發(fā)布時(shí)間：2018-03-01 09:33

  本文關(guān)鍵詞： 宮頸癌 樹突狀細(xì)胞 色素上皮衍生因子　出處：《第三軍醫(yī)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的 子宮頸癌(cervical cancer)是嚴(yán)重威脅女性健康的惡性腫瘤。目前，其臨床治療采用了手術(shù)、化療、放療、生物治療等多種手段，雖療效明確，但副作用大且復(fù)發(fā)率高(約35%)。已開發(fā)的各種基于HPV的預(yù)防性疫苗，因免疫型別，接種時(shí)機(jī)和遠(yuǎn)期效應(yīng)等因素的影響，與臨床需求尚有較遠(yuǎn)差距。因此，進(jìn)一步深化子宮頸癌的發(fā)病機(jī)制系統(tǒng)研究，以改進(jìn)現(xiàn)有的治療策略，或研發(fā)更為有效的新型治療手段對(duì)降低宮頸癌的死亡率極為重要。 高危乳頭瘤病毒(human papilloma virus，HPV)持續(xù)感染是子宮頸癌發(fā)生的明確致病因素，人類惡性腫瘤的形成不僅取決于腫瘤的惡性行為，還與機(jī)體的抗腫瘤免疫監(jiān)視功能相關(guān)。樹突狀細(xì)胞(dendritic cell，DC)作為人體內(nèi)最強(qiáng)大的專職抗原提呈細(xì)胞(antigen presenting cell，APC)，在機(jī)體抗腫瘤細(xì)胞免疫過程中居于中心地位。在識(shí)別局部危險(xiǎn)抗原后，定位于外周組織的未成熟DC(immature DC，imDC)分化成熟(matureDC，mDC)并定向遷移至引流淋巴結(jié)，激活免疫應(yīng)答，因此，DC分化成熟是特異性抗腫瘤免疫的關(guān)鍵環(huán)節(jié)。研究發(fā)現(xiàn)，成熟DC數(shù)量在宮頸浸潤(rùn)癌病灶中顯著下調(diào)；在離體實(shí)驗(yàn)，SiHa細(xì)胞(宮頸癌細(xì)胞系，HPV16+)培養(yǎng)上清顯著下調(diào)DC分化的能力。上述研究表明，宮頸癌發(fā)生、進(jìn)展過程中，存在病變局部組織DC分化受阻。 DC的分化行為涉及復(fù)雜的生理調(diào)節(jié)機(jī)制，局部微環(huán)境的細(xì)胞因子和各類活性物質(zhì)均參與DC分化的調(diào)節(jié)。新近研究發(fā)現(xiàn)，哺乳動(dòng)物體內(nèi)廣泛存在的炎性介質(zhì)--色素上皮衍生因子(pigmented epithelium-derived factor，PEDF)在一系列腫瘤(包括宮頸鱗癌組織)組織內(nèi)表達(dá)異常，其具有多種生物學(xué)特性，能夠促進(jìn)與DC具有相同細(xì)胞來源的巨噬細(xì)胞的活化和募集。本研究小組前期結(jié)果發(fā)現(xiàn)：鼠重組PEDF可顯著上調(diào)小鼠骨髓來源DC(BMDC)靶向跨膜遷移行為和表面標(biāo)志物CD11c表達(dá)水平。以上證據(jù)提示：宮頸局部重要活性物質(zhì)-PEDF可能是DC分化、成熟的重要調(diào)節(jié)因子，癌變組織局部PEDF缺失是DC分化、成熟受阻的重要因素。鑒于PEDF與DC密切的關(guān)系，我們?cè)噲D通過觀察宮頸浸潤(rùn)癌局部組織PEDF表達(dá)水平及樹突狀細(xì)胞的分布，初步探討PEDF對(duì)樹突狀細(xì)胞表型分化、合成分泌及抗原提呈等生物學(xué)功能的調(diào)節(jié)作用，將為進(jìn)一步明確PEDF、DC與宮頸癌發(fā)生免疫逃逸的作用，可能實(shí)施逆轉(zhuǎn)DC功能障礙，為重建患者免疫監(jiān)視效率以提高局部抗腫瘤免疫提供初步的實(shí)驗(yàn)依據(jù)，為宮頸癌治療提供新的臨床治療策略。 材料和方法： 一.宮頸癌局部PEDF表達(dá)及DC分布特點(diǎn)的研究：取正常宮頸組織(子宮肌瘤患者，10例)，不同分期宮頸浸潤(rùn)性鱗癌患者宮頸病變組織(Ib期10例、II期10例、III期6例)，Western blot檢測(cè)PEDF表達(dá)，免疫熒光檢測(cè)宮頸病變組織DC分布特點(diǎn)。 二. PEDF對(duì)小鼠BMDC分化和抗原提呈功能的影響：取6-8周正常C57BL/6小鼠脛骨和股骨骨髓，用紅細(xì)胞裂解液裂解紅細(xì)胞后，應(yīng)用重組鼠粒細(xì)胞-巨噬細(xì)胞集落刺激因子(Recombinant mouse gramulocyre-macrophage，rmGM-CSF)和重組人白介素4(Recombinant mouse Interleukin-4，rmIL-4)進(jìn)行體外誘導(dǎo)培養(yǎng)DCs，DCs分組如下： A組：DCs+PEDF(50ng/mL)；B組：DCs+PEDF(100ng/mL)；C組：DCs+PEDF(200ng/mL)；D組：DCs+LPS(1ug/mL)；E組：DCs+1640。從形態(tài)學(xué)上對(duì)細(xì)胞進(jìn)行觀察；通過流式細(xì)胞術(shù)(Flow CytoMetry，F(xiàn)CM)檢測(cè)各組DCs表達(dá)CD11c、CD80和CD86的變化；混合淋巴細(xì)胞反應(yīng)(mixed lymphocyte reaction,MLR)檢測(cè)DC刺激T淋巴細(xì)胞的能力；酶聯(lián)免疫吸附測(cè)定法(enzyme-linked immunosorbent assay,ELISA法)檢測(cè)其分泌IL-12的水平。 