【摘要】：目的：髓源性抑制細胞(Myeloid-derived suppressor cells,MDSCs)為未成熟但具免疫抑制能力的髓系細胞。MDSCs與腫瘤等疾病的發(fā)生密切相關,是免疫學領域近年來的熱點之一。MDSCs在細胞構成上具有高度異質性,但迄今的研究局限于單核細胞樣和粒細胞樣MDSCs兩個亞群。MDSCs是否還包括其它細胞群尤其是髓系發(fā)生早期的細胞一直是MDSC研究亟待解決的關鍵問題之一。據報道,小鼠胚胎干細胞和造血干細胞在體外可產生一種在分化潛能上相當于粒細胞/巨噬細胞祖細胞(GMPs)但呈現抑制活性的細胞群,提示體內GMPs可能具有免疫調節(jié)功能。本研究的目的是,通過系統地檢測髓系分化通路早期系列細胞群對T細胞的抑制能力,探明髓系祖細胞的免疫調節(jié)功能屬性并界定MDSC的發(fā)育起源。方法：1)小鼠Lewis肺癌細胞模型構建：接種對數生長期的LLC細胞于6-8周齡近交系C57BL/6J小鼠左腋皮下；4周后選擇所長腫瘤大小適宜(直徑約1.5cm)的動物用于實驗。2)骨髓細胞分離與制備：取脛骨與股骨骨髓,去除紅細胞,制備成骨髓單細胞懸液。3)細胞表型分析與分選：細胞用抗標志蛋白抗體(與熒光素分子結合)標記；通過流式細胞分析儀分析細胞表型和頻率,通過流式細胞分選儀分選純化特定細胞群。4)非特異性T細胞抑制實驗：實驗設置三個組別即待測細胞組、非免疫細胞(使用純化的巨核細胞/紅細胞祖細胞,MEP)對照組和空白對照組(以培液代替骨髓細胞)；骨髓細胞分別與經CFSE染色的C57BL/6J小鼠脾臟細胞在抗CD3/抗CD28抗體存在下共培養(yǎng)72小時；流式術測定CFSE熒光強度變化。5)特異性T細胞抑制實驗：實驗設計及方法與非特異性T細胞抑制實驗類似,不同之處是以經絲裂霉素c處理的BABL/c小鼠脾臟細胞(作為抗原提呈細胞)代替抗CD3/抗CD28抗體刺激C57BL/6J小鼠脾臟T細胞。6)統計方法：數據統計采用SPSS17.0統計軟件進行獨立樣品T檢驗分析。統計結果以平均值±標準差(standard deviation, SD)表示。結果：1)粒細胞/巨噬細胞祖細胞(GMPs)表現極強的免疫抑制能力。GMPs是MDSCs兩個亞群的前體細胞。本課題首先分析了GMPs對非特異和抗原特異性激活的T細胞增殖的影響。在非特異T細胞反應中,分離自荷瘤小鼠的GMPs (T-GMP,T:tumor)呈現極強的T細胞抑制活性。加入T-GMP后,T細胞的增殖指數為0.82±0.45%(分裂T細胞/總脾臟細胞百分比,三次獨立實驗,下同),顯著低于荷瘤小鼠MEP(T-MEP)添加組的22.40±2.09%(P0.01)和無骨髓細胞添加組(Medium)的(27.87±3.44%)(P0.001)。后兩者間則無明顯差異,亦即非免疫細胞MEP對T細胞增殖無抑制作用。出乎意料的是,分離自正常小鼠(注射PBS)的GMP (N-GMP, N:normal)也能有效地抑制T細胞增殖并表現出與T-GMP相當的抑制活性。N-GMP和N-MEP組T細胞的增殖指數分別為1.17±1.03%和23.10±2.45%(P0.01)。在特異性T細胞反應抑制實驗中,我們得到了類似的結果。T-GMP組、T-MEP組和Medium組的T細胞增殖指數分別為0.32±0.08%、3.15±0.52%和4.37士0.9%(0.32±0.08% VS 3.15±0.52%,P0.05；0.32±0.08%VS 4.37±0.9%,P0.01)。N-GMP組和N-MEP組的T細胞增殖指數分別為0.36±0.02%和3.76±0.63%(P0.01)。2)共同髓系祖細胞(CMPs)是髓系發(fā)生通路中最早的抑制細胞群。接下來檢測了GMPs上游的系列細胞群,包括共同髓系祖細胞(CMPs)、多能祖細胞(MPPs)和造血干細胞(HSCs)。結果顯示,分離自荷瘤小鼠CMP(T-CMP)和正常小鼠CMP (N-CMP)均能有效地抑制T細胞的增殖。在非特異T細胞反應中,T-CMP和N-CMP兩組T細胞的增殖指數分別為4.51±1.89%和5.21±2.91%,而相對應的T-MEP組和N-MEP組的T細胞增殖指數分別為26.70±2.09%和28.00±2.45%。在特異性T細胞反應中,T-CMP組和N-CMP組T細胞的增殖指數分別為1.36±0.15%和1.47±0.21%,而相對應的T-MEP組和N-MEP組的T細胞增殖指數分別為3.15±0.52%和3.76±0.63%。但是,無論是分離自正常小鼠還是荷瘤小鼠的MPPs和HSCs,均不表現T細胞抑制作用。3)早期髓系祖細胞具有比其下游細胞更強的抑制活性。我們進而比較了GMPs和CMPs與經典MDSCs的抑制能力。在相同的細胞比例(GMP或MDSC:脾臟細胞=1：2)下,GMP抑制能力明顯強于MDSCs(T-GMP VST-MDSC: 0.82±0.45%VS 7.61±1.51%, P0.05; N-GMP VS N-MDSC:1.17±1.03% VS 11.50±1.05%,P0.001)。來源于生理與腫瘤鼠的CMP(CMP:脾臟細胞=1：8)的免疫抑制能力同樣強于MDSCs (MDSC:脾臟細胞=1：8)(5.21±2.91 VS 28.00±.45%,P0.01,正常小鼠；4.51±1.89%VS 26.7±2.09%,P0.01,荷瘤小鼠)。4)骨髓造血干/祖細胞在腫瘤進程中呈現特征性的發(fā)育變化。最后,測定了造血干/祖細胞群在腫瘤狀態(tài)下豐度的變化。在荷瘤小鼠中,GMP占骨髓細胞的比例為1.120±0.071%,顯著高于生理狀態(tài)下的GMP含量(0.649±0.058%,三次以上獨立實驗,P0.01)。腫瘤CMP含量則低于生理狀態(tài)下的含量(0.111±0.019% VS0.233±0.032%,P0.01)。在生理和腫瘤狀態(tài)下,MPP的含量分別為0.033±0.003%和0.199±0.040%(P0.05),HSC的含量分別為0.033±0.007%和0.176±0.0136%(P0.01)。結論：1)髓系祖細胞GMPs和CMPs具有免疫抑制功能,并表現比其下游細胞(經典MDSCs)更強的抑制活性。這些發(fā)現不僅讓我們鑒定出了髓源性抑制細胞家族的兩個新成員并確定CMPs是髓系分化通路中最早出現的抑制性細胞群,而且折示它們在骨髓組織自穩(wěn)態(tài)維護以及防止自身免疫和過度免疫反應中可能起重要作用。2)髓系細胞(包括CMPs、GMPs和經典MDSCs)的免疫抑制功能是其內在功能特性,與病理狀態(tài)無關；但是,腫瘤環(huán)境可以刺激HSC增殖并促進其向髓系細胞尤其GMPs的分化。這些結果表明髓系抑制細胞是機體免疫調節(jié)網絡的固有組成部分,同時為其在腫瘤等病態(tài)下擴增的機理提供了新的解釋視角。
