ObjectiveTo humanize the variable region of the IL9-2G1 rabbit monoclonal antibody （IL9 2G1 mAb） and to evaluate its biological activity. MethodsThe humanized sequences of IL9-2G1 mAb were designed. The heavy chain variable region （VH） and light chain variable region （VL） of the antibody were humanized and cloned into vector backbones containing the constant region frameworks of human IgG1 and Ig Kappa subtypes. Protein purification was carried out using the Chinese hamster ovary cell （CHO） expression system. Enzyme-linked immunosorbent assay （ELISA） was used to qualitatively analyze the antigen-binding activity of the antibodies. After screening the optimal humanized antibody candidate， surface plasmon resonance （SPR） was performed to detect molecular affinity. Western blot was used to examine the effects of the rIL-9 group and the rIL-9 + hIL-9 mAb group on the phosphorylation of Janus kinase 1/signal transducer and activator of transcription 3 （JAK1/STAT3）， while ELISA was employed to assess the impact of the Th9 group and the Th9 + hIL-9 mAb group on interleukin-9 （IL-9） secretion. ResultsPurification and ELISA results showed that the heavy chain OH and light chain FL exhibited good compatibility and expression performance. SPR results indicated that the equilibrium dissociation constant （KD） of M501042-OHFL against the analyte M30903 was 1.33×10^-7 M， which was superior to the parental 2G1 antibody. The Western blot results showed that the levels of phosphorylated JAK1 （p-JAK1） and phosphorylated STAT3 （p-STAT3） were significantly decreased in the rIL-9 + hIL-9 mAb group compared with the rIL-9 group （P<0.01）. The ELISA results indicated that the secretion of IL-9 was significantly reduced in the Th9 + hIL-9 mAb group compared with the Th9 group （P<0.01）. ConclusionHumanization modification is performed on the variable region of IL9-2G1 rabbit monoclonal antibody， and the humanized antibody hIL-9 mAb OHFL with high affinity and strong binding stability is successfully constructed， which provides an experimental basis for subsequent research.

ObjectiveTo construct an interleukin-9 （Il9） gene knockout mouse model on a C57BL/6N background， establish a reliable genotyping method， and provide a standardized animal model for investigating Il9-related immune mechanisms and diseases. MethodsIl9 knockout mouse strain （C57BL/6N-Il9^em1Cya） was generated using conventional gene knockout technology. The method for DNA extraction from mouse tails was optimized to ensure template quality. Two sets of specific primers （F1/R1 and F1/R2） were designed to amplify target fragments by PCR， and mouse genotypes were determined by agarose gel electrophoresis. Specifically， primer pair F1/R1 targeted the knockout sequence with an expected product of 465 bp， while F1/R2 targeted the wild-type sequence with an expected product of 703 bp. Subsequently， Western blot was performed to verify the knockout efficiency of Il9 in primary immune cells and key organs of the offspring mice， and flow cytometry was used to assess the effects of Il9 deficiency on the function of the immune system in major immune cell populations. ResultsIl9 knockout mice were successfully bred and genotyped. PCR identification showed that homozygous mice （-/-） exhibited only a 465 bp band， wild-type mice （+/+） displayed only a 703 bp band， and heterozygous mice （+/-） showed both bands simultaneously. Meanwhile， Western blot analysis revealed significantly decreased IL-9 expression in various organs of the offspring mice （P<0.001）. Flow cytometric results demonstrated that Il9 deficiency exerted no obvious effects on the proportions of peripheral blood lymphocytes， splenic lymphocytes， and peritoneal macrophages. ConclusionAn Il9 gene knockout mouse model on a C57BL/6N background is successfully constructed. The established PCR genotyping system is highly efficient and accurate， which can effectively distinguish mice of different genotypes. This model provides a stable and reliable experimental basis for subsequent functional studies of Il9 and exploration of the mechanisms underlying Il9-related diseases.

Objective : To construct a humanized mouse model of the interleukin-9 receptor(IL-9R) coding DNA sequence(CDS) gene and to verify the genotype and IL-9R expression in mice. Methods : The CRISPR/Cas9 genome editing technology was used to replace the exon 2-7 fragment of the il-9r gene in mouse embryonic stem cells with the corresponding human IL-9R sequence. After verifying the completion of the gene fragment replacement, tetraploid embryos were constructed and microinjected back into the oviducts of surrogate mice. Through surrogacy by female mice, homozygous humanized mice were obtained. DNA was extracted from the homozygous humanized mice IL-9R CDS gene, and their genotypes were identified by agarose gel electrophoresis after PCR amplification. Western blot was used to detect the expression of IL-9R in the spleen and thymus of homozygous humanized mice with either wild-type(WT) or IL-9R gene humanization. Results : Gel electrophoresis after PCR amplification showed that mice with only a 1 805 bp band amplified using WT primers were wild-type, while mice with 2 553 bp and 2 340 bp bands amplified using 5KI and 3KI primers, respectively, were homozygous humanized mice with IL-9R CDS gene. Western blot results indicated that the tissues of homozygous humanized mice model with IL-9R CDS gene expressed IL-9R significantly. Conclusion The humanized mouse model with IL-9R CDS gene has been successfully constructed and characterized.

