【作者】 徐冰；

【导师】 顾为望；

【作者基本信息】 南方医科大学 ， 人体解剖与组织胚胎学， 2007， 博士

【摘要】 转移性是恶性肿瘤主要的生物学特性之一，也是导致肿瘤患者死亡的主要原因，即使对患者实施肿瘤切除，术后转移的发生也是影响手术疗效的主要障碍。肿瘤转移的发生发展是一个多因素、多步骤的复杂生物学过程。因此，研究肿瘤的转移，就必然要建立与临床表现相近的肿瘤转移动物模型。人体肿瘤移植于裸小鼠建立人癌异种移植动物模型为肿瘤的深入研究提供了可能。皮下移植和原位移植是目前常用的两种方法。原位移植由于获得与人体肿瘤生长相似的微环境，更利于肿瘤恶性行为的表达，因此，建立肿瘤转移动物模型大多采用原位移植的方法。肝癌、胃癌和肺癌是常见的肿瘤，利用这三种肿瘤进行研究具有一定的代表意义。本课题利用不同程度免疫缺陷小鼠观察了人肝癌原位移植肿瘤在宿主体内的生物学特性差异；模拟临床，建立了人肝癌和人肺癌术后转移动物模型；同时利用经体内筛选建立的人胃癌高转移动物模型以及建立的人胃癌细胞株MKN-45mc，对肿瘤转移的相关机制进行探讨。一、目的1．建立人肝癌NOD-SCID小鼠和裸小鼠原位移植模型，并通过两种模型生物学特性的比较，对不同类型免疫缺陷动物在人肝癌异种移植方面的应用价值进行初步探讨。2．建立人肝癌术后转移动物模型，观察并比较手术切除肿瘤后对人肝癌小鼠皮下移植模型和原位移植模型的影响。3．建立裸小鼠人肺癌术后转移模型，为研究肺癌的转移机制和术后抗转移治疗提供适宜的动物模型。4．比较体内筛选前后建立的人胃癌原位移植动物模型，以及人胃癌细胞株MKN-45mc和MKN-45生物学特性出现的差异，对转移的相关机制进行探讨。二、方法1．将组织结构完整的SMMC-LTNM瘤块植入NOD-SCID小鼠和裸小鼠肝脏内，建立人肝癌原位移植模型。观察成瘤率、肿瘤体积和重量、脏器的转移情况以及血清甲胎蛋白（AFP）、γ-谷氨酰转肽酶同工酶Ⅱ（γ-GTⅡ）等指标。2．将组织结构完整的HC-031瘤块植入NOD-SCID小鼠皮下和肝脏内，分别建立人肝癌皮下移植和原位移植模型。模拟临床肿瘤手术切除方法，切除皮下移植瘤和原位移植瘤，建立肝癌术后转移模型。观察肿瘤生长和动物的生存状况。通过病理解剖和组织病理学技术研究肿瘤在动物体内转移情况。3．将人非小细胞肺癌NCI-H460组织块植入裸小鼠皮下，建立皮下移植瘤模型。4w后，25只裸小鼠作为手术组切除肿瘤组织，建立术后转移模型，15只裸小鼠作为对照组。每两周两组各处死5只动物动态观察其远处各脏器转移情况。动物明显消瘦时结束实验。采用免疫组化方法检测MMP-2、OPN、CD44v6、CD62等蛋白在转移灶及原发灶的表达情况。4．用人胃癌MKN-45sci和MKN-45肿瘤组织块建立人胃癌原位移植动物模型，观察并比较两种模型的肿瘤生长和转移、血清肿瘤标志物及MMP-2、OPN、CD44v6、CD62与Timp-2的表达情况。取MKN-45sci肝转移灶形成的皮下移植瘤，用组织块法进行原代培养，建立MKN-45mc细胞株，观察肿瘤细胞的形态学、生长速度、体外侵袭能力、细胞周期变化，并与MKN-45细胞株进行比较。三、结果1．NOD-SCID小鼠人肝癌原位移植模型于5w可扪及肿瘤的生长，成瘤率为100％，11w肿瘤体积为（4.48±0.93）cm³，瘤重为（7.02±1.15）g，肺部转移率为53.85％；裸小鼠人肝癌原位移植模型于6w～7w可扪及肿瘤的生长，成瘤率为100％，11w肿瘤体积为（1.02±0.70）cm³，瘤重为（2.87±0.44）g，体内未见转移发生。NOD-SCID小鼠的瘤体积、瘤重、肺转移率均高于裸小鼠（P=0.000，P=0.000，P=0.011）。二者均保持AFP高分泌和γ-GT同工酶阳性的特性。2．人肝癌HC-031皮下移植模型和原位移植模型由于肿瘤负荷过大分别于11w和6w而出现濒死状态，荷瘤平均生存时间为75d和44d。病理解剖，皮下移植瘤模型未发现转移（0／7），原位移植瘤模型的肿瘤和邻近脏器侵袭明显，部分出现肺部转移（2／7）。皮下切瘤动物和肝原位切瘤动物分别于17w和12w出现恶病质现象，平均生存时间为154d和112d。病理解剖发现肺部均有肉眼可见癌性转移结节（7／7，7／7）。未切瘤动物和切瘤动物血清中AFP和γ-GTⅡ表达均为阳性。3．人肺癌NCI-H460裸小鼠皮下移植瘤10w时瘤体严重坏死，并且动物出现明显消瘦，解剖未发现转移。手术组裸小鼠于10w、12w各发现一例肺转移（1／5），14w时裸小鼠明显消瘦，肺转移率达100％（5／5），有肉眼明显可见的肺转移结节，其他脏器未见转移。免疫组化结果表明，MMP-2、OPN、CD44v6、CD62和Timp-2在肺转移灶内的表达明显高于原发灶，CD54和MMP-9在转移灶内的表达则低于原发灶。4．人胃癌原位移植模型MKN-45sci组动物的肝转移出现早且转移率高，4w左右肉眼即可看到肝转移结节（100％），同时伴有淋巴结转移（100％）、肺转移（71％）、脾转移（29％）和腹水；移植瘤体积和重量明显增加，分别达到（3089±1617）mm³和（2.66±1.32）g，动物出现恶病质。而4w时MKN-45组肝、肺、脾未见明显转移灶，仅2只动物有淋巴结转移；小鼠未见明显消瘦，移植瘤体积和重量为（275±90）mm³和（0.35±0.14）g。MKN-45sci组血清中NSE和CYFRA21-1的浓度均较高，且随着肿瘤生长时间的延长而增高。而MKN-45组CYFRA21-1始终呈现阴性，4w时NSE仅为76.9 ng／ml。免疫组化结果显示MMP-9、OPN抗体在MKN-45sci组原位移植瘤和转移灶呈强阳性反应，而在MKN-45组原位移植瘤反应较弱。CD44v6抗体在MKN-45sci组原位移植瘤和转移灶呈阳性反应。E-cadherin抗体在MKN-45sci组原位移植瘤呈阳性反应，肝转移灶呈阴性反应。建立的MKN-45mc细胞株的染色体为超三倍体细胞，具有人类恶性肿瘤细胞染色体的特点。细胞形态为典型的上皮样细胞，与MKN-45的形态学特点相似。流式细胞分析显示MKN-45mc与MKN-45细胞周期各时相比例分别为：G₀-G₁期62.51％／53.95％，S期9.46％／1452％，G₂-M期28.04％／31.53％，两株细胞DNA合成期的细胞比例均较高。MKN-45mc与MKN-45细胞倍增时间分别为34.8h和42.1h，前者的生长速度高于后者。MKN-45mc与MKN-45的36h体外过膜数分别为（60.38±8.86）／高倍视野和（32.50±17.26）／高倍视野，前者多于后者。四、结论1．建立了与临床表现相似的人肝癌动物模型。与裸小鼠相比，NOD-SCID小鼠在建立人肝癌的异种移植模型方面有更大的应用价值。2．模拟临床肿瘤切除方法，建立人肝癌术后转移动物模型。实验结果表明，荷瘤动物肿瘤切除后，生存期延长，有利于肿瘤转移发生。3．裸小鼠人肺癌术后转移模型模拟了临床肿瘤根除术后发生远处转移的过程，结果提示肺癌转移的发生可能与部分粘附分子的异常表达相关，同时为研究肺癌转移机制和术后抗转移治疗提供理想的动物模型。4．人胃癌原位移植动物模型体内筛选前后肿瘤的生物学特性不同，经筛选后肿瘤的生长和转移能力较未筛选的增强，其机理与肿瘤细胞粘附和降解能力得到强化有关。筛选后建立的细胞株生长速度、过膜能力比原细胞株增强，表明肿瘤转移与肿瘤细胞增殖和运动变形能力有关。更多还原

