常见人体肿瘤转移动物模型的建立及相关机理研究
发布时间：2018-08-29 13:07
【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)cm~3，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)cm~3，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)mm~3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)mm~3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：G_0-G_1期62.51％／53.95％，S期9.46％／1452％，G_2-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。
[Abstract]:Metastasis is one of the main biological characteristics of malignant tumors, and also the main cause of death of cancer patients. Even after tumor resection, the occurrence of postoperative metastasis is also a major obstacle to the efficacy of surgery. It is necessary to establish an animal model of tumor metastasis which is similar to the clinical manifestation. Human tumor transplantation in nude mice provides the possibility of establishing an animal model of human cancer xenotransplantation. Liver cancer, gastric cancer and lung cancer are common tumors, and the use of these three kinds of tumors has a certain representative significance. In this study, immunodeficiency mice were used to observe the orthotopic transplantation of human liver cancer in vivo. The animal models of human hepatocellular carcinoma and human lung cancer metastasis were established by simulating clinical conditions, and the high metastasis animal models of human gastric cancer and the human gastric cancer cell line MKN-45mc were established by screening in vivo.
First, the purpose
1. The orthotopic transplantation models of human hepatocellular carcinoma NOD-SCID mice and nude mice were established, and the application value of different immunodeficient animals in xenotransplantation of human hepatocellular carcinoma was preliminarily discussed by comparing the biological characteristics of the two models.
2. Establish the animal model of human liver cancer metastasis after operation, observe and compare the effect of tumor resection on subcutaneous transplantation model and orthotopic transplantation model of human liver cancer mice.
3. To establish a metastasis model of human lung cancer in nude mice, and provide an appropriate animal model for studying the metastasis mechanism and anti-metastasis therapy.
4. To compare the animal models of orthotopic transplantation of human gastric cancer established before and after screening in vivo and the differences of biological characteristics between human gastric cancer cell lines MKN-45mc and MKN-45, and to explore the mechanism of metastasis.
Two, method
1. SMMC-LTNM tumor with intact tissue was implanted into the liver of NOD-SCID mice and nude mice to establish orthotopic transplantation model of human hepatocellular carcinoma.
2. HC-031 tumor was implanted into NOD-SCID mice subcutaneously and in the liver to establish the models of subcutaneous transplantation and original transplantation of human hepatocellular carcinoma. Anatomical and histopathological techniques were used to study the metastasis of tumors in animals.
3. Human non-small cell lung cancer (NSCLC) NCI-H460 was implanted subcutaneously into nude mice to establish a subcutaneous transplanted tumor model. Four weeks later, 25 nude mice were used as surgical group to remove tumor tissues, and 15 nude mice as control group. Five animals in each group were killed every two weeks to observe the distant organ metastasis. Immunohistochemistry was used to detect the expression of MMP-2, OPN, CD44v6 and CD62 in metastatic and primary lesions.
4. The orthotopic transplantation model of human gastric cancer was established by using MKN-45sci and MKN-45 tumor tissue blocks. The growth and metastasis of tumor, the expression of serum tumor markers and MMP-2, OPN, CD44v6, CD62 and Timp-2 were observed and compared between the two models. MKN-45 MC cell line was established to observe the morphology, growth rate, invasion ability in vitro and cell cycle changes of tumor cells, and compared with MKN-45 cell line.
Three, the result
1. Human hepatocellular carcinoma orthotopic transplantation model in NOD-SCID mice could palpate the growth of tumor in 5 weeks, the tumor formation rate was 100%, the tumor volume was (4.48.93) cm~3, the tumor weight was (7.02 1.15) g, and the lung metastasis rate was 53.85%; human hepatocellular carcinoma orthotopic transplantation model in nude mice could palpate the growth of tumor in 6 to 7 weeks, the tumor formation rate was 100%, and the tumor volume was (1.02.7) at 11 weeks. The tumor volume, tumor weight and lung metastasis rate of NOD-SCID mice were higher than those of nude mice (P = 0.000, P = 0.000, P = 0.011). Both of them maintained the characteristics of high AFP secretion and positive gamma-GT isozyme.
