Objective To explore the predictive model and its value of irAEs based on peripheral blood markers.Methods The baseline clinical data,laboratory tests,and irAEs follow-up results of 825 malignant tumor patients treated with PD-1/PD-L1 antibodies in the First Affiliated Hospital of Kunming Medical University were retrospectively collected from December 2020 to December 2023.The patients were divided into irAEs group and non-irAEs group according to the presence or absence of irAEs.The differences between and within groups were analyzed by t-test,rank-sum test,chi-square test and Fisher exact probability method.LASSO,Ridge and Elastic-net logistic regressions were used to screen the predictors and establish the risk prediction models for irAEs.Results 136 patients experienced 178 irAEs,of which endocrine toxicity accounted for 42.64％,hepatitis 35.29％,pneumonia 20.58％,grade≥G3 accounted for 19.07％,involving more than two organs accounted for 24.26％of the total number of irAEs.Univariate analysis showed that baseline CD4+T cell count,IL-6,IL-17,TSH,GLB and ALB were associated with irAEs.GLB,ALB,IL-17 and TSH were selected as the important risk factors by Ridge,LASSO and Elastic-Net logistic regression.The results showed that the AUC of the three algorithms were over 0.800.The AUC of internal validation set by LASSO-Logistic was 0.800(95％CI 0.739～0.862).The AUC of external validation set was 0.800(95％CI 0.739～0.861)and the DCA curve results indicated the highest net return for this predictive model.Conclusion GLB,ALB,IL-17 and TSH are independent predictors of irAEs,and the predictive model of irAEs based on them is effective.

[Abstract] Objective: To investigate the effects of CRISPR/Cas9 gene editing mediated PD-1 knockdown on the proliferation, phenotype, IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys. Methods: gRNA targeting PD-1 gene of Cynomolgus monkey was designed, and the corresponding plasmid was constructed and extracted. peripheral blood mononuclear cells (PBMCs) of Cynomolgus monkeys were isolated, and plasmid DNAs were added for transfection by using Lonza 4D electrorotometer. FACS analysis and fluorescence microscopy were used to detect transfection efficiency at 48 h after transfection. Genomic DNA of T cells was extracted for PCR amplification and T7E1 digestion identification. The proliferation of T cells was induced under the stimulation of human CD3 antibody and IL-2, and the cell growth curve was drawn. PI staining flow cytometry was used to detect cell cycle and the expression levels of CD4 and CD8, and ELISA was used to detect the secretion of IFN- γ and IL-2. Results: At 48 h after transfection, the cells with green fluorescent protein expression in experimental group were observed under fluorescence microscopy with a transfection efficiency of (21.6±3.2)%. T7E1 enzyme digestion results showed that the PCR product of genomic DNA of cells in experimental group showed 3 bands after digestion, including the target cleavage bands(243，197 bp). Compared with non-transfected cells, the cells in experimental group exhibited slow proliferation, delayed colony formation, with small volume and weak refraction; the number of T cells at G0/G1 phase of the experimental group was significantly increased (P<0.05), while the number of cells at G2/M phase was significantly reduced (P<0.05); and the secretion levels of IFN-γ and IL-2 in the cells of the experimental group increased significantly (both P<0.05). However, the difference in the expression levels of CD4 and CD8 was not statistically significant between the two groups (both P>0.05). Conclusion: PD-1 gene knockout can arrest T cells in Cynomolgus monkey at G0/G1 phase, thereby inhibiting its proliferation and increasing the secretion of IFN-γ and IL-2 in the meanwhile.