【Abstract】 Metastasis is one of the most important biological characteristics on malignanttumor, and is the main cause of death for most cancer patients and thus is a majorobstacle to the successful treatment of such patients. Even if had tumour-reductivesurgery（TRS） for cancer patients, postoperative metastasis is the main obstruct thatimpact the curative effect of operation. The occurance and development of tumormetastasis is a complicated process of biological change finished by multiple factorsand steps. Therefore, it’s necessary to generate the animal model of tumor metastasiswhich is close to the clinical condition for study of tumor metastasis. The animalmodel of human carcinoma xenotransplantation, established by transplanting humancarcinoma into nude mice, provides the possibility to investigate tumor deeply. Bothsubcutaneous implantation and orthotopic transplantation are two common methodsto produce these models. Due to obtain microenvironment the same as the situationsin human body, orthotopic transplantation is easier to express the malignant tumorbehavior. Thus, orthotopic transplantation is the main method to establish animalmodel of tumor metastasis.Liver cancer, gastric cancer and lung cancer, the prevalentcarcinoma, have representative significance if research on them.In this study, weobserved the difference of the biological characteristics in parasitifer on orthotopic into the subscutaneousness and liver of NOD-SCID mice. Then having TRS for tumorof subcutaneous implantation and orthotopic transplantation, we established a modelof postoperative metastasis of HCC by imitating clinical operation method. Weobserved the tumor growth and animal survival condition, and investigated metastasisin vivo by pathological anatomy and histopathology technology.3. To establish the NCI-H460 subcutaneous metastasis model of nonsmall-celllung cancer （NSCLC） by using the histological intact tumor tissue. Four weeks later,40 nude mice were selected as operation group and had tumor resection to establishthe postoperative metastasis model at random. The rest were assigned to the controlgroup. Then five mice were sacrificed and dissected every two weeks to observe themetastasis in two groups. All of mice were sacrificed when animals were obviouslyslender, and subsequently detect the expression of MMP-2, OPN, CD44v6, CD62, etc,in metastases and primary tumor by immunohistochemistry （IHC）.4. We established orthotopic transplantation models of human gastric cancer innude mice using histologically intact tissue of MKN-45sci and MKN-45.The resultson growth, metastasis, markers in serum and expression of MMP-2, OPN, CD44v6,CD62 and Timp-2 of tumor in two models were observed. We established cell strainMKN-45mc, which was derived from a subcutaneous tumor that was grown