結(jié)果： 一、宮頸組織局部PEDF表達(dá)及DC分布特點(diǎn) 1. PEDF在宮頸浸潤(rùn)鱗癌組織的表達(dá)：宮頸浸潤(rùn)性鱗癌患者宮頸病變組織PEDF蛋白表達(dá)水平低于正常宮頸組織(P0.05)。 2.宮頸浸潤(rùn)鱗癌組織局部CD1a(+)細(xì)胞密度、CD83(+)細(xì)胞密度均降低(P0.05)。 二、PEDF對(duì)小鼠BMDC分化和抗原提呈功能的影響 1.形態(tài)學(xué)觀察：GM-CSF誘導(dǎo)培養(yǎng)小鼠BMDC至第5天時(shí)，細(xì)胞表面見少量毛刺樣突起，細(xì)胞聚集成團(tuán)，呈集落樣貼壁生長(zhǎng)；加入不同濃度的PEDF(50ng/ml、100ng/ml、200ng/ml)和LPS(陽性對(duì)照，1ug/ml)作用后，可見細(xì)胞集落增大、表面突起增多。 2.表型分化：以不同濃度的PEDF(50ng/ml、100ng/ml、200ng/ml)作用于GM-CSF誘導(dǎo)培養(yǎng)小鼠BMDC，，流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面分子CD11c表達(dá)水平顯著升高(PEDF50、100、200ng/ml，92.80±3.81%、87.43±7.98%、92.53±3.13%，P0.05)差異有統(tǒng)計(jì)學(xué)意義；CD80表達(dá)水平顯著升高(PEDF50、100、200ng/ml，92.77±3.50%、94.10±2.76%、93.30±3.22%，P0.05)差異有統(tǒng)計(jì)學(xué)意義；CD86表達(dá)水平顯著升高(PEDF50、100、200ng/ml，77.53±2.94%、78.27±1.99%、79.13±6.10%，P0.05)差異有統(tǒng)計(jì)學(xué)意義。 3.混合淋巴細(xì)胞反應(yīng)(MLR)：不同濃度的PEDF(50ng/ml、100ng/ml、200ng/ml)作用于GM-CSF誘導(dǎo)培養(yǎng)小鼠BMDC，混合淋巴反應(yīng)刺激指數(shù)(stimulation index，SI)表達(dá)值顯著升高(PEDF50、100、200ng/ml，1.44±0.11、1.61±0.40、1.59±0.29，P0.05)，差異有統(tǒng)計(jì)學(xué)意義。 4.分泌Il-12水平：不同濃度的PEDF(50ng/ml、100ng/ml、200ng/ml)作用于GM-CSF誘導(dǎo)培養(yǎng)小鼠BMDC，IL-12分泌水平顯著升高(PEDF50、100、200ng/ml，222.27±8.01、227.47±4.58、214.83±12.64pg/mL，P0.05)，差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論： 1.宮頸浸潤(rùn)鱗癌組織中PEDF表達(dá)水平顯著下調(diào)，DC分布密度下降，且分化成熟度降低，提示PEDF的表達(dá)水平可能與局部DC生物學(xué)功能異常相關(guān)。 2.PEDF顯著上調(diào)小鼠BMDC表面CD11c、CD80、CD86表達(dá)水平，上調(diào)Il-12分泌及激活T淋巴細(xì)胞增殖的能力，可作為宮頸局部樹突狀細(xì)胞免疫功能的正向調(diào)節(jié)劑。 3.PEDF下調(diào)導(dǎo)致DC免疫功能異�？赡苁菍m頸癌發(fā)生免疫逃逸的重要環(huán)節(jié)之一，其調(diào)節(jié)機(jī)制有待進(jìn)一步研究。為通過PEDF逆轉(zhuǎn)DC功能障礙，重建患者免疫監(jiān)視效率以提高局部抗腫瘤免疫作用提供了初步實(shí)驗(yàn)依據(jù)。
[Abstract]:Background and purpose
Cervical cancer (cervical cancer) is a serious threat to the health of women malignant tumor. At present, the clinical treatment with surgery, chemotherapy, radiotherapy, biological therapy and other means, although the effect is clear, but the side effects and the recurrence rate is high (about 35%). A variety of HPV has been developed based on the preventive vaccine, because immune type, affect the timing of vaccination and long term effects and other factors, and the clinical needs still far gap. Therefore, study of the pathogenesis of cervical cancer to further deepen the system, to improve the existing treatment strategies, or the development of more effective new treatment is very important for reducing the mortality rate of cervical cancer.
