本文選題：γδT細(xì)胞 + 胚胎胸腺器官培養(yǎng)(FTOC)　； 參考：《中國醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 前言 多能淋巴干細(xì)胞遷移至胸腺,從胸腺的皮質(zhì)逐漸移行到髓質(zhì),經(jīng)過陽性選擇和陰性選擇,逐步分化成熟為T細(xì)胞。T細(xì)胞表面的抗原受體(T cell receptor)TCR有兩類,一類是由α鏈和β鏈組成的TCRαβ,另一類是由γ鏈和δ鏈組成的TCRγδ。迄今為止,關(guān)于TCRγδ細(xì)胞分化成熟的過程以及生理功能、識(shí)別抗原的特點(diǎn)等方面還所知不多,遠(yuǎn)沒有TCRαβ細(xì)胞那樣詳盡。γδT細(xì)胞主要分布于皮膚和粘膜組織,數(shù)量較少,但它在抵御外界病原體侵害、免疫調(diào)節(jié)和腫瘤免疫等諸多方面都有著不可替代的作用。因此,γδT細(xì)胞也引起了眾多學(xué)者的關(guān)注。近幾年的研究表明:γδT細(xì)胞以胸腺內(nèi)和胸腺外兩條途徑發(fā)育。胸腺內(nèi)發(fā)育的依據(jù)是應(yīng)用骨髓干細(xì)胞在胸腺組織內(nèi)培養(yǎng)可以誘導(dǎo)分化為γδT細(xì)胞。Ciofani M等人在2007年的研究中發(fā)現(xiàn)大量的胸腺衍生信號(hào)和高度保守的分化途徑,促使T細(xì)胞逐步分化成熟。T細(xì)胞的發(fā)育是以級(jí)聯(lián)方式有序發(fā)生的,TCR基因的啟動(dòng)順序依次為TCR D(δ)-TCRG(γ)-TCRB(β)-TCRA(α),αβT細(xì)胞的分化不可能繞過TCRD(δ)-TCRG(γ)基因的表達(dá)而直接表達(dá)αβTCR。故推測αβT細(xì)胞來自于γδT細(xì)胞,γδT細(xì)胞應(yīng)處于固有免疫應(yīng)答和適應(yīng)性免疫應(yīng)答的中間過渡階段。過渡性免疫應(yīng)答理論認(rèn)為部分胸腺γδT細(xì)胞可分化為αβT細(xì)胞。我們以胎齡14.5d C57BL/6胎鼠為研究對象,采用胚胎胸腺器官培養(yǎng)(FTOC)、CFSE染色及流式細(xì)胞分選和檢測等技術(shù)來觀察胸腺γδT細(xì)胞的分化去向,為過渡性免疫應(yīng)答提供科學(xué)的理論依據(jù)。 實(shí)驗(yàn)材料 一、主要試劑 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;培養(yǎng)液Ⅰ:20%胎牛血清RPMI1640;培養(yǎng)液Ⅱ:20%成鼠血清RPMI1640(除血清不同外,兩種培養(yǎng)液其他成分相同)等。 二、主要儀器 體視顯微鏡;流式細(xì)胞儀;熒光顯微鏡;顯微外科鑷、剪;CO_2恒溫培養(yǎng)箱;倒置顯微鏡;臺(tái)式離心機(jī);小離心機(jī);超凈空氣凈化臺(tái);酶標(biāo)儀;超聲波細(xì)胞破碎儀80W等。 三、試驗(yàn)動(dòng)物 SPF級(jí)14.5d C57BL/6孕鼠(雌雄比例為2:1,晚8點(diǎn)合籠過夜,第二天檢查出現(xiàn)陰道栓的為第0.5d)。C57BL/6小鼠(5-6周齡)購于中國醫(yī)學(xué)科學(xué)院北京試驗(yàn)動(dòng)物中心,許可證編號(hào):SCXK(北京)2004-0001。 實(shí)驗(yàn)方法 一、體外胚胎胸腺器官培養(yǎng)(FTOC)方法的建立 將無菌明膠海綿置于24孔板中,將經(jīng)消毒處理過的無菌0.8μm微孔濾膜(剪成直徑約1ml的圓形)置于凝膠海綿上。分為兩個(gè)實(shí)驗(yàn)組,第Ⅰ組加1ml的培養(yǎng)液Ⅰ;第Ⅱ組加1ml的培養(yǎng)液Ⅱ。無菌取胎齡14.5d C57BL/6胎鼠胸腺,隨機(jī)分別放置在浸于不同培養(yǎng)液中的濾膜上,一個(gè)胎鼠的兩個(gè)胸腺小葉放入不同的實(shí)驗(yàn)組,每孔4個(gè)小葉。胚胎胸腺器官保持在氣液界面中,置37℃,5%體積分?jǐn)?shù)CO_2孵箱孵育,每隔3d更換培養(yǎng)液。隔天檢測胚胎胸腺細(xì)胞亞群隨體外培養(yǎng)時(shí)間延長的變化。 二、不同胎齡胎鼠體內(nèi)胸腺細(xì)胞表達(dá)γδTCR的時(shí)相 采用熒光抗體標(biāo)記、流式檢測技術(shù),分別檢測12.5d、13.5d、14.5d、15.5d、16.5d各胎齡C57BL/6胎鼠體內(nèi)胸腺細(xì)胞表達(dá)γδTCR的時(shí)相。 三、刺激因素的濃度及其促進(jìn)增殖的時(shí)相 刺激因素包括Mtb-Ag和IL-2。制備C57BL/6小鼠胸腺單細(xì)胞懸液,接種到2塊96孔培養(yǎng)板中,分為實(shí)驗(yàn)組和對照組,實(shí)驗(yàn)組分別加入5μg/ml、7μg/ml、9μg/ml三個(gè)不同濃度的刺激因素(Mtb-Ag)10μl,對照組加入等量的等滲0.1M的PBS。