Model Establishment Of Mouse Minimal Residual Leukemia (MRL) And The Study Of Its Biological Properties

In a hematologic complete remission after chemotherapy, a large portion of the leukemia cells remain out of sight. These cells are the components of an important clinical problem termed "minimal residual leukemia (MRL)". The remission induced by chemotherapy means that, after treatment with chemotherapy, the leukemic cells are less than 5% with bone marrow morphology examination. The leukemic cells in the body are approximatly 10¹² at the presentation of patient and may be as low as "zero"or as much as 10¹⁰ at complete remission depending on the diffierent individuals. Therefore, the residual leukemic cells, which are resistant to and escape from the chemotherapeutic agents, may be the root of occurrence and recurrence after complete remission. Thus, detectoin and elimination of MRL cells are critical. In our research, we chose the international standard domestic inbred DBA/2 mouse that its genetics background were close to human being, as the animal model. The L1210 leukemic cells were inoculated and then treated with chemotherapeutic agent—Cyclophosphamide (CTX), to establish the MRL model and to study the biological properties of MRL. Dose screening of MRL modelTo establish a cell line and a convenient and well-duplicated MRL model, moreover, to observe the relationship between cell number inoculating and mouse survival time, we chose the international standard domestic inbred DBA/2 mouse to inoculate the different numbers of L1210 leukemic cells. On the third day after 1×10⁶ leukemic cells per mouse inoculating, different doses of CTX were used for the chemotherapy. The results showed: 1. The inoculating of L1210 leukemic cells increased while the mouse survival time decreased; 2. The survival time increased following the increase of the doses of the CTX; 3. The survival time of the inoculated mouse treating with CTX at 125mg/kg was equal to the survival time of the mouse inoculated500 L. C. without treatment. So we chose the dose of 125mg/kg for inducing the model of MRL mouse.Model establishment of MRL and its biological propertiesTo study the biological properties successively, 1×10⁶ leukemic cells per mouse were inoculated and 125 mg/kg CTX was treated on the third day to induce the MRL model. Then, the MRL model mice were divided at random as control, MRLâ… ,MRLâ…¡and the MRLâ…¢group. The time points of examination of MRL I,MRLâ…¡and the MRLâ…¢groups were the 9,15 days and the day that the mice were at critical stage after chemotherapy. The objectives included: 1. white blood cell (WBC)counting on the next day after chemotherapy; 2. the pathological investigation of liver,spleen and bone borrow; 3. bone marrow smear examination; 4. experimental transplantation; 5. examinations of cell cycle, sub-group of T lymphocyte in spleen and H-2K^d expression by flow cytometry (FCM). The results indicated: 1. WBC in peripheral blood of MRL decreased to the lowest level on the 3-5 days after chemotherapy and then increased until the 9^th day that WBC reachde to the level before chemotherapy, no leukemic cells were found in peripheral blood, the survival time of the normal mice which inoculated the MRL cells were over 60 days; 2. The percentage of G₁ cycle was higher than the normal group; The percentage of G₂ cycle was lower than the normal group (P＜0.05); There was no significant difference between S cycle and that of normal group; In MRLâ…¢group, the percentage of G₁ and G₂ cycle were lower than the normal group and the S cycle was higher than the normal group; 3. The percentage of CD₄⁺, CD₈⁺ in MRLâ… , MRLâ…¡and MRLâ…¢group were lower than the normal group while the percentage of CD₄⁺/CD₈⁺ was significantly higher than the normal group; 4. There was no significant difference of the expression of H-2K^d between MRLâ… , MRLâ…¡and MRLâ…¢groups and the normal group (P＞0.05). Above all, these indicated that, a dynamic changes of cell cycle, sub-group of T lymphocyte in the spleen and the expression of H-2K^d were observed during the reccreence of MRL and the disease course.Study of the reccurence of MRLTo study the relationship between reccurence of MRL and the leukemia de novo which had no any treatment, we compared the mice in MRLâ…¢group and the mice with leukemia de novo at critical stage. Methods: 1. WBC counting; 2. Biopsy of liver,spleen and bone marrow for pathologic investigation ; 3. The weight of liver,spleen and tumor; 4. FCM detecting the cell CTX of bone marrow cells,sub-group of T lymphocyte in spleen and the expression of H-2K^d. Results showed: 1. The WBC counting and the percentage of Leukemic cells in peripheral blood of reccurence of MRL were higher than the leukemia de novo; 2. The weight percentage of spleen and tumor in the mice of MRL reccurence were higher than that of the leukemia de novo, but the weight percentage of liver was lower than that; 3. More leukemic cells were found in the liver,spleen and bone marrow of MRL reccurence; 4. The percentage of CD₄⁺, CD₈⁺ and the expression of H-2K^d of MRL reccurence was lower than that of the leukemia de novo; 5. The percentage of G₁ cycle of MRL reccurence was lower than the leukemia de novo;, the percentages of S,G₂ and G₂/G₁ were higher than the leukemia de novo. The results indicated that the body immunity of MRL reccurence was lower than the leukemia de novo; and the leukemic infiltration were more prominent.ConclusionsFrom the results, we draw the conclusions:1. A MRL model can be established by inoculating leukemic cells into mouse at a dose of 1×10⁶ cells per mouse and injecting CTX with a dose of 125mg/kg on the day 3.The number of residual leukemic cells in mouse are approximately 500.2. There were a dynamic changes of cell cycle, sub-group of T lymphocyte in the spleen and the expression of H-2K^d in MRL mouse at different disease courses and during the reccreence of leukemia.3.The body immunity of MRL reccurence mouse was lower than the leukemia de novo and the leukemic infiltration were more prominent.