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

【摘要】：背景和目的 子宮頸癌(cervical cancer)是嚴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，但副作用大且復發(fā)率高(約35%)。已開發(fā)的各種基于HPV的預防性疫苗，因免疫型別，接種時機和遠期效應等因素的影響，與臨床需求尚有較遠差距。因此，進一步深化子宮頸癌的發(fā)病機制系統(tǒng)研究，以改進現(xiàn)有的治療策略，或研發(fā)更為有效的新型治療手段對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，還與機體的抗腫瘤免疫監(jiān)視功能相關(guān)。樹突狀細胞(dendritic cell，DC)作為人體內(nèi)最強大的專職抗原提呈細胞(antigen presenting cell，APC)，在機體抗腫瘤細胞免疫過程中居于中心地位。在識別局部危險抗原后，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)量在宮頸浸潤癌病灶中顯著下調(diào)；在離體實驗，SiHa細胞(宮頸癌細胞系，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的分化行為涉及復雜的生理調(diào)節(jié)機制，局部微環(huán)境的細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)表達異常，其具有多種生物學特性，能夠促進與DC具有相同細胞來源的巨噬細胞的活化和募集。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志物CD11c表達水平。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，我們試圖通過觀察宮頸浸潤癌局部組織PEDF表達水平及樹突狀細胞的分布，初步探討PEDF對樹突狀細胞表型分化、合成分泌及抗原提呈等生物學功能的調(diào)節(jié)作用，將為進一步明確PEDF、DC與宮頸癌發(fā)生免疫逃逸的作用，可能實施逆轉(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驗依據(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表達及DC分布特點的研究：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PEDF表達，免疫熒光檢測宮頸病變組織DC分布特點。 二. PEDF對小鼠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，用紅細胞裂解液裂解紅細胞后，應用重組鼠粒細胞-巨噬細胞集落刺激因子(Recombinant mouse gramulocyre-macrophage，rmGM-CSF)和重組人白介素4(Recombinant mouse Interleukin-4，rmIL-4)進行體外誘導培養(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)學上對細胞進行觀察；通過流式細胞術(shù)(Flow CytoMetry，F(xiàn)CM)檢測各組DCs表達CD11c、CD80和CD86的變化；混合淋巴細胞反應(mixed lymphocyte reaction,MLR)檢測DC刺激T淋巴細胞的能力；酶聯(lián)免疫吸附測定法(enzyme-linked immunosorbent assay,ELISA法)檢測其分泌IL-12的水平。 結(jié)果： 一、宮頸組織局部PEDF表達及DC分布特點 1. PEDF在宮頸浸潤鱗癌組織的表達：宮頸浸潤性鱗癌患者宮頸病變組織PEDF蛋白表達水平低于正常宮頸組織(P0.05)。 2.宮頸浸潤鱗癌組織局部CD1a(+)細胞密度、CD83(+)細胞密度均降低(P0.05)。 二、PEDF對小鼠BMDC分化和抗原提呈功能的影響 1.形態(tài)學觀察：GM-CSF誘導培養(yǎng)小鼠BMDC至第5天時，細胞表面見少量毛刺樣突起，細胞聚集成團，呈集落樣貼壁生長；加入不同濃度的PEDF(50ng/ml、100ng/ml、200ng/ml)和LPS(陽性對照，1ug/ml)作用后，可見細胞集落增大、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誘導培養(yǎng)小鼠BMDC，，流式細胞術(shù)檢測細胞表面分子CD11c表達水平顯著升高(PEDF50、100、200ng/ml，92.80±3.81%、87.43±7.98%、92.53±3.13%，P0.05)差異有統(tǒng)計學意義；CD80表達水平顯著升高(PEDF50、100、200ng/ml，92.77±3.50%、94.10±2.76%、93.30±3.22%，P0.05)差異有統(tǒng)計學意義；CD86表達水平顯著升高(PEDF50、100、200ng/ml，77.53±2.94%、78.27±1.99%、79.13±6.10%，P0.05)差異有統(tǒng)計學意義。 3.混合淋巴細胞反應(MLR)：不同濃度的PEDF(50ng/ml、100ng/ml、200ng/ml)作用于GM-CSF誘導培養(yǎng)小鼠BMDC，混合淋巴反應刺激指數(shù)(stimulation index，SI)表達值顯著升高(PEDF50、100、200ng/ml，1.44±0.11、1.61±0.40、1.59±0.29，P0.05)，差異有統(tǒng)計學意義。 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)計學意義。 結(jié)論： 1.宮頸浸潤鱗癌組織中PEDF表達水平顯著下調(diào)，DC分布密度下降，且分化成熟度降低，提示PEDF的表達水平可能與局部DC生物學功能異常相關(guān)。 2.PEDF顯著上調(diào)小鼠BMDC表面CD11c、CD80、CD86表達水平，上調(diào)Il-12分泌及激活T淋巴細胞增殖的能力，可作為宮頸局部樹突狀細胞免疫功能的正向調(diào)節(jié)劑。 3.PEDF下調(diào)導致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)機制有待進一步研究。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驗依據(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：1551363