置37℃、5%體積分?jǐn)?shù)CO_2孵箱孵育,繼續(xù)孵育。在第3、6d,采用MTT比色法觀察不同濃度的Mtb-Ag對C57BL/6小鼠胸腺細(xì)胞的增殖變化。以刺激因素的最佳濃度和14.5d胎齡C57BL/6小鼠胸腺單細(xì)胞懸液體外共培養(yǎng),分別于第0、2、4、6、8 d標(biāo)記γ鏈熒光抗體,檢測胸腺γδT細(xì)胞的增殖時(shí)相。 四、純化胸腺γδT細(xì)胞與CFSE染色 制備14.5d胎齡C57BL/6小鼠胸腺單細(xì)胞懸液,體外應(yīng)用刺激因素培養(yǎng)2d后,標(biāo)記γ鏈熒光抗體,進(jìn)行流式分選純化,然后以CFSE最佳濃度染色。 五、胸腺γδT細(xì)胞的分化去向 排除γ鏈熒光抗體的干擾作用,將FCSE染色的胸腺γδT細(xì)胞加入胚胎胸腺器官的濾膜上進(jìn)行孵育,分別在第5、8d加入抗β鏈的熒光抗體,流式檢測其分化去向。 六、統(tǒng)計(jì)學(xué)分析 實(shí)驗(yàn)數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差表示,組間差異比較采用單因素方差分析(one-wayANOVA),用SPSS16.0軟件完成。P0.05有顯著性差異。 結(jié)果 FTOC系統(tǒng)中使用培養(yǎng)液Ⅰ能更好的接近于體內(nèi)前體T細(xì)胞的發(fā)育過程;14.5d胎齡小鼠體內(nèi)胸腺細(xì)胞表達(dá)γδTCR為總胸腺細(xì)胞數(shù)的15.07%,和12.5d、13.5d、15.5d、16.5d胎齡小鼠體內(nèi)胸腺細(xì)胞相比,所占比例最高;Mtb-Ag 7μg/ml和5μg/ml、9μg/ml相比較對小鼠胸腺細(xì)胞的增殖有明顯的促進(jìn)作用(p0.05);加入適宜濃度的刺激因素,體外培養(yǎng)第2d胸腺γδT細(xì)胞增殖達(dá)到高峰;用于分選的胸腺細(xì)胞的濃度為1×10~7個(gè)/ml共1ml,分選的陽性細(xì)胞為3%;30μm的CFSE是最佳試驗(yàn)濃度;第5d由CFSE染色的胸腺γδT細(xì)胞分化為αβT細(xì)胞的分化率為8.12%,第8d為3.36%。 討論 分子遺傳學(xué)研究證明,在胚胎期多能淋巴干細(xì)胞進(jìn)入胸腺時(shí),其受體基因還未發(fā)生重排,胚胎14.5d發(fā)現(xiàn)重排的TCRγ轉(zhuǎn)錄達(dá)高峰,TCRβ基因開始重排,TCRα基因重排晚于TCRβ基因2d。07年有學(xué)者發(fā)現(xiàn)TCRγ、δ主要在胚胎胸腺未成熟的T細(xì)胞膜上表達(dá),而TCRα、β主要在成熟T細(xì)胞膜上表達(dá)。14.5d胎鼠胸腺T細(xì)胞基本處于原始、未成熟階段,取其進(jìn)行體外培養(yǎng),隨著體外培養(yǎng)時(shí)間的推移,T細(xì)胞通過陽性和陰性選擇,不斷地分化為CD4~+CD8~+細(xì)胞,并進(jìn)一步分化成熟為CD4~+CD8~-和CD4~-CD8~+細(xì)胞,待體外胸腺培養(yǎng)8d后已有相當(dāng)數(shù)量的T細(xì)胞分化成熟為單陽性細(xì)胞。故14.5d胎齡鼠的胸腺細(xì)胞是進(jìn)行胸腺細(xì)胞發(fā)育研究的最佳起始時(shí)間。因此,本實(shí)驗(yàn)選用14.5d胎鼠胸腺器官采用FTOC技術(shù)進(jìn)行體外培養(yǎng)。在培養(yǎng)液的液相中我們發(fā)現(xiàn)大量的CD4~+CD8~+雙陽性細(xì)胞輸出胸腺,其機(jī)制還不明確,但為我們輸入CFSE染色的胸腺γδT細(xì)胞提供了胸腺空間微環(huán)境,在此環(huán)境中,CFSE染色的胸腺γδT細(xì)胞在第5、8d分化為αβT細(xì)胞的分化率分別為8.12%[812/(1×10~4個(gè)/ml×0.1ml)]和3.36%[336/(1×10~4個(gè)/ml×0.1ml)]。07年有學(xué)者研究發(fā)現(xiàn)胚胎豬胸腺的CD4~+γδT細(xì)胞代表過渡狀態(tài)和獨(dú)立亞型,不輸出胸腺;胸腺CD4~+γδT細(xì)胞能在適當(dāng)?shù)呐囵B(yǎng)條件下分化為TCRαβ細(xì)胞,這也為過渡性免疫應(yīng)答提供了科學(xué)的理論依據(jù)。 結(jié)論 經(jīng)純化與CFSE染色的14.5d胎鼠胸腺γδT細(xì)胞在體外胚胎胸腺器官中孵育可分化為αβT細(xì)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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392.9

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