2. The subcutaneous transplantation model and the orthotopic transplantation model of HC-031 were in the state of near death due to the overload of the tumor at 11th and 6th weeks, respectively. The average survival time of the transplanted tumor was 75 and 44 days. Pathological anatomy showed that no metastasis was found in the subcutaneous transplantation model (0/7). The tumor and adjacent organs of the orthotopic transplantation model had obvious invasion and partial lung metastasis (2). The average survival time was 154 D and 112 D in subcutaneous and in situ tumor-resected animals, respectively. The metastatic nodules (7/7, 7/7) were found in the lung by pathological dissection. The expression of AFP and gamma-GT II was positive in the serum of both non-resected and resected animals.
3. Human lung cancer NCI-H460 nude mice underwent subcutaneous transplantation of tumor for 10 weeks, and the tumor necrosis was severe, and the animals showed significant emaciation and no metastasis. One case of lung metastasis (1/5) was found in each of the nude mice in the operation group at 10 and 12 weeks, and the metastasis rate was 100% (5/5) in the nude mice at 14 weeks, with obvious metastasis nodules in the lung and no metastasis in other organs. Metastasis. Immunohistochemistry showed that the expressions of MMP-2, OPN, CD44v6, CD62 and Timp-2 were significantly higher in lung metastases than in primary lesions, while the expressions of CD54 and MMP-9 were lower in metastases.
4. Liver metastasis was early and high in MKN-45sci group, with lymph node metastasis (100%), lung metastasis (71%), spleen metastasis (29%) and ascites. The volume and weight of transplanted tumor increased significantly, reaching (3089+1617) mm~3 and (2.66+1.32) g, respectively. Cachexia was found in the mice, but no metastasis was found in the liver, lung and spleen of MKN-45 group at 4 weeks, only 2 animals had lymph node metastasis, and the mice did not show obvious emaciation. The serum NSE and CYFRA21-1 concentrations in MKN-45 SCI group were higher than those in MKN-45 group with (275+90) mm~3 and (0.35+0.14) G. The results of immunohistochemistry showed that MMP-9, OPN antibody and MKN-45sci were strongly positive in transplanted tumors and metastases, but weakly in MKN-45. CD44v6 antibody was positive in transplanted tumors and metastases in MKN-45sci. The established MKN-45mc cell line is a Hypertriploid cell with the characteristics of human malignant tumor cell chromosomes. The cell morphology is typical epithelioid cells, similar to the morphological characteristics of MKN-45. Flow cytometry analysis showed that MKN-45mc and MKN-45 cell cycle. The proportions of the two cell lines were 62.51%/53.95% in G_0-G_1 phase, 9.46%/1452% in S phase and 28.04%/31.53% in G_2-M phase, respectively. The doubling time of MKN-45mc and MKN-45 cells were 34.8 h and 42.1 h, respectively. The growth rate of the former was higher than that of the latter. The number of outer membrane of MKN-45mc and MKN-45 cells in 36 h was 60.46 h, respectively. 38 + 8.86) / high field of view and (32.50 + 17.26) / high power field of vision, the former is more than the latter.
Four. Conclusion
1. Establishment of human hepatocellular carcinoma animal model with similar clinical manifestations. Compared with nude mice, NOD-SCID mice have more application value in establishing xenograft model of human hepatocellular carcinoma.
2. To simulate the clinical tumor resection method and establish the animal model of human liver cancer metastasis after operation. The experimental results show that the survival time of tumor-bearing animals after tumor resection is prolonged, which is conducive to the occurrence of tumor metastasis.
3. The metastasis model of human lung cancer in nude mice simulates the process of distant metastasis after clinical tumor eradication. The results suggest that the metastasis of lung cancer may be related to the abnormal expression of some adhesion molecules, and provide an ideal animal model for studying the metastasis mechanism of lung cancer and anti-metastasis therapy.
