【作者】 孙伟；

【导师】 范跃祖；

【作者基本信息】 同济大学 ， 肿瘤学， 2005， 硕士

【摘要】 目的“血管生成拟态（VM）”是高侵袭恶性肿瘤与经典“肿瘤血管生成”完全不同、不依赖血管内皮细胞的全新肿瘤血管供血模式。胆囊癌作为手术切除率低、易复发转移、预后差的高恶性肿瘤，是否也存在VM，迄今未见报道。本研究通过人胆囊癌石蜡标本、体外细胞三维培养及其干预实验，初探胆囊癌是否存在VM，以及其形态学特征、分子机制、临床意义和NCTD对胆囊癌VM的影响和机制。方法①收集手术切除经病理确诊原发性胆囊癌、胆囊腺瘤、慢性胆囊炎石蜡标本74例、10例、10例和相关临床病理参数；应用HE染色、CD₃₁和PAS双重染色，观察胆囊癌是否存在VM，并作临床病理参数相关分析、Cox分析、Kaplan-Meier曲线绘制。②通过体外细胞二维培养、Transwell小室侵袭实验、胶原收缩实验、细胞毒实验及CD₃₁免疫荧光检测，明确GBC-SD、SGC-996细胞侵袭、迁移潜能，NCTD IC₅₀及HUVEC细胞鉴定。在此基础上，通过鼠尾Ⅰ型胶原三维培养进行胆囊癌VM体外模拟实验和药物干预实验，实验分GBC-SD细胞对照组、SGC-996细胞对照组、TIMP₁组、TIMP₂和NCTD组，应用HE和PAS染色、光学和电子显微镜观察三维培养组织切片体外模拟VM形态学。③应用SABC、Envision法免疫组化和ELISA放免方法测定人胆囊癌石蜡切片、三维培养组织切片和二、三维培养上清液中VM相关分子MMP₁、MMP₂、MMP₉、MT₁-MMP、VEGF、VE-cad和Ⅳ胶原蛋白表达，RT-PCR测定胆囊癌细胞表达内皮细胞相关基因。结果①在74例人胆囊癌中发现13.5％（10／74）存在VM，胆囊癌VM与病人性别、年龄、肿瘤部位、瘤体大小、分化程度、Nevin分期、浸润深度、局部淋巴转移无关；但与组织类型（χ²=10.241，P=0.017），生存期（χ²=5.7221，P=0.0168）有关，可能由于VM（+）阳性标本少，本组胆囊癌VM和肝脏转移无显著相关。②GBC-SD和SGC-996细胞的NCTD IC₅₀分别为56.18μg·ml^-1和22.66μg·ml^-1，GBC-SD细胞体外穿过Transwell小室侵袭能力（P=0.0013）和收缩迁移能力（P＜0.001）明显高于SGC-996细胞，故GBC-SD、SGC-996细胞分属高、低侵袭潜能细胞株。经HE和PAS染色，高倍、倒置光相差显微镜观察，鼠尾Ⅰ型胶原和Matrigel胶三维培养GBC-SD细胞密集如癌巢，48h即形成以肿瘤细胞为内衬的基质型单环或多环中央空洞网络状管道结构，14d管腔成熟；而低侵袭性SGC-996细胞始终未形成模拟VM的管道状结构。③相关分子表达，对胆囊癌石蜡切片检测显示：VM（+）、VM（-）胆囊癌细胞MMP₁、MMP₂、MMP₉、MT1-MMP、VEGF表达明显高于胆囊腺瘤、胆囊炎（P＜0.0001），VE-Cad、Ⅳ型胶原明显低于胆囊腺瘤、胆囊炎（P＜0.0001）；VM（+）胆囊癌MMP₂（F=5.74，P=0.0187）、MT1-MMP（F=8.78，P=0.0039）表达明显高，而VEGF（F=5.78，P=0.0182）、Ⅳ型胶原（F=5.80，P=0.0181）明显低于VM（-）胆囊癌，MMP₂、MT1-MMP与胆囊癌VM呈正相关，VEGF、Ⅳ型胶原与胆囊癌VM呈负相关；VM（+）胆囊癌MMP₁（F=0.27，P=0.6082）、MMP₉（F=2.80，P=0.0976）、VE-Cad（F=1.37，P=0.2450）表达与VM（-）胆囊癌无差别。对三维培养组织切片检测显示：GBC-SD细胞MMP₁、MMP₂、MMP₉、MT1-MMP、VEGF表达明显高于SGC-996细胞。MMP-1在VM（-）组表达与分化程度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随分化程度越低、Nevin分期越晚（S3～S5）、淋巴结及肝脏转移表达量增加，有显著差异（P＜0.05），在VM（+）组与临床病理指标无关（P＞0.05），同为肝脏转移组，VM（+）组MMP-1表达显著高于VM（-）组（P＜0.05）。MMP-2在VM（-）组表达与浸润深度、分化程度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随侵及浆膜、分化程度越低、Nevin分期越晚（S3～S5）、淋巴结及肝脏转移表达量增加，有显著差异（P＜0.05），在VM（+）组与临床病理类型腺癌有关（P＜0.05），同为淋巴结、肝脏转移组、中低分化腺癌、浸润深度，Nevin分期（S3～S5），VM（+）组MMP-2表达均显著高于VM（-）组（P＜0.05）。MMP-9在VM（-）组表达与浸润深度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随侵及浆膜、分化程度越低、Nevin分期越晚（S3～S5）、肝脏转移表达量增加，有显著差异（P＜0.05），在VM（+）组与浸润深度、Nevin分期、肝脏转移有关，并随侵及浆膜、Nevin分期越晚（S3～S5）、肝脏转移表达量增加，有显著差异（P＜0.05），同为侵及浆膜组，腺癌，Nevin分期（S3～S5）、肝脏转移，VM（+）组MMP-9表达均显著高于VM（-）组（P＜0．05）。MMP-14在无论VM（-）组和VM（+）组其表达与浸润深度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随侵及浆膜、Nevin分期越晚（S3～S5）、肝脏转移表达量增加，有显著差异（P＜0.05），同为侵及浆膜组，Nevin分期（S3～S5）、淋巴结及肝脏转移，VM（+）组MMP-14表达均显著高于VM（-）组（P＜0.05）。VEGF在VM（-）组表达与浸润深度、Nevin分期、淋巴结转移、肝脏转移密切相关，并随侵及浆膜、Nevin分期越晚（S3～S5）、肝脏转移表达量增加，有显著差异（P＜0.05），在VM（+）组仅肝脏转移组表达明显高于无肝脏转移，有显著差异（P＜0.05）。同一病理指标下，VM（-）组和VM（+）组表达VEGF无差异（P＞0.05），VE-cad在VM（-）组表达与浸润深度、Nevin分期、淋巴结转移、分化程度相关，并随侵及浆膜、Nevin分期越晚（S3～S5）、淋巴结转移、分化差表达量减少，有显著差异（P＜0.05），在VM（+）组与临床病理指标无关（P＞0.05），同一病理指标下，VM（-）组和Ⅷ（+）组表达VE-cad无差异（P＞0.05），Ⅳ胶原在VM（-）组表达与浸润深度、Nevin分期、分化程度、肝脏转移相关，并随侵及浆膜、Nevin分期越晚（S3～S5）、肝脏转移、分化差表达量减少，有显著差异（P＜0.05），在VM（+）组Ⅳ胶原表达与浸润深度、分化程度相关，并随侵及浆膜，分化差表达量减少，有显著差异（P＜0.05），同为淋巴结肝脏转移，VM（+）组Ⅳ胶原表达均显著低于VM（-）组（P＜0.05）。二、三维培养上清液ELISA检测显示：三维培养GBC-SD细胞MMP2蛋白表达较三维SGC-996细胞、二维GBC-SD明显增高（P＜0.05），而三维培养GBC-SD细胞MMP₉蛋白表达较三维SGC-996细胞、二维GBC-SD增高不显（P＞0.05），RT-PCR显示具有Ⅷ能力的GBC-SD可以强表达内皮细胞相关基因，而SGC-996仅弱或不表达④应用NCTD后，鼠尾Ⅰ型胶原三维培养48h，GBC-SD细胞即丧失形成单环或多环网络样结构能力，细胞稀疏、浮起、固缩，或聚集、核固缩碎裂，细胞凋亡、坏死；TIMP₂则起这种作用的时间明显延迟，TIMP₁无此作用。