釉质上皮型颅咽管瘤实验模型建立及IL-6对釉质上皮型颅咽管瘤细胞体外迁移特性的影响
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摘要
颅咽管瘤(Craniopharyngioma, CP)是一种沿颅咽管路径生长的颅内上皮来源的轴外肿瘤,多位于鞍区或鞍旁区域,是颅内最常见的先天性肿瘤之一。颅咽管瘤约占所有儿童颅内肿瘤发病率的6-13%,特别是在鞍区儿童肿瘤中占60%以上。1904年,奥地利神经病理学家Jakob Erdheim首次将颅咽管瘤作为一类单独存在的鞍区肿瘤进行了详细的描述,100多年过去了,国内外广大学者对颅咽管瘤进行了广泛而深入的研究,但限于该肿瘤研究平台的缺乏和技术条件的制约,目前对于其肿瘤起源、病理特征、肿瘤生物学特性等方面的认识仍有很大争议。
颅咽管瘤属于良性肿瘤,国际卫生组织(World Health Organization, WHO)肿瘤病理分类为Ⅰ级,按组织形态可分为釉质上皮型(Adamantinomatous craniopharyngioma, ACP)和鳞状乳头型(Squamous papillary craniopharyngioma, SPCP)两个亚型。相较于后者,釉质上皮型颅咽管瘤常形成指状突起侵袭周围脑组织,术中全切肿瘤困难,术后易复发,临床预后也差于后者。国外多个单中心大宗随访结果也提示釉质上皮型颅咽管瘤患者预后较鳞状乳头型差,但具体机制尚不清楚。多年来,由于缺乏稳定的体外及体内模型,国内外学者主要在组织学层面对釉质上皮型颅咽管瘤进行了大量研究,但相关基础研究进展缓慢；少数学者对釉质上皮型颅咽管瘤细胞进行了体外培养并应用于功能实验,但文中均未对肿瘤细胞特性进行相关描述,体内移植瘤模型也少有报道。我们有必要对釉质上皮型颅咽管瘤实验模型进一步探索,为深入了解该肿瘤生物学特性和发生发展规律提供新的研究手段。
我课题组前期研究中发现：颅咽管瘤,特别是釉质上皮型颅咽管瘤与周边正常神经组织常形成炎症粘连,导致术中难以全切肿瘤,术后肿瘤易复发,炎症程度与患者预后明显相关；自发的颅咽管瘤囊性结构破裂和术中囊液渗漏常可导致无菌性脑膜炎的发生；组织学检查发现釉质上皮型颅咽管瘤细胞周围常形成密实的胶质增生带和大量炎症细胞侵润。这些现象说明颅咽管瘤细胞或肿瘤间质细胞可能通过产生大量炎症细胞因子调节肿瘤炎症的发生发展,影响患者预后。Mori曾报道白细胞介素6(Inierleukin-6, IL6)在颅咽管瘤中异常表达,可能是导致颅咽管瘤炎症的重要炎性介质,影响患者预后；但限于缺乏可靠的实验手段,作者并未继续在体外或体内进行相关功能实验深入研究,其具体作用机制尚不清楚。
IL6是一种重要的多功能细胞因子,参与调节造血、免疫与炎症反应,与细胞凋亡、增殖、损伤修复等密切相关,也参与调节中枢神经和心血管系统的重要生理功能。同时,IL6在大肠癌、乳腺癌等多种上皮性肿瘤以及脑胶质瘤等中枢神经肿瘤中均有高表达,且与肿瘤预后密切相关。IL6可以通过肿瘤细胞“自分泌”或肿瘤微环境中间质细胞“旁分泌”等途径,与相应受体结合形成复合体导致异常信号通路的激活,进而促进肿瘤发生、肿瘤新生血管形成,肿瘤迁移侵袭能力提高。然而,IL6在釉质上皮型颅咽管瘤中的具体作用尚不清楚。由此,我们假设：釉质上皮型颅咽管瘤细胞及其微环境中间质成分是否也能通过“自分泌”或“旁分泌”的方式分泌IL6,促使肿瘤细胞特性发生改变,从而影响患者预后?
