period from January 2012 to December 2016 were retrospectively analyzed.Patients were divided into four groups: the interstitial cystitis(IC)group(n=28), the neurogenic bladder(NB)group(n=36), the overactive bladder syndrome(OAB)group(n=13)and the idiopathic dysuria(ID)group(n=14). Different sets of evaluation parameters were used for different diseases.Patients’ baseline data and data in stage I(test phase)and stage Ⅱ(permanent SNM)were recorded, statistically analyzed and compared.Results:Ninety-one people underwent SNM treatment.Of them, 53 patients received permanent implants(stage Ⅱ), and the total conversion rate of stage I to stage Ⅱ was 58.2%(53/91). Patients receiving permanent implants(stage Ⅱ)had a preoperative period ranging from 3 months to 30 years, and were followed up for 2 to 58 months after treatment, with an average follow-up of 19.6 months.The improvement rates in stage I for urinary urgency, daily urination frequency, daily nocturnal urination frequency, maximum urine volume, daily average urine volume, daily urine leakage frequency, and quality of life score were 35.4%, 31.6%, 33.7%, 32.6%, 49.2%, 43.2% and 13.2%, respectively.The improvement rates in stage Ⅱ for urinary urgency, daily urination frequency, daily nocturnal urination frequency, maximum urine volume, daily average urine volume, daily urine leakage frequency, and quality of life score were 43.2%, 40.0%, 37.8%, 50.5%, 70.5%, 70.4% and 43.2%, respectively.Three adverse events occurred, including 1 case of recurrent symptoms, 1 case of moderate infection, and 1 case of electrical lead dislocation.Conclusions:Sacral nerve stimulation has definitive and consistent curative effects on LUTD in elderly people.The follow-up time should be extended to further study the safety of sacral nerve stimulation.

OBJECTIVE: explore the expression of miR-155-5p in Wilms tumor and its effect in regulating the proliferation, migration and apoptosis of Wilms tumor cells. METHODS: Specimens of tumor tissues and paired adjacent tissues were obtained from 40 patients with Wilms tumor for detection of the expression levels of miR-155-5p using RT-qPCR. Wilms tumor cell line G401 was transfected with miR-155-5p mimics and miR-155-5p inhibitor to induce miR-155-5p over-expression and its inhibition, respectively, and the changes in the cell proliferation, migration and apoptosis were assessed using cell counting kit-8 (CCK-8), wound healing assay and fl ow cytometry. RESULTS: RT-qPCR showed that the expression of miR-155-5p decreased significantly in Wilms tumor tissues as compared with normal kidney tissues and was significantly associated with TNM stage ( < 0.05). In G401 cells, over-expression of miR-155-5p significantly inhibited the cell proliferation and migration and promoted cell apoptosis ( < 0.05), and down-regulation of miR-155-5p obviously enhanced the proliferation and migration and suppressed apoptosis of the cells ( < 0.05). CONCLUSIONS miR-155-5p is down-regulated in Wilms tumor and its expression level is correlated with TNM stage. miR-155-5p participates in the progression of Wilms tumor by inhibiting the proliferation and migration and promoting apoptosis of the tumor cells, and may serve as a novel biomarker for diagnosis, therapy and prognostic evaluation of Wilms tumor.
Apoptosis ; Cell Movement ; Cell Proliferation ; Humans ; Kidney Neoplasms ; genetics ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; Wilms Tumor ; genetics

Objective: To investigate the effect factors of liver enzymes elevation by monitoring the liver function changes before and after intraportal islet transplantation. Methods: 16 diabetic patients who received intraportal islet transplantation in our hospital were analyzed. The levels of aspartic aminotransferase (AST), alanine aminotransferase (ALT)and total bilirubin (TBil)were monitored after islet transplantation. Results: Among those 16 diabetic patients who received intraportal islet transplantation, 11 patients showed an increased AST and 8 patients showed an increased ALT, among which a 2.5-fold increase in AST was observed in 4 patients and over 1.5-fold elevation of ALT was observed in 3 patients. The level of TBil were in the normal range before and after transplantation in all patients. Transplanted tissue volume of islet was the main factor for significantly increased AST (P<0.05) in this study. It is also shown that the change in portal pressure is related to the AST elevation after islet transplantation. Conclusions The amount of transplanted islet tissue volume is related to the liver enzymes elevation after intraportal islet transplantation. Therefore, the improvement of the purity of islet to reduce the amount of transplanted tissue could be benefit to prevent the liver injury after islet transplantation. Meanwhile, the purified islets should be injected as slowly as possible to maintain a stable portal pressure.

