基于间充质干细胞的小鼠急性移植物抗宿主病骨髓微环境损伤体外细胞模型的建立与评价策略研究

Establishment and Evaluation Strategy of an in Vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease

目的:建立小鼠急性移植物抗宿主病(aGVHD)骨髓微环境损伤的体外细胞模型并进行评价。方法:以6-8周龄雌性C57BL/6N小鼠作为骨髓和淋巴细胞的供体,6-8周龄雌性BALB/c小鼠作为aGVHD模型的受鼠。受鼠接受致死剂量(8.0 Gy,72.76 cGy/min)γ射线全身照射后6-8 h内,尾静脉输注供鼠来源骨髓细胞(1×10^(7)/只),建立骨髓移植(BMT)组小鼠模型(n=20);尾静脉输注供鼠来源骨髓细胞(1×10^(7)/只)及脾脏淋巴细胞(2×10^(6)/只),建立小鼠aGVHD模型(n=20)。造模后d 7麻醉小鼠,摘取眼球取血,静置离心后吸取血清。分离小鼠间充质干细胞(MSC),分别用添加了2%、5%和10%的BMT组血清和相同浓度的aGVHD组血清的培养基进行培养。通过成纤维细胞集落形成实验(CFU-F)评价两组血清对MSC集落形成能力的影响;通过CD29和CD105免疫荧光染色评价两组MSC表面分子表达的差异;通过实时荧光定量PCR(RT-qPCR)检测MSC自我更新相关基因Oct-4、Sox-2和Nanog的表达情况。结果:成功建立起可以模拟小鼠aGVHD骨髓微环境损伤的体外细胞模型。CFU-F实验表明,在培养后d 7,与BMT组相比,aGVHD血清浓度为2%和5%时,MSC集落形成能力显著降低(P<0.05);在培养后d 14,与BMT组相比,不同aGVHD血清浓度组MSC集落形成能力均显著降低(P<0.05)。免疫荧光染色实验表明,MSC表面分子CD29^(+)和CD105^(+)细胞百分比在不同aGVHD血清浓度组较BMT组均降低,在aGVHD血清浓度为10%时差异最为显著(P<0.001,P<0.01)。RT-qPCR检测结果显示,aGVHD血清浓度组MSC自我更新相关基因Oct-4、Sox-2和Nanog表达下降,在aGVHD血清浓度为10%时差异最为显著(P<0.01,P<0.001,P<0.001)。结论:不同浓度的小鼠aGVHD血清和小鼠MSC共同培养,发现添加不同浓度的小鼠aGVHD血清MSC的自我更新能力受损程度不同,为开展aGVHD骨髓微环境损伤领域研究提供了新的工具。

Objective:To establish a mesenchymal stem cell(MSC)-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease(aGVHD).Methods:Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors,and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients.The recipient mouse received a lethal dose(8.0 Gy,72.76 cGy/min)of total bodyγirradiation,and injected with donor mouse derived bone marrow cells(1×10^(7)/mouse)in 6-8 hours post irradiation to establish a bone marrow transplantation(BMT)mouse model(n=20).In addition,the recipient mice received a lethal dose(8.0 Gy,72.76 cGy/min)of total bodyγirradiation,and injected with donor mouse derived bone marrow cells(1×10^(7)/mouse)and spleen lymphocytes(2×10^(6)/mouse)in 6-8 hours post irradiation to establish a mouse aGVHD model(n=20).On the day 7 after modeling,the recipient mice were anesthetized and the blood was harvested post eyeball enucleation.The serum was collected by centrifugation.Mouse MSCs were isolated and cultured with the addition of 2%,5%,and 10%recipient serum from BMT group or aGVHD group respectively.The colony-forming unit-fibroblast(CFU-F)experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC.The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining.In addition,the expression of self-renewal-related genes including Oct-4,Sox-2,and Nanog in MSC was detected by real-time fluorescence quantitative PCR(RT-qPCR).Results:We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD.CFU-F assay showed that,on day 7 after the culture,compared with the BMT group,MSC colony formation ability of aGVHD serum concentrations groups of 2%and 5%was significantly reduced(P<0.05);after the culture,at day 14,compared with the BMT group,MSC colony formation ability in different aGVHD serum concentration was significantly reduced(P<0.05).The immunofluorescence staining showed that,compared with the BMT group,the proportion of MSC surface molecules CD29^(+)and CD105^(+)cells was significantly dereased in the aGVHD serum concentration group(P<0.05),the most significant difference was at a serum concentration of 10%(P<0.001,P<0.01).The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4,Sox-2,and Nanog was decreased,the most significant difference was observed at an aGVHD serum concentration of 10%(P<0.01,P<0.001,P<0.001).Conclusion:By co-culturing different concentrations of mouse aGVHD serum and mouse MSC,we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability,which providing a new tool for the field of aGVHD bone marrow microenvironment damage.