The establishment of a mouse tumor model of human diffuse large B-cell lymphoma can be applied to (1) in-depth study of the pathogenesis of lymphoma; (2) evaluation of therapeutic drugs; and (3) study of the conditions and tumor characteristics for the establishment of a mouse model of human diffuse large B-cell lymphoma.
Operation method
subcutaneous implantation
Principle
The subcutaneous inoculation of 107 cells in SCID mice successfully established a diffuse large B-cell lymphoma (DLBCL) transplantation tumor model in humans, with a tumorigenicity rate of 70%, and histological manifestations of the tumors were similar to those of human DLBCL.
Materials and Instruments
SPF grade SCID mice Move I. Cell culture Caveat After culturing SUDHL-4 cells under different conditions, 10% was found to be a relatively suitable serum concentration. It has been reported in the literature that Iscove' sMEM is recommended for the in vitro culture of human DLBCL cell line, and this experiment proved that the use of RPMI1640 cultured with 10% FBS resulted in good cell growth, moderate passaging speed, and cell viability of 4-5 generations, with a high inoculation success rate, and is suitable for other experiments, such as flow assay. Common Problems Although DLBCL is classified as an independent disease by WHO, it shows obvious heterogeneity in epidemiology, clinical features, histomorphology, molecular genetics and immunophenotype, response to treatment and prognosis, etc. The immunophenotype of DLBCL is very complex, and has a close relationship with prognosis. The study of protein expression associated with tumor cell development is not only of great significance in the diagnostic typing, prognostic evaluation and clinical treatment of DLBCL, but also helps to elucidate the pathogenesis of lymphomas and the development of specific targeted therapeutic agents. Although a few studies on the human-derived DLBCL cell line SUDHL-4 have been reported in the literature, the present experiments investigated the biological characteristics and expression of relevant markers of SUDHL-4 cells in vitro culture, and established a stable animal model of human DLBCL based on SUDHL-4 cells. For more product details, please visit Aladdin Scientific website.
RPMI 1640 culture medium Penicillin Glutamine Streptomycin CD19 APC Antibody CD20 PE Antibody CD24 PE Antibody Goat Anti-Rabbit IgG FITC
Flow Cytometer Centrifuge Water Bath Culture Flasks Refrigerator Syringes Tubes Sealing Film Micropipettes
1. SUDHL-4 is a human GCB-like DLBCL cell line. The initial density of 2. 5x105 /ml cells was placed in T25 cell culture flasks containing 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomycin and 30 ug/ml glutamine in RPMI1640 culture medium, and cultured in an incubator with saturated humidity at 37℃, 5% CO2. 2.
2. After the first passaging for 2-3 d, transfer to T75 cell culture flasks, and then to T150 cell culture flasks as appropriate according to the growth of the cells.Preparation of flow cytometry samples
1. Take SUDHL-4 cells in logarithmic growth phase, rinse them with serum-free PBS for 2 times, and then make cell suspension (1 x107/ml ) in PBS/2% FBS. 2.
2. Add the appropriate antibody according to the concentration recommended in the instruction manual and incubate at 4℃ for 15 min, avoiding light; if intracellular staining is required, fix the cells with 1% paraformaldehyde, then treat them with a membrane-breaking agent (0.25% Saponin) and add the antibody for staining at the same time, and incubate at 4℃ for overnight, avoiding light.
3. Cells were rinsed with PBS/2% FBS for 1 time and suspended in 200 ul of PBS for measurement. 4.
4. 3x105 cells were measured in each sample by flow cytometry and the results were analyzed by FlowJo software.Laboratory Animals
1. SPF grade SCID mice, female, 5 weeks old, weighing 16-20 g, were randomly grouped and kept in an SPF grade mouse house with constant temperature (20-26℃) and humidity (50%-56%). 2.
2. The mice were placed in laminar flow racks with lids, the air was filtered by medium efficiency filtration, fed with standard pellet feed, and all items in contact with the mice were sterilized beforehand.Cell inoculation
1. SUDHL-4 cells in logarithmic growth phase were taken, rinsed with serum-free PBS for 2 times and suspended in serum-free PBS. 2.
2. 10 mice in the experimental group were subcutaneously inoculated with 0.1 ml of cell suspension containing 107 cells on one side of the ribs; 3 mice in each of the 2 tumorigenic control groups were subcutaneously inoculated with 0.1 ml of cell suspension containing 5 × 106 cells and 1 x 106 cells on the right side of the ribs; and 0.1 ml of cell suspension containing 5 × 106 cells and 1 x 106 cells were subcutaneously inoculated into the right rib cage in the normal control group (10 mice in each group). PBS.
3. Mice were not treated before inoculation.V. Indicator Tests of Hormonal Mice
1. Observe the general condition, tumor formation and tumor growth of mice every day during the experiment period.
2. Body weight, tumor length, diameter and height are measured daily and tumor volume is calculated (calculation method: π/6 x length x width x height); when the tumor reaches 1,200 mm3, it will be considered as the end point of humane. 3.
3. After the mice were anesthetized and killed by pulling the neck, the tumor formation was first observed on all parts of the body surface; then the animals were dissected and the metastasis of internal organs and lymph nodes was observed.
4. Remove the tumors, fix them in 10% neutral formalin solution, and then take paraffin sections and observe them with HE staining.Statistical processing
The experimental data were expressed as mean ± standard deviation (X ± SD) and processed by SPSS11.5 statistical software package, and one-way analysis of variance (ANOVA) was used for comparing the means of the samples in each group.