胸腺γδT细胞的分化去向

发布时间：2018-06-08 04:49

本文选题：γδT细胞 + 胚胎胸腺器官培养(FTOC)　；参考：《中国医科大学》2009年硕士论文

【摘要】： 前言多能淋巴干细胞迁移至胸腺,从胸腺的皮质逐渐移行到髓质,经过阳性选择和阴性选择,逐步分化成熟为T细胞。T细胞表面的抗原受体(T cell receptor)TCR有两类,一类是由α链和β链组成的TCRαβ,另一类是由γ链和δ链组成的TCRγδ。迄今为止,关于TCRγδ细胞分化成熟的过程以及生理功能、识别抗原的特点等方面还所知不多,远没有TCRαβ细胞那样详尽。γδT细胞主要分布于皮肤和粘膜组织,数量较少,但它在抵御外界病原体侵害、免疫调节和肿瘤免疫等诸多方面都有着不可替代的作用。因此,γδT细胞也引起了众多学者的关注。近几年的研究表明:γδT细胞以胸腺内和胸腺外两条途径发育。胸腺内发育的依据是应用骨髓干细胞在胸腺组织内培养可以诱导分化为γδT细胞。Ciofani M等人在2007年的研究中发现大量的胸腺衍生信号和高度保守的分化途径,促使T细胞逐步分化成熟。T细胞的发育是以级联方式有序发生的,TCR基因的启动顺序依次为TCR D(δ)-TCRG(γ)-TCRB(β)-TCRA(α),αβT细胞的分化不可能绕过TCRD(δ)-TCRG(γ)基因的表达而直接表达αβTCR。故推测αβT细胞来自于γδT细胞,γδT细胞应处于固有免疫应答和适应性免疫应答的中间过渡阶段。过渡性免疫应答理论认为部分胸腺γδT细胞可分化为αβT细胞。我们以胎龄14.5d C57BL/6胎鼠为研究对象,采用胚胎胸腺器官培养(FTOC)、CFSE染色及流式细胞分选和检测等技术来观察胸腺γδT细胞的分化去向,为过渡性免疫应答提供科学的理论依据。实验材料一、主要试剂 APC-抗CD4;PE-抗CD8;Cy5-抗CD3;CFSE;羊抗鼠的一抗sc-1778 TCRγ:IgG和Texa Red-抗TCRγ二抗;羊抗鼠的一抗sc-9101 TCRβ:IgG和Texa Red-抗TCRβ二抗;DMSO;Mtb-Ag;IL-2;培养液Ⅰ:20%胎牛血清RPMI1640;培养液Ⅱ:20%成鼠血清RPMI1640(除血清不同外,两种培养液其他成分相同)等。二、主要仪器体视显微镜;流式细胞仪;荧光显微镜;显微外科镊、剪;CO_2恒温培养箱;倒置显微镜;台式离心机;小离心机;超净空气净化台;酶标仪;超声波细胞破碎仪80W等。三、试验动物 SPF级14.5d C57BL/6孕鼠(雌雄比例为2:1,晚8点合笼过夜,第二天检查出现阴道栓的为第0.5d)。C57BL/6小鼠(5-6周龄)购于中国医学科学院北京试验动物中心,许可证编号:SCXK(北京)2004-0001。实验方法一、体外胚胎胸腺器官培养(FTOC)方法的建立将无菌明胶海绵置于24孔板中,将经消毒处理过的无菌0.8μm微孔滤膜(剪成直径约1ml的圆形)置于凝胶海绵上。分为两个实验组,第Ⅰ组加1ml的培养液Ⅰ;第Ⅱ组加1ml的培养液Ⅱ。无菌取胎龄14.5d C57BL/6胎鼠胸腺,随机分别放置在浸于不同培养液中的滤膜上,一个胎鼠的两个胸腺小叶放入不同的实验组,每孔4个小叶。胚胎胸腺器官保持在气液界面中,置37℃,5%体积分数CO_2孵箱孵育,每隔3d更换培养液。隔天检测胚胎胸腺细胞亚群随体外培养时间延长的变化。二、不同胎龄胎鼠体内胸腺细胞表达γδTCR的时相采用荧光抗体标记、流式检测技术,分别检测12.5d、13.5d、14.5d、15.5d、16.5d各胎龄C57BL/6胎鼠体内胸腺细胞表达γδTCR的时相。三、刺激因素的浓度及其促进增殖的时相刺激因素包括Mtb-Ag和IL-2。制备C57BL/6小鼠胸腺单细胞悬液,接种到2块96孔培养板中,分为实验组和对照组,实验组分别加入5μg/ml、7μg/ml、9μg/ml三个不同浓度的刺激因素(Mtb-Ag)10μl,对照组加入等量的等渗0.1M的PBS。置37℃、5%体积分数CO_2孵箱孵育,继续孵育。在第3、6d,采用MTT比色法观察不同浓度的Mtb-Ag对C57BL/6小鼠胸腺细胞的增殖变化。以刺激因素的最佳浓度和14.5d胎龄C57BL/6小鼠胸腺单细胞悬液体外共培养,分别于第0、2、4、6、8 d标记γ链荧光抗体,检测胸腺γδT细胞的增殖时相。四、纯化胸腺γδT细胞与CFSE染色制备14.5d胎龄C57BL/6小鼠胸腺单细胞悬液,体外应用刺激因素培养2d后,标记γ链荧光抗体,进行流式分选纯化,然后以CFSE最佳浓度染色。五、胸腺γδT细胞的分化去向排除γ链荧光抗体的干扰作用,将FCSE染色的胸腺γδT细胞加入胚胎胸腺器官的滤膜上进行孵育,分别在第5、8d加入抗β链的荧光抗体,流式检测其分化去向。六、统计学分析实验数据采用均数±标准差表示,组间差异比较采用单因素方差分析(one-wayANOVA),用SPSS16.0软件完成。P0.05有显著性差异。结果 FTOC系统中使用培养液Ⅰ能更好的接近于体内前体T细胞的发育过程;14.5d胎龄小鼠体内胸腺细胞表达γδTCR为总胸腺细胞数的15.07%,和12.5d、13.5d、15.5d、16.5d胎龄小鼠体内胸腺细胞相比,所占比例最高;Mtb-Ag 7μg/ml和5μg/ml、9μg/ml相比较对小鼠胸腺细胞的增殖有明显的促进作用(p0.05);加入适宜浓度的刺激因素,体外培养第2d胸腺γδT细胞增殖达到高峰;用于分选的胸腺细胞的浓度为1×10~7个/ml共1ml,分选的阳性细胞为3%;30μm的CFSE是最佳试验浓度;第5d由CFSE染色的胸腺γδT细胞分化为αβT细胞的分化率为8.12%,第8d为3.36%。讨论分子遗传学研究证明,在胚胎期多能淋巴干细胞进入胸腺时,其受体基因还未发生重排,胚胎14.5d发现重排的TCRγ转录达高峰,TCRβ基因开始重排,TCRα基因重排晚于TCRβ基因2d。