[Abstract]:Objective: Myeloid-derived support cells (MDSCs) are immature but immunosuppressed myeloid cells. MDSCs are closely related to the occurrence of diseases such as tumors, and is one of the hot spots in the field of immunology. MDSCs are highly heterogeneous in cell formation, but studies to date are limited to both monocyte and granulocyte-like MDSCs. Whether MDSCs also include other cell populations, especially the early-stage cells of the myeloid lineage, have been one of the key issues to be addressed in the MDSC study. It has been reported that mouse embryonic stem cells and hematopoietic stem cells can in vitro produce a cell population that is equivalent to granulocyte/ macrophage progenitor cells (GMPs) in differentiation potential but exhibit inhibitory activity, suggesting that GMPs in vivo may have an immunomodulatory function. The purpose of this study was to investigate the ability of the early series of cell populations to inhibit T cells in the early series of myeloid differentiation and to explore the functional properties of myeloid progenitor cells and to define the development origin of MDSCs. Methods:1) The mouse Lewis lung cancer cell model was constructed: the LLC cells in the logarithmic growth phase were subcutaneously implanted in the left axilla of the C57BL/ 6J mice at 6-8 weeks of age; after 4 weeks, the animals with the appropriate tumor size (about 1.5 cm in diameter) were selected for the experiment.2) The bone marrow cells were isolated and prepared: taking the tibia and the femur bone marrow, removing red blood cells to prepare a single cell suspension of bone marrow;3) cell phenotypic analysis and sorting: labeling the cell with an anti-marker protein antibody (in combination with a fluorescein molecule); analyzing the cell phenotype and frequency by flow cytometry; and sorting and purifying a specific cell group by flow cytometry.4) the non-specific T cell inhibition experiment: three groups of cells to be tested, non-immune cells (using purified megakaryocyte/ erythrocyte progenitor cells, MEPs) control group and blank control group (replacing bone marrow cells with culture liquid); bone marrow cells were co-cultured with CFSE-stained C57BL/ 6J mouse spleen cells for 72 hours in the presence of anti-CD3/ anti-CD28 antibody; flow cytometry was used to determine the change of CFSE fluorescence intensity. The experimental design and method were similar to those of non-specific T cell inhibition, except that the spleen cells of the BABL/ c mice treated with mitomycin c (as antigen-presenting