Objective:To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell (OCS-LC).Methods:(1) A2780 cells were induced into OCS-LC in serum-free medium, and authenticating their stem-like properties by sphere-forming test, differentiation test and CD 133 marker detection. (2) Exosomes from ascites and A2780 cell were extracted by ultracentrifugation, then authenticating them. The morphology of exosomes was observed by the transmission electron microscope, exosome particle size was measured by nanoparticle tracking analysis (NTA). The expressions of heat shock protein 70 (HSP-70), CD 63 and CD 9 were detected by western blot. (3) The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC. The groups were divided into control group, ascites-derived exosomes (ADE) group (ADE+OCS-LC group), and cells-derived exosomes (CDE) group (CDE+OCS-LC group). The sphere-forming ability was evaluated by sphere-forming cycle, maximum sphere diameter and sphere-forming rate of each group; the expression of CD 133 was detected by immunofluorescence staining under microscope; quantitative real-time (qRT)-PCR was used to detected the expression levels of octamer-4 (Oct-4), Nanog mRNA of the signature genes in the stem cells of each group; the metastasis ability of each group was measured by transwell assay. Results:(1) Identification of OCS-LC: sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming. Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium. Flow cytometry showed that the proportion of CD 133 positive ( CD133+) cells in OCS-LC group was (18.9±0.9)%, significantly higher than that of control group (0.6±0.5)% ( t=38.570, P<0.01). (2) Under transmission electron microscope, clear lipid bilayer structure was observed in ADE and CDE, and one side presented a concave hemispheric or cup like structure. NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm, with an average diameter of 67.2 nm. Western blot analysis showed that the specific protein molecules HSP-70, CD 63 and CD 9 were positive. (3) Three groups′ OCS-LC could continue to grow into spheres, and the group of ADE+OCS-LC showed two growth modes, most of the cells continued to grow into spheres, while a small part of cells grew in adherent differentiation. The sphere-forming rate of OCS-LC in the control group, ADE+OCS-LC group, and CDE+OCS-LC group were (1.05±0.20)%, (4.15±0.10)%, and (10.45±0.25)%, the sphere-forming cycle of OCS-LC in the three groups were (15.3±1.5), (10.3±0.6), and (6.7±0.6) days, and the maximum diameters of OCS-LC were (100.3±3.2), (145.2±5.1) and (170.0±2.1) μm, respectively. And the differences were statistically significant (all P<0.05). After co-culture of exosomes with OCS-LC, the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group (all P<0.05). Immunofluorescence staining showed that the number of CD 133 green fluorescent chromophore cells in OCS-LC groups [(46.2±2.1)%, (58.4±2.2)%] was significantly higher than that in the control group [(26.6±1.5)%] after the addition of exosomes in co-culture, the positive rate of CD 133 was higher than that in the control group( F=187.588, P<0.05). The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24, 4.03±0.31, compared with that in control group (1.04±0.12), the differences were statistically significant ( F=134.932, P<0.05). The mRNA expression levels of Nanog were 1.57±0.32, 2.66±0.15, which were significantly higher than that in the control group (1.00±0.07), and the differences were statistically significant ( F=49.329, P<0.05). And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group (all P<0.05). The number of invasive cells in the three groups of OCS-LC were: control group 30±5, ADE+OCS-LC group 102±4, CDE+OCS-LC group 210±7, and there were statistically significant differences among three groups ( F=820.800, P<0.05). Compared with the control group, the number of invaded cells in the co-culture group were significantly increased ( P<0.05), and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group ( t=23.202, P<0.05). Conclusions:Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC, and promote the invasion of tumor cells. Moreover, CDE is superior to ADE.

Objective:To investigate the dynamic changes of mitochondrial fission and fusion in the heart of cardiac arrest (CA) rats after return of spontaneous circulation (ROSC), and to explore the role of mitochondrial fission and fusion in the myocardial injury after ROSC.Methods:Healthy male SD rats were randomly random number assigned into the post-resuscitation (PR) 4 h ( n=12), PR 24 h ( n=12), PR 72 h ( n=12), and sham groups ( n=6). The rat CA model was induced by asphyxia, and cardiopulmonary resuscitation (CPR) was performed 6 min after CA. The protein expressions of mitochondrial Drp1, Fis1, Mfn1, and Opa1 were determined by Western blot in each group at 4, 24 and 72 h after ROSC. The mRNA expressions of Drp1, Fis1, Mfn1, and Opa1 were determined by RT-PCR. Myocardial ATP content and mitochondrial respiratory function were measured. The histopathologic changes of myocardial tissue were observed under light microscope. One-way analysis of variance (ANOVA) was use to compare quantitative data, and LSD- t test was used for comparison between groups. Results:Compared with the sham group, the protein and mRNA expressions of Drp1 and Fis1 were increased (all P<0.05) and the protein and mRNA expressions of Mfn1 and Opa1 were decreased (all P<0.05) in the PR 4 h and PR 24 h groups. However, there were no statistical differences in the protein and mRNA expressions of Drp1, Fis1, Mfn1, and Opa1 in the PR 72 h group compared with the sham group (all P>0.05). Compared with the sham group, the levels of ATP content [(4.53±0.76) nmol/g protein vs. (8.57±0.44) nmol/g protein and (5.58±0.58) nmol/g protein vs. (8.57±0.44) nmol/g protein] and mitochondrial respiratory control rate [(2.47±0.38) vs. (3.45±0.32) and (2.97±0.24) vs. (3.45±0.32)] were obviously decreased in the PR 4 h and PR 24 h groups (all P<0.05). There were no statistically significant differences in the ATP content [(7.73±0.95) nmol/g protein vs. (8.57±0.44) nmol/g protein] and mitochondrial respiratory control ratio [(3.39±0.34) vs. (3.45±0.32)] between the PR 72 h group and the sham group (all P>0.05). The pathological damage of myocardial tissue was obvious in the PR 4 h group, and was improved significantly in the PR 72 h group. Conclusions:The imbalance of mitochondrial fission and fusion is probably involved in the pathological process of myocardial injury after ROSC, which may be related to mitochondrial dysfunction.