07年有学者发现TCRγ、δ主要在胚胎胸腺未成熟的T细胞膜上表达,而TCRα、β主要在成熟T细胞膜上表达。14.5d胎鼠胸腺T细胞基本处于原始、未成熟阶段,取其进行体外培养,随着体外培养时间的推移,T细胞通过阳性和阴性选择,不断地分化为CD4~+CD8~+细胞,并进一步分化成熟为CD4~+CD8~-和CD4~-CD8~+细胞,待体外胸腺培养8d后已有相当数量的T细胞分化成熟为单阳性细胞。故14.5d胎龄鼠的胸腺细胞是进行胸腺细胞发育研究的最佳起始时间。因此,本实验选用14.5d胎鼠胸腺器官采用FTOC技术进行体外培养。在培养液的液相中我们发现大量的CD4~+CD8~+双阳性细胞输出胸腺,其机制还不明确,但为我们输入CFSE染色的胸腺γδT细胞提供了胸腺空间微环境,在此环境中,CFSE染色的胸腺γδT细胞在第5、8d分化为αβT细胞的分化率分别为8.12%[812/(1×10~4个/ml×0.1ml)]和3.36%[336/(1×10~4个/ml×0.1ml)]。07年有学者研究发现胚胎猪胸腺的CD4~+γδT细胞代表过渡状态和独立亚型,不输出胸腺;胸腺CD4~+γδT细胞能在适当的培养条件下分化为TCRαβ细胞,这也为过渡性免疫应答提供了科学的理论依据。结论经纯化与CFSE染色的14.5d胎鼠胸腺γδT细胞在体外胚胎胸腺器官中孵育可分化为αβT细胞。
[Abstract]:Preface
The pluripotent lymphoid stem cells migrate to the thymus and move from the cortex of the thymus to the medulla. After positive selection and negative selection, the antigen receptor (T cell receptor) TCR, which is gradually differentiated and mature to the surface of T cell.T cells, has two categories, one is the TCR a beta composed of alpha chain and beta chain, and the other is TCR gamma delta composed of gamma chain and delta chain. So far, There is not much knowledge about the process of differentiation and maturation of TCR - y delta cells, the physiological functions, and the identification of antigen, far from the details of TCR alpha beta cells. The number of gamma delta T cells is mainly distributed in the skin and mucous tissue, and the number is less, but it has not been replaced in many aspects such as resistance to external pathogens, immune regulation and tumor immunity. Therefore, the gamma delta T cells have also aroused the attention of many scholars. In recent years, it has been shown that gamma delta T cells develop in two ways in the thymus and outside the thymus. The basis of the development of the thymus gland is that the application of bone marrow stem cells in the thymus tissue can induce the differentiation into the.Ciofani M cell of the gamma delta T cells and so on in 2007. The thymus derived signal and highly conserved differentiation pathway promote the development of T cells to gradually differentiate and mature.T cells in order of cascade, and the sequence of TCR genes in sequence is TCR D (delta) -TCRG (y) -TCRB (beta) -TCRA (a), and the differentiation of alpha beta T cells can not bypass the expression of TCRD (delta) -TCRG (gamma) gene and directly express alpha beta TCR.. It is presumed that the alpha beta T cells are derived from the gamma delta T cells, and that the gamma delta T cells should be in the intermediate stage of the inherent immune response and