| 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.
|
PDF Full Text Request