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Tscl对巨噬细胞生存和功能的影响

发布时间:2018-07-03 01:06

  本文选题:间充质干细胞 + 胚胎干细胞 ; 参考:《北京协和医学院》2015年博士论文


【摘要】:目的:多能干细胞包括胚胎干细胞(ES)和诱导多能干细胞(iPS),作为一类具有无限自我更新和多向分化潜能的细胞群体,能进一步分化为多种类型的细胞。MSC临床应用前景广阔,是细胞替代治疗和组织工程的首选种子细胞,是移植领域和自身性免疫疾病治疗的研究热点。探索全反式维甲酸(RA)和SB431542对胚胎干细胞(ES)和诱导多能干细胞(iPS)分化为间充质干细胞(MSC)样细胞的影响。方法:将培养的鼠ES和iPS根据不同的分化条件分为对照组、SB431542。组及RA组不同浓度组,对照组为DMEM完全培养基,SB431542组为DMEM完全培养基中含10 I.tM的SB431542,RA组的DMEM完全培养基中含RA浓度分别为0.05、0.10、0.20、0.40 nM。分化培养4 d后观察细胞形态,流式细胞术检测细胞表面标志物CDl05和干细胞抗原1(Sca-1)的表达,以CD105+细胞和Sca-1-细胞的比例确定iPS和ES向MSC分化的程度。结果:1)小鼠ES和iPS在ESGRO-2i培养基里生长较好。ES分化4 d后,对照组和SB431542组ES和iPS分化成内皮样细胞,在含有不同浓度RA的培养基中ES和iPS可有效分化为纤维状细胞;2)ES分化4 d后,SB431542组Sca-1+细胞比例显著高于对照组(P0.05),CD105+细胞比例与对照组比较差异无统计学意义(P0.05)。在RA浓度为0.05、0.10、0.20、0.40 nM组中,ES分化的CD105+细胞比例显著高于对照组和SB431542组(P0.05),Sca-1+细胞比例显著高于对照组(P0.05),但与SB431542组比较显著降低(P0.05);RA浓度为0.20 nM组的CD105+和Sca-1+细胞比例显著高于RA浓度为0.05、0.10、0.40 nM组(P0.05);3)iPS分化4 d后,SB431542组CD105+细胞和Sca-1+细胞比例显著高于对照组(P0.05)。在RA浓度为0.05、0.10、0.20、0.40 nM组中,iPS分化的CD105+细胞比例显著高于对照组和SB431542组(P0.05),Sca-1+细胞比例显著高于对照组(P0.05),但与SB431542组比较均显著降低(P0.05);RA浓度为0.20nM组Sca-1+细胞比例显著高于RA浓度为0.05、0.10、0.40 nM组(P0.05),RA浓度为0.20 nM组CD105+细胞比例显著高于RA浓度为0.05、0.10 nM组(P0.05),但与RA浓度为0.40 nM组差异无统计学意义(P0.05)。结论:利用RA可以促进多能干细胞向MSC样细胞分化,分化方法较为简单,分化周期较短,为多能干细胞向MSC样细胞的分化提供了一定的方法基础。背景:结节硬化复合物1(Tscl)和结节硬化复合物2(Tsc2)结合形成一个复合物,这个复合物可以通过哺乳动物雷帕霉素靶蛋白1(mTORC1)来调节细胞代谢和能量的稳定,TSC1和TSC2功能的丢失可引起mTORC1活性的升高,进而引起细胞体积增大、增殖能力增强。有研究称Tscl调节巨噬细胞M1/M2的极化,但对于Tscl对巨噬细胞的功能和自稳的精确调控作用尚不明确,本研究我们揭示Tscl对于调节巨噬细胞的生存、生长、迁移和吞噬功能具有十分重要的作用。材料与方法:我们将由Lysozyme启动子控制表达Cre重组酶的LysMCre鼠与Tsc1flox/flox鼠交配获得髓系细胞特异性的Tscl条件性敲除鼠(LysMCreTsc1flox/flox);PCR鉴定鼠的基因型,western blot鉴定鼠巨噬细胞Tscl蛋白的敲除情况;巨噬细胞的凋亡和生长由流式细胞术、RT-PCR进行检测;巨噬细胞精氨酸酶活化由QuantiChromTM arginase assay kit进行检测;M1和M2相关的基因的mRNA的表达由RT-PCT进行检测;巨噬细胞对T细胞活化的功能通过CD4+T细胞和巨噬细胞体外共培养3天后CD4+T细胞的活化进行鉴定;巨噬细胞的吞噬功能由VybrantTM phagocytosis assay试剂盒进行检测;巨噬细胞的迁移由Transwell实验进行检测;脾脏细胞和淋巴结细胞Foxp3的染色是通过Treg检测试剂盒进行。结果:Tsc1 KO鼠巨噬细胞凋亡增加、细胞变大,腹腔注射Rapa可部分抑制Tscl缺陷引起的巨噬细胞的凋亡和细胞生长;在稳定和炎症状态下,Tscl缺陷的巨噬细胞在稳定和诱导作用下M1和M2特性均较WT巨噬细胞高,抑制mTORC1部分逆转了Tscl缺陷引起的巨噬细胞的M2和M1的变化;Tscl缺陷的巨噬细胞CCR2和CCR5趋化受体表达降低、迁移能力下降、趋化因子升高,抑制mTORC1部分逆转了Tscl缺陷引起的巨噬细胞的趋化因子和趋化受体的变化;另外,Tscl缺陷的巨噬细胞的吞噬能力和产生活性氧(ROS)的能力增加;与Tsc1缺陷的巨噬细胞共培养的CD4+T细胞的增殖和活化功能减弱。结论:1)Tscl促进巨噬细胞的生存、抑制巨噬细胞的凋亡,并且这两种作用是与]mTORC1的活性有关。CD71和CD98可能参与了Tsc1调节的巨噬细胞的生长;2)Tsc1促进巨噬细胞M1和M2的特性,Tsc1缺陷的巨噬细胞在稳定状态下,分别增高了M1和M2的特性。在LPS和IL-4刺激下,Tsc1缺陷的巨噬细胞分布增高了M1和M2的极化特性;3)Tsc1促进巨噬细胞的迁移,Tscl缺陷的巨噬细胞的CCR2和CCR5的趋化受体表达降低,CCL2趋化引起的迁移能力降低。另外,Tsc1缺陷的巨噬细胞趋化因子表达升高。Tscl抑制巨噬细胞的吞噬和ROS的产生,促进CD4+T细胞的活化;4) Tscl KO鼠引起自发的淋巴组织增生性的疾病。脾脏和淋巴结增大,CD4+T细胞和CD8+T细胞活化严重。另外,鼠的体重没有明显的变化。
[Abstract]:Objective : To investigate the effect of pluripotent stem cells ( ES ) on embryonic stem cells ( ES ) and differentiation of pluripotent stem cells ( ES ) into mesenchymal stem cells ( MSC ) - like cells .
( 2 ) After 4 days of ES differentiation , the proportion of Sca - 1 + cells was significantly higher than that in the control group ( P0.05 ) . In the group with RA concentration of 0.05 , 0.10 , 0.20 and 0.40 nM , the percentage of CD105 + cells differentiated in ES was significantly higher than that in the control group ( P0.05 ) , but the proportion of Sca - 1 + cells was significantly higher than that of the control group ( P0.05 ) , but the ratio of Sca - 1 + cells was significantly lower than that of the control group ( P0.05 ) ;