因此,基于釉质上皮型颅咽管瘤基础研究的需要以及IL6在该肿瘤中可能存在的重要生物学作用,本研究采用原代培养的方法建立人釉质上皮型颅咽管瘤有限细胞系,明确其生物学性状；采用裸小鼠皮下种植细胞悬液的方法探讨釉质上皮型颅咽管瘤体内模型建立的可能性；同时,采用抗体芯片技术检测不同炎症分级肿瘤组织中炎症细胞因子的表达,外源性IL6刺激釉质上皮型颅咽管瘤细胞,明确IL6对肿瘤细胞生物学特性的作用,并对其相关作用机制进行初步分析,旨在阐述炎症与颅咽管瘤间的相互关系,为临床治疗颅咽管瘤提供新的思路。
第一章第一节釉质上皮型颅咽管瘤细胞模型的建立与检测
目的：探讨有效的釉质上皮型颅咽管瘤细胞原代培养方法,检测体外培养的肿瘤细胞自身生物学特性。
方法：采用胰酶消化法建立釉质上皮型颅咽管瘤有限细胞系,并通过滤膜分次过滤法纯化细胞；倒置显微镜、HE染色观察生长情况及细胞形态,免疫细胞化学方法鉴定肿瘤表型,噻唑蓝比色法绘制细胞生长曲线,并用流式细胞技术分析肿瘤细胞周期。
结果：本次原代培养成功率为88.90%,其中原代细胞可传代率为81.3%。人釉质上皮型颅咽管瘤细胞在含血清培养基中呈贴壁生长,肿瘤细胞常成小团状生长,体积大,胞浆丰富,可见少量气泡状结构,细胞呈不规则多边形“上皮样”形态,胞核成圆形,体积较大。免疫细胞化学鉴定Pan-ck在釉质上皮型颅咽管瘤细胞胞浆内表达,证实其上皮性来源。肿瘤细胞体外增殖缓慢,但第1-5天细胞增殖率均有差异(F=190.748,P<0.0001),细胞周期显示肿瘤细胞均在正常二倍体范围内,未见异倍体细胞出现,肿瘤细胞多处于G0-G1期,46.8±3.61%；部分处于G2-M期,37.3±1.51%；少部分处于S期,15.9±2.77%。
结论：采用有血清贴壁培养法可以建立釉质上皮型颅咽管瘤有限细胞系模型。通过滤膜分次过滤等特殊处理方法,可有效地对釉质上皮型颅咽管瘤进行分离、培养和扩增,有利于后续实验的进行；釉质上皮型颅咽管瘤细胞呈现Pan-CK阳性表型,细胞主要处于G0-G1期,体外增殖缓慢,倍体分析未见异倍体细胞。
第一章第二节人釉质上皮型颅咽管瘤裸鼠皮下移植瘤模型的初步建立
目的：在前期釉质上皮型颅咽管瘤有限细胞系建立的基础上,初步建立人釉质上皮型颅咽管瘤的裸鼠皮下移植瘤模型。
方法：采用胰酶消化法将新鲜的釉质上皮型颅咽管瘤组织原代培养,常规传代,然后将纯化的第三代颅咽管瘤细胞制成细胞悬液,接种到BALB/c裸小鼠背部皮下或腋下,观察接种成瘤后肿瘤组织生长情况和特点,明确肿瘤基本性质。
结果：利用体外培养的釉质上皮型颅咽管瘤细胞种植于裸小鼠皮下可以建立该肿瘤移植瘤模型。颅咽管瘤细胞可以在裸鼠皮下存活,接种后8天可见有移植瘤形成,背部成瘤率约为37.5%,病理切片显示肿瘤样鳞状上皮细胞团增生。
结论：通过颅咽管瘤细胞原代培养,裸鼠皮下种植已纯化的肿瘤细胞悬液可以建立颅咽管瘤移植瘤动物模型,有可能作为颅咽管瘤基础研究的有效手段。但目前该模型尚不稳定,成瘤率较低,需要更多的研究优化该移植瘤建立的条件。
第二章第一节颅咽管瘤上皮间质转化相关蛋白的表达及临床病理联系
目的：检测颅咽管瘤,特别是肿瘤正常组织交界处上皮间质转化相关蛋白(Vimentin, E-cadherin, β-catenin)表达,联系临床病理和预后进行相关分析。
方法：运用免疫组化和免疫荧光方法检测42例颅咽管瘤中Vimentin, E-cadherin, β-catenin的表达,联系临床病理特征和预后进行相关分析。
结果：肿瘤整体和肿瘤正常组织交界处,釉质上皮型和鳞状乳头型颅咽管瘤两亚型间Vimentin, E-cadherin表达均有显著差别(X2=11.181,P=0.004,,=6.931,P=0.031；X2,=17.393,P<0.001,X2=8.501,P=0.014)；肿瘤正常组织交界处,β-catenin核/浆表达在两亚型肿瘤中有显著差别(X2=19.765,P<0.001)。整体范围内,Vimentin和E-cadherin表达与肿瘤复发、术后体重和下丘脑功能紊乱相关(X2=23.834,P<0.001,r=0.643,P<0.001,r=0.637,P<0.001；X2=16.526,P<0.001,r=-0.579,P<0.001,r=-0.714,P<0.001)；肿瘤正常组织交界处,Vimentin和E-cadherin/β-catenin表达与肿瘤复发、术后体重和下丘脑功能紊乱相关(X=19.782,P<0.001,r=0.642,P<0.001,r=0.573,P=0.001；X2=11.475,P=0.003,r=-0.54,P<0.001,r=-0.626,P<0.001；X2=16.572,P<0.001,r=-0.54,P<0.001,r=-0.506,P=0.001)
结论：本研究首次证实颅咽管瘤组织中Vimentin, E-cadherin和β-catenin表达与肿瘤预后相关；上皮间质转化有可能是影响颅咽管瘤进程的重要机制之一。
第二章第二节IL-6对釉质上皮型颅咽管瘤细胞体外迁移特性的影响
目的：探讨IL6对釉质上皮型颅咽管瘤细胞迁移特性的影响及其相关作用机制。
方法：回顾性分析炎症等级与颅咽管瘤临床病理特点的相关性；采用抗体芯片检测不同炎症等级的釉质上皮型颅咽管瘤组织的差异炎症细胞因子；筛选IL6刺激颅咽管瘤细胞,检测其对肿瘤细胞体外迁移特性的影响,初步探讨相关作用机制。
结果：颅咽管瘤组织炎性与肿瘤病理类型(X2=6.603,P=0.037)、全切率(X2=8.188,P=0.017)、钙化(y=0.326,P=0.022)和术后患者HSS评分(γ,=0.376,P=0.008)相关；IL6等多种炎症因子在不同炎症等级肿瘤组织中差异表达；IL6-IL6Ra-GP130在部分颅咽管瘤中表达；肿瘤囊液、肿瘤细胞和肿瘤相关成纤维细胞上清中可检测出sIL6R的表达；IL6促进釉质上皮型颅咽管瘤细胞体外迁移能力,并有浓度依赖性(F=112.390,P<0.0001；F=126.654,P<0.0001)；IL6可以增强肿瘤细胞Vimentin的表达,减弱E-cadherin的表达。
结论：颅咽管瘤组织炎性与肿瘤病理类型、全切率、钙化程度和术后患者下丘脑功能相关,炎症反应重的患者大块钙化出现率高、肿瘤全切率低、患者术后HSS评分高；不同炎症等级的釉质上皮型颅咽管瘤中炎症细胞因子表达存在差异；IL6可能通过“经典”和“反式”途径诱导釉质上皮型颅咽管瘤细胞发生EMT转变,增强细胞体外迁移能力。
Craniopharyngioma is an intracranial extra-axial epithelial tumor of sellar or parasellar region, which grows along the cranial pharyngeal tube..Craniopharyngioma is one of most common intracranial congenital tumor which accounts for6-13%of all intracranial tumors and constitutes60%tumors of the sellar region in children. Since the first description of craniopharyngioma as a distinct tumor in the pituitary region by Austrian neuropathologist Jakob Erdheim in1904; many extensive and in-depth study on craniopharyngioma has been conducted. As yet, more than100years later, expert consensus on the origin, pathological features and biological characteristics of this formidable neoplasm is still lacking due to the lack of the tumor research platform and technical conditions.