OBJECTIVE: To investigate the predictive value of pulse infusion index (PPI) in the short-term prognosis of patients with sepsis-induced acute kidney injury (AKI). METHODS: A retrospective cohort study was conducted. The clinical data of patients with sepsis-induced AKI admitted to intensive care unit (ICU) of the First Affiliated Hospital of Soochow University from July 2021 to December 2022 were enrolled. The basic information of the patients were collect, including age, gender, site of infection, underlying disease, mean arterial pressure (MAP) and heart rate (HR) at admission, as well as the use of mechanical ventilation and vasoactive drugs, and norepinephrine (NE) dosage. Laboratory indicators, sequential organ failure assessment (SOFA) score and PPI within 24 hours of admission were also recorded, and the patient's prognosis during ICU hospitalization was also recorded. The differences in clinical data between the patients of two groups with different prognosis were compared. Spearman correlation method was used to analyze the correlation between PPI and SOFA score. Binary multivariate Logistic regression analysis was used to screen independent risk factors for death during ICU hospitalization in sepsis patients with AKI. Receiver operator characteristic curve (ROC curve) was plotted to evaluate the predictive value of PPI for the short-term prognosis of patients with sepsis-induced AKI. RESULTS: A total of 102 patients with sepsis-induced AKI were enrolled, of which 70 patients in the survival group and 32 patients in the death group, with ICU mortality of 31.4. Compared with the survival group, SOFA score, HR, procalcitonin (PCT), serum creatinine (SCr), and NE dosage in the death group were significantly increased [SOFA score: 11.22±2.48 vs. 8.56±2.01, HR (bpm): 103.80±12.47 vs. 97.41±9.73, PCT (μg/L): 9.22 (5.24, 17.84) vs. 6.19 (3.86, 7.71), SCr (μmol/L): 163.2 (104.7, 307.9) vs. 125.5 (89.3, 221.0), Lac (mmol/L): 2.81 (1.95, 4.22) vs. 2.13 (1.74, 2.89), NE usage (μg×kg^-1×min^-1): 0.7 (0.4, 1.1) vs. 0.5 (0.2, 0.6), all P < 0.05], while PPI was significantly lower than that in survival group [0.83 (0.42, 1.55) vs. 1.70 (1.14, 2.20), P < 0.01]. Spearman correlation analysis showed that based on SOFA score, PPI was closely related to the severity of patients with sepsis-induced AKI (r = -0.328, P < 0.05). Binary multivariate Logistic regression analysis showed that PPI [odds ratio (OR) = 0.590, 95% confidence interval (95%CI) was 0.361-0.966, P = 0.002], SOFA score (OR = 1.406, 95%CI was 1.280-1.545, P < 0.001), PCT (OR = 2.061, 95%CI was 1.267-3.350, P = 0.006) were independent risk factors of the short-term prognosis of patients with sepsis-induced AKI. ROC curve analysis showed that the area under the ROC curve (AUC) of PPI for death during ICU hospitalization in patients with sepsis-induced AKI was 0.779 (95%CI was 0.686-0.855, P < 0.001), which superior to PCT (AUC = 0.677, 95%CI was 0.577-0.766, P = 0.004), and similar to SOFA score (AUC = 0.794, 95%CI was 0.703-0.868, P < 0.001). When the cut-off value of PPI was 0.72, the sensitivity was 50.0%, and the specificity was 97.1%. CONCLUSIONS PPI has a good predictive value for the short-term prognosis of patients with sepsis-induced AKI during ICU hospitalization.
Humans ; Heart Rate ; Retrospective Studies ; ROC Curve ; Sepsis/complications* ; Prognosis ; Procalcitonin ; Acute Kidney Injury/etiology* ; Intensive Care Units