into fromliver metastasis of MKN-45sci. We observed cell morphology, cell growth velocity,invasive power in vitro, change of cell cycle on cell strain MKN-45mc, and comparedwith strain MKN-45Results:1. The growth rate of the tumor was 100％in the orthotopic transplantationmodel of human hepatocellular carcinoma in NOD-SCID mice. The tumor inNOD-SCID mice was palpable at the 5th week. The volume, the weight and the lungmetastasis rate of the tumor was （4.48±0.93）cm³, （7.02±1.15）g and 53.85％in NOD-SCID mice at the 11th week respectively. Growth rate of tumor was 100％inthe orthotopic transplantation model of human hepatocellular carcinoma in nude mice.The tumor was palpable between the 6th week and the 7th week. The volume, theweight of the tumor was（1.02±0.70）cm³ and （2.87±0.44）g in nude mice at the 11thweek respectively, while no metastasis. The volume, the weight, the metastasis rate ofthe tumor in NOD-SCID mice were all significantly higher than those in nude mice（P=0.000, P=0.000, P=0.011）. The two kinds of animals all kept the characteristics:highly secrete AFP highly and positiveγ-GTⅡ.2. The models of subcutaneous implantation and orthotopic transplantation wereon the brink of death because of overload of tumor at the 11th and 6th week,separately. The average survival time of the two kinds of models is 75d and 44drespectively. By pathological anatomy, metastasis （0/7） was not observed in model ofsubcutaneous implantation. Metastasis in lung （2/7） was observed in the model oforthotopic transplantation and the tumor obviouslly invaded the adjacent organs. Theanimals after TRS to the model of subcutaneous implantation and orthotopictransplantation were on the brink of death at the 17w and 12w respectively. Theaverage survival time of them is 75d and 44d, respectively. Metastasis nodes in lungcould be found with naked eyes （7/7,7/7）. The expression of AFP andγ-GTⅡin theserum of animals via both resection and non-resection was positive.3. Mice in control group, non-metastasis in each stage, were the expression ofobvious emaciation and tumor necrosis after 10 weeks. While in the operation group,the metastasis rate was 20％（1/5） in lung when mice were sacrificed at 10 and 12weeks. The metastasis rate was 100％（5/5） in lung when the mice were sacrificed at14 weeks with non-metastasis in other organs. The IHC results indicate that theexpression of MMP-2, OPN, CD44v6, CD62 and Timp-2 in metastases were higherthan in primary tumor, while the expression of CD54 and MMP-9 in metastases were lower than that in primary tumor.4. The metastasis in liver in MKN-45sci group was earlier occurrence and higherpercentage. At the 4th week, the metastasis percentage in liver, lymphonode, lung,spleen and abdominal dropsy is 100％, 100％, 71％, 29％and 29％, respectively. Thevolume and weight of transplantation tumor obviously increased to （3089+1617）mm³and （2.66±1.32）g. The cachexia occurred to the animal in MKN-45sci group at the4th week. Whereas the