Objective:To construct patient-derived xenograft (PDX) models from head and neck squamous cell carcinoma (HNSCC) patients, to explore the effect of immune reconstitution timing on the PDX modeling and immune microenvironment in humanized immune system mice (huHSC-NCG-hIL15), and to provide a reliable animal model for research on the mechanisms of head and neck squamous carcinoma and for studies on immune therapy drug interventions.Methods:This study enrolled 28 HNSCC patients (25 laryngeal carcinomas, 3 hypopharyngeal carcinomas). PDX models were established in Balb/c nude (nu) mice, NSG mice, and humanized immune system-reconstituted huHSC-NCG-hIL15 mice. Fresh HNSCC samples were transplanted into Balb/c nu and NSG mice to generate PDX models, with subsequent analysis of success-associated factors. One successfully established PDX tumor was subsequently implanted into humanized immune system-reconstituted huHSC-NCG-hIL15 mice. Tumor transplantation was performed at distinct immune reconstruction timepoints (2 vs. 7 weeks post-reconstitution), and tumor growth patterns were monitored. Flow cytometry and multiplex immunohistochemical staining were utilized to characterize immunological profiles in peripheral lymphoid organs and tumor microenvironments. Hematoxylin-eosin (HE) staining was employed to assess histomorphological concordance between primary patient tumors and PDX model tissues. Results:HNSCC PDX models were successfully established. NSG mice exhibited a higher and more stable tumor take rate compared to Balb/c nu mice (pilot study: 4/10 vs. 3/10 cases; mean take rate 60%-80% vs. 20%-60 %). The PDX success rate in NSG mice was 46.4% (13/28). In the huHSC-NCG-hIL15 mice model with immune reconstitution at 7 weeks, tumors grew significantly faster, and the PDX modeling process was shorter (617 mm3 at day 70 in 7-week cohort vs.280 mm3 in 2-week cohort). Flow cytometry analysis of the immune microenvironment showed that at 7 weeks of immune reconstitution, the proportions of B cells in the spleen and tumor tissues(2-week vs. 7-week: spleen 16.2% vs. 61.7%, tumor 26.0% vs. 38.8%) and myeloid cells in the spleen (2-week vs. 7-week: spleen 47.2% vs. 88.1 %) were significantly higher, while mice at 2 weeks post-reconstitution showed a higher proportion of T cells (2-week vs. 7-week: spleen 13.2% vs. 9.3%, tumor 4.8% vs. 2.5%). HE results demonstrated that the tumor tissues in PDX models maintained a high degree of morphological similarity to the primary tumors in both NSG and huHSC-NCG-hIL15 mouse models. Conclusion:The HNSCC PDX modeling protocol demonstrates operational feasibility and high reproducibility, establishing this model as a robust platform for mechanistic and immunotherapeutic studies.