4. The biological characteristics of human gastric cancer orthotopic transplantation animal models were different before and after screening in vivo. The growth and metastasis ability of the tumor after screening was stronger than that of the non-screening. The mechanism was related to the enhancement of adhesion and degradation ability of tumor cells. Tumor metastasis is related to cell proliferation and movement deformability.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R-332;R730.2
本文编号：2211300
[Abstract]:Metastasis is one of the main biological characteristics of malignant tumors, and also the main cause of death of cancer patients. Even after tumor resection, the occurrence of postoperative metastasis is also a major obstacle to the efficacy of surgery. It is necessary to establish an animal model of tumor metastasis which is similar to the clinical manifestation. Human tumor transplantation in nude mice provides the possibility of establishing an animal model of human cancer xenotransplantation. Liver cancer, gastric cancer and lung cancer are common tumors, and the use of these three kinds of tumors has a certain representative significance. In this study, immunodeficiency mice were used to observe the orthotopic transplantation of human liver cancer in vivo. The animal models of human hepatocellular carcinoma and human lung cancer metastasis were established by simulating clinical conditions, and the high metastasis animal models of human gastric cancer and the human gastric cancer cell line MKN-45mc were established by screening in vivo.
First, the purpose
1. The orthotopic transplantation models of human hepatocellular carcinoma NOD-SCID mice and nude mice were established, and the application value of different immunodeficient animals in xenotransplantation of human hepatocellular carcinoma was preliminarily discussed by comparing the biological characteristics of the two models.
2. Establish the animal model of human liver cancer metastasis after operation, observe and compare the effect of tumor resection on subcutaneous transplantation model and orthotopic transplantation model of human liver cancer mice.
3. To establish a metastasis model of human lung cancer in nude mice, and provide an appropriate animal model for studying the metastasis mechanism and anti-metastasis therapy.
4. To compare the animal models of orthotopic transplantation of human gastric cancer established before and after screening in vivo and the differences of biological characteristics between human gastric cancer cell lines MKN-45mc and MKN-45, and to explore the mechanism of metastasis.
Two, method
1. SMMC-LTNM tumor with intact tissue was implanted into the liver of NOD-SCID mice and nude mice to establish orthotopic transplantation model of human hepatocellular carcinoma.
2. HC-031 tumor was implanted into NOD-SCID mice subcutaneously and in the liver to establish the models of subcutaneous transplantation and original transplantation of human hepatocellular carcinoma. Anatomical and histopathological techniques were used to study the metastasis of tumors in animals.
3. Human non-small cell lung cancer (NSCLC) NCI-H460 was implanted subcutaneously into nude mice to establish a subcutaneous transplanted tumor model. Four weeks later, 25 nude mice were used as surgical group to remove tumor tissues, and 15 nude mice as control group. Five animals in each group were killed every two weeks to observe the distant organ metastasis. Immunohistochemistry was used to detect the expression of MMP-2, OPN, CD44v6 and CD62 in metastatic and primary lesions.
4. The orthotopic transplantation model of human gastric cancer was established by using MKN-45sci and MKN-45 tumor tissue blocks. The growth and metastasis of tumor, the expression of serum tumor markers and MMP-2, OPN, CD44v6, CD62 and Timp-2 were observed and compared between the two models. MKN-45 MC cell line was established to observe the morphology, growth rate, invasion ability in vitro and cell cycle changes of tumor cells, and compared with MKN-45 cell line.
Three, the result
1. Human hepatocellular carcinoma orthotopic transplantation model in NOD-SCID mice could palpate the growth of tumor in 5 weeks, the tumor formation rate was 100%, the tumor volume was (4.48.93) cm~3, the tumor weight was (7.02 1.15) g, and the lung metastasis rate was 53.85%; human hepatocellular carcinoma orthotopic transplantation model in nude mice could palpate the growth of tumor in 6 to 7 weeks, the tumor formation rate was 100%, and the tumor volume was (1.02.7) at 11 weeks. The tumor volume, tumor weight and lung metastasis rate of NOD-SCID mice were higher than those of nude mice (P = 0.000, P = 0.000, P = 0.011). Both of them maintained the characteristics of high AFP secretion and positive gamma-GT isozyme.
