| Bone marrow (BM) suppression is the most serious side-effect caused by chemotherapy. Chemotherapy leads to decrease of blood and BM cell numbers. In response to BM damage of chemotherapy, BM regeneration is initiated. Within 3-4 weeks, the damaged BM is repaired through hematopoiesis. We propose that the expression of BM regeneration regulatory genes (BM-TRRGs) determines the speed of BM regeneration after chemotherapy insults. We used 5-fluorouracil (5-Fu) mouse chemotherapy model to identify BM-TRRGs. After 5-Fu injection, like in humans, the mouse BM is severely damaged and regeneration is initiated. Within 2-3 weeks, the damaged BM is fully recovered through active hematopoiesis.Using gene expression mirochips, we analyzed the expression patterns of 39000 genes using total mRNA obtained from mouse BM after 5-Fu treatment. The expression of CXCL9, also named monokine induced by IFN-γ(Mig) , and its receptor CXCR3 were identified with expression patterns fit with our hypothesis. ELISA analysis of mouse serum after 5-Fu treatment showed that Mig protein level in serum was increased by 10-fold at day 7 after 5-Fu injection, which is consistent with the gene expression data. The gene and protein expression studies indicate that CXCL9/Mig may be involved in regulation of BM regeneration.The function of Mig in regulation of BM regeneration was examined by 1) transient overexpression of the protein through plasmid muscle electroporation in normal mice and 5-Fu treated mice; 2) systemically use of the recombinant murine Mig (rMuMig) in normal and 5-Fu treated mice; 3) anti-Mig blocking antibody injection to block endogenous Mig in 5-Fu treated mice. The effects of BM regeneration were analyzed by measuring the peripheral blood and BM cell counts, BM tissue section, cell cycle analysis, hematopoietic colony assay, and mouse survival rate after 5-Fu.We constructed the eukaryote expressing plasmid pcDNA-Mig, which contain the cDNA sequence of the mouse Mig gene. The plasmid was introduced to the mouse tibia muscle by electroporation. The expression of the mouse Mig in sera after electroporation was measured by ELISA method. We found that Mig expression lasted for 7 days. During the entire experiment, the mice with pcDNA-Mig showed no significant difference in peripheral white blood compared to the control pcDNA3.1 mice. But there were significant decrease in bone marrow cell counts in the pcDNA-Mig mice. HE stained BM slices showed that at day 5 and 10 more sinus appeared in pcDNA-Mig group mice, with the average diameter larger than normal.To study the BM protection role of Mig, we injected 5-Fu at 200 mg/kg 7 days after plasmid electroporation into normal mice. Note the day of 5-Fu injection as day 0, mice were sacrificed at day 0, 3, 7, 11, 14 to count the peripheral white blood and bone marrow cells, as well as tissue section of the femurs. The cell counts showed that at day 11 pcDNA-Mig group was higher than pcDNA3.1 group in both white blood and bone marrow cells. Histology showed that at day 11 pcDNA-Mig group had more hematopoietic cells than the control group. Hematological data analysis suggests that Mig overexpression before 5-Fu treatment accelerates the BM recovery after 5-Fu treatement, which indicate that Mig may function as BM protector in chemotherapy.To further study the role of Mig in protection of BM from chemotherapy damage, we produced the recombinant mouse Mig protein (rMuMig). The murine Mig is a low molecular protein of 12 kDa with no glycosylation. The prokaryotic expression system was chosen to make the protein using the pET expression system. The vector was pET28a and the host strain was E. coli BL21. IPTG was used to induce the expression of recombinant protein. We constructed the prokaryotic expressing plasmid pET28-m, which contained the coding sequence of mature murine Mig protein. Then we optimized the expression of the rMuMig and decided that when the bacteria culture reached OD600=0.8 to start the induction by 1mM IPTG. The collected bacteria went through sonication, Urea denaturation, dilution before the final cation-exchange chromatography by S Sepharose. When the conductivity reached 75mS/cm, rMuMig was eluted from the column. SDS-PAGE showed the purity was