本文選題：臍血　切入點(diǎn)：樹(shù)突狀細(xì)胞　出處：《鄭州大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景和目的 近年來(lái),隨著人口老齡化及人類(lèi)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(shì)。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(duì)機(jī)體損傷較大,在殺傷癌細(xì)胞的同時(shí),往往也伴隨著正常組織細(xì)胞的損傷。隨著人們對(duì)腫瘤發(fā)生發(fā)展機(jī)制的進(jìn)一步認(rèn)識(shí)和腫瘤免疫分子生物學(xué)和生物工程技術(shù)的進(jìn)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(shù)突狀細(xì)胞(dendritic cells, DCs)因具有抗原呈遞能力和活化初始T細(xì)胞的能力而成為腫瘤免疫治療的研究熱點(diǎn)。但DCs在腫瘤患者外周血和組織中含量較少,體外擴(kuò)增也較難達(dá)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(yīng)及機(jī)制,有助于為臍血來(lái)源的DCs及細(xì)胞毒性T細(xì)胞的臨床應(yīng)用打下基礎(chǔ)。 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(gè)核細(xì)胞(Human Umbilicus blood mononuclear cell, HUBMC),將培養(yǎng)瓶靜置2小時(shí),取貼壁生長(zhǎng)細(xì)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(shù)突狀細(xì)胞(Human Umbilicus blood dendritic cell, HUBDC)。取對(duì)數(shù)生長(zhǎng)期的BGC823腫瘤細(xì)胞,用反復(fù)凍融法取得腫瘤細(xì)胞提取物作為腫瘤抗原,負(fù)載HUBDCs,促使HUBDCs成熟。于普通光學(xué)顯微鏡下觀察HE染色后的HUBDCs形態(tài),并用流式細(xì)胞儀對(duì)其進(jìn)行表型鑒定。將HUBDCs中的非貼壁細(xì)胞采用尼龍毛柱法分離出T細(xì)胞并鑒定其純度。觀測(cè)負(fù)載抗原成熟的HUBDCs對(duì)T細(xì)胞的活化增殖能力(混合淋巴細(xì)胞反應(yīng))。采用MTT比色法檢測(cè)活化的T細(xì)胞對(duì)BGC823靶細(xì)胞的殺傷。動(dòng)物實(shí)驗(yàn)部分,首先建立荷瘤動(dòng)物模型,將BGC823細(xì)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(jī)分成四組,治療方案如下：A組：體外負(fù)載抗原的HUBDCs和未激活T細(xì)胞；B組：體外經(jīng)HUBDCs誘導(dǎo)活化的特異性T細(xì)胞(CTL); C組：負(fù)載BGC823抗原的HUBDCs; D組：未經(jīng)HUBDCs致敏的新分離T細(xì)胞。觀察裸鼠體內(nèi)腫瘤的生長(zhǎng)情況并分別記錄。 結(jié)果 用細(xì)胞因子GM-CSF、SCF和IL-4配伍能誘導(dǎo)人臍血貼壁單個(gè)核細(xì)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(shù)枝狀DCs形態(tài),流式分析儀檢測(cè)細(xì)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(dǎo)前有顯著升高。其中,CD86表達(dá)率為40.59%±3.27%,CD54為59.21%±6.32%,CD11c表達(dá)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(dǎo)前相比,差異具有統(tǒng)計(jì)學(xué)意義P0.01。同種混合淋巴細(xì)胞反應(yīng)顯示：HUBDCs體外可以使抗原特異性T細(xì)胞活化增殖為效應(yīng)T細(xì)胞(cytotoxic T lymphocytes, CTL),且CTL對(duì)靶細(xì)胞BGC823可以產(chǎn)生特異性的抑制及殺傷。在動(dòng)物實(shí)驗(yàn)中,成功建立人胃癌BGC823荷瘤裸鼠模型。按實(shí)驗(yàn)方案分別對(duì)各組治療后,注射負(fù)載抗原HUBDCs +新分離T細(xì)胞的A組和注射特異性CTL的B組都有明顯抑瘤效應(yīng),至30天時(shí)A組腫瘤大小為：211mm3B組為：153mm3,B組抑瘤效應(yīng)更加顯著；C組單獨(dú)注射負(fù)載抗原的HUBDCs, D組注射未致敏新分離的T細(xì)胞均無(wú)明顯抑瘤效果,至30天時(shí)C組,D組腫瘤大小分別為：1093 mm3和1022mm3。C組D組腫瘤細(xì)胞仍快速生長(zhǎng)。 結(jié)論 1)利用細(xì)胞因子能從人臍血單個(gè)核細(xì)胞(HUBMC)中誘導(dǎo)出具有典型形態(tài)、表型和具有活化T細(xì)胞功能的HUBDCs; 2)成熟人臍血DC與T細(xì)胞共同移植,能顯著增強(qiáng)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(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

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