【作者】 丁惠；

【导师】 王泽华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2013， 博士

【摘要】 第一部分宫颈癌组织中巨噬细胞的存在及其分布特点目的探讨肿瘤相关巨噬细胞(tumor-associated macrophages, TAMs)在宫颈癌组织中的存在及其分布特点。方法免疫组化双染法检测61例早期宫颈浸润癌标本,27例CIN标本和29例正常宫颈标本中CD68阳性的巨噬细胞和D2-40阳性的微淋巴管的存在和分布,并对宫颈癌中巨噬细胞的分布与临床病理特征间进行分析。结果1.巨噬细胞在宫颈癌癌巢中的数量明显多于在正常宫颈组织的数量(P<0.05)。在癌间质中的数量明显多于在正常宫颈组织和CIN组织中的数量(P均<0.05),而在癌周或癌旁中的数量和正常宫颈组织相比无明显差异(P>0.05)。2.微淋巴管在宫颈癌组织中主要位于癌间质和癌周,而在癌巢区罕见。微淋巴管在癌间质中的分布明显高于正常宫颈组织和CIN组织(P均<0.05)。3.对巨噬细胞密度(macrophage density, MD)和微淋巴管密度(lymphatic vessel density, LVD)的Pearson相关分析发现癌间质中的TAMs和癌间质中的LVD(P=0.0002),以及癌间质中的TAMs和癌周的LVD存在显著正相关(P=0.0322)。在癌巢TAMs和癌间质LVD,癌周TAMs和癌周LVD,或癌旁TAMs和癌旁LVD间未发现明显相关性(P>0.05)。4.有淋巴转移的宫颈癌患者癌问质MD水平显著高于无淋巴转移的宫颈癌患者的癌间质MD水平(P=0.033)。肿瘤长径大于等于2cm的宫颈癌患者癌间质MD水平显著高于肿瘤长径小于2cm者(P=0.006)。TAMs数量与病例年龄、FIGO分期、侵犯深度、组织分化、或组织学类型间无明显相关性(P>0.05)。结论宫颈癌间质中的TAMs明显增加,且与淋巴管密度和淋巴转移相关。第二部分宫颈癌微环境中巨噬细胞的来源目的探讨宫颈癌肿瘤间质中增加的TAMs的来源。方法1.用transwell小室实验观察宫颈癌细胞Hela、Siha和C33A对经佛波酯诱导THP-1单核细胞转化而成的巨噬细胞的迁移影响,模拟巨噬细胞的募集。2.在宫颈癌组织中利用免疫组化和免疫荧光的方法用Ki67抗体原位检测宫颈癌组织中的巨噬细胞的增殖活性。结果1.巨噬细胞体外募集实验结果显示同空白对照组和正常宫颈上皮细胞CRL2614组相比,宫颈癌细胞系组巨噬细胞的迁移明显增加(P均<0.001)。2.免疫组化双染法和免疫荧光法检测宫颈癌组织中巨噬细胞的Ki67增殖活性的实验结果一致显示,CD68阳性着色的细胞与Ki67阳性着色细胞无重叠,表明宫颈癌组织中的巨噬细胞不具有增殖活性。结论宫颈癌细胞可以促使附近的巨噬细胞发生定向迁移,宫颈癌癌间质中增加的TAMs可能部分来源于对邻近区域已存在的巨噬细胞的募集。第三部分TAMs对宫颈癌细胞的侵袭迁移影响目的探讨TAMs对宫颈癌细胞的侵袭迁移能力影响。方法1.用铺有Matrigel基质胶的1transwell小室实验观察TAMs对宫颈癌细胞Hela、Siha和C33A的侵袭能力的影响。2.用transwell小室实验观察TAMs对宫颈癌细胞Hela、Siha和C33A的迁移能力影响。结果1.宫颈癌细胞的transwell侵袭实验结果显示：在TAMs的影响下,相比空白对照组,Hela组、Siha组和C33A组细胞穿透基质胶和微孔运动到对侧的侵袭数目均明显增加(P均<0.001)。2.宫颈癌细胞的transwell迁移实验结果显示：在TAMs的影响下,相比空白对照组,Hela组、Siha组和C33A组细胞穿透基质胶和微孔运动到对侧的迁移数目均明显增加(P均<0.001)。结论TAMs可以促进宫颈癌细胞的侵袭迁移能力。第四部分TAMs促宫颈癌淋巴管的形成目的探讨TAMs对宫颈癌淋巴管形成的影响和机制。方法1.分别收集正常宫颈上皮细胞、宫颈癌细胞、巨噬细胞和巨噬细胞-宫颈癌细胞共培养的培养上清作为条件培养基,通过人淋巴管内皮细胞HLEC的体外成管实验,了解不同细胞对淋巴管形成的影响。2.建立“巨噬细胞-宫颈癌细胞”直接共培养模型,ELISA法检测共培养上清中IL-1p和IL-8的分泌。3.通过免疫磁珠分离“巨噬细胞-宫颈癌细胞”直接共培养模型中的细胞,用RT-PCR法检测巨噬细胞和宫颈癌细胞在共培养前后淋巴管生成相关因子(VEGF-C/-D/-A、IL-1β和IL-8)的变化。4.建立模拟宫颈癌微环境的“巨噬细胞-宫颈癌细胞-淋巴管内皮细胞”直接共培养模型和作为对照的“巨噬细胞-正常宫颈上皮细胞-淋巴管内皮细胞”直接共培养模型,研究宫颈癌微环境对淋巴管内皮细胞的表面受体VEGFR-3的表达影响。结果1. HLEC的管腔样结构的形成数量在普通LEC培养基,CRL2614条件培养基,巨噬细胞条件培养基,Hela条件培养基,Siha条件培养基,C33A条件培养基中无明显差别(P>0.05)。只有在巨噬细胞-宫颈癌细胞共培养条件培养基中生长的HLEC形成的管腔样结构才明显增加(P<0.001)。2. ELISA结果显示同正常宫颈上皮细胞CRL2614、单独培养的巨噬细胞、单独培养的宫颈癌细胞系相比,巨噬细胞-宫颈癌细胞直接共培养模型中分泌的IL-1p和IL-8水平均明显增加(P均<0.001)。3. RT-PCR实验结果显示IL-1p和IL-8在与巨噬细胞共培养之后的宫颈癌细胞系中的mRNA表达均明显增加(P均<0.05)。与宫颈癌细胞系共培养之后的巨噬细胞中VEGF-C和VEGF-A的mRNA表达明显增加,VEGF-D的表达在Hela组显示增加,IL-1p在Hela和Siha组的表达增加(P均<0.05)。4.对比“巨噬细胞-正常宫颈上皮细胞-HLEC共培养模型”中的HLEC,“巨噬细胞-宫颈癌细胞-HLEC共培养模型”中的HLEC的VEGFR-3的mRNA表达明显增加(P均<0.05)。结论微环境中的巨噬细胞通过与宫颈癌细胞的相互作用促进淋巴管的形成。更多还原

