树突状细胞生物学功能变化与宫颈癌发病关系的研究

发布时间：2018-03-01 09:33

  本文关键词： 宫颈癌 树突状细胞 色素上皮衍生因子　出处：《第三军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景和目的 子宫颈癌(cervical cancer)是严重威胁女性健康的恶性肿瘤。目前，其临床治疗采用了手术、化疗、放疗、生物治疗等多种手段，虽疗效明确，但副作用大且复发率高(约35%)。已开发的各种基于HPV的预防性疫苗，因免疫型别，接种时机和远期效应等因素的影响，与临床需求尚有较远差距。因此，进一步深化子宫颈癌的发病机制系统研究，以改进现有的治疗策略，或研发更为有效的新型治疗手段对降低宫颈癌的死亡率极为重要。 高危乳头瘤病毒(human papilloma virus，HPV)持续感染是子宫颈癌发生的明确致病因素，人类恶性肿瘤的形成不仅取决于肿瘤的恶性行为，还与机体的抗肿瘤免疫监视功能相关。树突状细胞(dendritic cell，DC)作为人体内最强大的专职抗原提呈细胞(antigen presenting cell，APC)，在机体抗肿瘤细胞免疫过程中居于中心地位。在识别局部危险抗原后，定位于外周组织的未成熟DC(immature DC，imDC)分化成熟(matureDC，mDC)并定向迁移至引流淋巴结，激活免疫应答，因此，DC分化成熟是特异性抗肿瘤免疫的关键环节。研究发现，成熟DC数量在宫颈浸润癌病灶中显著下调；在离体实验，SiHa细胞(宫颈癌细胞系，HPV16+)培养上清显著下调DC分化的能力。上述研究表明，宫颈癌发生、进展过程中，存在病变局部组织DC分化受阻。 DC的分化行为涉及复杂的生理调节机制，局部微环境的细胞因子和各类活性物质均参与DC分化的调节。新近研究发现，哺乳动物体内广泛存在的炎性介质--色素上皮衍生因子(pigmented epithelium-derived factor，PEDF)在一系列肿瘤(包括宫颈鳞癌组织)组织内表达异常，其具有多种生物学特性，能够促进与DC具有相同细胞来源的巨噬细胞的活化和募集。本研究小组前期结果发现：鼠重组PEDF可显著上调小鼠骨髓来源DC(BMDC)靶向跨膜迁移行为和表面标志物CD11c表达水平。以上证据提示：宫颈局部重要活性物质-PEDF可能是DC分化、成熟的重要调节因子，癌变组织局部PEDF缺失是DC分化、成熟受阻的重要因素。鉴于PEDF与DC密切的关系，我们试图通过观察宫颈浸润癌局部组织PEDF表达水平及树突状细胞的分布，初步探讨PEDF对树突状细胞表型分化、合成分泌及抗原提呈等生物学功能的调节作用，将为进一步明确PEDF、DC与宫颈癌发生免疫逃逸的作用，可能实施逆转DC功能障碍，为重建患者免疫监视效率以提高局部抗肿瘤免疫提供初步的实验依据，为宫颈癌治疗提供新的临床治疗策略。 材料和方法： 一.宫颈癌局部PEDF表达及DC分布特点的研究：取正常宫颈组织(子宫肌瘤患者，10例)，不同分期宫颈浸润性鳞癌患者宫颈病变组织(Ib期10例、II期10例、III期6例)，Western blot检测PEDF表达，免疫荧光检测宫颈病变组织DC分布特点。 二. PEDF对小鼠BMDC分化和抗原提呈功能的影响：取6-8周正常C57BL/6小鼠胫骨和股骨骨髓，用红细胞裂解液裂解红细胞后，应用重组鼠粒细胞-巨噬细胞集落刺激因子(Recombinant mouse gramulocyre-macrophage，rmGM-CSF)和重组人白介素4(Recombinant mouse Interleukin-4，rmIL-4)进行体外诱导培养DCs，DCs分组如下： A组：DCs+PEDF(50ng/mL)；B组：DCs+PEDF(100ng/mL)；C组：DCs+PEDF(200ng/mL)；D组：DCs+LPS(1ug/mL)；E组：DCs+1640。从形态学上对细胞进行观察；通过流式细胞术(Flow CytoMetry，FCM)检测各组DCs表达CD11c、CD80和CD86的变化；混合淋巴细胞反应(mixed lymphocyte reaction,MLR)检测DC刺激T淋巴细胞的能力；酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA法)检测其分泌IL-12的水平。 结果： 一、宫颈组织局部PEDF表达及DC分布特点 1. PEDF在宫颈浸润鳞癌组织的表达：宫颈浸润性鳞癌患者宫颈病变组织PEDF蛋白表达水平低于正常宫颈组织(P0.05)。 2.宫颈浸润鳞癌组织局部CD1a(+)细胞密度、CD83(+)细胞密度均降低(P0.05)。 二、PEDF对小鼠BMDC分化和抗原提呈功能的影响 1.形态学观察：GM-CSF诱导培养小鼠BMDC至第5天时，细胞表面见少量毛刺样突起，细胞聚集成团，呈集落样贴壁生长；加入不同浓度的PEDF(50ng/ml、100ng/ml、200ng/ml)和LPS(阳性对照，1ug/ml)作用后，可见细胞集落增大、表面突起增多。 2.表型分化：以不同浓度的PEDF(50ng/ml、100ng/ml、200ng/ml)作用于GM-CSF诱导培养小鼠BMDC，，流式细胞术检测细胞表面分子CD11c表达水平显著升高(PEDF50、100、200ng/ml，92.80±3.81%、87.43±7.98%、92.53±3.13%，P0.05)差异有统计学意义；CD80表达水平显著升高(PEDF50、100、200ng/ml，92.77±3.50%、94.10±2.76%、93.30±3.22%，P0.05)差异有统计学意义；CD86表达水平显著升高(PEDF50、100、200ng/ml，77.53±2.94%、78.27±1.99%、79.13±6.10%，P0.05)差异有统计学意义。 3.混合淋巴细胞反应(MLR)：不同浓度的PEDF(50ng/ml、100ng/ml、200ng/ml)作用于GM-CSF诱导培养小鼠BMDC，混合淋巴反应刺激指数(stimulation index，SI)表达值显著升高(PEDF50、100、200ng/ml，1.44±0.11、1.61±0.40、1.59±0.29，P0.05)，差异有统计学意义。 4.分泌Il-12水平：不同浓度的PEDF(50ng/ml、100ng/ml、200ng/ml)作用于GM-CSF诱导培养小鼠BMDC，IL-12分泌水平显著升高(PEDF50、100、200ng/ml，222.27±8.01、227.47±4.58、214.83±12.64pg/mL，P0.05)，差异有统计学意义。 结论： 1.宫颈浸润鳞癌组织中PEDF表达水平显著下调，DC分布密度下降，且分化成熟度降低，提示PEDF的表达水平可能与局部DC生物学功能异常相关。 2.PEDF显著上调小鼠BMDC表面CD11c、CD80、CD86表达水平，上调Il-12分泌及激活T淋巴细胞增殖的能力，可作为宫颈局部树突状细胞免疫功能的正向调节剂。 3.PEDF下调导致DC免疫功能异常可能是宫颈癌发生免疫逃逸的重要环节之一，其调节机制有待进一步研究。为通过PEDF逆转DC功能障碍，重建患者免疫监视效率以提高局部抗肿瘤免疫作用提供了初步实验依据。
[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锛

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