Craniopharyngiomas are benign tumors which are classified into, adamantinomatous variant and squamous papillary variant by WHO. The adamantinomatous craniopharyngioma often form finger-like protrution invades the surrounding brain tissue, leading to difficult to total resection of the tumor, postoperative recurrence and worse clinical prognosis compared to the latter variant. The results of several single-center studies have shown that the prognosis of adamantinomatous craniopharyngioma is worse than the squamous papillary variant, but the mechanism is not clear. Although many scholars had undertaken extensive research on the adamantinomatous craniopharyngioma in the organizational level, the progress of basic research of adamantinomatous craniopharyngioma is slow due to the lack of stable in vitro and in vivo models. Several scholars cultured the adamantinomatous craniopharyngioma in vitro and used in functional experiments, however, characteristics of tumor cells was not mentioned in the study. At the same time, few in vivo xenograft model of adamantinomatous craniopharyngioma are reported. The contemporary understanding of the biological characteristics of this tumor can be further expanded by establishment of the adamantinomatous craniopharyngioma experimental model.
We previously found that the formation of inflammatory adhesions of adamantinomatous craniopharyngioma with the surrounding normal brain tissue is closely related to tumor total resection, postoperative recurrence, prognosis of patients in follow up; the spontaneous rupture of craniopharyngioma cysts and cyst fluid spillage during surgery can lead to aseptic meningitis; histologically, intense fibrillary gliosis and infiltration of inflammatory cells are frequently present in the surrounding parenchyma. These observations suggest that tumor cells may produce certain inflammatory cytokines that might play an important role in tumor progression and prognosis of patients. Mori et al has demonstrated IL-6as an important inducer of craniopharyngioma associated inflammation affecting the prognosis of patients, however the elucidation of the role of IL-6in craniopharyngioma in vitro is not yet reported due to the lack of technical conditions.
IL-6is multifunctional cytokine that contributes to the regulation of hemopoiesis, immunity and inflammatory response, also contributes to regulate physiological function of central nervous and cardiovascular system, and associates with cell survival, proliferation and wound healing. High expression of IL-6can be found in colorectal cancer, breast cancer and other epithelial tumors, as well as glioma and other central nervous system tumors, which is closely related with prognosis. Tumor cells or mesenchymal cells in tumor microenvironment can secrected IL6, through autocrine or paracrine secretion. IL-6after binding to its receptors forms a complex to activate the abnormal signaling pathways, contributing to the promotion of tumorigenesis, tumor angiogenesis, tumor migration and invasion. However, the role of IL-6in adamantinomatous craniopharyngioma is still not clear. We contemplate that the IL-6secretion by adamantinomatous craniopharyngioma or mesenchymal cells in tumor microenvironment through either autocrine or paracrine secretion might promote characteristics change in tumor cells and thus affect the prognosis of this patients?
In this study, we established the adamantinomatous craniopharyngioma finite cell line by primary culture in vitro as the necessity of the scientific reserch of craniopharyngioma and further investigate the biological characters of the tumor cells in order to elucidate the role of IL-6in craniopharyngioma. Additionally, we established the xenotransplanted tumor model of human adamantinomatous craniopharyngioma by implanting the adamantinomatous craniopharyngioma cells into the nude mice subcutaneously. The antibody array technology was used to investigate the expression of inflammatory cytokines in the different inflammation grade of the adamantinomatous craniopharyngioma tissue; which was followed by the stimulation of the adamantinomatous craniopharyngioma in vitro by IL-6to investigate the role of IL6on biological characters of tumor cells. Based on the findings of this experiment the possible mechanism of IL-6on craniopharyngioma cells is discussed. The aim of our study is to elucidate the relationship between inflammation and craniopharyngioma that might aid in development of clinical treatment for craniopharyngioma.
Chapter I Section I Cell culture and characterization of adamantinomatous craniopharyngioma
Objective:To investigate an efficient method of primary adamantinomatous craniopharyngioma cells culture and observe the behaviour of adamantinomatous craniopharyngioma cells in vitro.
Methods:The specimen of adamantinomatous craniopharyngioma were treated with trypsin and cultured in vitro, and purified by filter membrane. The cell morphology was observed by inverted microscope and hematoxylin eosin staining. The expression of Pan-CK was examined by immunocytochemistry. The MTT assay and flow cytometry were employed to study the cell proliferation and cell cycle.
