基于类器官技术的小鼠急性移植物抗宿主病肠道损伤模型建立与评价策略研究

Establishment and Evaluation of Intestinal Injury Model of Mouse Acute Graft Versus Host Disease Based on An Organoid Technology

目的:借助急性移植物抗宿主病(aGVHD)模型小鼠血清和肠类器官培养技术,建立模拟aGVHD肠道损伤的体外模型并探索类器官水平aGVHD肠道损伤的规律。方法:以20-22 g雌性C57BL/6小鼠和20-22 g雌性BALB/c小鼠分别作为骨髓移植的供鼠和受鼠。受鼠接受致死剂量(8.0 Gy)的γ射线全身照射后的4-6 h内,输注供鼠来源骨髓细胞(1×10^(7)/只,n=20)及脾脏有核细胞(5×10^(6)/只,n=20)混合液共0.25 ml,建立aGVHD小鼠模型(n=20)。于造模后d 7麻醉aGVHD小鼠,摘取眼球取血,静置离心后收取血清。分离C57小鼠小肠隐窝,在模拟体内肠道干细胞生长增殖的3D培养体系中培养。分别在类器官培养基中加入5%、10%和20%体积比例的aGVHD血清,培养3d,倒置显微镜下观察小肠类器官的形成过程,比较小肠类器官形态。为了进一步评估,收集aGVHD肠道类器官并观察其病理变化。结合HE染色进行肠类器官形态评价;结合Alcian Blue染色,观察aGVHD小肠类器官分泌功能;结合免疫组化技术,分析血清浓度在5%、10%和20%条件下肠类器官中Lgr5^(+)和Clu^(+)肠干细胞分布及变化规律。结果:成功建立了体外模拟aGVHD肠道损伤类器官模型。HE染色结果显示,5%浓度血清组肠道类器官形态明显较10%和20%组完整,随aGVHD血清浓度的升高,肠道类器官形态破坏加重。5%aGVHD血清组小肠类器官数量最多,出芽率最高,病理评分最低;20%aGVHD血清组形态破坏严重,没有出芽,病理评分最高(t=3.668、4.334、5.309,P<0.05)。Alcian blue染色结果显示,20%aGVHD血清组肠道类器官分泌功能较5%组和10%组类器官弱,未见杯状细胞分布及其分泌黏液染色。免疫组化结果表明,5%组的aGVHD血清组的肠类器官中Lgr5^(+)细胞数量较10%aGVHD和20%的aGVHD血清组肠道类器官多,基本未见Clu^(+)细胞,相反,20%aGVHD血清组Lgr5^(+)细胞破坏严重,无法观察,Clu^(+)细胞比例明显升高。结论:aGVHD血清浓度影响体外小肠类器官数量、功能和肠干细胞的数量。血清浓度越高类器官发生损伤风险越大,揭示了aGVHD场景中肠类器官的形成和功能规律,为开展aGVHD肠损伤领域研究提供了新的工具。

Objective: To establish an intestinal organoid model that mimic acute graft versus host disease(aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of a GVHD intestine in vitro by advantage of organoid technology. Methods: 20-22 g female C57BL/6 mice and 20-22g female BALB/c mice w ere used as donors and recipients for bone marrow transplantation,respectively. Within 4-6 h after receiving a lethal dose( 8. 0 Gy) of γ ray total body irradiation,a total of 0. 25 ml of murine derived bone marrow cells( 1 × 10^(7)/mice,n = 20) and spleen nucleated cells( 5 × 10^(6)/mice,n = 20) w as infused to establish a mouse model of a GVHD( n = 20). The a GVHD mice w ere anesthetized at the 7th day after transplantation,and the veinal blood w as harvested by removing the eyeballs,and the serum w as collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice w ere harvested and cultivated in 3D culture system that maintaining the grow th and proliferation of intestinal stem cells in vitro. In our experiment,5%,10%,20% proportions of a GVHD serum w ere respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids w ere observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups w ere analyzed. For further evaluation,the a GVHD intestinal organoids w ere harvested and their pathological changes w ere observed. Combined w ith HE staining,intestinal organ morphology evaluation w as performed. Combined w ith Alcian Blue staining,the secretion function of a GVHD intestinal organoids w as observed. The distribution and changes of Lgr5^(+)and Clu^(+)intestinal stem cells in intestinal organoids w ere analyzed under the conditions of 5%,10% and 20% serum concentrations by immunohistochemical stainings. Results: The results of HE staining show ed that the integrity of intestinal organoids in the 5% concentration serum group w as better than that in the 10% and 20% groups. The 5% concentration serum group show ed the highest number of organoids,the highest germination rate and the low est pathological score among experimental groups,w hile the 20% group exhibited severe morphological destruction and almost no germination w as observed,and the pathological score w as the highest among all groups( t = 3. 668,4. 334,5. 309,P < 0. 05). The results of Alican blue staining show ed that the secretion function of intestinal organoids in serum culture of a GVHD in the 20%group w as w eaker than that of the 5% group and 10% of the organoids,and there w as almost no goblet cells,and mucus w as stainned in the 20% a GVHD serum group. The immunohistochemical results show ed that the number of Lgr5^(+)cells of intestinal organoids in the 5% group w as more than that of the intestinal organoids in the 10% a GVHD serum group and 20% a GVHD serum group. Almost no Clu^(+)cells w ere observed in the 5% group. The Lgr5^(+)cells in the 20%group w ere seriously injuried and can not be observed. The proportion of Clu^(+)cells in the 20% group significantly increased. Conclusion: The concentration of a GVHD serum in the culture system can affect the number and secretion function of intestinal organoids as w ell as the number of intestinal stem cells in organoids. The higher the serum concentration,the greater the risk of organoid injury,w hich reveal the characteristics of the formation and functional change of aGVHD intestinal organoids,and provide a novel tool for the study of intestinal injury in aGVHD.