adaptive immune response. The transitional immune response theory suggests that the partial thymus Delta T cells can differentiate into alpha beta T cells. We take fetal 14.5d C57BL/6 fetal mice as the research object, using embryonic thymus organ culture (FTOC) and CFSE staining. And the technology of flow cytometry sorting and detection to observe the differentiation direction of thymus gamma delta T cells, and provide a scientific theoretical basis for transitional immune response.
Experimental materials
First, major reagents
APC- anti CD4; PE- anti CD8; Cy5- anti CD3; CFSE; one anti sc-1778 TCR Gamma: IgG and Texa Red- against CD8 two. The same).
Two, the main instrument
Stereoscopic microscope; flow cytometry; fluorescence microscope; microsurgical forceps, scissors; CO_2 constant temperature incubator; inverted microscope; table centrifuge; small centrifuge; ultra clean air purification table; enzyme labeling instrument; Ultrasonic Cell Crusher 80W and so on.
Three, experimental animals
SPF 14.5d C57BL/6 pregnant rats (female and male 2:1, night at 8 points late, second days to examine the occurrence of vaginal suppository as 0.5d).C57BL/6 mice (5-6 weeks old) purchased at the Beijing experimental animal center of the Chinese Academy of Medical Sciences, license number: SCXK (Beijing) 2004-0001.
Experimental method
Establishment of an in vitro embryo thymus organ culture (FTOC) method
The aseptic gelatin sponge was placed in 24 Kong Banzhong, and the sterile 0.8 m microporous filter membrane, which was cut into a circular diameter of about 1ml, was placed on the gel sponge. It was divided into two experimental groups, the first group and the 1ml culture medium I, the second group and the 1ml culture medium II. The sterile fetal age 14.5d C57BL/6 fetal rat thymus was randomly placed in the different culture. On the filter membrane in the nutrient solution, two thymic lobules of a fetal rat were placed in a different experimental group and 4 lobules per pore. The embryonic thymus organs were kept in the gas-liquid interface, placed at 37 degrees C, 5% volume fraction CO_2 incubated and replaced every 3D.
Two, the time of expression of gamma delta TCR in thymocytes of fetal rats in different gestational ages.
The expression of 12.5d, 13.5d, 14.5d, 15.5d and 16.5d was detected by fluorescent antibody labeling and flow cytometry in thymocytes of fetal rats of different gestational ages.
Three, the concentration of stimulant and the time to promote proliferation.