【Abstract】 Part Ⅰ The presence and distribution characters of TAMs in cervical cancer tissuesObjectiveTo explore the presence and distribution characters of tumor-associated macrophages (TAMs) in cervical cancer tissues.MethodsImmunohistochemistry was used for detection of D2-40-positive lymphatic endothelial cells and CD68-positive macrophages in117cervix tissues including sixty-one invasive carcinomas of uterine cervix, twenty-seven cervical intraepithelial neoplasms (CIN), and twenty-nine normal cervix. And the relationship between the number of TAMs and the clinicopathological features in61human cervical cancers were analyzed.Results1. Macrophages in cervical cancer were enriched in tumor nests and stroma when compared with normal cervix and CIN (P<0.05), whereas no significant differences among peritumor, paratumor and normal cervix were found (P>0.05).2. Lymphatic vessels in cervical cancer were mainly located in the tumor stroma and peritumor, but rarely observed in cancer nests. LVD in tumor stroma was apparently higher than that in normal cervix and CIN (all P<0.05).3. Pearson tests showed there was a significant positive correlation between stromal TAMs and stromal LVD (P=0.0002), and between stromal TAMs and peritumor LVD (P=0.0322). Significant correlation was not found between tumor nests TAMs and stromal LVD, between peritumor TAMs and peritumor LVD, or between paratumor TAMs and paratumor LVD (P>0.05).4. Stromal MD in cases with lymphatic metastasis were increased, when compared with those without lymphatic metastasis (P=0.033). And there were increased stromal TAMs in tumors2cm or greater in diameter, when compared with that in those smaller than2cm in diameter (P=0.006). There was no significant correlation between TAMs and age, FIGO stage, invasion depth, histological differentiation, or histological type.ConclusionIncreased stromal TAMs are significantly correlated with LVD and lymphatic metastasis in cervical cancer. Part Ⅱ The source of TAMs in cervical cancer microenviromentObjectiveTo explore the source of TAMs in the tumor microenviroment of cervical cancer.Methods1. The influence of cervical cancer cells (Hela, Siha or C33A) on migration of PMA-treated THP-1macrophages was examed by transwell assays, which imitated the macrophages recruitment in vitro.2. The proliferation activity of macrophages in cervical cancer tissues was evaluated by double staining with CD68antibody and Ki67antibody by immunohistochemistry (IHC) and immunofluorescence (IF) stains.Results1. Cervical cancer cells were more potential to promote the migration of macrophages towards them compared with blank control and normal cervix epithelium cell line CRL2614(all P<0.001).2. Both IHC and IF showed that macrophages marked by CD68in cervical cancer tissues were Ki67negative. ConclusionCervical cancer cells promote the directional migration of existed macrophages, and part of the increased TAMs in tumor nests and stroma might be owned to recruitment of the preexisting macrophages in the surrounding. Part Ⅲ The influence of TAMs on the invasion and migration of cervical cancer cellsObjectiveTo explore the influence of