| Background and objectRadiotherapy is an important mean in the clinical abdominal cancer treatment,but the side effects such as intestinal injury will reduce the quality of life significantly and limit the effective radiation dose,and there is no effective treatment yet [1,2].Nuclear terror attack and nuclear power plant leakage also cause intestinal and bone radiation injury.It is great challenges for clinical treatment and nursing.Traditional model of radiation injury of intestinal epithelial cells in vitro rely on the 2D culture of immortalized progenitor cell lines and colon cancer cell lines.They can not accurately represent intestinal stem cells activity in vivo because of some gene mutations and its weak sensitivity to radiation.Intestinal stem cells are located at the bottom of the crypt,including Lgr5 stem cells and relatively quiescent intestinal stem cells upon the paneth's cells.The daughters of stem cells proliferate and migrate along the crypt-villus axis.Lgr5 stem cells die rapidly after irradiation,viable stem cells and the cells repossess stemness entering the cell cycle to complete the crypt villus after cycle arrest.However,stem cell cultured in vitro is a difficult problem in stem cell research and clinical application.Hans Clevers laboratory successfully developed a system in vitro,three dimensional intestinal organoid culture system,for stem cell sresearch in 2009.The system use matrigel provides the support of cell matrix,and simulate the key signal pathway in vivo by different kinds of cytokines.In order to solve the problems of radiation-induced intestinal injury,researchers looking forword to variety of cytokines,small molecular compounds and so on.However,the high cost in vivo limits the feasibility of these cytokines and small molecular compounds.Intestinal organoid culture makes it become possible.We developed a screening platform based on intestinal stem cell irradiation model in vitro,then screened different kinds of cytokines and small molecular compounds.After that,we confirmed the effects through exprement in vivo to distinguish the targets.Our reaserch provides a new platform and clue for the research of the new measures to reduce the side effects of radiotherapy and chemotherapy in clinical treatment and nursingMethods1.Establishment of an in vitro model of radiation-induced intestinal injurySmall intestinal crypts from C57BL/6(WT)and P53-/-mice were isolated and cultured in matrigel 3D system.The crypt clones were irradiated by X ray at a dose of 4-10 Gy after different times of embedding.The growth and death of crypt clones were observed and imaged continuously;MTT staining was applyed and then we counted the survival rate of the same genotype crypts at different times after irradiation.Total RNA was extracted and the expression of Bax,Bbc3,P21,Mdm2,Bcl2l1,Lgr5,Olfm4 and Ascl2 were detected through quantitative real-time PCR(RT-PCR).2.Dmog,IL22 and FingolimodHCl were used to distinguish their targets on intestinal injury through radiation-induced intestinal injury in vitro.The crypt clones were irradiated by X ray at a dose of 6Gy 48 h after seeding,then treated with Dmog at different concentrations 6h after irradiation,IL22 and FingolimodHCl at different concentrations 3h before irradiation.The growth and death of crypt clones were observed and imaged continuously,the survival rates of crypts on day 5 were counted.The crypts clones were radiated at a dose of 6Gy 48 h after embedding of WT with IL22 administration,then total RNA was extracted from those crypts clones.The relative expression of Lgr5 and P53 target genes in small intestinal stem cells at different times were detected by RT-PCR.C57BL/6 mices were irradiated TBI at dose of 10 Gy or 12 Gy,Dmog and FingolimodHCl were treated before or after irradiation at different concentrations.The body weight and mental state of mices were observed after irradiation every day.Mices were sacrificed 84 h after irradiation of 10 Gy and 96 h after irradiation of 12 Gy,the intestine were photoed and being compared in control group and that in treated group.HE staining was used to obverse the absence of crypts and the integrity of villi.Brdu IHC was used to evaluate the proliferation of small intestinal crypts.Combining the results in vivo and in vitro experiments,the targets of cytokines and small molecule compounds were preliminarily distinguished.Results1.The crypt cultured in 3D system can represent the crypt-villus in vivoThe research successfully established 3D culture system for intestinal crypt cells.The crypts clone generated from WT and P53-/-mice bud at 24 h after seeding,formed the organiods after 96-120 h.Thourgh 6Gy X ray,the budding of WT crypt clone on day 2-4 were totally blocked;but the crypt genegrated from P53-/-mice continued their growth and budding.Moreover,the survival rate of WT crypt clones was significantly lower than that of P53-/-crypt clones.RT-PCR tests indicated that the transcription of genes of P53 pathway increased significantly after irradiation.However,the expression of stem cell marker genes decreased obviously 24 h after irradiation exposure.The crypts cultured in 3D system can mimic the death and regeneration depending on P53 in vivo,suggesting a suitable model for radiation protection drugs screening.2.The crypt from C57BL/6 mice were irradiated by X ray at a dose of 6Gy 48 h after seeding;Then treated with Dmog at different concentrations 6h after irradiation.The survival rate of crypts in control was(49±4)%;the survival rate of crypts in 200?M group was(57±5)%;the survival rate of crypts in 500?M group was(56±5)%;the survival rate of crypts in 1000?M group was(60±7)%;the survival rate of crypts in 1000?M group was higher than that in control group(P<0.05).Treated with IL22 at different concentrations 3h before irradiation,the survival rate of crypts in 1ng/ml was(69±7)%;the survival rate of crypts in 3ng/ml was(82±9)%;the survival rate of crypts in 5ng/ml was(85±9)%;the survival rate of crypts in 10ng/ml was(60±5)%;the survival rate of crypts in treated group was higher than that in control group(P<0.05).Treated with FingolimodHCl at different concentrations 3h before irradiation,the survival rate of crypts in 0.5?M group was(52±10)%;the survival rate of crypts in 1?M group was(71±5)%;the survival rate of crypts in 2?M group was(20±6)%;the survival rate of crypts in 4?M group was(11±2)%;the survival rate of crypts in 1?M group was higher than control group(P <0.05).These results indicate IL22 and Dmog and Fingolimod HCl can protect the crypts form irradiation.C57BL/6 mice were irradiated at dose of 10 Gy or 12 Gy,Dmog were treated 6h after irradiation and FingolimodHCl were treated 1h before irradiation in different concentrations.Mouses in Dmog treatment group had no difference to that in control groups.The anatomy observation: FingolimodHCl were used 1h before irradiation with 5mg/kg/d can reduce the swelling and decrease intestinal mucus of intestinal.HE staining: The villus integrity was better than that in the control group;and the amount of crypts was much more than that in the control group.Brdu IHC: The proliferation of crypt in treatment group was much more than that in the control group(P<0.05).Conclusion1.The crypts cultured in 3D system can mimic radiation-induced intestinal injury in vivo,suggesting it is a suitable model for radiation protection drugs screening.The results confirmed that the intestinal stem cells in 3D culture system are consistent with P53 dependent cell death and cell cycle arrest and mitotic catastrophe in vivo.2.IL22 can be used to positive control for radiation protections screening target on stem cells.Radiation-induced intestinal injury rely on the stem cells in 3D culuture in vitro can distinguish the effect on stem cells or others.The establishment of this system provides a new platform for the study of the new measures to reduce the side effects of radiotherapy in the clinical treatment and nursing. |
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