本文選題：臍血　切入點：樹突狀細胞　出處：《鄭州大學》2011年碩士論文　論文類型：學位論文

【摘要】：研究背景和目的 近年來,隨著人口老齡化及人類生存環(huán)境的惡化,惡性腫瘤的發(fā)病率和病死率均呈現(xiàn)明顯升高的趨勢。目前臨床腫瘤的治療普遍還是采用手術(shù)、放療和化療等傳統(tǒng)方法。但這些方法難以清除體內(nèi)的微小腫瘤病灶且對機體損傷較大,在殺傷癌細胞的同時,往往也伴隨著正常組織細胞的損傷。隨著人們對腫瘤發(fā)生發(fā)展機制的進一步認識和腫瘤免疫分子生物學和生物工程技術(shù)的進展,腫瘤生物治療迅速發(fā)展,成為腫瘤治療的第四種治療模式。樹突狀細胞(dendritic cells, DCs)因具有抗原呈遞能力和活化初始T細胞的能力而成為腫瘤免疫治療的研究熱點。但DCs在腫瘤患者外周血和組織中含量較少,體外擴增也較難達到理想的治療用劑量。因此,探討從健康產(chǎn)婦臍血分離誘生DCs的方法,研究其體內(nèi)外的抗腫瘤效應及機制,有助于為臍血來源的DCs及細胞毒性T細胞的臨床應用打下基礎。 方法 采集健康足月分娩產(chǎn)婦臍血50-80ml,分離出人臍血單個核細胞(Human Umbilicus blood mononuclear cell, HUBMC),將培養(yǎng)瓶靜置2小時,取貼壁生長細胞,在含15%胎牛血清1640培養(yǎng)液中加入GM-CSF、IL-4和SCF,培養(yǎng)誘生人臍血樹突狀細胞(Human Umbilicus blood dendritic cell, HUBDC)。取對數(shù)生長期的BGC823腫瘤細胞,用反復凍融法取得腫瘤細胞提取物作為腫瘤抗原,負載HUBDCs,促使HUBDCs成熟。于普通光學顯微鏡下觀察HE染色后的HUBDCs形態(tài),并用流式細胞儀對其進行表型鑒定。將HUBDCs中的非貼壁細胞采用尼龍毛柱法分離出T細胞并鑒定其純度。觀測負載抗原成熟的HUBDCs對T細胞的活化增殖能力(混合淋巴細胞反應)。采用MTT比色法檢測活化的T細胞對BGC823靶細胞的殺傷。動物實驗部分,首先建立荷瘤動物模型,將BGC823細胞注射于裸鼠并觀察成瘤情況,記錄腫瘤大小。將荷瘤裸鼠隨機分成四組,治療方案如下：A組：體外負載抗原的HUBDCs和未激活T細胞；B組：體外經(jīng)HUBDCs誘導活化的特異性T細胞(CTL); C組：負載BGC823抗原的HUBDCs; D組：未經(jīng)HUBDCs致敏的新分離T細胞。觀察裸鼠體內(nèi)腫瘤的生長情況并分別記錄。 結(jié)果 用細胞因子GM-CSF、SCF和IL-4配伍能誘導人臍血貼壁單個核細胞向DC分化,加入腫瘤抗原提取物能促使HUBDCs成熟,HE染色光鏡下觀察HUBDCs形態(tài)符合典型毛刺狀、樹枝狀DCs形態(tài),流式分析儀檢測細胞表型：MHC-Ⅰ、MHC-Ⅱ、CD86(協(xié)同刺激分子)、CD54(黏附分子)、CD11c均較誘導前有顯著升高。其中,CD86表達率為40.59%±3.27%,CD54為59.21%±6.32%,CD11c表達率為67.01%±5.17%,MHC-Ⅰ和MHC-Ⅱ分別為：42.37%±10.11%和56.31%±6.76%,與誘導前相比,差異具有統(tǒng)計學意義P0.01。同種混合淋巴細胞反應顯示：HUBDCs體外可以使抗原特異性T細胞活化增殖為效應T細胞(cytotoxic T lymphocytes, CTL),且CTL對靶細胞BGC823可以產(chǎn)生特異性的抑制及殺傷。在動物實驗中,成功建立人胃癌BGC823荷瘤裸鼠模型。按實驗方案分別對各組治療后,注射負載抗原HUBDCs +新分離T細胞的A組和注射特異性CTL的B組都有明顯抑瘤效應,至30天時A組腫瘤大小為：211mm3B組為：153mm3,B組抑瘤效應更加顯著；C組單獨注射負載抗原的HUBDCs, D組注射未致敏新分離的T細胞均無明顯抑瘤效果,至30天時C組,D組腫瘤大小分別為：1093 mm3和1022mm3。C組D組腫瘤細胞仍快速生長。 結(jié)論 1)利用細胞因子能從人臍血單個核細胞(HUBMC)中誘導出具有典型形態(tài)、表型和具有活化T細胞功能的HUBDCs; 2)成熟人臍血DC與T細胞共同移植,能顯著增強荷瘤裸鼠的抗腫瘤能力。
[Abstract]:Background and purpose of research
In recent years, with the population aging and deterioration of the living environment of mankind, the incidence and mortality rate of malignant tumors showed a significant increasing trend. The current clinical treatment of tumors generally or with surgery, radiotherapy and chemotherapy and other traditional methods. But it is difficult to remove small tumor lesions in vivo and the damage to the body is larger in these methods, killer cancer cells at the same time, often accompanied by normal tissue damage. With the progress of the mechanism of the occurrence and development of the further understanding of molecular biology and tumor immunity and tumor biological engineering technology, biological treatment of tumor development, become the fourth method for cancer treatment. Dendritic cells (dendritic cells, DCs) research hotspot in tumor immunity the treatment has become the antigen-presenting ability and the ability to activate naive T cells. But the DCs in the peripheral blood and tissues containing less In vitro expansion is also difficult to achieve an ideal therapeutic dose. Therefore, exploring the method of inducing DCs from umbilical cord blood of healthy women, studying its anti-tumor effect and mechanism in vivo and in vitro, is helpful to lay a foundation for the clinical application of cord blood derived DCs and cytotoxic T cells.
Method