[Abstract] Objective: To establish a method for in vitro isolation and culture of T lymphocytes from peripheral blood of cynomolgus monkeys that induced by human CD3 antibody based on the foundation of protein homology of CD3 from human, cynomolgus monkey and porcine. Methods: The amino acid sequences of human, cynomolgus monkeys and porcine CD3 proteins were obtained from NCBI, and the sequence, homology and phylogenetic tree were analyzed by DNAMAN software. Western blotting was used to detect the expression of CD3 protein on T cell membranes from the three species. PBMCs of healthy cynomolgus were isolated and divided into three groups: group A was stimulated with anti-human CD3Ab alone, group B was stimulated with IL-2 alone, and group C was costimulated with human CD3Ab and IL-2. Cell morphology and growth status were observed under inverted microscope and the cell growth curve was plotted. Cell viability was detected by trypan blue staining and the expressions of CD3, CD4 and CD8 on T cell surface were detected by flow cytometry. Results: The homology of the amino acid sequence of human CD3 protein to cynomolgus monkey and porcine were 86.9% and 65.6% respectively. The expression levels of CD3 protein on cynomolgus and porcine T cell membrane were 79% and 17% contrast to human, respectively. Cells of group A did not proliferate. Proliferation, viability and CD3 expression [(93.8±3.6)% vs (70.3±4.7)%, P<0.01] in T cells of group C were significantly higher than those in group B. Growth curve of T cells in group C showed an S-shape, which is consistent with Logistic growth curve. T cells in group C exhibited high purity and expressed high level CD3; moreover, the CD8+T cell took a high proportion. Conclusion: The membrane of T lymphocytes from peripheral blood of cynomolgus can express CD3 protein that highly homological to human. Co-stimulation of human CD3Ab, IL-2 and 1% PHAcan induce the proliferation and differentiation of T lymphocytes of cynomolgus, and obtain T lymphocytes with good growth status, high proliferation ability and high purity.

Objective To explore the relationship between MspⅠpolymorphism of CYP1A1 gene and susceptibility to breast cancer in Yi nationality in Yunnan province. Methods The gene polymorphism of restriction enzyme of 3′-terminal of CYP1A1 was detected by PCR-RFLP in 60 healthy Yi women , 51 Yi women with breast cancer, 235 healthy Han women, and 250 Han women with breast cancer. Results The distribution frequency of CYP1A1-MspⅠgenotypes was significantly higher in Yi women with breast cancer (51.0%) than in the healthy Yi women (33.3%) (P < 0.05), an the allele C had a higher frequency in women with breast cancer than in healthy controls (P < 0.05). Significant differences of frequencies were found in genotypes TT, TC and CC between women with breast cancer and healthy controls (P < 0.05). The risks of TC and CC for breast cancer were 1.19 and 1.95 folds respectively to TT genotype. But as compared with Yi women , the distribution frequency of CYP1A1-MspⅠ genotypes did not differ between Han women with breast cancer and in the healthy control (P > 0.05), and there were no differences in three genotype frequencies (P > 0.05). Conclusions Gene polymorphism of CYP1A1 genotypes may be associated with the risk of breast cancer in Yi nationality but not in Han nationality in Yunnan. The mutation of CYP1A1 gene may increase the incidence of breast cancer in Yi nationality.

Objective To construct and identify the siRNA eukaryotic expression vector targeting gene CXC chemo-kine receptor-4 and explore its role in invasion process of breast cancer cells in vitro. Methods Two siRNAs were designed and synthesized according to the coding sequence of CXCR4 gene and cloned into eukaryotic expression plasmid pGE-1-U6/kna. The constructed CXCR4-siRNA expression vector was transfected into MDA-MB-231 cells by liposome. Western blot was used to evaluate the suppression of CXCR4 expression in different groups. The inva-sion and migration of MDA-MB-231 cells were evaluated by cell invasion assay in vitro. Results Enzyme digestion and DNA sequencing confirmed that the CXCR4-siRNA expression vector was successfully constructed. After trans-fection,the CXCR4-siRNA obviously suppressed the expression of CXCR4 compared with control groups and the ability of cell migration was decreased markedly. Conclusion CXCR4-siRNA expression vector can effectively sup-press CXCR4 expression in the breast cancer cells and decrease potential of cell invasion, which may provide a no-vel strategy for gene therapy of breast cancer metastasis.

Objective To construct and identify the siRNA eukaryotic expression vector targeting gene CXC chemokine receptor-4 and explore its role in invasion process of breast cancer cells in vitro.Methods Two siRNAs were designed and synthesized according to the coding sequence of CXCR4 gene and cloned into eukaryotic expression plasmid pGE-1-U6/kna.The constructed CXCR4-siRNA expression vector was transfected into MDA-MB-231 cells by liposome.Western blot was used to evaluate the suppression of CXCR4 expression in different groups.The invasion and migration of MDA-MB-231 cells were evaluated by cell invasion assay in vitro.Results Enzyme digestion and DNA sequencing confirmed that the CXCR4-siRNA expression vector was successfully constructed.After transfection,the CXCR4-siRNA obviously suppressed the expression of CXCR4 compared with control groups and the ability of cell migration was decreased markedly.Conclusion CXCR4-siRNA expression vector can effectively suppress CXCR4 expression in the breast cancer cells and decrease potential of cell invasion,which may provide a novel strategy for gene therapy of breast cancer metastasis.