2. The subcutaneous transplantation model and the orthotopic transplantation model of HC-031 were in the state of near death due to the overload of the tumor at 11th and 6th weeks, respectively. The average survival time of the transplanted tumor was 75 and 44 days. Pathological anatomy showed that no metastasis was found in the subcutaneous transplantation model (0/7). The tumor and adjacent organs of the orthotopic transplantation model had obvious invasion and partial lung metastasis (2). The average survival time was 154 D and 112 D in subcutaneous and in situ tumor-resected animals, respectively. The metastatic nodules (7/7, 7/7) were found in the lung by pathological dissection. The expression of AFP and gamma-GT II was positive in the serum of both non-resected and resected animals.
3. Human lung cancer NCI-H460 nude mice underwent subcutaneous transplantation of tumor for 10 weeks, and the tumor necrosis was severe, and the animals showed significant emaciation and no metastasis. One case of lung metastasis (1/5) was found in each of the nude mice in the operation group at 10 and 12 weeks, and the metastasis rate was 100% (5/5) in the nude mice at 14 weeks, with obvious metastasis nodules in the lung and no metastasis in other organs. Metastasis. Immunohistochemistry showed that the expressions of MMP-2, OPN, CD44v6, CD62 and Timp-2 were significantly higher in lung metastases than in primary lesions, while the expressions of CD54 and MMP-9 were lower in metastases.
4. Liver metastasis was early and high in MKN-45sci group, with lymph node metastasis (100%), lung metastasis (71%), spleen metastasis (29%) and ascites. The volume and weight of transplanted tumor increased significantly, reaching (3089+1617) mm~3 and (2.66+1.32) g, respectively. Cachexia was found in the mice, but no metastasis was found in the liver, lung and spleen of MKN-45 group at 4 weeks, only 2 animals had lymph node metastasis, and the mice did not show obvious emaciation. The serum NSE and CYFRA21-1 concentrations in MKN-45 SCI group were higher than those in MKN-45 group with (275+90) mm~3 and (0.35+0.14) G. The results of immunohistochemistry showed that MMP-9, OPN antibody and MKN-45sci were strongly positive in transplanted tumors and metastases, but weakly in MKN-45. CD44v6 antibody was positive in transplanted tumors and metastases in MKN-45sci. The established MKN-45mc cell line is a Hypertriploid cell with the characteristics of human malignant tumor cell chromosomes. The cell morphology is typical epithelioid cells, similar to the morphological characteristics of MKN-45. Flow cytometry analysis showed that MKN-45mc and MKN-45 cell cycle. The proportions of the two cell lines were 62.51%/53.95% in G_0-G_1 phase, 9.46%/1452% in S phase and 28.04%/31.53% in G_2-M phase, respectively. The doubling time of MKN-45mc and MKN-45 cells were 34.8 h and 42.1 h, respectively. The growth rate of the former was higher than that of the latter. The number of outer membrane of MKN-45mc and MKN-45 cells in 36 h was 60.46 h, respectively. 38 + 8.86) / high field of view and (32.50 + 17.26) / high power field of vision, the former is more than the latter.
Four. Conclusion
1. Establishment of human hepatocellular carcinoma animal model with similar clinical manifestations. Compared with nude mice, NOD-SCID mice have more application value in establishing xenograft model of human hepatocellular carcinoma.
2. To simulate the clinical tumor resection method and establish the animal model of human liver cancer metastasis after operation. The experimental results show that the survival time of tumor-bearing animals after tumor resection is prolonged, which is conducive to the occurrence of tumor metastasis.
3. The metastasis model of human lung cancer in nude mice simulates the process of distant metastasis after clinical tumor eradication. The results suggest that the metastasis of lung cancer may be related to the abnormal expression of some adhesion molecules, and provide an ideal animal model for studying the metastasis mechanism of lung cancer and anti-metastasis therapy.
4. The biological characteristics of human gastric cancer orthotopic transplantation animal models were different before and after screening in vivo. The growth and metastasis ability of the tumor after screening was stronger than that of the non-screening. The mechanism was related to the enhancement of adhesion and degradation ability of tumor cells. Tumor metastasis is related to cell proliferation and movement deformability.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R-332;R730.2
【引证文献】
相关期刊论文 前1条
1 钱晓玲;郭勇;;丹参酮ⅡA对肿瘤转移影响的实验展望[J];深圳中西医结合杂志;2010年06期
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