above 99%. The chemotaxis assay demonstrated that the ED50 of our purified rMuMig was 30ng/ml. Then we immunized rat to get anti-Mig polyclonal antibody serum.We first examined the role of rMuMig in normal mice. We tried 3 doses of rMuMig - 0.15, 1.5, and 15μg/kg. PBS of equal volume was injected in control group. All mice of the 4 groups were injected consecutively for 5 days and once a day. The date starting the injection was noted as day 0 and data of the mice were collected at day 0, 5, 10, 15. The difference between the rMuMig groups and the control group on peripheral white blood cells was only observed on day 5 between the 15μg/kg group and the control. For the bone marrow cells, the two groups of 1.5 and 15μg/kg had lower cell counts at day 5 and recovered at day 10 and 15. Histology of 15μg/kg group showed more sinus and larger diameters of vessels than that of the control group. The results were similar to that of mice after plasmid transfection.We further studied the effect of rMuMig on the 5-Fu treated mice. Two ways of administration were taken: administration of rMuMig before and after the 5-Fu treatment. PBS was injected as control. After treated with 250mg/kg 5-Fu the mice in the control group had a survival rate of only 10%, while administration of rMuMig before 5-Fu could protect more than 50% of mice from dying. But if rMuMig was administrated after 5-Fu treatment, no mice survived. The peripheral white blood and bone marrow cells were counted at day 0, 3, 7, 11, and 14 after 5-Fu treatment. At ay 7, 11 and 14, rMuMig group mice had more cells than control. Especially at day 14, the bone marrow of rMuMig group mice had already recovered to near the normal level, while that of control group was only 1/10 of the normal level. Histology revealed that bone marrow of rMuMig group was suppressed less severe at day 7. At day 11 and 14, more hematopoietic cells and regular blood vessels were observed in the rMuMig treated mice than that of control group.The anti-Mig antibody was used to neutralize elevated expression of endogenous Mig after 5-Fu treatment. The anti-rMuMig polyclonal antibody raised in rat was administrated to mice after 5-Fu treatment consecutively for 10 days once a day. At day 0, 3, 7, 11, and 14 mice were sacrificed to count the WBC and BM cells, and histology femur tissue sections were examined. The result was similar to that of administration of rMuMig before 5-Fu treatment. The survival rate was up-regulated to 60% compared with 10% of the control preimmune serum group. And the mice injected with polyclonal antibody serum had a better and more rapid BM recovery than the control.From the experiments of rMuMig and its antibody we conclude that: prior administration of rMuMig before 5-Fu protects mouse bone marrow form the chemotherapy damage; using anti-Mig antibody after 5-Fu enhance the bone marrow recovery.To further study the mechanism of the protection effect of rMuMig, we administrated rMuMig to mice during the BM regeneration phase after 5-Fu treatment. We found that rMuMig inhibited bone marrow cells transition to S-phase of cell cycle, suggesting that the protection role of rMuMig is mediated by slowing down the BM cell cycle rendering the cells less vulnerable to cell-cycle specific chemotherapy drugs, like 5-Fu.Human Mig is 82% identical to mouse Mig. We propose that human Mig may play similar roles as mouse Mig. We expressed and purified the recombinant human Mig (rHuMig). The prokaryotic expressing plasmid pET28-h was constructed, which contained the coding sequence of mature human Mig protein. When the bacteria culture reached 0.8 at OD600, the protein expression was induced using 1mM IPTG. Protein purification process was carried out similar to rMuMig. SDS-PAGE showed the purity was above 95%.In summary, this work purified the recombinant murine Mig, studied the function of Mig in regeneration of bone marrow after chemotherapy, which suggest that Mig may be an important BM protector against chemotherapy induced BM side effect. Moreover, we also expressed and purified recombinant human Mig, which should facilitate the studies of human Mig as a BM protector in new protein drug development. |
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