摘要
背景
电离辐射诱导机体发生急、慢性炎性反应,进而破坏机体免疫平衡,并表现为淋巴细胞中各细胞亚群的比例失衡,Treg细胞作为淋巴细胞的重要免疫调节亚群,在辐射照射后细胞数目、比例及表型的改变将直接影响机体免疫平衡,为此近年来已有学者对Treg细胞辐射敏感性方面开展了初步研究,但随着对Treg细胞研究的进一步深入,明确了许多与Treg细胞表型和功能相关的转录因子与表面分子,而这些分子的差异表达往往与炎性反应类型和所在的器官不同而不同,其中哪些因子在辐射诱导的炎性反应中发挥作用尚未见相关研究报道。
目的
分析不同剂量丫射线照射对淋巴细胞及其Treg亚群细胞数和细胞比例的影响,并在此基础上明确2Gyγ射线照射对Treg细胞凋亡率、增殖能力及Treg细胞表型的影响,进而分析Treg细胞凋亡与表型改变之间的关系和靶向作用于差别表达表型因子的microRNA的表达变化,探讨表型改变及其潜在作用的基因水平调控机制,为辐射诱导免疫失衡的预防与恢复治疗提供理论依据。
方法
1.利用不同剂量(0.25Gy,0.5Gy,0.75Gy,1Gy,2Gy和5Gy)60Coy射线全身照射小鼠,分离胸腺和脾脏中的淋巴细胞并进行计数,再利用FACS检测Treg细胞亚群的比例变化；
2.2Gy γ射线照射离体小鼠胸腺淋巴细胞,在照后9h利用FACS检测Treg细胞及Tcon细胞中7-AAD的结合水平；
3.2Gy γ射线全身照射小鼠,在照后1、4和10d分离胸腺和脾脏淋巴细胞,利用FACS检测Treg细胞及Tcon细胞Annexin-V和Ki-67的表达,检测Treg细胞核心转录因子FOXP3的表达,检测Treg细胞表型因子CD39、CD103等的表达,检测nTreg细胞特异性转录因子Helios的表达,检测凋亡相关因子Bcl-2、 Bax的表达；
4.检索靶向作用于CD39的microRNAs,并利用real-time PCR对检索到的microRNAs进行扩增分析,扩增底物为从分选的CD4+T细胞中提取的RNA；
5.分选CD4+CD25+Treg细胞,利用ATP检测试剂盒检测Treg细胞内ATP水平。
结果
1.小鼠受照后胸腺细胞和脾脏细胞呈剂量依赖性减少,在照后4d降至最低(F=118.08、144.01,p<0.05)。较高剂量(1-5Gy)照射后,胸腺中Treg细胞数随时间逐渐减少,而脾脏中该细胞亚群逐渐恢复；而较低剂量照射后胸腺中Treg细胞数有所增加。与0Gy组相比较,Treg细胞比例随剂量增加而增加(F胸腺=5.16、89.44、3.01,p<0.05；F脾脏=52.02、32.13、27.45,p<0.05)；
2.离体Treg细胞自然存活率低于Tcon细胞,2Gy γ射线照射后,二者存活率均减小,后者减小幅度显著(t=-14.03,p<0.05)；
3.全身受照后4d, Treg细胞凋亡率显著增加,且高于Tcon细胞；不同器官中Treg细胞凋亡率和增殖水平改变不同,一方面胸腺Treg细胞凋亡率低于脾脏,另一方面胸腺中Ki-67+Treg细胞比例在受照后1d先减小,至照后4d又增加,而脾脏中该比例先增加,至照后4d相对对照组有所恢复；
4.受照后FOXP3+Treg细胞占CD4+T细胞的比例增加,单个细胞中FOXP3的MFI变化在胸腺中下调,在脾脏中上调,但均在受照后10d改变最为显著(t=3.22,-5.34；p<0.05)；而Helios的表达相对稳定,胸腺与脾脏中的变化规律基本一致；
5.受照后Treg细胞中CD39和CD103的表达具有显著差异,其中CD39+Treg细胞比例增加,单个Treg细胞中CD39的MFI在胸腺中上调而在脾脏中下调；CD103+Treg细胞比例变化在脾脏中先下调后上调,在胸腺中于受照后4d出现一过性上调,单个Treg细胞CD103的MFI在胸腺中下调,而在脾脏中上调；
6.胸腺Treg细胞中Bcl-2的MFI变化无统计学意义,而Bax的MFI呈上调趋势,Bax/Bcl-2比值亦上调(t=-3.43,p<0.05)；脾脏中的变化规律相反,Bcl-2的MFI持续上调,而Bax的MFI变化无统计学意义,Bax/Bcl-2比值下调t=2.40,p<0.05)；单个Treg细胞中CD39的MFI变化规律与Bax/Bcl-2比值变化规律一致；
7.利用microRNA数据库检索了靶向作用于CD39的microRNAs,检索结果有6个,然后利用real-time PCR进行扩增分析发现miR-31的表达变化与CD39的MFI变化规律基本一致。
结论
1.不同剂量照射对淋巴细胞及其Treg细胞亚群细胞数的影响不同,其中较高剂量诱导Treg细胞数减少,而低剂量照射诱导其增加；淋巴细胞与Treg细胞亚群之间具有不同的时间响应；
2.离体Treg细胞对辐射具有抗性,但辐射可诱导全身照射的Treg细胞凋亡率显著增加,且辐射诱导Treg细胞凋亡率与增殖能力的改变随受照后时间和所处器官的不同而具有不同规律,表明Treg细胞的活性受所处微环境的影响显著。
3.Bax和Bcl-2分别在胸腺和脾脏的辐射诱导Treg细胞凋亡过程中发挥主要作用,Bax/Bcl-2比值的改变可以反映Treg细胞的凋亡水平,且Bax和Bcl-2参与的Treg细胞凋亡机制中受CD39的正向调节；
4.辐射可诱导FOXP3的表达改变,Treg细胞具有可塑性；同时辐射可诱导Treg细胞表型改变,其中CD39和CD103是辐射诱导Treg细胞表达差异的两个重要表面因子, CD103介导辐射诱导炎性反应中Treg细胞的迁移,而CD39的表达差异则主要参与Treg细胞的抗辐射诱导凋亡；
5.CD39的表达水平变化受外在微环境的影响,也有基因水平的调节,其中miR-31对其发挥负向靶向调节作用。
Background
Acute or chronic inflammation can be induced by ionizing radition, which will damage the immune balance. The important immune regulatory subset is Treg, its change in number, percentage or phenotype will directly influence the equilibrium. For this reason, many studies were recently carried out on radition sensitivity of Treg cells. As the studies on Treg cells were going deeply into, many transcription factors and surface molecules were revealed which were related to Treg cell phenotype and function. However there were differential expression of these proteins in different type of imflammation and infectious tissues. To reveal which protein will be play a role in radiation indueced inflammation, many studies should be carried out.