High risk human papillomavirus (human papilloma, virus, HPV) persistent infection is clear the pathogenic factors of cervical cancer, and malignant behavior of human malignant tumor depends not only on the tumor, but also associated with the anti tumor immune surveillance function. Dendritic cells (dendritic cell DC) antigen as the most powerful. A cell (antigen presenting, cell, APC) play a central role in the cellular immune anti-tumor process. In recognition of partial dangerous antigens, immature DC located in peripheral tissues (immature, DC, imDC) the maturity (matureDC, mDC) and migrate to the draining lymph node, the activation of the immune response, so DC, is a key link in the differentiation and maturation of specific antitumor immunity. The study found that the number of mature DC in invasive cervical carcinoma were significantly down regulated in; in vitro experiments, SiHa cells (human cervical carcinoma cell line, HPV16+) training The ability to clear over the down-regulation of DC differentiation. The research shows that the occurrence of cervical cancer, the progress of the process, there are local lesion tissue DC differentiation was blocked.
The differentiation behavior of DC involves complex physiological regulatory mechanisms, regulation of cytokines and various kinds of active substance of local micro environment are involved in the differentiation of DC. Recent studies showed that medium - inflammation exists widely in mammalian pigment epithelium derived factor (pigmented epithelium-derived, factor, PEDF) in a series of tumors (including cervical squamous cell carcinoma) abnormal expression within the organization, which has a variety of biological characteristics, can promote cell activation and recruitment with the same source and DC macrophages. The research team found: early results of recombinant rat PEDF upregulated mouse bone marrow derived DC (BMDC) level targeting transmembrane migration behavior and surface marker CD11c expression. The above evidence suggests that the cervical part an active material -PEDF DC may be an important regulator of the differentiation, maturation, cancerous tissue is the lack of local PEDF DC differentiation, mature blocked by the The relationship between PEDF and DC. In view of the close, we try to observe the distribution and expression of PEDF and invasive cervical cancer tissue dendritic cells, preliminary study of PEDF on dendritic cell differentiation, secretion and regulation of antigen presentation and other biological functions, to further clarify the role of PEDF, immune escape DC with cervical cancer, possible reversal of DC dysfunction, immune surveillance for patients with reconstruction in order to improve the efficiency of local anti-tumor immunity provide a preliminary experimental basis, to provide a new therapeutic strategy for clinical treatment of cervical cancer.
Materials and methods:
A study of cervical cancer. The expression of PEDF and DC local distribution characteristics: take the normal cervical tissues (10 patients with myoma of uterus), the different stages of cervical invasive squamous cell carcinoma patients with cervical lesions (10 cases, Ib 10 cases, II III 6 cases), the expression of Western blot detected PEDF, immunofluorescence detection of cervical the distribution characteristics of DC lesions.
Two. PEDF on the differentiation of mouse BMDC and antigen presenting function effect: 6-8 weeks of normal C57BL/6 mouse tibia and femur bone marrow, with red blood cell lysis of red blood cells after application of recombinant mouse granulocyte macrophage colony stimulating factor (Recombinant mouse, gramulocyre-macrophage, rmGM-CSF) and recombinant human interleukin 4 (Recombinant mouse Interleukin-4 rmIL-4, DCs) were induced and cultured in vitro, DCs group A: DCs+PEDF (50ng/mL); group B: DCs+PEDF (100ng/mL); group C: DCs+PEDF (200ng/mL); group D: DCs+LPS (1ug/mL); group E: DCs+1640. cells were observed in morphology; by flow cytometry (Flow CytoMetry, FCM) to detect the DCs expression of CD11c, CD80 and CD86 change; mixed lymphocyte reaction (mixed lymphocyte reaction, MLR) detection ability of DC to stimulate T lymphocyte; enzyme linked immunosorbent assay (enzyme- Linked immunosorbent assay, ELISA method) test the level of its secretion of IL-12.
Result錛

本文編號(hào)：1551363