Summary

2'-脱氧核苷在裸体小鼠中治疗鼠胚胎胸腺的肾下囊移植

Published: July 19, 2019
doi:

Summary

我们提供一种简单而有效的方法,将治疗E18.5胸腺素的2’脱氧腺苷移植到裸鼠的肾胶囊中。该方法应辅助研究胸腺上皮细胞功能和T细胞成熟。

Abstract

胸腺是重要的中央免疫器官,在T细胞的发育和分化中起着至关重要的作用。胸腺移植是研究胸腺上皮细胞功能和T细胞在体内成熟的重要方法。在这里,我们将描述在我们的实验室中使用的实验方法,将2′-脱氧腺苷(以耗尽供体的淋巴细胞)治疗胚胎胸腺移植到贫血裸鼠的肾脏胶囊中。此方法既简单又高效,不需要特殊技能或设备。通过这一简单方法获得的结果表明,移植胸腺能有效支持受体的T细胞生产。此外,还将进一步阐明与议定书有关的若干要点。

Introduction

胸腺是中央免疫器官,胸腺胸腺细胞内经过正阴性选择,成为成熟的T细胞1,2。异常阳性或阴性选择分别导致免疫缺陷或自身免疫性疾病3,4。因此,胸腺器官移植是研究供体胸腺中T细胞选择过程的重要途径。这种方法在分析由基因突变介导的胸腺上皮功能时尤为重要,基因突变在突变5时引起胚胎致命表型。

为了研究受体的T细胞在移植胸腺中的成熟,有必要消耗胸腺内的供体淋巴细胞。为此,胚胎14天、15天或16天(E14,E15,E16)胸腺通常选择6,7。从更成熟的阶段胸腺也可以成功地耗尽捐赠者的淋巴细胞,通过治疗2′-脱氧腺苷。然而,关于消耗淋巴细胞和使用较老胸腺培养的详细方案之前没有描述8,9。虽然移植协议已被几个研究10,11引入,但进一步修改和改进这些协议是必要的。

我们的协议分为两部分:(i) 在含有2’脱氧腺苷的培养物中,通过培养物从晚期E18.5胸腺中消耗T淋巴细胞。(二) 将培养的胸腺移植到接受者体内。在此过程中,我们开发了一种简单的方法,将大组织(E18.5胸腺)输送到肾胶囊中,降低了肾损伤的几率。在关注后期胸腺的同时,我们的方案也可以直接用于或修改胸腺移植,用于不同发育阶段或其他类似大小的组织。

Protocol

所提出的方案符合暨南大学伦理委员会关于动物护理的指导方针。 注:所用材料列在”材料表”中。 1.胚胎胸腺的分离 在实验前对所有手术器械进行高压灭菌,用70%乙醇对工作台/机进行灭菌。 使用二氧化碳,麻醉和安乐死怀孕的雌性小鼠(成功交配后18.5天)。然后用70%乙醇擦拭腹部区域。注: 在这里,我们交配Insm1<sup…

Representative Results

这里我们展示的分离的E18.5胸腺包含两个完整的叶 (图1)。此外,我们显示头皮静脉针被剪切,形成输液管的斜面 (图 2)。接下来,我们还展示了在受体小鼠生长8周后移植在肾胶囊(图3A)和胸腺中所状的胸腺位置的代表性图像(图3B)。为了确定T细胞是否在移植胸腺的裸小鼠中产生,在移植的胸腺和外周血液中,我们使?…

Discussion

胚胎胸腺肾下囊移植是研究胸腺上皮细胞功能和T细胞在体内成熟过程的重要方法。虽然对胚胎胸腺器官培养和移植有若干实验性研究6,7,我们的方案提供了一个简单的替代程序,对小鼠胚胎胸腺培养和肾下囊移植较老的胸腺组织。

我们的协议通过合并几个不同的修改6,7,10,11来改进以前的协议。

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了暨南大学对S.J.的启动包和中国广州科技项目(授权号201704020209至S.J.)的支持。我们感谢Amy Botta(多伦多约克大学生物学系,加拿大M3J 1P3)对手稿进行校对和编辑。

Materials

0.5% Povidone iodine Shanghai Likang Distinfectant Hi-Tech Co.Ltd 20171113
0.9% Sodium Chloride Injection Shandong Qilu Pharmaceuyical Co.Ltd 2C17112101
1 mL Sterile syringe Solarbio YA1090
2’-Deoxyguanosine MEC HY-17563 1M in DMSO, 1:800 using (final 1.25mM)
24 Well Plate Corning Incorporated Costar 3524
4-0 Surgical suture needles with thread NingBo Cheng-He Microsurgical Instruments Factory China YY0166-2002
60mm Cell Culture Dish Corning Incorporated 430166
70% ETOH LIRCON 20181221
APC anti-mouse CD8a antibodies Biolegend 100711 1:100
Bent-tip fine forceps, JZ 10 cm Shanghai Medical Devices Group Co.,Ltd. JD1060 To sterilize before use
Cefmetazole Sodium for Injection Sichuan Hexin Pharmaceutical co,Ltd 17062111 079 6mg in 0.5ml 0.9% NaCl solution, 7.5ul/g body weight
Dissecting scissors, JZ 10 cm Shanghai Medical Devices Group Co.,Ltd. JC2303 To sterilize before use
Fetal bovine serum (FBS) GIBCO 10270-106
Fine forceps, JZ 10 cm Shanghai Medical Devices Group Co.,Ltd. JD1050 To sterilize before use
Flow cytometry BD FACSCanto II
Flunixin meglumine MACLIN F810147 1mg in 1ml 0.9% NaCl solution,2ul/g body weight
Forceps, Dumont#5 World Precision Instruments 14098 To sterilize before use
Infrared lamp OTLAN MT-810
Needle holder, JZ 14 cm Shanghai Medical Devices Group Co.,Ltd. J32010 To sterilize before use
PE anti-mouse CD4 Biolegend 100511 1:100
Penicillin-Streptomycin mixture GIBCO 15140122 1:100
Pentobarbital sodium salt Sigma P3761 1.5% solution in PBS, 75ug/g body weight
RPMI1640 Medium GIBCO C14-11875-093
Scalp vein needle Shanghai Kindly Medical Instruments Co., Ltd XC001
Spring scissors VANNAS S11014-12 To sterilize before use
stereomicroscope OLYMPUS SZ61
Sterile 15cm cotton swab Guangzhou Haozheng 20150014
Sterile gauze 5 cm x 7 cm-8P Guangzhou Haozheng 20172640868
Sterile PBS (1x) GENOM GNM20012
Tissue forceps, JZ 12.5 cm Shanghai Medical Devices Group Co.,Ltd. J41010 To sterilize before use

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Wang, J., Chen, G., Cui, Q., Song, E., Tao, W., Chen, W., Wang, C., Jia, S. Renal Subcapsular Transplantation of 2′-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice. J. Vis. Exp. (149), e59657, doi:10.3791/59657 (2019).

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