發(fā)布時(shí)間：2018-06-23 15:37

  本文選題：S1PR5 + NK細(xì)胞��； 參考：《中國人民解放軍醫(yī)學(xué)院》2015年博士論文

【摘要】：研究背景：異基因造血干細(xì)胞移植(allogeneic hematopoietic stem cell transplantation, allo-HSCT)技術(shù)已經(jīng)成為治療血液系統(tǒng)疾病和某些實(shí)體腫瘤的重要方法之一。急性移植物抗宿主病(acute graft versus host disease, aGVHD)仍是影響移植成功率的重要因素。NK細(xì)胞是allo-HSCT后最早恢復(fù)免疫重建的細(xì)胞,目前的研究已經(jīng)證實(shí)NK細(xì)胞可以抑制aGVHD的發(fā)生但不影響移植物抗腫瘤作用。鞘氨醇-1-磷酸受體5(sphingosine-1-phosphate receptor 5, S1PR5)是近年來發(fā)現(xiàn)的表達(dá)于NK細(xì)胞表面的一種G蛋白耦聯(lián)受體,能夠介導(dǎo)NK細(xì)胞由骨髓和淋巴結(jié)向其他組織的遷移,進(jìn)而發(fā)揮免疫學(xué)功能。我們前期的臨床研究發(fā)現(xiàn)allo-HSCT患者中S1PR5表達(dá)水平與aGVHD的發(fā)生率密切相關(guān)。研究目的：主要通過動(dòng)物試驗(yàn)探討S1PR5對(duì)aGVHD的影響及相關(guān)作用機(jī)制。研究方法及結(jié)果：①應(yīng)用CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat and cas9 protein)技術(shù)構(gòu)建S1PR5基因敲除(S1PR5-/-)的小鼠；通過qPCR技術(shù)及蛋白免疫印跡雜交技術(shù)分別在RNA水平及蛋白水平上成功驗(yàn)證了S1PR5-/-純合子小鼠中S1PR5被敲除。②流式細(xì)胞術(shù)分別檢測(cè)S1PR5-/-和WT C57BL/6小鼠體內(nèi)骨髓、脾臟、淋巴結(jié)及外周血中淋巴細(xì)胞亞群分布,結(jié)果發(fā)現(xiàn)S1PR5-/-小鼠體內(nèi),骨髓(P=0.002)、淋巴結(jié)(P=0.016)中NK細(xì)胞比例較WT小鼠明顯增高,但是脾臟(P=0.000)、外周血(P=0.010)中NK細(xì)胞比例較WT小鼠明顯降低。③分別將供者WT(對(duì)照組)或者S1PR5-/-(實(shí)驗(yàn)組)C57BL/6小鼠移植物(脾細(xì)胞+骨髓細(xì)胞)經(jīng)尾靜脈注射入致死劑量照射后的受者BALB/c小鼠體內(nèi),建立aGVHD模型，，隨訪小鼠體重、aGVHD評(píng)分及生存時(shí)間。移植后觀察60天,發(fā)現(xiàn)實(shí)驗(yàn)組小鼠較對(duì)照組小鼠生存率明顯降低(P=0.019)；aGVHD評(píng)分明顯升高(P=0.010)；兩組體重減輕變化沒有統(tǒng)計(jì)學(xué)差異(P=0.119)。④移植后第4天分別取受者小鼠的骨髓、脾臟、淋巴結(jié)及外周血,應(yīng)用流式細(xì)胞術(shù)檢測(cè)不同部位淋巴細(xì)胞亞群分布。移植后第4天,實(shí)驗(yàn)組小鼠骨髓中NK細(xì)胞含量明顯高于對(duì)照組(P=0.036)；而在脾臟、淋巴結(jié)及外周血中,兩組間的NK細(xì)胞含量沒有明顯差異。實(shí)驗(yàn)組小鼠骨髓T細(xì)胞含量明顯低于對(duì)照組小鼠中T細(xì)胞量(P=0.023)；在脾臟、淋巴結(jié)及外周血中,兩組間T細(xì)胞含量均沒有明顯差異。⑤移植后第30天分別取出受者小鼠的骨髓、脾臟、淋巴結(jié)及外周血,應(yīng)用流式細(xì)胞術(shù)檢測(cè)不同組織中淋巴細(xì)胞亞群分布。實(shí)驗(yàn)組小鼠骨髓中NK細(xì)胞含量明顯高于對(duì)照組骨髓中NK細(xì)胞含量(P=0.037)；在脾臟、淋巴結(jié)及外周血中,兩組NK細(xì)胞含量沒有明顯差異。實(shí)驗(yàn)組小鼠外周血中T細(xì)胞含量明顯高于對(duì)照組外周血中T細(xì)胞含量(P=0.031)；在骨髓、脾臟及淋巴結(jié)中,兩組間T細(xì)胞含量沒有明顯差異。研究結(jié)論：① CRISPR/Cas9技術(shù)是建立基因敲除小鼠模型有效而快捷的方法之一；② S1PR5影響NK細(xì)胞的在小鼠體內(nèi)的分布；③供者小鼠中S1PR5表達(dá)缺失能夠加重受者小鼠的aGVHD:④ S1PR5表達(dá)缺失致使NK細(xì)胞由骨髓遷出受阻，外周循環(huán)中對(duì)aGVHD起保護(hù)作用的NK細(xì)胞減少,這可能是受者S1PR5表達(dá)缺失導(dǎo)致aGVHD加重的重要機(jī)制。
[Abstract]:Background: allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become one of the most important methods for the treatment of hematological diseases and some solid tumors. Acute graft versus host disease (acute graft versus host disease, aGVHD) is still an important factor affecting the success rate of transplantation. .NK cells are the earliest cells to restore the immune reconstruction after allo-HSCT. Current studies have shown that NK cells can inhibit the occurrence of aGVHD but do not affect the anti-tumor effect of the grafts. The -1- phosphoric acid receptor 5 (sphingosine-1-phosphate receptor 5, S1PR5) is a G protein coupled receptor expressed on the surface of NK cells in recent years. It can mediate the migration of NK cells from bone marrow and lymph nodes to other tissues, and then play immunological functions. Our previous clinical studies have found that the expression level of S1PR5 in allo-HSCT patients is closely related to the incidence of aGVHD. Objective: To investigate the effect of S1PR5 on aGVHD and the mechanism of its related action through animal experiments. And results: (1) CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat and cas9 protein) was used to construct S1PR5 gene knockout mice. The distribution of lymphocyte subsets in bone marrow, spleen, lymph node and peripheral blood in S1PR5-/- and WT C57BL/6 mice was detected by