Collected from healthy full-term delivery maternal cord blood 50-80ml, isolated from human umbilical cord blood mononuclear cells (Human Umbilicus blood mononuclear cell, HUBMC), the flask standing 2 hours, adherent cells in GM-CSF medium containing 15% fetal bovine serum IL-4 and SCF, 1640 training, training induced human umbilical cord blood dendritic cells (Human Umbilicus blood dendritic cell, HUBDC). BGC823 cells in logarithmic growth phase, the load of HUBDCs by freeze-thaw method to obtain tumor cell extracts as tumor antigen, HUBDCs, to mature. In the ordinary optical microscope after HE staining, the morphology of the HUBDCs, and the phenotype was identified by flow cytometry. The HUBDCs in non adherent cells by nylon fiber isolated T cells and identify the purity. Observe the load on T cell activation and proliferation of mature HUBDCs antigen (mixed lymphocyte reaction) by MTT. Colorimetric detection of activated T BGC823 cells to kill target cells. Animal experiment, first to establish tumor bearing animal model, BGC823 cells were injected into nude mice and the tumor growth was observed and recorded. The size of tumor bearing nude mice were randomly divided into four groups, treatment group were as follows: A: in vitro antigen loaded HUBDCs and not the activation of T cells; group B: in vitro induced by HUBDCs activation of specific T cells (CTL); group C: load BGC823 antigen HUBDCs; D group: without HUBDCs sensitized isolated T cells. Tumor growth of and were recorded.
Result
Cell factor GM-CSF, and SCF and IL-4 can induce human umbilical cord blood adherent mononuclear cells to differentiate into DC, with tumor antigen extracts can promote HUBDCs maturation and HE staining of HUBDCs with typical morphology of burr, dendritic DCs morphology, flow cytometry analyzer to detect cell surface type: MHC- 1, MHC- 2, CD86 (costimulatory molecules), CD54 (CD11c, adhesion molecules) were induced significantly increased. Among them, the expression rate of CD86 was 40.59% + 3.27%, CD54 = 59.21% + 6.32%, the expression rate of CD11c was 67.01% + 5.17%, MHC- I and MHC- II respectively: 42.37% + 10.11% and 56.31% + 6.76%, compared with the induction the difference has statistical significance P0.01. showed that allogeneic mixed lymphocyte reaction: HUBDCs in vitro can make antigen-specific T cell activation and proliferation of effector T cells (cytotoxic T lymphocytes, CTL), and CTL BGC823 on target cells to produce specific inhibit and kill Injury. In animal experiments, establishment of human gastric cancer BGC823 in nude mice model. According to the experimental scheme in each group after treatment, B group were injected with antigen loaded HUBDCs + T cells isolated A injection group and the specific CTL has obvious anti-tumor effect, and 30 days in A group, tumor size: 211mm3B group 153mm3, group B: the antitumor effect of C group was more significant; a single injection of antigen loaded HUBDCs, D group were injected with unsensitized new isolated T cells had no obvious inhibitory effect on tumor growth, to 30 days in C group, D group, tumor size was 1093 mm3 and 1022mm3.C group D group of tumor cells is still fast growth.
conclusion
1) the use of cytokines to induce a typical form, phenotype and HUBDCs that has the function of activating T cells from human umbilical cord blood mononuclear cells (HUBMC).
2) the combined transplantation of DC and T cells in mature human umbilical cord blood can significantly enhance the anti-tumor ability of nude mice.