Objective:To explore the perioperative safety and postoperative short-and long-term efficacy of stomach-partitioning gastrojejunostomy (SPGJ) with distal selective vagotomy (DSV) for treating benign gastric outlet obstruction (GOO).Methods:The clinical data of 26 benign GOO patients treated by Beijing Friendship Hospital, Capital Medical University from January 2019 to July 2023 were retrospectively analyzed. There were 20 males (76.9%) and 6 females (23.1%), aged from 25 to 75 years, with an average age of (55.8±13.6) years, and an average body mass index (BMI) of (20.1±3.4) kg/m 2. There were 12 cases in SPGJ-DSV group and 14 cases in SPGJ group. The main outcome was the gastrointestinal quality of life index (GIQLI) 1 year after surgery in both groups. Independent sample t-test was used to test the difference between the continuous variables with normal distribution. The comparison between groups of non-normal distribution continuous variables was tested by Mann-Whitney U test. Counting data were compared using Chi-square test or Fisher exact test. Results:There were no significant differences between the two groups in terms of operative time ( P=0.071), intraoperative blood loss ( P=0.422), time to pass gas ( P=0.538), time to liquid intake ( P=0.386), postoperative hospitalization ( P=0.431), complications within 30 days after surgery ( P=0.999), and postoperative GOOSS grade ( P=0.483). Among them, postoperative DGE occurred in one patient in each of the two groups, both of which were grade A. In the follow-up results, compared with the SPGJ group, SPGJ-DSV group had a significant advantage in GIQLI score, and the difference was statistically significant ( P=0.028). The incidence of gastric ulcer, reflux esophagitis, bile reflux and gastritis in SPGJ-DSV group was 8.3%, 8.3%, 8.3% and 58.3%, while that in SPGJ group was 35.7%, 21.4%, 21.4% and 57.1%, respectively, but there was no statistical significance between groups. Conclusion:In the treatment of benign GOO patients, SPGJ with DSV did not significantly increase the difficulty of laparoscopic procedures, operative time and intraoperative blood loss. Moreover, it showed a significant advantage in gastrointestinal quality of life 1 year after surgery. In addition, the incidence rates of gastric ulcers and reflux esophagitis were lower in the SPGJ-DSV group 1 year after surgery, but further confirmation is needed in large sample studies.

Objective:To investigate the high-risk factors affecting the prognosis of patients with pT 1-2N 1M 0 after mastectomy, establish a nomogram prediction model, perform risk stratification, and screen the radiotherapy benefit populations. Methods:Clinical data of 936 patients with pT 1-2N 1M 0 breast cancer undergoing mastectomy in the Fourth Hospital of Hebei Medical University from January 2010 to December 2016 were retrospectively analyzed and 908 cases had complete follow-up data. They were divided into the radiotherapy (RT) group ( n=583) and non radiotherapy (NRT) group ( n=325) according to the radiotherapy. After propensity score matching (PSM) was performed 1 vs. 1, 298 cases were assigned into the RT group and 298 in the NRT group. Overall survival (OS) and disease-free survival (DFS) were compared between two groups using log-rank test. Nomogram prediction model was established, the survival differences were compared among different risk groups, and the radiotherapy benefit populations were screened. Results:Univariate analysis showed that the 5- and 8-year OS and DFS in the RT group were significantly better than those in the NRT group (both P<0.001). Multivariate analysis showed that age, tumor quadrant, number of lymph node metastases, T staging, and Ki-67 level were the independent prognostic factors for OS. Age, tumor quadrant, and T staging were the independent prognostic factors for DFS. The OS nomogram analysis showed that the OS of patients in the high-risk group was significantly improved by post-mastectomy radiotherapy (PMRT) ( P=0.001), while PMRT did not show an advantage in the low- and medium-risk groups ( P=0.057, P=0.099). The DFS nomogram analysis showed that DFS was significantly improved by PMRT in patients in the medium- and high-risk groups ( P=0.036, P=0.001), whereas the benefits from PMRT were not significant in the low-risk group ( P=0.475). Conclusions:For patients with pT 1-2N 1M 0 breast cancer after mastectomy, age ≤ 40 years, tumor located in the inner quadrant or central area, T 2 staging, 2-3 lymph node metastases, Ki-67>30% are the high-risk factors affecting clinical prognosis. The nomogram prediction model can screen the populations that can benefit from PMRT, providing reference for clinical decision-making.