M2 Type Macrophage Plays A Role In The Restore The Small Intestine With Irradiated Injury

Radiation therapy is one of the three major means of cancer treatment. The patients with preoperative or postoperative radiotherapy can effectively cure the tumor, reduced risk of tumor recurrence. But radioactive bowel injury is an important clinical problem during radiation therapy of the abdominal and pelvic tumor because intestinal epithelium radiation sensitivity is high. The patients often appear nausea, vomiting, abdominal pain, diarrhea, and absorb nutrition disorder, weight loss, even stool, intestinal obstruction, intestinal basket and other lesions. It not only seriously affect the patient’s quality of lifebut also hinder the radiation treatment for abdominal pelvic tumors.Mammal’s intestinal epithelium is composed of mono-layer columnar epithelium. Differentiation of epithelial cells mainly happens in the villi and epithelial surface; proliferation of epithelial cells mainly occurs in the crypt. Crypt stem cells can divide into stem cells and TA cells, TA cells will transfer to villi and differentiate. to absorb cell line(intestinal cells) and secretory cell line(goblet cells and endocrine cells). So intestinal mono-layer columnar epithelium is composed of four types of intestinal epithelial cell line, they are all originated from stem cells and the basal crypt stem cells play an important role in maintaining intestinal epithelium balance.1-2 Intestinal epithelial injury during intestinal infection, irradiation and idiopathic inflammatory bowel disease would induce epithelial cell apoptosis and the destruction of the epithelial barrier3. The intestinal epithelial injury would also activate regeneration program: crypt stem cell proliferates, move to the differentiation of area and make continuous compact structure of epithelium4. The apoptotic,effete epithelium cellis far from crypt stem cells, how the effete epithelium timely notify the stem cells to proliferate? Maybe mobile immune cells do the work. Macrophages are one part of the innate immune system, many of them locate in the bowel 5. They locate beneath the epithelial barrierand participate in intestinal immune response. But primary culture of bowel macrophages is difficult. Tanja cultured macrophages with epithelial cells, but after 7 days only a small amount of co-cultivation macrophages survived. Hiroki et al. studied the transmit signals between the macrophages and the epithelial cells, they preferred to culture Caco- 2 with THP 1 cell line, not the primary macrophages.Mature macrophages mainly derive from bone marrow and reach the organization with peripheral blood circulation, its microenvironment can occur macrophage polarization. Currently macrophage polarization is divided into two main types: M1 type mainly involve in the immune response, phagocytose harmful microbes,and produce inflammatory cytokines IL-6, NO to improve the body’s defense against the invasion of harmful substances; M2 type mainly involve in the damaged tissue and secrete anti-inflammatory cytokines to repair and remodel tissue, reduce the body’s inflammatory response.So, in this thesis, first, the intestinal macrophages polarized type was identified; then, the culture media of the apoptotic epithelial CT26 cells simulated Raw264.7 cell to polarize to M2; when M2 Raw264.7 cocultured with sorted crypt intestinal stem cells, stem cells proliferation was studied in depth. This thesis provides a theoretical basis of M2 macrophage for the repair small intestine with irradiated injure.Part One: Polarized mice intestinal macrophage and roles in irradiate injureObjective: Intestinal macrophages function in radiation injury.Methods: C57 BL / 6 mouse received 2Gy and 8Gy whole body irradiation. At 72 and 96 hour after irradiation, mouse intestinal macrophages were collected and acquired with FACSort.Total RNA was extracted with RNeasy Mini Spin Kit.To analyze polarization of macrophages,PCR amplifications were performed with Primer specific for inos 、IL-10.Normal C57BL/6 mouse received 200μl Clodronate liposomes and PBS liposomes via intraperitoneal separately at 1 day, 5 days, 7 days 9 days to deplete part of intestinal macrophages. On the eighth and the eleventh day, we acquired CD68+ cell