OBJECTIVE: To investigate the transcriptome profile of genital tubercles (GTs) in male SD rats and explore the mechanism of hypospadias induced by Di (2-ethylhexyl) phthalate (DEHP). METHODS: Forty time-pregnant SD rats were randomly divided into 4 equal groups, namely GD16 group and GD19 group (in which the male GTs were collected on gestation day[GD]16 and GD19 for RNA-seq, respectively), control group and DEHP exposure group (with administration of oil and 750 mg/kg DEHP by gavage from GD12 to GD19, respectively).In the control and DEHP exposure groups, the GTs were collected from the male fetuses on GD19.5, and scanning electron microscopy and HE staining were used to observe the morphological changes.The differentially expressed genes (DEGs) in the GTs were screened using lllumina HiSeq 2000 followed by GO and KEGG enrichment analyses to characterize the transcriptome profile.Immunofluorescence assay was performed to verify the DEGs (Mafb) identified by RNA-seq results.Immunofluorescence assay and Western blotting were used to examine the expression levels of Mafb in the penile tissue. RESULTS: A total of 1360 DEGs were detected in the GTs between GD16 group and GD19 group by RNA-seq.Among these genes, 797 were up-regulated and 563 were down-regulated.These DEGs were mainly enriched in the cell adhesion plaque signaling pathway, axon guidance signaling pathway, and extracellular matrix receptor signaling pathway.Compared with that in GD16 group, Mafb was significantly up-regulated in GD19 group, which was consistent with the sequencing results.Mafb and β-catenin were significantly down-regulated in DEHP-exposed group compared with the control group ( < 0.01). CONCLUSIONS Mafb expression increases progressively with the development of GTs in male SD rats.DEHP exposure causes significant down-regulation of Mafb and β-catenin, suggesting that β-catenin signaling pathway that affects Mafb is related to DEHP-induced hypospadias in SD rats.
Animals ; Diethylhexyl Phthalate ; Female ; Gene Expression Profiling ; Humans ; Hypospadias ; MafB Transcription Factor ; Male ; Oncogene Proteins ; Phthalic Acids ; Pregnancy ; Rats ; Rats, Sprague-Dawley

We explored the improved method to prepare decellularized kidney scaffold and provide experimental basis for kidney tissue engineering and renal pathology and toxicology in vitro research. We perfused rat kidneys with PBS (group control) and prepared the decellularized kidney scaffolds with sodium dodecyl sulfate (SDS) (group S), Triton X-100 combined with SDS (group TS), and Triton X-100 combined with SDS after repeated freezing and thawing (group FTS) in different flow velocity. Meanwhile we measured their fluid distributions and vascular resistance. We examined the degree of decellularization of acellular scaffolds by HE, DAPI staining and DNA quantification. We examined the retention of main composition and structural integrity of decellularized scaffolds by Masson, PAS and immunohistochemical staining. We also detected the ultrastructure, cytotoxicity and the level of growth factor of the scaffolds by scanning electron microscope, MTT and ELISA, respectively. The results showed that the time of decellularization in group FTS was less than that in group S and TS. The vascular resistance of scaffolds decellularized at 10 mL/min flow velocity was lower. The fluid distribution in groups S, TS and FTS was different from that in control group. No residual cell was detected by HE and DAPI staining. DNA content was less than 50 ng/mg. Masson, PAS and immunohistochemical staining results showed that there was extracellular collagen, polysaccharide, type I collagen, type IV collagen, fibronectin and laminin in the decellularized scaffolds, and the scanning electron microscope result showed the scaffolds had the honeycomb structure. The cytotoxicity level of decellularized scaffolds was between grade 0 to 1. The level of VEGF, EGF, IGF-1 and PDGF-BB in group FTS were significantly higher than those in group S and TS. In concluding, combining freeze-thawing with perfusion can produce more ideal and effective whole organ decellularized scaffold of rat kidney, and make a foundation for the study of kidney tissue engineering and in vitro pathology and toxicology of kidney.
Animals ; Collagen ; Extracellular Matrix ; Freezing ; Kidney ; Perfusion ; Rats ; Tissue Engineering ; Tissue Scaffolds

Objective To retrospectively compare the efficacy of Serva NB1 collagenase with Vitacyte GOLD collagenase on islet isolation of pancreas.Methods All the human pancreata were obtained from Chinese organ donors.In GMP laboratory,the pancreata were trimmed and distended with Serva NB1 collagenase (Serva NB1,n =12) or Vitacyte GOLD collagenase (Vitacyte GOLD,n =5) and digested according to a modified Ricordi semi-automatic protocol,and the digestion duration was recorded.The digested islets were then collected and washed,followed by the continuous density purification in a Cobe 2991 cell separator.The islet yield,purity,viability and glucose-stimulated insulin release (GSI) were determined each time after purification.Quantity and quality of isolated islets were determined by digestion efficacy.Results The digestion duration in Vitacyte GOLD collagenase group was significantly shorter than in Serva NB1 collagenase group to achieve the same digestion endpoint (P＜ 0.05).The islets yields of different sizes were variable between the two groups.The Vitacyte GOLD collagenase digestion produced more islets with a diameter range of 50-100 μm than the ServaNB1 collagenase digestion (P＜0.05),but the latter yielded more islets with a diameter range of 251-300 μm and 301-350μm (P＜0.05).There was no significant difference in total islets yields,viability,and GSI between two collagenase digestions (P＞0.05).Conclusion Both Vitacyte GOLD collagenase and Serva NB1 collagenase can be used for the clinical islet isolation in China.