Results:The success rate of primary culture and passage culture is88.9%and81.3%. Human adamantinomatous craniopharyngioma cells showed adherent growth in serum-containing medium. The tumor cells were large with abundant cytoplasm were large and characteristically demonstrated assemebled growth. A small amount of bubble like structure was seen in the cytoplasm. The tumor cells demonstrated slow growth, the growth rate of cells in1st-5th day is statistically significant difference (F=190.748..P<0.0001). The cells were diploid and depicted irregular polygonal and epithelioid form. The tumor cells in G0-G1,46.8±3.61%; in G2-M,37.3±1.51%; in S,15.9±2.77%.
Conclusion:The finite adamantinomatous craniopharyngioma cell line could be cultured in a serum medium and isolated, cultured and expanded effectively by the membrane sub-filtering techinque, which was beneficial for the follow-up experiments. The adamantinomatous craniopharyngioma cells demonstrated the Pan-CK positive phenotype, slow growth mainly in the G0-G1phase with no aneuploid cells.
Chapter I Section II
Establishment of subcutaneous xenotransplanted tumor model of human adamantinomatous craniopharyngioma in nude mice
Objective:Establishment of subcutaneous xenotransplanted tumor model of human adamantinomatous craniopharyngioma in mude mice on the basis of the primary adamantinomatous craniopharyngioma cells culture previously.
Methods:Craniopharyngioma cells were primarily cultured by trypsin digestion. The purified cells in third passage were then implanted subcutaneously into the nude mice. The specimens were studied by microscope to ensure the characteristics of the xenotransplanted tumor.
Results:The subcutaneous xenotransplanted tumor can be established by implanting the adamantinomatous craniopharyngioma cells into the nude mice subcutaneously. Adamantinomatous craniopharyngioma cells can survive in the BALB/c nude mice. The xenograft can be found on8days after implantation. The tumor formation rate at the back was37.5%. The disorderly growth of the squamous epithelial tumor cells can be found under microscope in subcutaneous tissue of nude mice.
Conclusion:The subcutaneously xenotransplanted tumor model of human adamantinomatous craniopharyngioma in nude mice was successfully established and it might be beneficial to condunct further studies on this tumor. However, the model is not stable, and the rate of tumor formation is too low. More research is needed to optimize the conditions of the xenotransplanted tumor establishment.
Chapter II Section I Epithelial-mesenchymal transition and clinico-pathological correlation in craniopharyngioma
Objective:To assess the immunophenotypic changes associated with epithelial mesenchymal transition (EMT) in craniopharyngioma, especially at the tumour invasive front, and correlate the findings with clinicopathological features and patient outcomes.
Methods:42craniopharyngiomas were, subjected to the detection of EMT makers (vimentin, E-cadherin and β-catenin) by immunohistochemistry. The relationships between expression of these markers and various clinico-pathological indicators and clinical outcomes of these tumors were analyzed.
Results:There was statistically significant difference in the expression of vimentin and E-cadherin between adamantinomatous and papillary variants in whole tumor and at the tumor invasive front (χ2=11.181, P=0.004,χ2=6.931,P=0.031;χ2=17.393, P<0.001, χ2=8.501, P=0.014). There was statistically significant difference in the cytoplasmic or nuclear accumulation of β-catenin between the two variants above at the tumor invasive front (χ2=19.765, P<0.001). The expression of vimentin and E-cadherin but β-catenin in whole-tumour sections were associated with tumor recurrence, postoperative weight and hypothalamic disturbances (χ2=23.834, P<0.001, r=0.643, P<0.001, r=0.637, P<0.001; χ2=16.526, P<0.001, r=-0.579, P<0.001, r=-0.714,P<0.001), and the expression of vimentin and E-cadherin/β-catenin at the tumor invasive front were colligated with tumor recurrence, postoperative weight and hypothalamic disturbances (χ2=19.782, P<0.001, r=0.642, P<0.001, r=0.573, P=0.001;χ2=11.475, P=0.003, r=-0.54, P<0.001, r=-0.626, P<0.001;χ2=16.572, P<0.001, r=-0.54, P<0.001, r=-0.506, P=0.001).
Conclusions:Our study exemplifies, for the first time, the potential prognostic implications of vimentin, E-cadherin and β-catenin expression in craniopharyngiomas. Epithelial-mesenchymal transition may represent a crucial mechanism in the progression of craniopharyngiomas.
Chapter ⅡSection Ⅱ The effection of Interleukin-6on the migration characteristics of adamantinomatous craniopharyngioma cells in vitro
Objective:To elucidate the effect and mechanism of the IL-6on the migration characteristics of adamantinomatous craniopharyngioma cells in vitro.
.Methods; We demonstrated the inflammation and clinico-pathological correlation in craniopharyngioma previously. In this study, antibody array assay was used to assess the expression of different inflammatory molecules in adamantinomatous craniopharyngioma tissues at different inflammatory levels. Further, the effect and mechanism of IL-6on adamantinomatous craniopharyngioma cell migration in vitro was investigated.
Results:The inflammation of craniopharyngioma was associated with pathological classification (χ2=6.603, P=0.037), the rate of total resection (χ2=8.188, P=0.017), calcification (γ=0.326, P=0.022) and postoperative HSS (y=0.376, P=0.008). The expression of IL-6and other inflammatory factors varied in tumor tissue at different level of inflammatory exposure. Expressions of IL6-IL6Ra-GP130were detected by immunohistochemistry and soluble IL-6R in the cystic fluid and supernatant by craniopharyngioma cells and fibroblasts were determined by ELISA. Migration in adamantinomatous craniopharyngioma was promoted by IL-6treatment in a dose-dependent manner (F=112.390, P﹤0.0001; F=126.654, P﹤0.0001). The expression of Vimentin was increased and E-cadherin was decreaseed by IL-6treatment.
