The Effects And Mechanism Of TIGAR On The Differentiation Of Bmi1~+ Stem Cells And The Repair Of Radiation-induced Intestine Damage

Objective: As the rapidly development of the nulear technology and widely application of radiotherapy in oncotherapy.Prevention and treatment of the acute radiation injury have become an important issue in the field of nuclear security.In present study,we investigated the effects of TP53-induced glycolysis and apoptosis regulatory proteins(TIGAR)in regulating the differentiation of the small intestinal crypt Bmi1+ stem cells which have a high radiation resistance and multi-directional differentiation potential by using lineage tracer technology.Exploring the feasibility of TIGAR could promote the restoration of the radiation-induced intestine damage and protect mice against acute intestinal type radiation sickness.Methods: TIGAR was overexpressed in mouse intestinal mucosa by injection of TIGAR over-expressing adenovirus(Ad-Tigar-EGFP)via the superior mesenteric artery(SMA).Western blot was used to test the levels of TIGAR and GFP in the intestinal mucosa.HE staining was perfomed to measure the number of intestinal crypt and the length of villus before and after radiation exposure.The proliferation and viability of intestinal crypt stem cells was monitored by Ki67 immunohistochemical staining.Survival analysis was used to evaluate the protective function of TIGAR overexpression on irradiated mice.The effects of TIGAR were also examined ex vivo by using organoids transfected with Ad-Tigar-EGFP,and immunofluorescence technique was used to detect the effects of TIGAR overexpression on the proliferation ability of crypt organoids.Western blot assay was performed to reveal the correlation between TIGAR and BMI1 expression.Finally,we performed lineage tracing technology to detect the proliferation ability of Bmi1 positive stem cells and their progeny in Bmi1 Cre ER/+,Rosa26 m Tm G/+ mice,and showed the correlation between TIGAR and Bmi1 + stem cell proliferation.Results:(1)The proteins of TIGAR are mainly distributed in the small intestine,and expressing levels in duodenum were higher than that of ileum.After the SMA injection of methylene blue dye,it mainly distributed in the jejunum and ileum,therefore,adenovirus was administrated the same way.Exogenous injection Ad-Tigar-EGFP can increase the levels of TIGAR in the jejunum and ileum in mice significantly(P < 0.05).(2)Injection of Ad-Tigar-EGFP can alleviate the damage of the small intestine induced by radiation,leading to the increase of crypt number and villi length significantly(P < 0.05).The immunohistochemical staining showed more Ki67-positive cells in Ad-Tigar-EGFP group than control group,suggesting that Ad-Tigar-EGFP group.We also found that TIGAR over-expressing can improve the survival rate of full abdomen X-ray irradiation in mice.(3)The immunofluorescence technique confirmed that Ad-Tigar-EGFP could express in organoids stably more than 6 days,and the peak time was the fourth days.A large number of organoids were lost after 2 days at lethal dose X-ray irradiation in the control group(Ad-EGFP),and all the organoids were died post-IR of 5 days.Compared with the Ad-EGFP,the survival rate were higher in Ad-Tigar-EGFP group at various time points,indicating that TIGAR over-expressing can reduce the mortality of organoids after X-ray irradiation,and maintain the proliferation activity of organoids.The correlation of the expression level of TIGAR and BMI1 was found different bowel mucosa layer.The lineage tracer technology was used to detect the proliferation of Bmi lineage cells in organoids post-10 Gy X-ray irradiation,finding that TIGAR over-expressing can promote differentiation and proliferation ability of the Bmi1+ stem cells in irradiated organoids,thus promotes organoids proliferation.Conclusion: Compared to caudal vein and intraperitoneal injection,the SMA injection of Ad-Tigar-EGFP can reduce the first effect effectively,then making the TIGAR over-expressing successfully in the intestine.It was found that the over-expressing protein of TIGAR exogenously can alleviate intestinal injury caused by X-ray irradiation and increase the survival rate of mice in vivo experiment.Using organoids culturing system and lineage tracer technique,it was illustrated that the molecular mechanisms of TIGAR can accelerate Bmi1+ stem cell proliferation,differentiation,and repair the radiation damaged crypts.