Objective
To analyze the influence of different dose γ-ray irradiation on the number of lymphocytes and their Treg subset, and to explicit the radiation induced changes of apoptosis, proliferation and phenotype of Treg cells. Then to further analyze the relation between apoptosis and differently expressed phenotype factor, and do the expression of microRNAs which target effect on the factor. Then disscuss the mechanism of which the phenotype factor is regulated during its transcription, which will estabilish the theoretical foundation to revover the immune imbalance induced by ionizing radiation.
Methods
1. Mice were administered with whole body irradiation of γ-rays at different doses, and lymphocytes were separated from thymus and spleen, then the number of total cells were counted and the percentages of CD4+T and CD4+FOXP3+CD25+Treg lymphocytes were analyzed using FCM.
2. The lymphocytes in vitro were irradiated by2Gy γ-ray irradiation and the7-AAD combining rate of Treg and Tcon were detected using FCM in9h after exposure.
3. The Annexin-V and Ki-67expression level of Treg and Tcon cells ex vivo were detected as well as the expression level of the transcription factor FOXP3and Helios, the surface molecules CD39and CD103, and apoptosis related proteins Bcl-2and Bax using FCM.
4. The microRNAs target effect on CD39were retrieved through different database, the their amplification in Treg cells were analysed using real-time PCR.
5. CD4+CD25+Treg cells were sorted and their intracellular ATP concentration were measured using ATP concentration assay kit and microplate reader.
Results
1. The lymphocyte numbers in thymus and spleen decreased in dose-dependent manner and reached to the minimum at4d after irradiation,(F=118.08,144.01,.P<0.05); Exposure to higher dose(more than1Gy) decreased Treg number time-dependently in thymus, however increased it in spleen; On the contrary, exposure to lower dose(less than0.75Gy) increased Treg number in thymus; Besides the percentage of Treg cells increased dose dependently(in thymus F=5.16,89.44,3.01, P<0.05; in spleen F=52.02,32.13,27.45, P<0.05).
2. The survival rate of both Treg and Tcon cells in vitro decreased after irradiation and the latter one decreased more significantly although it was higher in control group.
3. The Treg cell apoptosis rate increased significantly and was higher than the one of Tcon cells in4d after irradiation. The apoptosis and proliferation rate of Treg cells were different in different tissues. For proliferation, the percentage of Ki-67+Treg cells in thymus was decreased in1d and reversed in4d, however the trendency was inverse in spleen.
4. The percentage of FOXP3+Treg cells increased after irradiation and the MFI of FOXP3in Treg cell was decreased in thymus and increase in spleen. Both striking changes were happened in10d after irradiation. Besides the expression level of Helios kept relatively stable.
5. The CD39and CD103of Treg cells were differently expressed after irradiation. The percentage of CD39+Treg cells increased and CD39expression level of Treg cell was upregulated in thymus but inverse in spleen. The changes of the percentage of CD103+Treg cells were complicated than CD39, it was firtly increased then reverse in spleen, however there was a transient increase in thymus in4d. and CD103expression level of Treg cell was down regulated in thymus but inverse in spleen.
6. It was Bax that its MFI significantly increased but the Bcl-2in thymus and it was reverse in spleen that the MFI of Bcl-2increased, which resulted in the ratio of Bax/Bcl-2increase in thymus and inverse in spleen.
7. Six microRNAs were found which target effect on CD39, and the expression changing trendency of miR-31was similar with the one of CD39.
Conclusion
1. The cell number changes of lymphocytes and their Treg subset in dose dependent manner, and there are different time-response between lymphocytes and their Treg subsets.
2. Treg cells in vitro were resistant to irradiation, however the apoptosis rate of the ex vivo ones increased significantly after irradiation. The proliferation rate of Treg cells induced by radiation changes in time and tissue dependent manner, which revealed that the survival of Treg cells are significantly influenced by the microenvironment.
3. FOXP3of Treg cells expressed differently after irradiation as well as the phenotype factors CD39and CD103. The CD39is related to the radiation induced apoptosis and CD103helps Treg cells immigration.
4. Bax and Bcl-2respectively take part in the apoptosis program of Treg cells from thymus and spleen. And CD39may play a role in the program.
5. The expression of CD39can be regulated by genes in which miR-31may play a negative role.
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