Conclusion:The inflammation in craniopharyngioma was significantly associated with pathological classification. Additionally this inflammation was associated with the rate of total resection, calcification and postoperative HSS. Antibody arrays demonstrated a significant change in cytokine profiles in patients with different degrees of inflammation. IL-6might promot migration in vitro via classic-and trans-signaling pathways by inducing EMT in adamantinomatous craniopharyngioma.
颅咽管瘤属于良性肿瘤,国际卫生组织(World Health Organization, WHO)肿瘤病理分类为Ⅰ级,按组织形态可分为釉质上皮型(Adamantinomatous craniopharyngioma, ACP)和鳞状乳头型(Squamous papillary craniopharyngioma, SPCP)两个亚型。相较于后者,釉质上皮型颅咽管瘤常形成指状突起侵袭周围脑组织,术中全切肿瘤困难,术后易复发,临床预后也差于后者。国外多个单中心大宗随访结果也提示釉质上皮型颅咽管瘤患者预后较鳞状乳头型差,但具体机制尚不清楚。多年来,由于缺乏稳定的体外及体内模型,国内外学者主要在组织学层面对釉质上皮型颅咽管瘤进行了大量研究,但相关基础研究进展缓慢；少数学者对釉质上皮型颅咽管瘤细胞进行了体外培养并应用于功能实验,但文中均未对肿瘤细胞特性进行相关描述,体内移植瘤模型也少有报道。我们有必要对釉质上皮型颅咽管瘤实验模型进一步探索,为深入了解该肿瘤生物学特性和发生发展规律提供新的研究手段。
我课题组前期研究中发现：颅咽管瘤,特别是釉质上皮型颅咽管瘤与周边正常神经组织常形成炎症粘连,导致术中难以全切肿瘤,术后肿瘤易复发,炎症程度与患者预后明显相关；自发的颅咽管瘤囊性结构破裂和术中囊液渗漏常可导致无菌性脑膜炎的发生；组织学检查发现釉质上皮型颅咽管瘤细胞周围常形成密实的胶质增生带和大量炎症细胞侵润。这些现象说明颅咽管瘤细胞或肿瘤间质细胞可能通过产生大量炎症细胞因子调节肿瘤炎症的发生发展,影响患者预后。Mori曾报道白细胞介素6(Inierleukin-6, IL6)在颅咽管瘤中异常表达,可能是导致颅咽管瘤炎症的重要炎性介质,影响患者预后；但限于缺乏可靠的实验手段,作者并未继续在体外或体内进行相关功能实验深入研究,其具体作用机制尚不清楚。
IL6是一种重要的多功能细胞因子,参与调节造血、免疫与炎症反应,与细胞凋亡、增殖、损伤修复等密切相关,也参与调节中枢神经和心血管系统的重要生理功能。同时,IL6在大肠癌、乳腺癌等多种上皮性肿瘤以及脑胶质瘤等中枢神经肿瘤中均有高表达,且与肿瘤预后密切相关。IL6可以通过肿瘤细胞“自分泌”或肿瘤微环境中间质细胞“旁分泌”等途径,与相应受体结合形成复合体导致异常信号通路的激活,进而促进肿瘤发生、肿瘤新生血管形成,肿瘤迁移侵袭能力提高。然而,IL6在釉质上皮型颅咽管瘤中的具体作用尚不清楚。由此,我们假设：釉质上皮型颅咽管瘤细胞及其微环境中间质成分是否也能通过“自分泌”或“旁分泌”的方式分泌IL6,促使肿瘤细胞特性发生改变,从而影响患者预后?
因此,基于釉质上皮型颅咽管瘤基础研究的需要以及IL6在该肿瘤中可能存在的重要生物学作用,本研究采用原代培养的方法建立人釉质上皮型颅咽管瘤有限细胞系,明确其生物学性状；采用裸小鼠皮下种植细胞悬液的方法探讨釉质上皮型颅咽管瘤体内模型建立的可能性；同时,采用抗体芯片技术检测不同炎症分级肿瘤组织中炎症细胞因子的表达,外源性IL6刺激釉质上皮型颅咽管瘤细胞,明确IL6对肿瘤细胞生物学特性的作用,并对其相关作用机制进行初步分析,旨在阐述炎症与颅咽管瘤间的相互关系,为临床治疗颅咽管瘤提供新的思路。
第一章第一节釉质上皮型颅咽管瘤细胞模型的建立与检测
目的：探讨有效的釉质上皮型颅咽管瘤细胞原代培养方法,检测体外培养的肿瘤细胞自身生物学特性。
方法：采用胰酶消化法建立釉质上皮型颅咽管瘤有限细胞系,并通过滤膜分次过滤法纯化细胞；倒置显微镜、HE染色观察生长情况及细胞形态,免疫细胞化学方法鉴定肿瘤表型,噻唑蓝比色法绘制细胞生长曲线,并用流式细胞技术分析肿瘤细胞周期。
结果：本次原代培养成功率为88.90%,其中原代细胞可传代率为81.3%。人釉质上皮型颅咽管瘤细胞在含血清培养基中呈贴壁生长,肿瘤细胞常成小团状生长,体积大,胞浆丰富,可见少量气泡状结构,细胞呈不规则多边形“上皮样”形态,胞核成圆形,体积较大。免疫细胞化学鉴定Pan-ck在釉质上皮型颅咽管瘤细胞胞浆内表达,证实其上皮性来源。肿瘤细胞体外增殖缓慢,但第1-5天细胞增殖率均有差异(F=190.748,P<0.0001),细胞周期显示肿瘤细胞均在正常二倍体范围内,未见异倍体细胞出现,肿瘤细胞多处于G0-G1期,46.8±3.61%；部分处于G2-M期,37.3±1.51%；少部分处于S期,15.9±2.77%。
结论：采用有血清贴壁培养法可以建立釉质上皮型颅咽管瘤有限细胞系模型。通过滤膜分次过滤等特殊处理方法,可有效地对釉质上皮型颅咽管瘤进行分离、培养和扩增,有利于后续实验的进行；釉质上皮型颅咽管瘤细胞呈现Pan-CK阳性表型,细胞主要处于G0-G1期,体外增殖缓慢,倍体分析未见异倍体细胞。
第一章第二节人釉质上皮型颅咽管瘤裸鼠皮下移植瘤模型的初步建立
目的：在前期釉质上皮型颅咽管瘤有限细胞系建立的基础上,初步建立人釉质上皮型颅咽管瘤的裸鼠皮下移植瘤模型。