cells) were used in place of the anti-CD3/ anti-CD28 antibody to stimulate the spleen T cells of C57BL/ 6J mice. The data statistics are analyzed by using the SPSS17.0 statistical software for independent sample T test. The statistical results are represented by standard deviation (SD). Results:1) The granulocyte/ macrophage progenitor cells (GMPs) showed strong immunosuppression ability. GMPs are the precursor cells of two subpopulations of MDSCs. The effects of GMPs on the proliferation of non-specific and antigen-specific activated T cells were analyzed. In the non-specific T cell reaction, the GMPs (T-GMP, T: tumor) isolated from the tumor-bearing mice showed strong T-cell inhibitory activity. After the addition of T-GMP, the proliferation index of T-cells was 0.82-0.45% (percentage of divided T-cells/ total spleen cells and three independent experiments, the same below), which was significantly lower than that of the control group (22.40-2.09% (P0.01) and (27.87-3.44%) of the non-bone marrow cell-added group (P0.001). There was no significant difference between the two groups, that is, the non-immune cell MEP had no inhibitory effect on T cell proliferation. Surprisingly, GMP (N-GMP, N: normal) isolated from normal mice (injected PBS) can also effectively inhibit T cell proliferation and exhibit a comparable inhibitory activity as T-GMP. The proliferation index of T cells in N-GMP and N-MEP group was 1.17, 1.03% and 23.10-2.45%, respectively (P0.01). In the specific T cell reaction inhibition assay, similar results were obtained. The T-cell proliferation index of T-GMP group, T-MEP group and medium group was 0.32-0.08%, 3.15-0.52% and 4.37-0.9% (0.32-0.08% VS 3.15-0.52%, P0.05; 0.32-0.08% VS 4.37-0.9%, P0.01). The T-cell proliferation index of N-GMP group and N-MEP group was 0.36-0.02% and 3.76-0.63% (P0.01). The series of cell populations upstream of GMPs were then tested, including common myeloid progenitor cells (CMPs), pluripotent progenitor cells (MPPs) and hematopoietic stem cells (HSCs). The results showed that both CMP (T-CMP) and normal mouse CMP (N-CMP) were effective in the inhibition of T cell proliferation. In the non-specific T cell reaction, the proliferation index of T cells in T-CMP and N-CMP was 4.51% 1.89% and 5.21% 2.91%, respectively, while the corresponding T-MEP group and N-MEP group had a T-cell proliferation index of 26.70% 2.09% and 28.00-2.45%, respectively. In the specific T-cell reaction, the proliferation index of T-and N-group T cells was 1.36-0.15% and 1.47-0.21%, respectively, while the corresponding T-MEP group and N-MEP group had a T-cell proliferation index of 3.15-0.52% and 3.76-0.63%, respectively. However, neither the MPPs nor the HSCs isolated from