OBJECTIVE: To investigate the effect of Piwil2-induced cancer stem-like cell (Piwil2-iCSC)-derived exosomes on the proliferation,migration and invasion of human umbilical cord blood-derived mesenchymal stem cells (hucMSCs). METHODS: Piwil2-iCSC-derived exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy,nanoparticle tracking analysis and Western blotting.Exosome uptake assay was used to identify the pathway that Piwil2-iCSCderived exosomes utilized.HucMSCs were divided into control group,PBS intervention group and exosome intervention group,and CCK-8 assay,wound healing assay,Transwell assay,Western blotting and cell karyotype analysis were used to observe the proliferation,migration,invasion,expression levels of MMP2 and MMP9 proteins,and chromosome structure of hucMSCs. RESULTS: The diameter of Piwil2-iCSC-derived exosomes ranged from 50 nm to 100 nm,and most of them were oval or spherical capsules rich in CD9,CD63 and Piwil2 proteins.Exosomal uptake assay showed that the exosomes executed theirs functions after entering the cells.Compared with the control cells and PBS-treated cells,hucMSCs treated with the exosomes showed significantly increased number of proliferating cells (<0.05) with accelerated healing rate (<0.05 at 24 h;<0.01 at 48 h),increased invasive cells (<0.01),enhanced protein expressions of MMP2(<0.05 PBS group;<0.01 control group) and MMP9(<0.05),but their karyotype still remained 46XY without any abnormalities. CONCLUSIONS Piwil2-iCSC-derived exosomes can promote the proliferation,migration and invasion but does not cause cancer-like heterogeneity changes in hucMSCs.
Argonaute Proteins ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Exosomes ; physiology ; Fetal Blood ; cytology ; Humans ; Karyotyping ; Mesenchymal Stem Cells ; pathology ; Neoplasm Invasiveness ; Neoplastic Stem Cells ; Umbilical Cord ; Wound Healing

Objective To investigate the antibiotic resistance pattern and molecular epidemiology of carbapenem-resistant Pseudomonas aeruginosa.Methods The antimicrobial susceptibility was measured by agar dilution method for the 104 strains of carbapenem-resistant P.aeruginosa (CRPA) collected from Huashan Hospital.The homology between these strains was evaluated by pulsed field gel electrophoresis (PFGE).Results Of thel04 CRPA strains,85.6％ were resistant to meropenem and 98.1％ to imipenem.These strains also showed various percentages of resistance to amikacin (18.3％),gentamicin (40.4％),ceftazidime (26.9％),cefepime (21.2％),ciprofloxacin (44.2％),levofloxacin (50.0％),piperacillin-tazobactam (19.2％),cefoperazone-sulbactam (26.9％),ticarcillin-clavulanic acid (52.9％),aztreonam (26.9％),and colistin (5.8％).PFGE analysis showed that these strains were divided into 48 types,belonging to 9 clones.Only 3 strains were non-typeable.Clone A was the primary epidemic strain (41.6％,42/101),which was mainly isolated from Neurosurgery,Geriatrics and General Ward.Clone B accounted for 5.9％ (6/101) of the strains.Conclusions Multiple clones of carbapenem resistant Pseudomonas aeruginosa were prevalent in Huashan hospital.Effective infection control approaches should be adopted to prevent the development and the further spreading of antimicrobial resistance.

Objective To investigate the clinical characteristics of the infections caused by carbapenem-resistant Pseudomonas aeruginosa (CRPA) for better prevention and treatment of CRPA infections. Methods A retrospective study was conducted to compare the features of CRPA infections (n=85) and carbapenem-susceptible P. aeruginosa (CSPA) infections (n=94) treated in Huashan Hospital from January 1, 2013 to December 31, 2013. Results? The?proportion?of?CRPA?infections?was?significantly?higher?than CSPA in Neurosurgery (40.0% versus 16.0%) and Intensive Care Unit (22.4%, 9.6%). Traumatic brain injury (30.6%) and vascular accidents (21.2%) were the main underlying diseases in CRPA patients, which was higher than in CSPA patients (11.7%and?8.5%,?respectively).?CRPA?infection?was?associated?with?significantly?higher?incidence?of?fever,?altered?mental?status,?and?severe hypoproteinemia than CSPA infection. Multiple bacterial infection was found in more CRPA patients (45.9%, 39/85) than in CSPA patients (24.5%, 23/94). Fewer CRPA patients showed positive treatment response (44.7%, 38/85) than CSPA patients (78.7%, 74/94). CRPA was associated with significantly more cases of disease progression (55.3%, 47/85) and more deaths (16.5%, 14/85) than CSPA (21.3% and 1.1%, respectively). Logistic regression analysis indicated that stay in Department of Neurosurgery, prior carbapenem use, peripherally inserted central catheter, nasal feeding, and mechanical ventilation were the risk factors for CRPA infection. Conclusions No specific clinical manifestation is associated with CRPA infection, which poses a therapeutic challenge and results in unfavorable prognosis. Rational use of antibacterial agents and appropriate supporting treatments are essential for control of CRPA in Huashan Hospital.