方法：采用胰酶消化法将新鲜的釉质上皮型颅咽管瘤组织原代培养,常规传代,然后将纯化的第三代颅咽管瘤细胞制成细胞悬液,接种到BALB/c裸小鼠背部皮下或腋下,观察接种成瘤后肿瘤组织生长情况和特点,明确肿瘤基本性质。
结果：利用体外培养的釉质上皮型颅咽管瘤细胞种植于裸小鼠皮下可以建立该肿瘤移植瘤模型。颅咽管瘤细胞可以在裸鼠皮下存活,接种后8天可见有移植瘤形成,背部成瘤率约为37.5%,病理切片显示肿瘤样鳞状上皮细胞团增生。
结论：通过颅咽管瘤细胞原代培养,裸鼠皮下种植已纯化的肿瘤细胞悬液可以建立颅咽管瘤移植瘤动物模型,有可能作为颅咽管瘤基础研究的有效手段。但目前该模型尚不稳定,成瘤率较低,需要更多的研究优化该移植瘤建立的条件。
第二章第一节颅咽管瘤上皮间质转化相关蛋白的表达及临床病理联系
目的：检测颅咽管瘤,特别是肿瘤正常组织交界处上皮间质转化相关蛋白(Vimentin, E-cadherin, β-catenin)表达,联系临床病理和预后进行相关分析。
方法：运用免疫组化和免疫荧光方法检测42例颅咽管瘤中Vimentin, E-cadherin, β-catenin的表达,联系临床病理特征和预后进行相关分析。
结果：肿瘤整体和肿瘤正常组织交界处,釉质上皮型和鳞状乳头型颅咽管瘤两亚型间Vimentin, E-cadherin表达均有显著差别(X2=11.181,P=0.004,,=6.931,P=0.031；X2,=17.393,P<0.001,X2=8.501,P=0.014)；肿瘤正常组织交界处,β-catenin核/浆表达在两亚型肿瘤中有显著差别(X2=19.765,P<0.001)。整体范围内,Vimentin和E-cadherin表达与肿瘤复发、术后体重和下丘脑功能紊乱相关(X2=23.834,P<0.001,r=0.643,P<0.001,r=0.637,P<0.001；X2=16.526,P<0.001,r=-0.579,P<0.001,r=-0.714,P<0.001)；肿瘤正常组织交界处,Vimentin和E-cadherin/β-catenin表达与肿瘤复发、术后体重和下丘脑功能紊乱相关(X=19.782,P<0.001,r=0.642,P<0.001,r=0.573,P=0.001；X2=11.475,P=0.003,r=-0.54,P<0.001,r=-0.626,P<0.001；X2=16.572,P<0.001,r=-0.54,P<0.001,r=-0.506,P=0.001)
结论：本研究首次证实颅咽管瘤组织中Vimentin, E-cadherin和β-catenin表达与肿瘤预后相关；上皮间质转化有可能是影响颅咽管瘤进程的重要机制之一。
第二章第二节IL-6对釉质上皮型颅咽管瘤细胞体外迁移特性的影响
目的：探讨IL6对釉质上皮型颅咽管瘤细胞迁移特性的影响及其相关作用机制。
方法：回顾性分析炎症等级与颅咽管瘤临床病理特点的相关性；采用抗体芯片检测不同炎症等级的釉质上皮型颅咽管瘤组织的差异炎症细胞因子；筛选IL6刺激颅咽管瘤细胞,检测其对肿瘤细胞体外迁移特性的影响,初步探讨相关作用机制。
结果：颅咽管瘤组织炎性与肿瘤病理类型(X2=6.603,P=0.037)、全切率(X2=8.188,P=0.017)、钙化(y=0.326,P=0.022)和术后患者HSS评分(γ,=0.376,P=0.008)相关；IL6等多种炎症因子在不同炎症等级肿瘤组织中差异表达；IL6-IL6Ra-GP130在部分颅咽管瘤中表达；肿瘤囊液、肿瘤细胞和肿瘤相关成纤维细胞上清中可检测出sIL6R的表达；IL6促进釉质上皮型颅咽管瘤细胞体外迁移能力,并有浓度依赖性(F=112.390,P<0.0001；F=126.654,P<0.0001)；IL6可以增强肿瘤细胞Vimentin的表达,减弱E-cadherin的表达。
结论：颅咽管瘤组织炎性与肿瘤病理类型、全切率、钙化程度和术后患者下丘脑功能相关,炎症反应重的患者大块钙化出现率高、肿瘤全切率低、患者术后HSS评分高；不同炎症等级的釉质上皮型颅咽管瘤中炎症细胞因子表达存在差异；IL6可能通过“经典”和“反式”途径诱导釉质上皮型颅咽管瘤细胞发生EMT转变,增强细胞体外迁移能力。
Craniopharyngioma is an intracranial extra-axial epithelial tumor of sellar or parasellar region, which grows along the cranial pharyngeal tube..Craniopharyngioma is one of most common intracranial congenital tumor which accounts for6-13%of all intracranial tumors and constitutes60%tumors of the sellar region in children. Since the first description of craniopharyngioma as a distinct tumor in the pituitary region by Austrian neuropathologist Jakob Erdheim in1904; many extensive and in-depth study on craniopharyngioma has been conducted. As yet, more than100years later, expert consensus on the origin, pathological features and biological characteristics of this formidable neoplasm is still lacking due to the lack of the tumor research platform and technical conditions.