flow cytometry. The results showed that the proportion of NK cells in S1PR5-/- mice, bone marrow (P=0.002) and lymph node (P=0.016) was significantly higher than that of WT mice, but the proportion of NK cells in the spleen (P=0.000) and peripheral blood (P=0.010) was larger than that of WT. Mice were significantly reduced. (3) the donor WT (control group) or S1PR5-/- (experimental group) C57BL/6 mice (spleen cells + bone marrow cells) were injected into the BALB/c mice after the tail vein injection into the lethal dose irradiated mice. The aGVHD model was established, and the weight, aGVHD score and survival time of the mice were followed up. After 60 days after transplantation, the mice in the experimental group were found. The survival rate of the control group was significantly lower (P=0.019) and the aGVHD score increased significantly (P=0.010), and there was no significant difference in weight loss between the two groups (P=0.119). (4) the bone marrow, spleen, lymph node and peripheral blood of the recipient mice were taken on the fourth day after transplantation, and the lymphocyte subsets in different parts were detected by flow cytometry. Fourth The content of NK cells in the bone marrow of the mice in the experimental group was significantly higher than that of the control group (P=0.036), but there was no significant difference in the content of NK cells between the two groups in the spleen, lymph nodes and peripheral blood. The content of T cells in the bone marrow of the mice in the experimental group was significantly lower than that of the T cells in the control group (P=0.023); in the spleen, lymph nodes and peripheral blood, the T cells in the spleen, lymph nodes and peripheral blood contained T cells in the experimental group. The bone marrow, spleen, lymph node and peripheral blood of the recipient mice were removed thirtieth days after transplantation. The distribution of lymphocyte subgroups in different tissues was detected by flow cytometry. The content of NK cells in the bone marrow of the experimental group was significantly higher than that in the bone marrow of the control group (P=0.037), and in the spleen, lymph nodes and peripheral blood, the bone marrow of the mice in the experimental group was significantly higher than that of the control group. In the blood, there was no significant difference in the content of NK cells in the two groups. The content of T cells in the peripheral blood of the experimental group was significantly higher than the content of T cells in the peripheral blood of the control group (P=0.031). There was no significant difference in the content of T cells between the two groups in the bone marrow, spleen and lymph nodes. The conclusion: (1) the CRISPR/ Cas9 technique was effective and fast for the establishment of a gene knockout mouse model. One of the methods of Czechoslovakia (S1PR5) affects the distribution of NK cells in mice; (3) the lack of S1PR5 expression in the donor mice can aggravate the deletion of the aGVHD: (S1PR5) expression in the recipient mice, resulting in the removal of the NK cells from the bone marrow, and the decrease in the NK cells that protect the aGVHD in the peripheral circulation, which may result in the S1PR5 expression loss of the recipient leading to aGV. The important mechanism of HD aggravation.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R457.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 管立勛;蔡博;高麗;李曉紅;李猛;王莉莉;高春記;;異基因造血干細(xì)胞移植后外周血NK細(xì)胞S1PR5表達(dá)變化對(duì)移植物抗宿主病的影響[J];中國實(shí)驗(yàn)血液學(xué)雜志;2012年02期

本文編號(hào)：2057610