表明，NCTD有抑制体外模拟胆囊癌VM能力，TIMP₂，TIMP₁无阻止体外模拟胆囊癌VM能力。通过三维培养组织切片和上清液检测，1／2CI₅₀NCTD不仅使MMP₂、MT1-MMP，而且使MMP₁、MMP₉、VEGF阳性的GBC-SD细胞减少、染色变浅，蛋白表达下降；且随时间延长，GBC-SD细胞MMP₂、MMP₉蛋白表达明显受抑制（P＜0.05），表明，NCTD不仅有效抑制GBC-SD细胞MMP₂、MT1-MMP，而且抑制MMP₁、MMP₉、VEGF、RT-PCR显示NCTD可以有效抑制ANG-1、ANG-2、EPHA2、VEGF表达，从而通过多靶点作用抑制和破坏体外模拟胆囊癌VM的形成和成熟。结论人原发性胆囊癌存在VM；胆囊癌VM与组织学类型、生存期相关。高侵袭潜能GBC-SD细胞在三维培养中具有形成以癌细胞为内衬的单环或多环网络结构的体外模拟VM能力。其分子机制涉及MMP₂、MT1-MMP，VEGF、EPHA2、Ⅳ型胶原等相关分子。NCTD可有效抑制体外模拟胆囊癌VM；其机制可能与NCTD通过多靶点抑制上述相关分子表达，从而发挥阻遏、破坏形成体外模拟胆囊癌VM的作用。更多还原

【Abstract】 Objective Vasculogenic mimicry （VM） is a new vessel mode and extravascular fluid pathway for blood supply of aggressive malignant tumor, with its no-reliance on endothelium cells and its difference from classical angiogenesis. It is not known if there is VM in human gallbladder carcinomas, a highly lethal and aggressive malignant tumor with unresectable, early metastasis or invasion and poor prognosis. Based on this viewpoint, we studied the VM in human gallbladder carcinomas, and its morphological character, molecular mechanisms and clinical significance, and the effect of NCTD on VM of gallbladder carcinomas and its mechanisms in vitro.Methods①74 carcinomas, 10 adenomas and 10 chronic inflammatory lesions of the gallbladder underwent operation and confirmed histopathologically, and their clinical- pathlogical dates were studied. VM in human gallbladder carcinomas and its correlation with clinical-pathlogical dates was observed and analyzed by HE dye, CD₃₁ and PAS dyes and light microscope. Cox multiple factor and postoperative 5-year survival rate（Kaplan-Meier survival curve） evalution.②The invasiveness, movement and NCTD’ IC₅₀ of GBC-SD cells and SGC-996 cells were studied by two-dimensional cultures, Transwell invasive experiment, collagen gel contraction experiment and cell toxic test in vitro. HUVEC was identified by CD₃₁ immunofluorescence. The VM models of gallbladder carcinomas established by rat-tail collagen I three-dimensional cultures of GBC-SD cells were used to evaluate the therapy effect of NCTD for VM of gallbladder carcinomas in vitro. The expriments were randomly divided into the control groups of GBC-SD cells and SGC-996 cells, TIMP₁ group, TIMP₂ group and NCTD group, and were taken different treatment. The morphological changes of the VM models of gallbladder carcinomas in vitro were observed by HE and PAS dyes, light and electron microscope.③Expression of the VM related molecules such as MMP₁, MMP₂, MMP₉, MT₁-MMP, VEGF, VE-cad and collagen IV on sections from formalin-fixed paraffin-embedded blocks of human gallbladder carcinomas in vivo, on sections and in supernates from the two and three-dimensional culture tissues of GBC-SD cells and GBC-SD cells in vitro was determined by streptavidin-biotin complex method（SABC）, Envision method and ELISA method. RT-PCR was performed in the regions of all tumor cells to observe the gene expression level of endothelial cells.Results①13.5% （10/74） of human gallbladder carcinomas were found to contain vasculogenic mimicry （VM）, namely intratumoral, tumor cell-lined extracellular matrix （ECM）-rich, PAS-positive and vasculogenic-like network patterns that is associated with histological type （χ²=10.241, P=0.017） and poor overall survival （χ²=5.7221, P=0.016）. VM is also not correlated with sex、age、location and diameter of carcinoma、differentiation degree、Nevin stage、serosal invasion、lymph node metastatis. because the sample number with VM（+） was not sufficient, VM is not associated with liver metastatis.