【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前10條

1 宋文剛,陳憲銳,李雅林,徐英萍,吳聰,秦慶亮;IL-18基因修飾的DC腫瘤融合瘤苗的抗腫瘤效應[J];第四軍醫(yī)大學學報;2003年24期

2 鐘志宏;鄢俊;陳國安;袁利亞;;耐受性樹突狀細胞誘導特異性免疫耐受的研究進展[J];廣東醫(yī)學;2009年06期

3 裴雪濤，，王立生，徐黎，吳祖澤;CD34~+造血祖細胞及其亞群（CD34~+CD38~+和CD34~+CD38~-[J];高技術(shù)通訊;1996年01期

4 張臨友,張學峰;Flt3L免疫擴增T、B、NK、DC細胞的研究進展[J];國外醫(yī)學(免疫學分冊);2003年05期

5 方傳龍;謝遵江;賈立敏;賀業(yè)春;劉穎;劉麗;;樹突狀細胞在黏膜免疫模型小鼠中的分布及E-cadherin和ICAM-1表達的研究[J];解剖學報;2006年03期

6 劉季芳;祁巖超;楊波;盧敏瑩;潘東曉;申鴻卓;;兩種不同抗原致敏方式負載DC疫苗對Lovo細胞的體外殺傷作用[J];山東醫(yī)藥;2010年07期

7 李勝富,馮剛,步宏,李幼平,張杰,楊宇如,盧一平;成人外周血來源樹突狀細胞體外誘導培養(yǎng)及成熟調(diào)控[J];生物醫(yī)學工程學雜志;2002年02期

8 王鶴樺;潘興華;高士爭;龐榮清;楊勇琴;屈璐;梁虹;;人骨髓來源的樹突狀細胞的誘導擴增及鑒定[J];細胞與分子免疫學雜志;2006年04期

9 王新利;李卿;高婷;付立葉;王揚;蔣濤;姜又紅;;樹突狀細胞分泌的exosomes體外誘導特異性CTL抗腎癌的初步研究[J];中國醫(yī)科大學學報;2010年01期

10 李寶金;殷曉煜;呂明德;王亮;向邦德;;IL-12基因修飾樹突狀細胞體外誘導免疫殺傷肝癌細胞[J];中華肝膽外科雜志;2005年12期

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