Craniopharyngiomas are benign tumors which are classified into, adamantinomatous variant and squamous papillary variant by WHO. The adamantinomatous craniopharyngioma often form finger-like protrution invades the surrounding brain tissue, leading to difficult to total resection of the tumor, postoperative recurrence and worse clinical prognosis compared to the latter variant. The results of several single-center studies have shown that the prognosis of adamantinomatous craniopharyngioma is worse than the squamous papillary variant, but the mechanism is not clear. Although many scholars had undertaken extensive research on the adamantinomatous craniopharyngioma in the organizational level, the progress of basic research of adamantinomatous craniopharyngioma is slow due to the lack of stable in vitro and in vivo models. Several scholars cultured the adamantinomatous craniopharyngioma in vitro and used in functional experiments, however, characteristics of tumor cells was not mentioned in the study. At the same time, few in vivo xenograft model of adamantinomatous craniopharyngioma are reported. The contemporary understanding of the biological characteristics of this tumor can be further expanded by establishment of the adamantinomatous craniopharyngioma experimental model.
We previously found that the formation of inflammatory adhesions of adamantinomatous craniopharyngioma with the surrounding normal brain tissue is closely related to tumor total resection, postoperative recurrence, prognosis of patients in follow up; the spontaneous rupture of craniopharyngioma cysts and cyst fluid spillage during surgery can lead to aseptic meningitis; histologically, intense fibrillary gliosis and infiltration of inflammatory cells are frequently present in the surrounding parenchyma. These observations suggest that tumor cells may produce certain inflammatory cytokines that might play an important role in tumor progression and prognosis of patients. Mori et al has demonstrated IL-6as an important inducer of craniopharyngioma associated inflammation affecting the prognosis of patients, however the elucidation of the role of IL-6in craniopharyngioma in vitro is not yet reported due to the lack of technical conditions.
IL-6is multifunctional cytokine that contributes to the regulation of hemopoiesis, immunity and inflammatory response, also contributes to regulate physiological function of central nervous and cardiovascular system, and associates with cell survival, proliferation and wound healing. High expression of IL-6can be found in colorectal cancer, breast cancer and other epithelial tumors, as well as glioma and other central nervous system tumors, which is closely related with prognosis. Tumor cells or mesenchymal cells in tumor microenvironment can secrected IL6, through autocrine or paracrine secretion. IL-6after binding to its receptors forms a complex to activate the abnormal signaling pathways, contributing to the promotion of tumorigenesis, tumor angiogenesis, tumor migration and invasion. However, the role of IL-6in adamantinomatous craniopharyngioma is still not clear. We contemplate that the IL-6secretion by adamantinomatous craniopharyngioma or mesenchymal cells in tumor microenvironment through either autocrine or paracrine secretion might promote characteristics change in tumor cells and thus affect the prognosis of this patients?
In this study, we established the adamantinomatous craniopharyngioma finite cell line by primary culture in vitro as the necessity of the scientific reserch of craniopharyngioma and further investigate the biological characters of the tumor cells in order to elucidate the role of IL-6in craniopharyngioma. Additionally, we established the xenotransplanted tumor model of human adamantinomatous craniopharyngioma by implanting the adamantinomatous craniopharyngioma cells into the nude mice subcutaneously. The antibody array technology was used to investigate the expression of inflammatory cytokines in the different inflammation grade of the adamantinomatous craniopharyngioma tissue; which was followed by the stimulation of the adamantinomatous craniopharyngioma in vitro by IL-6to investigate the role of IL6on biological characters of tumor cells. Based on the findings of this experiment the possible mechanism of IL-6on craniopharyngioma cells is discussed. The aim of our study is to elucidate the relationship between inflammation and craniopharyngioma that might aid in development of clinical treatment for craniopharyngioma.
Chapter I Section I Cell culture and characterization of adamantinomatous craniopharyngioma
Objective:To investigate an efficient method of primary adamantinomatous craniopharyngioma cells culture and observe the behaviour of adamantinomatous craniopharyngioma cells in vitro.
Methods:The specimen of adamantinomatous craniopharyngioma were treated with trypsin and cultured in vitro, and purified by filter membrane. The cell morphology was observed by inverted microscope and hematoxylin eosin staining. The expression of Pan-CK was examined by immunocytochemistry. The MTT assay and flow cytometry were employed to study the cell proliferation and cell cycle.
Results:The success rate of primary culture and passage culture is88.9%and81.3%. Human adamantinomatous craniopharyngioma cells showed adherent growth in serum-containing medium. The tumor cells were large with abundant cytoplasm were large and characteristically demonstrated assemebled growth. A small amount of bubble like structure was seen in the cytoplasm. The tumor cells demonstrated slow growth, the growth rate of cells in1st-5th day is statistically significant difference (F=190.748..P<0.0001). The cells were diploid and depicted irregular polygonal and epithelioid form. The tumor cells in G0-G1,46.8±3.61%; in G2-M,37.3±1.51%; in S,15.9±2.77%.