②IC₅₀ of NCTD for GBC-SD cells and SGC-996 cells was 56.18μg·ml^-1 and 22.66μg·ml^-1 respectively. The potential capacity of Transwell invasiveness （P=0.0013） and collagen gel contraction （P＜0.001） in GBC-SD cells was increased significantly than those of SGC-996 cells in vitro. GBC-SD cells and SGC-996 cells were so called as high- and low-invasive cell lines. Nest-like tumor cells and the network patterns composed of tumor cell-lined, patterned matrix type, loop or back-to-back morn loops network were detected in three-dimensional cultures of GBC-SD cells on matrigel and collagenⅠmatrix by HE and PAS dyes, light and electron microscope. These network patterns were initially formed at 48h, maturated at 14 days. But, SGC-996 cells were found to be incapable of forming throughout the vasculogenic-like network in three-dimensional cultures.③Expression of MMP₁, MMP₂, MMP₉, MT₁-MMP and VEGF was increased significantly in carcinomas with or without VM than adenomas and inflammatory lesions of the gallbladder （P＜0.0001） in vivo. Moreover, expression of VE-cad and collagenⅣwas decreased significantly in carcinomas with or without VM than adenomas and inflammatory lesions of the gallbladder （P＜0.0001）. Expression of MMP₂ （F=5.74, P=0.0187） and MT₁-MMP （F=8.78, P=0.0039） in the gallbladder carcinomas with VM was increased significantly than that of the gallbladder carcinomas without VM, that is showed positive correlation between expression of MMP₂, MT₁-MMP and VM of gallbladder carcinomas. Expression of VEGF （F=5.78, P=0.0182） and collagenⅣ（F=5.80, P=0.0181） in the gallbladder carcinomas with VM was decreased significantly than that of the gallbladder carcinomas without VM, that is cued negative correlation between expression of VEGF, collagenⅣand VM of gallbladder carcinomas. Moreover, there is no correlation on expression of MMP₁ （F=0.27, P=0.6082）, MMP₉ （F=2.80, P=0.0976） and VE-Cad （F=1.37, P=0.2450） between the gallbladder carcinomas with VM and the gallbladder carcinomas without VM. In the examinations on sections from the three-dimensional culture tissues in vitro, expression of MMP₁、MMP₂、MMP₉、 MT₁-MMP was increased significantly in GBC-SD cells than SGC-996 cells. Moreover, expression of VEGF was also increased significantly in GBC-SD cells than SGC-996 cells, the expression of MMP-1 was closely correlated with cell differention、Nevin stage、lymph node metastases、liver metastatis in case of gallbladder carcinomas without VM. it’s expression was significantly higher than that without lymph node and liver metastatis、high differention、Nevin stage（S1～S2） （P＜0.05）. there was no correlation between MMP-1 protein expression and pathologic index in case of gallbladder carcinomas with VM（p＞0.05）. In the same condition of liver metastatis, MMP-1 protein expression with VM was higher than that of without VM （P＜0.05）. the expression of MMP-2 was markedly correlated with cell differention、Nevin stage、lymph node metastases、liver metastatis、serosal invasion in case of gallbladder