Effect Of Kupffer Cells In The Experimental Hepatocarcinoenesis Of Kunming Mouse

OBJECTIVE Hepatocellular carcinoma (HCC) is the most common malignant tumor in China and worldwide, the morbidity is ascending year by year and has occupied 5 percent above in the malignant tumors. With the advancement of the modern medicine, for the late few years, some new methods were employed to HCC, such as biologic immunotherapy, liver transplantation, radio frequency, high focused ultrasound and etc, and some effects were gained, it is the biologic immunotherapy that became the studying focus to many reserchers as its peculiar preponderance especially, but HCC has the property of high malignance and poor prognosis, the 5 year survival rate is less than 5 percent in the patients complicated symptoms. Therefore, wo should lucubrate the correlated mechanism of occurrence and development of HCC further in order to study new therapeutic measure and target which has become hot spot of the turmor research now. The mononuclear phagacytic system(MPS) is important ingredient of organism immune defence, activated macrophage through phagocytosis, antibody dependent cell mediated cytotoxicity (ADCC), cytokine of excreting cell and so on to dissolve and kill tumour cell. The Carl von Kupffer described the astromacrophage in the liver first at 1886,and named after Kupffer cells (KCs). KCs is the dominated macrophages and are significant to restrain tumour generation, vegetation and metastasis. Therefore, this experiment use the specific suppressant Gdcl3 of MPS function to restraint the function of KCs and to observe the effect to the experimental hepatocarcinoenesis in the Kunming mouse that can confirm the defence function of KCs to the generation, vegetation of the HCC and approach correlated mechanism, so we can provid relative theory for new therapeutic measure and target.METHODS Sixty cases of 4 weeks old male Kunming mouses were vaccinated in the the anteroright hepatic under liver capsule with 0.2ml (2×105) H22 cell suspension and were randomly divided into two groups(each group 30): gadolinium chloride treatment group (GCG) and hepatocellular carcinoma group (HCCG). The 0.25% Gdcl3 solution (10mg/Kg) was infused into vena caudalis of mouses in GCG before seed for 1 week . The isotonic Na chloride was infused into vena caudalis of the mouses of HCCG by one time per 3 days. Simultaneously, seted normal control group (NCG) with 20 healthy Kunming mouses of the same age. Ten mouses taken out of each group were observed the natural death time and drawed survival curve, the remain mouses each group were killed to observer the follow index when the experiment was carried out for 3 weeks: the weight of the tumor, tumor vegetation condition in light microscope, ultramicrostructure of KCs in electron microscope, histochemical stain to identify the distribution of KCs and counted KCs number, ELISA mensurated the level of the blood-serum TNF-α, RT-PCR mensurated the express level of TNF-αmRNA of liver tissue .RESULTS (1) There were not denth in the mouses of NCG, Kaplan-Meier survive curve analysised by SPSS13.0 displayed the live time of HCCG and GCG were 40.5±3.5d and 30.5±2.5d respectively. Log-rank test indicated that the live time of NCG,HCCG and GCG decurtated gradually, the distinction had significance (P<0.05). (2)Macroscopic observation: the bulk of carcinoma of GCG exceed that of HCCG obviously, quality difference was significance (P <0.05). (3) HE stain show the morphosis of hepatic lobules of NCG was normal, the periportal and central vein structure was clear, cellocord rowed regularitily. The cells array was disorder and the atypia of organization was obvious in liver cancer tissue and the infiltration of inflammatory cells was evident in live tissue aroud liver cancer in HCCG and GCG . Stellate ganglion was found in GCG. (4) From the electron microscope, in normal human liver tissue, the KCs are well-shaped, their phagocytic activity are not strengthen obviously and phagolysosome intracytoplasm is few. With the infiltrations of neutrophils, lymphocytes and eosinophile granulocytes, cellular swelling, nuclearmegaly, increased phagosomes and lysosomes are obvious in KCs. The KCs in HCCG is activiated obviously in around cancer tissue, their membranes linke to cancer cells with cellular swelling, densed chromatin, augmentated and irregular shaped nuclear and generous endoplasmic reticulum intracytoplasm. But the function of KCs of GCG does not show to be activated obviously. (5) Immunohistochemical staining confirm that the KCs is orbicular-ovate, astero or fusiform,the quantity of KCs of the around cancer tissue and far from cancer tissue in the HCCG and GCG are 9.40±2.37pc./PH, 9.50±2.17pc./PH and 9.20±1.87 pc./PH, 9.00±2.10 pc./PH respectively, the quantity of KCs in NCG was 4.80±1.31 pc./PH. There were no KCs in cancer tissue. The quantities in tissues around and apart from cancer are more than in normal liver tissue (P<0.01). (6) ELISA was employed to evaluate the level of blood serum TNF-αof NCG, GCG and HCCG respectively: 1.635±0.237 ng/L, 3.964±0.205 ng/L, 4.977±0.357 ng/L, the TNF-αlevel of GCG and HCCG higher than NCG (P<0.05), HCCG compared with GCG, P<0.05. RT-PCR result: the express level of TNF-αmRNA of liver tissue in NCG, GCG and HCCG was heighten gradually. Semiquantitative analysis was that relative amount of mRNA in NCG,GCG and HCCG respectively 1.6, 3.5 and 8.0. The distinction was significance(P<0.05).CONCLUSIONS⑴The Gdcl3 pretreatment had no effect on quantity of KCs, but restrain the function of KCs obviously.⑵The function of KCs were restrain, which result that the generation of TNF-αwas decrease, the tumor immunity function degress obviously, result to growth acceleration of tumour and metastasisible and the live time of trial mouses decurtated.⑶The research that strengthen the protect function of KCs to liver cells and restrain it malignant transformation will become emphasis and hot spot of the further study.