RÉSUMÉ
ObjectiveThrough improving the potential of vascular endothelial growth factor receptor (VEGFR)-humanized mouse model (hKDR+/+) with C57BL/6N background to allow the growth of different mouse tumor cell lines, to establish novel tumor-bearing mouse models which can support in vivo tumorigenesis of different mouse tumor cell lines and be used to evaluate drugs targeting VEGFR.MethodsFirstly, a method to evaluate the in vivo activity of antibody targeting VEGFR based on the hKDR+/+ humanized mouse model was established. Recombinant activating gene 1 (Rag1) knockout mice (Rag1-/-) were established using CRISPR/Cas9 technology. Then these Rag1-/- mice were crossed with hKDR+/+ mice to get a double gene modified homozygous hKDR+/+/Rag1-/- mouse model by screening. Finally, tumor cell lines derived from different mouse strains were tested on the double gene-modified mouse model to compare the differences in tumor growth. ResultsAntibodies designed for VEGFR showed significant anti-tumor activity in hKDR+/+ mice, which significantly reduced tumor volume and weight compared with the PBS group (P<0.01, P<0.05). The number of B cells and T cells in the peripheral blood of Rag1-/- mice and hKDR+/+/Rag1-/- mice decreased (P<0.05, P<0.001). Tumors were observed in hKDR+/+/Rag1-/-, Rag1-/-, wild-type, and hKDR+/+ mice after 7 d of inoculation of MC38 cells derived from C57BL/6 mice. Tumors were only observed in groups of hKDR+/+/Rag1-/- and Rag1-/- mice, but not in the wild-type and hKDR+/+ mice after 10 d of inoculation with CT26 cells derived from BALB/c mice. After 3 weeks of inoculation, the tumor volume of hKDR+/+/Rag1-/- mice was significantly larger than that of Rag1-/- mice (P<0.01). ConclusionRag1 knockout mice were obtained and a novel hKDR+/+/Rag1-/- double genes modified mouse model was further screened. The tumor cell lines from different mouse strain origins were more prone to growth in mice with Rag1 gene deficiency. The results suggest that the reduced immune response of hKDR+/+ humanized mice will improve the capacity of supporting the growth of mouse tumor lines in the model. As a result, more tumor-bearing mouse models may be constructed for the evaluation of drugs targeting VEGFR in this way.
RÉSUMÉ
@#[Abstract] Objective: To explore the application value of human IL-15 transgenic NCG mice (NCG-hIL-15 mice) in preclinical evaluation of chimeric antigen receptor modified NK (CAR-NK) cell therapy for tumor treatment. Methods: qPCR and WB were performed to detect the expression of human IL-15 in the bone marrow and main organs (spleen, liver, lung, kidney and pancreas) of transgenic mice. After being transfused with human PBMC-derived NK (PB-NK) cells, the NCG-hIL-15 mice and control NCG mice were continuously monitored for the in vivo amplification of NK cells and the changes in body weight and survival time. Flow cytometry was used to detect the differential expressions of activated receptors and inhibitory receptors in amplified NK cells. WB was used to detect the expressions of perforin and granzyme-B. NCG-hIL-15 mice or NCG mice bearing MIAPaca-2 cell transplanted tumor were treated with anti-MUC1-CAR-NK cell reinfusion; then, the CAR-NK cell survival in different groups of mice was detected by Flow cytometry, and the survival time of tumor bearing mice was recorded and tumor growth was detected by in vivo imaging. Results: The results indicated that PB-NK cells could proliferate stably within 10 weeks in NCG-hIL-15 mice without obvious graft versus host diseases (GVHD) during the observation period. The in vivo-expanded human NK cells maintained the original expression patterns of various surface molecules, including KARs and KIRs. Compared with the NK cells in NCG mice, the NK cells in NCG-hIL-15 mice contained significantly higher amounts of granzyme-B and perforin (all P<0.05). CAR-NK cells showed significantly increased survival rate and stronger tumor-inhibitory effect in NCG-hIL-15 mice as compared with those in control NCG mice, resulting in significantly prolonged survival in NCG-hIL-15 mice (all P<0.01). Conclusion: NCG-hIL-15 mouse model has potential application value in preclinical trial and biological evaluation of NK cell-based immunotherapy.
RÉSUMÉ
Objective • To establish a rapid method to evaluate the activity of agonistic antibody using OX40 (tumor necrosis factor receptor superfamily member 4)/FcγR (Fcγ receptor)-humanized mice. Methods • Bone marrow cells from OX40-humanized mice and FcγR-humanized mice were collected and mixed with equal ratio. Then the mixed bone marrow cells were administrated into irradiated wild-type mice through the tail veins. The reconstruction efficiency of the immune system was confirmed by detecting the expression of hOX40 and hFcγR in the immune cells of chimera mice. After the chimera mice were generated successfully, they were used to evaluate the immunostimulatory activity of anti-hOX40 antibodies to CD4+ or CD8+ T cells. The results of flow cytometry were statistically analyzed. The unpaired t-test was used to compare the means between the two groups, and oneway ANOVA was used to compare the means between multiple groups. Results • Flow cytometry analysis showed that wild-type recipient mice were efficiently reconstituted with hFcγR expressing cells and hOX40 expressing cells to generate OX40/FcγR-humanized bone marrow chimera mice. In these mice, B cells and myeloid cells expressed hFcγRs (P<0.05), and T cells expressed hOX40 upon in vitro stimulation (P<0.05). When these mice were used to evaluate the immunostimulatory activity of anti-hOX40 antibody, significant expressions of IFN-γ and hOX40 were observed (P<0.05). Conclusion • OX40/FcγR-humanized bone marrow chimera mice are generated based on hFcγR expressing cells and hOX40 expressing cells, suggesting a rapid method to build a mouse model with both hFcγR and hOX40 expression. These mice are suitable for evaluating the immunostimulatory activity of agonistic human anti-hOX40 antibodies.