TAMs on the invasion and migration of cervical cancer cells.Methods1. The influence of TAMs on invasion of cervical cancer cells (Hela, Siha or C33A) was assessed by transwell assays with Matrigel supplied.2. The influence of TAMs on migration of cervical cancer cells (Hela, Siha or C33A) was assesses by transwell assays.Results1. The number of invading cervical cancer cells increased in presence of TAMs, compared with blank (P<0.001).2. The number of migrating cervical cancer cells increased in presence of TAMs, compared with blank (P<0.001).ConclusionTAMs promote the invasion and migration of cervical cancer cells. Part IV The effect of TAMs and human cervical cancer cells on lymphangiogenesisObjectiveTo detect the effect of TAMs and human cervical cancer cells on lymphangiogenesis.Methods1. The tube formation assays in vitro of HLEC were performed in different culture supernatants, including supernatants of CRL2614, cervical cancer cells, macrophages, and macrophage-cervical cancer cell coculture.2. ELISA assays were performed to determine the levels of IL-1β and IL-8in the conditioned media from macrophage-cervical cancer cell coculture.3.The mRNA expression of imflammatory factors (IL-1β and IL-8) and VEGFs (VEGF-C, VEGF-A and VEGF-D) in macrophages and cervical cancer cells cultured alone or cocultured were analyzed by RT-PCR.4. The mRNA expressions of VEGFR-3in HLEC isolated from "macrophage-cervical cancer cell-HLEC model" or "macrophage-CRL2614-HLEC model" was analyzed by RT-PCR.Results1. There was no significant difference in the number of tube-like structures formed by HLEC between cells cultured in common LEC medium and those cultured in conditioned media from CRL2614, macrophages, Hela, Siha or C33A (P>0.05). Only the conditioned medium from cervical cancer cells-macrophages cocluture resulted in a obvious increase in number of tube-like structures of HLEC (P<0.001).2. The levels of IL-1β and IL-8were obviously increased in the macrophage-cervical cancer cell coculture supernatants, when compared with that in culture supernatants of CRL2614, macrophages, or cervical cancer cells(all P<0.001).3.RT-PCR showed IL-1β and IL-8increased obviously in cervical cancer cells (Hela, Siha or C33A) cocultured with macrophages, respectively compared with Hela, Siha or C33A (all P<0.05). VEGF-C and VEGF-A increased in macrophages cocultured with cervical cancer cells (Hela, Siha or C33A) compared with macrophages cultured alone. VEGF-D showed increased in Hela group and IL-1β also was found increased in Hela or Siha groups (all P<0.05).4. The mRNA expression of VEGFR-3and PDPN were increased in HLEC cocultured with macrophages and Hela/Siha/C33A compared with those cocultured with macrophages and CRL2614(P<0.05).ConclusionMacrophages and tumor cells act synergistically to promote lymphangiogenesis in cervical cancer.更多还原