of thymus, lymph, intestine, spleen, bone marrow cells by FACSort and demonstrate the best time of removing macrophages.The eighth day after administration, mouse received 8Gy whole body irradiation and sacrificed to get the intestinal samples for HE staining 、CD68 and claudin-5 immunohistochemical staining 、distance between nuclei surrounding epithelium at 72 hours and 96 hours after irradiation,we observed the intestinal irradiation.Results: The intestinal macrophages percentage decreased at 72 hours after 2 Gy irradiation(25.8%, 33.7%, 30.8% vs 59.2%、60.6%、60.3%,control p<0.05) and recovered gradually at 96 hour(49.9%, 44%, 49.9% vs 25.8%, 33.7%, 30.8%, p<0.05); percentage decreased at 72 hours after 8 Gy irradiation(40.9%, 36%, 42.9% vs 59.2%、60.6%、60.3%,control p<0.05)) and did not obviously recover(40.2%, 40.5%, 39.3% vs 40.9%, 36%, 42.9%, p>0.05). Thus macrophages are present in the mouse intestine of the control group and the irradiation group; mice was injected with Clodronate liposomes and PBS liposomes and found macrophage cell of thymus, lymph, intestine, spleen, bone marrow number began to decline at the eighth days after the administration and further declined on the eleventh days, restored the level of the eighth day,so selected the eleventh days after the administration as the best time of dispelling macrophages. Pathological analysis showed that mouse were radiated at 8Gy does, intestinal damage in mouse of less macrophages were more obvious than the PBS liposomes group; distance between nuclei results showed the arrangement of epithelial cells in irradiate group is disorder than the control group; the epithelium of PBS liposomes group began to recover gradually and proliferation at 96 hour after 8Gy irradiation, nuclear arrangement had a relatively complete form than 72 hour; the arrangement of epithelial cells was the most disorder in mouse of Clodronate liposomes after 8Gy irradiation and appeared nuclear spacing increase, nuclear pyknosis necrosi,epithelial injury was more serious, internuclear distance further increased; the result of Claudin5 showed that PBS liposomes and Clodronate liposomes groups both had barrier damage at 72 h after 8Gy irradiation and had no obvious difference, mouse with Clodronate liposomes had the intestinal tissue damage and the damage degree was significantly higher than the PBS liposomes group.Conclusion: In the mouse intestinal tract,macrophage exists in the M2 type, and type does not change after irradiation and play a protective role in the intestinal irradiation.Part Two: M2 type macrophages in vitro promote Lgr5 intestinal stem cell proliferationObjective:M2 macrophages play a role to Lgr5 intstinal stemMethods: CT26 cells –colon cancer cells of mice received the dose of 6Gy and 15 Gy irradiation, measured apoptosis by flow cytometry of Annexin V/PI assay. RAW264.7 cells marcrophages of mice were induced polarization type with supernatant of apoptosis,and confirmed the polarization type via Elisa analysis of IL-10 and IL-12. Lgr5-EGFP-IRES-cre ERT2 mouse intestinal crypts and intestinal stem cells were isolated and cultured; the polarized RAW264.7 cells were co-culture with Lgr5 intestinal stem cells and Transwell cultured and detect the fluorescence intensity of GFP via multifunctional microplate reader.Results: The CT26 cells apoptosis assay showed apoptosis rate was 66.9% at 72 hours after 15 Gy irradiation; RAW264.7 cells were cultured with supernatant of apoptosis and found IL-10highIL-12 low at the third day. RAW264.7 occured polarized and became M2 macrophages; In the conditions of no Noggin, EGF, R- spondin stem cell growth factor, Lgr5 intestinal stem cells were co-culture with the control group and the M2 type RAW264.7,found M2 macrophages secrete EGF factor instead of the EGF of culture medium and could promote growth and proliferation of stem cells.Conclusion: M2 type macrophages promote Lgr5 intestinal crypt stem cell proliferation by secreting EGF factor.In summary, M2 macrophages exist in the intestinal tract and have no change in the condition of irradiation.M2 macrophages can secrete EGF factor and promote the proliferation of Lgr5 crypt stem cells. The lack of the intestinal M2 type macrophages can delay intestinal radiation damage repair.