| Accidental or intended radiation exposure presents a serious and on-going threat.Intestine is very sensitive to ionizing radiation and hard to be recurred because of thedamage of the intestinal structure and function impairment. The acute damage of intestinalstem cells (ISCs) and ISCs niche is the fundamental reason for barriers of crypt-villus’regeneration, and it is also the pathological mechanism of irradiation-induced intestinalepithelium damage. Thus, the effective treatments should be proposed according to thepathological mechanism of acute irradiation-induced intestinal epithelium damage.Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells thathave the ability of self-renewal and multipotency, which could differentiate into cells ofthe mesodermal lineages and other embryonic lineages, including adipocytes, osteocytes,chondrocytes, hepatocytes, neurons, muscle cells, epithelial cells, etc. The advantages ofMSCs has been explored in numerous studies, MSCs could migrate to injured, inflamedtissues from blood and exert therapeutic effects. As well as the immunosuppressive, anti-inflammatory and trophic effects of MSCs were described in recent years. These propertiesmake MSCs an ideal candidate cell type for tissue engineering, regenerative medicine andautoimmune disease treatment. Rspo1(R-spondin1) is a novel secreted protein thatarguments Wnt/β-catenin signaling by directly bind to Lgr5and Lgr6receptors on surfaceof ISCs and competitively inhibit DKK1, a inhibitor of Wnt/β-catenin signaling. For this,Rspo1can stimulate proliferation and differentiation of ISCs through the Wnt signalingpathway. In addition, human Rspo1can be used in mouse model, with potent and specificproliferative effects on intestinal crypt cells.Therefore, we established a mouse model of intestinal epithelium irradiation damageand observed the feature of pathological injuries. Meanwhile, we constructed amesenchymal stem cell line that stably expressed functional human Rspo1. The Rspon1-transfected MSCs were transplanted into mice with intestinal epithelium irradiationdamage to restore the normal architecture of crypt-villus through the immunosuppressive effects of MSCs together with Rspo1/β-catenin signal activation. This study is promised toprovide a new idea for rescuing the intestinal epithelium damage.Part1: Establishment of intestinal epithelium irradiation damage mouse modeland the feature of pathological injuriesPurpose: To establish a mouse model of intestinal epithelium irradiation damage andobserve the feature of pathological injuries.Method: Mice were grouped according to the abdominal irradiation dosage of10Gy,12Gy,14Gy,16Gy and18Gy to identify the minimum lethal dose of irradiation. Lgr5-EGFP-ires-CreERT2mice were abdominal irradiated by18Gy. The weight of miceeveryday were recorded, intestinal tissues were observed at0.5day,3.5day,6day toinvestigate pathological changes such as length of intestinal villi, number of crypt and thechange of the Lgr5+intestinal stem cells.Results:18Gy was adopted as the minimum lethal dose for abdominal irradiation.Compared with the untreated group, the weight loss, villus short, crypt number reductionand the number of Lgr5+intestinal stem cells deceased rapidly in the short term.Conclusion: A mouse model of intestinal epithelium irradiation damage wasestablished with abdominal irradiation of18Gy. This model closely imitates thepatholigical changes of intestinal epithelium irradiation damage and provides a good modelfor the study of the sublethal irradiation.Part2: Construction of Rspo1gene transducted mesenchymal stem cell line andidentification of the Biological ActivityPurpose: To construct a gene transfected mesenchymal stem cell line that stablyexpressed Rspo1.Methods: The full length human Rspo1cDNA was Subcloned into retroviralexpressing vector pEGZ-Term. The recombinant plasmid together with its helper virusvector was cotransfected into the package cell293T.The C3H10T1/2cells were infectedwith the supernatant of the transfected293T cells, and then were selected with G418. TheG418resistant cells were harvested for screening their C3H10/Rspo1expression byRT-PCR and flow cytometry. The biological effect of supernatant was analyzed by cellcounting.Results: The pEGZ-Term/Rspo1vector was constructed successfully and a cell line,named C3H10/Rspo1, which stably expressed Rspo1was obtained and its supernatant could promote colon cancer cell line SW480proliferation.Conclusion: In our study, a stable gene transfected mesenchymal stem cell line thatexpressed Rspo1was established providing a valuable tool to explore the novel strategy torescue the intestinal epithelium damage..Part3: Repair of mouse intestinal epithelium irradiation damage with Rspo1Transfected Mesenchymal Stem CellsPurpose: to restore the mouse with intestinal epithelium damage by the protectiveeffect of MSCs and the proliferative effect of Rspo1.Methods: Flow cytometry was adopted to detect the expression of chemokinereceptors and immunomodulatory molecules on the surface of C3H10T1/2. Intracellularstaining was used to detect the immunosuppressive factors and proinflammatory cytokinessecreted by MSCs. ELISA was used to detect the PGE2levels secreted by C3H10T1/2supernatants. Mice were abdominal irradiated at18Gy and divided into4groups, whichwere injected with PBS, C3H10T1/2, C3H10/mock and C3H10/Rspo1respectively. Thesurvival and the weight of mice everyday were recorded.Results: C3H10T1/2expresses chemokine receptor CXCR4and immunomodulatorymolecules such as PD-L1, B7H3and B7H4. Immunosuppressive factors such as TGF-β,IL-10, IL-4and PGE2are secreted by C3H10T1/2. For the mice with intestinal epitheliumirradiation damage, all members of the PBS group dead within two weeks and the survivalrate of the C3H10/Rspo1group is higher than that of the C3H10T1/2group. The weightloss rate of the C3H10/Rspo1group is also slower than that of the C3H10T1/2group.Conclusion: C3H10/Rspo1can migrate into intestinal epithelium of irradiation-induced damage, protect damaged ISCs through releasing immunosuppressive factors andimmunomodulatory molecules. Moreover, it can secrete the wnt agonist Rspo1to repair ofirradiation-induced intestinal epithelium by activating wnt signaling pathway. |