Objective:To construct patient-derived xenograft (PDX) models from head and neck squamous cell carcinoma (HNSCC) patients, to explore the effect of immune reconstitution timing on the PDX modeling and immune microenvironment in humanized immune system mice (huHSC-NCG-hIL15), and to provide a reliable animal model for research on the mechanisms of head and neck squamous carcinoma and for studies on immune therapy drug interventions.Methods:This study enrolled 28 HNSCC patients (25 laryngeal carcinomas, 3 hypopharyngeal carcinomas). PDX models were established in Balb/c nude (nu) mice, NSG mice, and humanized immune system-reconstituted huHSC-NCG-hIL15 mice. Fresh HNSCC samples were transplanted into Balb/c nu and NSG mice to generate PDX models, with subsequent analysis of success-associated factors. One successfully established PDX tumor was subsequently implanted into humanized immune system-reconstituted huHSC-NCG-hIL15 mice. Tumor transplantation was performed at distinct immune reconstruction timepoints (2 vs. 7 weeks post-reconstitution), and tumor growth patterns were monitored. Flow cytometry and multiplex immunohistochemical staining were utilized to characterize immunological profiles in peripheral lymphoid organs and tumor microenvironments. Hematoxylin-eosin (HE) staining was employed to assess histomorphological concordance between primary patient tumors and PDX model tissues. Results:HNSCC PDX models were successfully established. NSG mice exhibited a higher and more stable tumor take rate compared to Balb/c nu mice (pilot study: 4/10 vs. 3/10 cases; mean take rate 60%-80% vs. 20%-60 %). The PDX success rate in NSG mice was 46.4% (13/28). In the huHSC-NCG-hIL15 mice model with immune reconstitution at 7 weeks, tumors grew significantly faster, and the PDX modeling process was shorter (617 mm3 at day 70 in 7-week cohort vs.280 mm3 in 2-week cohort). Flow cytometry analysis of the immune microenvironment showed that at 7 weeks of immune reconstitution, the proportions of B cells in the spleen and tumor tissues(2-week vs. 7-week: spleen 16.2% vs. 61.7%, tumor 26.0% vs. 38.8%) and myeloid cells in the spleen (2-week vs. 7-week: spleen 47.2% vs. 88.1 %) were significantly higher, while mice at 2 weeks post-reconstitution showed a higher proportion of T cells (2-week vs. 7-week: spleen 13.2% vs. 9.3%, tumor 4.8% vs. 2.5%). HE results demonstrated that the tumor tissues in PDX models maintained a high degree of morphological similarity to the primary tumors in both NSG and huHSC-NCG-hIL15 mouse models. Conclusion:The HNSCC PDX modeling protocol demonstrates operational feasibility and high reproducibility, establishing this model as a robust platform for mechanistic and immunotherapeutic studies.

Objective:To speculate the effect of birth spacing on the pelvic floor type Ⅰ and Ⅱ fiber muscle strength of postpartum women with parities of two in different delivery modes.Methods:Totally 2 361 parturients who were investigated in Xuzhou Central Hospital from June 2016 to December 2020 were included in the questionnaire, clinical examination and pelvic floor surface electromyography assessment. According to the interval years between two parities and the pelvic floor typeⅠ and Ⅱ fiber muscle strength under different modes of delivery, curve fitting function equation was performed using curve regression method. The accuracy of the equation was verified by the receiver operating characteristic curve and the maximum area under the curve, and calculating the relative error rate.Results:A total of 2 357 parturients were included in the study and were divided into 4 groups based on delivery modes, women with both normal vaginal delivery were assigned to group A (589 cases); women with a first vaginal delivery and a second cesarean section were assigned to group B (480 cases); women with both cesarean deliveries were assigned to group C (1 273 cases); women with a first cesarean section and a second vaginal delivery were assigned to group D (15 cases). All of the curve fitting results were quadratic curves, and the appropriate interval years were selected when the muscle strength of type Ⅰ muscle fibers was>35 μV and that of type Ⅱ muscle fibers was>40 μV: 6-8 years in the group A, 5-10 years in the group B, and 1-11 years in the group C. The peak values of the quadratic curve were as follows: 7-8 years in the group A, 7-8 years in the group B, and 6 years in the group C. The maximum area under the curve of the function equations were all>0.6 (all P<0.05), the average relative error rate was 4.909%. Conclusions:The pelvic floor function of postpartum women with parities of two increases firstly and then decreases over time, showing a quadratic curve shape. In order to protect the pelvic floor function, the appropriate interval of birth spacing is 6-8 years.

Objective An analytical approach has been developed for determination of sildenafil blood with high performance liquid chromatography.Methods Sildenafil was extracted by solid phase extraction cartridges from blood and was separated by resered-phase gradient chromatography with DAD detection at 230nm.The analytical column was Nova-pak C18(150×3.9mm),5.0μm and the guard column was Phenomenex C18(ODS,4.0×3.0mm,Octadecyl).The mobile phase Was gradient mixture of acetonitrile(A) and 0.25% acetonitrile aqueous solution including 0.06% trifluoroacetic acid and 0.06% triethylamine (B).Results Linearity Was checked over the concentration range 1.5～80.0μg/mL.Limits of detection was 5.0ng.Average recovery was 82.45%.Conclusion This method issimple.exact and rapid.