carcinomas without VM. Increased expression of MMP-2 was observed in the case of lower differention、Nevin stage（S3～S5）、lymph node metastases、liver metastatis、serosal invasion （P＜0.05）. In the same condition of liver metastatis, cell differention, Nevin stage（S3～S5）、lymph node metastases、lower differention MMP-2 protein expression with VM was higher than that of without VM （P＜0.05）. a significant relationship was observed between MMP-9 expression without VM and Nevin stage、lymph node metastases、liver metastatis、serosal invasion. Increased expression of MMP-9 was observed in the case of lower differention、Nevin stage（S3～S5）、lymph node metastases、liver metastatis、serosal invasion （P＜0.05）. the expression of MMP-9 was also markedly correlated with Nevin stage、liver metastatis、serosal invasion in case of gallbladder carcinomas with VM （P＜0.05）. In the same condition of liver metastatis、Nevin stage（S3～S5）、serosal invasion, histological type MMP-9 protein expression with VM was higher than that of without VM （ P＜0.05 ）. the expression of MMP-14 was significantly related to the Nevin stage、liver metastatis、serosal invasion、lymph node metastases whether VM or not. Increased expression of MMP-14 was observed in the case of Nevin stage（S3～S5）、lymph node metastases、liver metastatis、serosal invasion （P＜0.05）. In the same condition of liver metastatis、Nevin stage（S3～S5）、serosal invasion, lymph node metastases MMP-9 protein expression with VM was higher than that of without VM （P＜0.05）. VEGF was closely correlated with Nevin stage、lymph node metastases、liver metastatis、serosal invasion in case of gallbladder carcinomas without VM. Increased expression of VEGF was observed in the case of Nevin stage（S3～S5）、liver metastatis、serosal invasion （P＜0.05）. the expression of VEGF was also markedly correlated with liver metastatis in case of gallbladder carcinomas with VM （P＜0.05）. there was not any different expression of VEGF between VM and without VM according to the same condition, a significant relationship was observed between VE-cad expression without VM and Nevin stage、lymph node metastases、liver metastatis、serosal invasion、differentiation degree, decreased expression of VE-cad was observed in the case of Nevin stage（S3～S5）、lymph node metastases、lower differentiation、serosal invasion （P＜0.05）. there was no correlation between VE-cad protein expression and pathologic index in case of gallbladder carcinomas with VM（p＞0.05）. there was not any different expression of VE-cad between VM and without VM according to the same condition. There was correlation betweenⅣcollagen and Nevin stage、liver metastatis、serosal invasion、or differentiation degree. decreased expression ofⅣcollagen was observed in the case of Nevin stage（S3～S5）、lower differentiation、serosal invasion、liver metastatis（P＜0.05） without VM. the expression ofⅣcollagen was also markedly correlated with serosal invasion、differentiation degree in case of gallbladder carcinomas with VM （P＜0.05）. In the same condition of liver metastatis、lymph node metastasesⅣcollagen expression without VM was higher than that of with VM （P＜0.05）, examinations on supernates from the cell cultures in vitro, expression of MMP₂ in GBC-SD cells cultured by collagen 1 matrix was increased significantly, it’s