OBJECTIVETo explore the the optimal condition for establishing immune deficiency mouse(BALB/c) model with CLL via subcutaneous inoculation of human chronic lymphocytic leukemia (CLL) cells at different inoculative locations and different cell concentrations.

METHODSFirstly, Two CLL cell lines (MEC-1-GFP and HG3-GFP)with the green fluorescent protein (GFP) were established by lentivirus system respectively, and then the MEC-1-GFP cells (5×10/ml) were inoculated into forelimb, hindlimb and abdomen to observe the tumorigenesis. Secondly, the MEC-1-GFP and HG3-GFP cells with same density (5×10/ml) were inoculated into forelimb to compare the time and rate of tumor formation. Thirdly, the MEC-1-GFP cells (1×10/ml) and HG3-GFP cells (5×10/ml) were inoculated into forelimb to compare the time and rate of tumor formation at different inoculative density. After observation for 5 weeks, the peripheral blood was collected and treated with EDTA and erythrocytolysin, then the of GFP positive cells were detected by flow cytomety. Meanwhile, the tumor-bearing mice were killed, and the tumors were isolated and cut into slices for histopathological examination.

RESULTSMEC-1-GFP and HG3-GFP cell lines were successfully established, and after inocutation of MEC-1-GFP cells with 5×10/ml the xenograt tumors were formated in forelimb, hindlimb and abdomen of mice, especially in the forelimb with a higher tumorigenic rate. In addition, the inoculation of same density of MEC-1-GFP and HG3-GFP cells (5×10/ml) also resulted in xenograft in forelimb, and the tumorigenic rate reached to 80% after 5 weeks. Moreover, the inocutation of MEC-1-GFP and HG3-GFP cells with 1×10and 5×10/ml respectively also effectively resulted to xenograft tumor in forelimb. The flow cytometry showed that there was no MEC-1-GFP and HG3-GFP cells in peripheral blood, while histopathological examination demonstrated CLL cell metastasis towards peritoneal cavity.

CONCLUSIONThe BALB/c nude mouse model is successfully established by subcutaneous injection of MEC-1-GFP and HG3-GFP cells. This model is a useful tool to explore the pathogenic mechanism.