RÉSUMÉ
Objective: To construct a humanized mouse models with human immune system and human tumor growth, and to provide the basis for human tumor immunological research and evaluation on the efficacy of tumor immunotherapeutic durgs. Methods: The humanized mice with human immune system reconstruction were established by intravenous injection of human CD34 fetal liver cells combined with implantation of human fetal thymic tissue under the renal capsule of NOD/SCID IL 2rg -/- after sub-lethal total body irradiation. The peripheral blood was collected, and the immune cell reconstitution was monitored every 3 weeks from the 3th week after humanization, including the ratios of CD45+, CD33, CD3, CD3 CD4+, CD3+ CD8, and CD56 + cells. The human A375 melanoma cells were subcutaneously inoculated at the 15th week after humanization, and the tumor volume was measured every 5 d.. Six weeks after tumor inoculation, the mice were sacrificed and the composition of human immune cells as well as their phenotypes in peripheral blood, bone marrow, lymph node, spleen and tumor tissue were measured by flow cytometry. The composition of human immune cells as well as their phenotypes in tumor tissue were measured by immunohistochemistry. Results: At the 15th week after humanization, the ratio of human T cells was (33. 53 ± 8. 57) % and the ratio of human B cells was (43. 33 ± 10. 05) % in the peripheral blood of the humanized mice. The human melanoma grew slowly during the first 3 weeks, and the human tumor sizes expanded rapidly and reached (1 564 ± 334. 3) mm3 at the 6th week after tumor inoculation. The immunohistochemical and flow cytometry analysis results showed that a large number of human T cells, which mainly composed by CD8+ T subsets, infiltrated in the human melanoma. The ratio of CD8/CD4 in tumor tissue was significantly higher than those in the peripheral blood, bone marrows, lymph nodes and spleen tissue (P< 0. 01). The levels of PD-1 molecules expressed in tumor-infiltrated human CD4 T and CD8 T cells were higher than those in the peripheral blood and immune organs. Conclusion: A humanized mouse models with human immune system and allogeneic human melanoma is successfully established. The high levels of human immune cells, including human T cells and B cells, can be detected in the peripheral blood, lymph organs, and human tumor tissue in this mouse model.
RÉSUMÉ
BACKGROUND: Humanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function. METHODS: Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34+ hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS: Most of the NSG mice tolerated the regimen well. The percentage of hCD45+ and CD19+ cells rose significantly in a time-dependent manner. The median percentage of hCD45+cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percentage of hCD45+ cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19+ cells in BM at weeks 8 and 12 was 21.5% and 39%, respectively (P=0.04). Similarly, the median percentage of hCD19+ cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19+ B cells in PB was 23% at week 12. At week 8, hCD3+ T cells were barely detectable, while hCD7+ was detected in the BM and spleen. The percentage of hCD3+ T cells was 2‒3% at week 12 in the BM, spleen, and PB of humanized NSG mice. CONCLUSION: We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45+ cells than earlier studies without any significant toxicity. And human CD45+ cell engraftment at week 8 was comparable to that of week 12.
Sujet(s)
Animaux , Humains , Souris , Lymphocytes B , Moelle osseuse , Busulfan , Sang foetal , Cellules souches hématopoïétiques , Rate , Lymphocytes T , Cordon ombilicalRÉSUMÉ
Liver diseases post great threats to human public health globally.Lacking of appropriate small animal models largely impeded the translational studies on human liver diseases, especially on viral hepatitis and related cirrhosis, hepatocellular carcinoma, etc.By human hepatocyte transplantation, the liver-humanized mice have significantly contribu-ted to the researches of human liver diseases.This review summarizes the currently widely used and representative human-ized mouse models, including uPA, FAH, TK-NOG, AFC8 mice and their applications in studies of human liver diseases.
RÉSUMÉ
OBJECTIVE: To discuss the application of humanized mice model in drug metabolism and toxicity. METHODS: By sorting foreign scientific literature, the history of the development of humanized mice model and its application in drug metabolizing enzymes and toxicity studies were reviewed and summarized to provide support for early drug development. RESULTS and CONCLUSION: The humanized mice model could reduce interspecies differences and has been increasingly used in drug metabolism and safety evaluation in foreign countries. Transgenic mice can be applied to studying the metabolism of drugs metabolized by certain enzyme. In the chimeric mice body, the disposal of drugs could be more systematic and comprehensive mirrored, predicting this model has enormous potential in drug discovery and development.