metastasis in liver, lung and spleen in MKN-45 group was notobvious at the same time other than lymphonode in two mice. The volume and weightof transplantation tumor were （275±90）mm³ and （0.35±0.14）g. The animal inMKN-45 group was not obvious emaciation. The density of NSE and CYFRA21-1 inserum in MKN-45sci group was much greater, which could enhance with theprolongation of the time that tumor grew. But CYFRA21-1 in MKN-45 group wasnegative all the time, and NSE was merely 76.9ng/ml at the 4th week. The IHCresults indicated, the expression of MMP-9 and OPN was higher in transplantationtumor and metastasis in MKN-45sci group, but lower in transplantation tumor inMKN-45 group; CD44v6 in transplantation tumor and metastasis in MKN-45sci ispositive; the expression of E-cadherin in MKN-45sci was deteced in orthotopictransplantation tumor, and not tested in metastasis in liver. The chromosome of cellstrain MKN-45mc established embodied the characteristics of that of humammalignant tumor cell, which was hypertriploid. Its characteristics of cell morphologywas similar to MKN-45, which was typical epitheliod cell. The phase proportion ofcell circle of MKN-45mc and MKN-45 was 62.51％/53.95％in G₀-G₁, 9.46％/14.52％in S, 28.04％/31.53％in G₂-M respectively via flow cytometry. The cell quantity oftwo cell strains was more greater in DNA synthesis phase. The cell growth velocity ofMKN-45mc was higher than that of MKN-45, whose double time was 34.8h and42.1h. The number of cell penetrating through membrane in MKN-45mc was greater than that in MKN-45 in 36 hours, which was （60.38±8.86） and （32.50±17.26） inhighpower field.Conclusion:1. We have established the animal model of HCC that is similar to the clinic. Theapplication significance in NOD-SCID mice is greater than that in nude mice as far asestablishing xenotransplantation model of HCC.2. We establish animal model of postoperative metastasis of human HCC byTRS in clinic. The results show that resection is easier to the development ofmetastasis in the animal bodies bearing cancer due to prolongation of life span.3. This animal model simulates the metastasis after clinical operation, and theIHC results indicated that metastasis is in correlation with some adhesion molecules.This kind of model provides valuable data to study mechanism of NSCLC metastasisand treatment of human NSCLC.4. The biological characteristics on orthotopic transplantation models of humangastric cancer in animal is different after filtration or non-filtration.The capability oftumor growth and metastasis which has been filtered is greater than which not filtered,which mechanism could be associated with strengthening adhesion and degradationof tumor cells. The growth velocity and invasive power in vitro of the cell strainwhich has been filtered is greater than the cell strain which not filtered, whichindicates that the ability of cell metastasis has relationship with the ability of cellmultiplication and metamorphosis.更多还原