The stimulation factors included Mtb-Ag and IL-2. preparation of C57BL/6 mouse thymus single cell suspension, inoculated into 2 96 hole culture plates, divided into experimental group and control group. The experimental group added 5 mu g/ml, 7 mu g/ml, 9 micron g/ml at three different concentrations (Mtb-Ag) 10 mu L, the control group added equal amount of 0.1M PBS. for 37 C, 5% volume fraction CO_2 incubator. In 3,6d, the proliferation of thymus cells of different concentrations of Mtb-Ag on C57BL/6 mice was observed by MTT colorimetry. The optimal concentration of stimulation factors and the co culture of 14.5d fetal C57BL/6 mouse thymus single cell suspension were co cultured, and the 0,2,4,6,8 D labeled gamma chain fluorescent antibody was used to detect the proliferation phase of the thymus Delta T cells.
Four, purification of thymus Delta T cells and CFSE staining
The thymic single cell suspension of 14.5d fetal age C57BL/6 mice was prepared. After the stimulation of 2D in vitro, the gamma chain fluorescent antibody was labeled and purified by flow sorting, and then stained with the best concentration of CFSE.
Five, differentiation and direction of thymus Delta T cells
The interference of gamma chain fluorescent antibody was excluded, and FCSE stained thymus Delta T cells were incubated on the filter membrane of the embryonic thymus organs. The fluorescent antibody against beta chain was added in 5,8d, and the direction of differentiation was detected by flow cytometry.
Six, statistical analysis
The experimental data were expressed by means of mean + standard deviation. The difference between groups was compared by one-way ANOVA (one-wayANOVA), and there was significant difference between SPSS16.0 and.P0.05.
Result
The use of culture fluid I in FTOC system can better close to the development of T cells in the body of the body; the thymus cells in the 14.5d fetal mice express 15.07% of the total number of thymus cells, and the proportion of the thymus cells in 12.5d, 13.5d, 15.5d, 16.5d fetal mice is the highest, Mtb-Ag 7 mu g/ml and 5 mu g/ml, 9 mu g/ml phase is compared to mice The proliferation of thymus cells was significantly promoted (P0.05); the proliferation of 2D thymus Delta T cells in vitro cultured in vitro was peak; the concentration of thymic cells in vitro was 1 x 10~7 /ml 1ml, the selected positive cells were 3%; 30 mu m CFSE was the best test concentration; 5D CFSE stained thymus Delta T cells The differentiation rate of alpha beta T cells was 8.12% and 8D was 3.36%.
discuss
Molecular genetics studies have shown that the receptor gene has not rearranged when the pluripotent lymphoid stem cells enter the thymus at the embryonic stage, the TCR gamma transcription of the rearrangement of the embryo 14.5d is peak, the TCR beta gene begins to rearrange, and the TCR alpha gene rearrangement later than the TCR beta gene in 2d.07 has been found to be TCR gamma, and the delta is mainly expressed on the immature T cell membrane of the embryonic thymus. And TCR alpha, beta mainly expressed on the mature T cell membrane on the.14.5d fetal rat thymus T cells, which were basically in the original, immature stage, and were cultured in vitro. With the time of culture in vitro, T cells were selectively differentiated into CD4~+CD8~+ cells through positive and negative selection, and further differentiated into CD4~+CD8~- and CD4~-CD8~+ cells. A considerable number of T cells have been differentiated and mature into single positive cells after 8D culture in the outer thymus. Therefore, the thymus cells of 14.5d fetal mice are the best starting time for the study of thymic cell development. Therefore, the thymus organs of 14.5d fetal mice were cultured in vitro by FTOC technology. We found a large number of CD4~+CD8 in the liquid phase of the culture. The mechanism of ~ + double positive cells output thymus is not clear, but it provides a thymic space microenvironment for the thymus Delta T cells which we enter CFSE staining. In this environment, the differentiation rate of CFSE stained thymus Delta T cells in 5,8d to alpha beta T cells is 8.12%[812/ (1 x 10~ 4 /ml x 0.1ml)] and 3.36%[336/ (1 * 10~4) In the past 7 years, some scholars have found that the CD4~+ gamma delta T cells in the thymus of the embryo of the embryo represent the transition state and independent subtype, and do not output the thymus. The thymus CD4~+ gamma delta T cells can differentiate into TCR alpha beta cells under appropriate culture conditions, which also provides scientific theoretical basis for the transitional immune response.
conclusion
The purified CFSE staining of 14.5d fetal mouse thymus gamma delta T cells can be differentiated into alpha beta T cells in vitro.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.9

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