the normal mice or the tumor-bearing mice did not show a T-cell inhibition.3) The early myeloid progenitor cells had a stronger inhibitory activity than the downstream cells. In turn, we compared the ability of GMPs and CMPs to suppress the classical MDSCs. Under the same ratio of cells (GMP or MDSC: spleen cells = 1:2), the inhibition of GMP was significantly stronger than that of MDSCs (T-GMP VST-MDSC: 0.82, 0.45% VS 7.61, 1.51%, P0.05; N-GMP VS N-MDSC: 1.17, 1.03% VS 11.50, 1.05%, P0.001). The immunosuppression ability of CMP (CMP: spleen cells = 1:8) derived from physiological and tumor mice was also stronger than that of MDSCs (MDSC: spleen cells = 1:8) (5.21% 2.91 VS 28.00).45%, P 0.01, normal mice; 4.51% 1.89% VS 26.7% 2.09%, P 0.01, Tumor-bearing mice).4) The bone marrow hematopoietic stem/ progenitor cells present a characteristic development change in the tumor process. Finally, the changes of the abundance of the hematopoietic stem/ progenitor cell population in the tumor were determined. In the tumor-bearing mice, the proportion of GMP in the bone marrow cells was 1.120-0.071%, which was significantly higher than that in the physiological state (0.649-0.058%, more than three independent experiments, P0.01). The content of CMP was lower than that in physiological state (0.111-0.019% VS.233-0.032%, P0.01). In the physiological and tumor state, the content of MPP was 0.033-0.003% and 0.199-0.040% (P0.05). The content of HSC was 0.033-0.007% and 0.176-0.0136%, respectively (P0.01). Conclusion:1) The myeloid progenitor cells GMPs and CMPs have an immunosuppressive function and show stronger inhibitory activity than its downstream cells (classical MDSCs). These findings not only allowed us to identify two new members of the myeloid-derived suppressor cell family and to determine that the CMPs were the earliest inhibitory cell populations in the myeloid differentiation pathway, and it is shown that they may play an important role in the self-stable maintenance of the bone marrow tissue and the prevention of autoimmune and hyperimmune responses.2) The immunosuppressive function of myeloid cells (including CMPs, GMPs and the classical MDSCs) is its intrinsic function, not related to the pathological state; however, The tumor environment can stimulate the proliferation of HSC and promote its differentiation into the myeloid cells, especially GMPs. These results show that the myeloid-inhibiting cell is an integral part of the immunomodulatory network of the organism, and provides a new interpretation view for the mechanism of its amplification in the condition of tumor.
【學位授予單位】：廣西師范大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R392

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