Conclusion:The finite adamantinomatous craniopharyngioma cell line could be cultured in a serum medium and isolated, cultured and expanded effectively by the membrane sub-filtering techinque, which was beneficial for the follow-up experiments. The adamantinomatous craniopharyngioma cells demonstrated the Pan-CK positive phenotype, slow growth mainly in the G0-G1phase with no aneuploid cells.
Chapter I Section II
Establishment of subcutaneous xenotransplanted tumor model of human adamantinomatous craniopharyngioma in nude mice
Objective:Establishment of subcutaneous xenotransplanted tumor model of human adamantinomatous craniopharyngioma in mude mice on the basis of the primary adamantinomatous craniopharyngioma cells culture previously.
Methods:Craniopharyngioma cells were primarily cultured by trypsin digestion. The purified cells in third passage were then implanted subcutaneously into the nude mice. The specimens were studied by microscope to ensure the characteristics of the xenotransplanted tumor.
Results:The subcutaneous xenotransplanted tumor can be established by implanting the adamantinomatous craniopharyngioma cells into the nude mice subcutaneously. Adamantinomatous craniopharyngioma cells can survive in the BALB/c nude mice. The xenograft can be found on8days after implantation. The tumor formation rate at the back was37.5%. The disorderly growth of the squamous epithelial tumor cells can be found under microscope in subcutaneous tissue of nude mice.
Conclusion:The subcutaneously xenotransplanted tumor model of human adamantinomatous craniopharyngioma in nude mice was successfully established and it might be beneficial to condunct further studies on this tumor. However, the model is not stable, and the rate of tumor formation is too low. More research is needed to optimize the conditions of the xenotransplanted tumor establishment.
Chapter II Section I Epithelial-mesenchymal transition and clinico-pathological correlation in craniopharyngioma
Objective:To assess the immunophenotypic changes associated with epithelial mesenchymal transition (EMT) in craniopharyngioma, especially at the tumour invasive front, and correlate the findings with clinicopathological features and patient outcomes.
Methods:42craniopharyngiomas were, subjected to the detection of EMT makers (vimentin, E-cadherin and β-catenin) by immunohistochemistry. The relationships between expression of these markers and various clinico-pathological indicators and clinical outcomes of these tumors were analyzed.
Results:There was statistically significant difference in the expression of vimentin and E-cadherin between adamantinomatous and papillary variants in whole tumor and at the tumor invasive front (χ2=11.181, P=0.004,χ2=6.931,P=0.031;χ2=17.393, P<0.001, χ2=8.501, P=0.014). There was statistically significant difference in the cytoplasmic or nuclear accumulation of β-catenin between the two variants above at the tumor invasive front (χ2=19.765, P<0.001). The expression of vimentin and E-cadherin but β-catenin in whole-tumour sections were associated with tumor recurrence, postoperative weight and hypothalamic disturbances (χ2=23.834, P<0.001, r=0.643, P<0.001, r=0.637, P<0.001; χ2=16.526, P<0.001, r=-0.579, P<0.001, r=-0.714,P<0.001), and the expression of vimentin and E-cadherin/β-catenin at the tumor invasive front were colligated with tumor recurrence, postoperative weight and hypothalamic disturbances (χ2=19.782, P<0.001, r=0.642, P<0.001, r=0.573, P=0.001;χ2=11.475, P=0.003, r=-0.54, P<0.001, r=-0.626, P<0.001;χ2=16.572, P<0.001, r=-0.54, P<0.001, r=-0.506, P=0.001).
Conclusions:Our study exemplifies, for the first time, the potential prognostic implications of vimentin, E-cadherin and β-catenin expression in craniopharyngiomas. Epithelial-mesenchymal transition may represent a crucial mechanism in the progression of craniopharyngiomas.
Chapter ⅡSection Ⅱ The effection of Interleukin-6on the migration characteristics of adamantinomatous craniopharyngioma cells in vitro
Objective:To elucidate the effect and mechanism of the IL-6on the migration characteristics of adamantinomatous craniopharyngioma cells in vitro.
.Methods; We demonstrated the inflammation and clinico-pathological correlation in craniopharyngioma previously. In this study, antibody array assay was used to assess the expression of different inflammatory molecules in adamantinomatous craniopharyngioma tissues at different inflammatory levels. Further, the effect and mechanism of IL-6on adamantinomatous craniopharyngioma cell migration in vitro was investigated.
Results:The inflammation of craniopharyngioma was associated with pathological classification (χ2=6.603, P=0.037), the rate of total resection (χ2=8.188, P=0.017), calcification (γ=0.326, P=0.022) and postoperative HSS (y=0.376, P=0.008). The expression of IL-6and other inflammatory factors varied in tumor tissue at different level of inflammatory exposure. Expressions of IL6-IL6Ra-GP130were detected by immunohistochemistry and soluble IL-6R in the cystic fluid and supernatant by craniopharyngioma cells and fibroblasts were determined by ELISA. Migration in adamantinomatous craniopharyngioma was promoted by IL-6treatment in a dose-dependent manner (F=112.390, P﹤0.0001; F=126.654, P﹤0.0001). The expression of Vimentin was increased and E-cadherin was decreaseed by IL-6treatment.
Conclusion:The inflammation in craniopharyngioma was significantly associated with pathological classification. Additionally this inflammation was associated with the rate of total resection, calcification and postoperative HSS. Antibody arrays demonstrated a significant change in cytokine profiles in patients with different degrees of inflammation. IL-6might promot migration in vitro via classic-and trans-signaling pathways by inducing EMT in adamantinomatous craniopharyngioma.
引文
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