expression was significantly higher than that of three--dimensional cultures of SGC-996 cells and the two-dimensional cultures of GBC-SD cells（P＜0.05）, expression of MMP₉ in GBC-SD cells cultured by collagen 1 matrix was not increased significantly, it’s expression was not significantly higher than that of three--dimensional cultures of SGC-996 cells and the two-dimensional cultures of GBC-SD cells（P＞0.05）, gene expression of endothelial cells were strongly observed in three--dimensional cultures of GBC-SD cells, negative or slight exoression were observed in three--dimensional cultures of SGC-996 cells④After treatment with NCTD, GBC-SD cells in three-dimensional cultures on rat-tail collagenⅠat 48h wasn’t able completely to form loop or loops vasculogenic-like network; sparseness, floating, shrinkage and aggregation of cell, nuclear shrinkage, chromosome condensation, karyorrhexis, apoptosis, necrosis in some GBC-SD cells were observed by light and electric microscope. Moreover, TIMP₂ exert the inhibitory effect on GBC-SD cells lingeringly when being compared with NCTD （5d vs. 48h）, TIMP₁ hasn’t the effect. It is shown that inhibitory effect of NCTD for the simulated VM of gallbladder carcinomas is more powerful and earlier than TIMP₂. TIMP1 and TIMP2 couldan’t prevent the formation of vasculogenic mimicry. The reduced cell amounts, the shallow dye and the decreased expression of not only MMP_2-, MT1-MMP-positive GBC-SD cells but also MMP_1-, MMP_9-, VEGF-positive GBC-SD cells were observed by examination for sections and supernates from the cell culture tissues after treatment with 1/2CI₅₀ NCTD in vitro. As the effect time of NCTD prolonged, the expression of MMP₂ and MMP₉ proteins of GBC-SD cells was inhibited significantly, which is also showed obvious difference on expression of MMP₂ and MMP₉ between the three-dimensional cultures of GBC-SD cells and the two-, three-dimensional cultures of SGC-996 cells and the two-dimensional cultures of GBC-SD cells, which isn’t able to form the simulated VM of gallbladder carcinomas. It has been demonstrated that NCTD inhibit effectively not only MMP₂ and MT1-MMP of GBC-SD cells, but also MMP₁, MMP₉, VEGF. further pullback and destroy the form and mature of the simulated VM of gallbladder carcinomas by the more target-point effects. NCTD could effiectively prevent the expression of ANG-1, ANG-2, EPHA2, VEGEConclusions Human gallbladder carcinomas were found to have vasculogenic mimicry that is associated with histological type, liver metastasis and poor overall survival. High-invasive potential GBC-SD cells were able to form tumor cell-lined, loop or more loops and simulated VM network of gallbladder carcinomas in three-dimensional cultures of collagenⅠmatrix. Its mechanisms correlate with the expression change of related-molecules such as MMP₂ and MT1-MMP, might also correlate with the expression change of MMP₁, MMP₉, VEGF, EPHA2 and collageⅣ. NCTD can inhibit effectively the simulated VM of gallbladder carcinomas in vitro. Its effect mechanisms was found to associate whith the inhibition of above-mentioned related-molucule expressions by more target-point effects, consequently exert the therapitic effect in the form and mature of the simulated VM of gallbladder carcinomas.更多还原