The percentage of CD105 + and Sca - 1 + cells at RA concentration of 0.20 nM was significantly higher than that in RA concentration of 0.05 , 0.10 , 0.40 nM ( P0.05 ) ;
3 ) The proportion of CD105 + cells and Sca - 1 + cells in SB4315group was significantly higher than that in control group ( P0.05 ) after 4 days of differentiation . The percentage of CD105 + cells in the differentiation of the cells was significantly higher than that in the control group ( P0.05 ) , but the proportion of Sca - 1 + cells was significantly higher than that of the control group ( P0.05 ) .
Conclusion : Tscl can be used to regulate the cell metabolism and energy stabilization . The results suggest that Tscl regulates the cell metabolism and energy stabilization by the mammalian rapamycin target protein 1 ( mTORC1 ) .
Apoptosis and growth of macrophages were detected by flow cytometry and RT - PCR .
The activation of macrophages was detected by the Quantizer assay kit .
The expression of mRNA of genes related to M1 and M2 was detected by RT - PCT ;
The activation of CD4 + T cells was confirmed by co - culture of CD4 + T cells and macrophages in vitro .
The phagocytic function of macrophages was detected by the vybrantTM assay kit ;
The migration of macrophages was detected by Transwell experiment ;
Results : The apoptosis and cell growth of macrophages induced by Tscl deficiency were partially inhibited by intraperitoneal injection of Rapa .
In stable and inflammatory conditions , the macrophages of Tscl - deficient macrophages were higher in both M1 and M2 than those of WT macrophages , and the inhibition of mTORC1 partially reversed the changes of M2 and M1 in macrophages caused by Tscl deficiency ;
Tscl - deficient macrophages CCR2 and CCR2 chemokine receptor expression decreased , the migration ability decreased , the chemokine increased , and the inhibition of mTORC1 partially reversed the changes of chemokine and chemokine receptors in macrophages caused by Tscl deficiency ;
In addition , the phagocytic ability of Tscl - deficient macrophages and the ability to produce reactive oxygen ( ROS ) increased ;
Conclusion : 1 ) Tscl promotes the survival of macrophages and inhibits the apoptosis of macrophages .
2 ) Tsc1 promoted the characteristics of macrophages M1 and M2 , and Tsc1 - deficient macrophages increased M1 and M2 respectively in steady state . Under the stimulation of LPS and IL - 4 , the distribution of Tsc1 - deficient macrophages increased the polarization characteristics of M1 and M2 .
3 ) Tscl promotes the migration of macrophages , the expression of CCR2 and CCR2 of Tscl - deficient macrophages decreases , and the migration ability of CCL2 tends to decrease . In addition , the expression of Tscl - deficient macrophages chemokine expression increased . Tscl inhibited the phagocytosis of macrophages and the production of ROS , and promoted the activation of CD4 + T cells .
4 ) Tscl KO mice induced spontaneous lymphoproliferative diseases . The spleen and lymph nodes increased , CD4 + T cells and CD8 + T cells were activated severely .
【学位授予单位】:北京协和医学院
【学位级别】:博士
【学位授予年份】:2015
【分类号】:R392

【共引文献】

相关期刊论文 前10条

1 刘晔;王育t,

本文编号:2091689


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