| PartⅠ:The inhibitory effect of MEIN on tumor using murine cancer modelsThe main components of MEIN are whey protein peptides and lactic acid bacteria, which are nutritional function food with anti-inflammatory function. Whey protein includes a-Lactalbumin, β-Lactoglobullin, serum immunoglobulins and so on, which are alkalinizated, ultrafiltrated, lyophilized and decomposed by trypsin to be whey protein peptides. Lactic acid bacteria includes L-bulgaricus and S-thermophilus. The production fermented by lactic acid bacteria is easier to absorbed.Two commonly used cancer cell lines, Lewis and C26, were used to build Lewis lung cancer model and C26mouse solid tumor model.In Lewis lung cancer model, mice were randomly divided into two groups of20each, male and female in half. From the inoculation day, mice were fed with either MEIN diet or control diet AIN-93M for16days. For Lewis lung tumor-bearing mice, food intake, body weight, tumor weight and tumor volume were recorded as the major indicators. Serum cytokine levels including Interleukin-6(IL-6), Interleukin-10(IL-10), Monocyte Chemoattractant Protein-1(MCP-1), Interferon-γ (IFN-γ), Tumor Necrosis Factor (TNF), Interleukin-12p70(IL-12p70), Interleukin-2(IL-2), Interleukin-4(IL-4), and Interleukin-5(IL-5) protein levels were determined using BD Cytometric Bead Array kits.In Lewis lung cancer model, the food intake and body weight change of mice in MEIN group did not show significant difference compared with control group (P>0.05); the mean tumor weight in control group was higher than that in MEIN group, the difference of mean tumor weight was statistically significant (P<0.05). The mean tumor volume measured at days8,10,12,16was significantly larger in MEIN group than that in control group (P<0.05). The tumor inhibition rate of MEIN was51.52%. The concentration of serum IL-6and MCP-1in the control group was significantly higher than that in MEIN group, and the difference was statistically significant(P<0.05).In C26cancer model, mice were randomly divided into four groups of24each, male and female in half. From the inoculation day, mice were fed with MEIN diet or control diet AIN-93M for16days, two groups which were fed with MEIN diet or contol diet were injected with cyclophosphamide. For C26rumor-bearing mice, food intake, body weight, tumor weight, rumor volume, liver weight, spleen weight and epididymis fat were recorded; the tumor histopathology and immunohisto-chemistry analysis were performed; serum and tumor tissue cytokine levels were determined using Cytometric Bead Array Method. In C26cancer model, the food intake and body weight change of treated-mice did not show significant difference compared with control group(P>0.05). Compared with the control group, the mean tumor volume of the MEIN+CTX group and CTX group was significantly different in day10, day12, day14and day16, the tumor volume of MEIN group was significantly different in day10and day12(P<0.05).Histopathologic examination showed that tumor malignancy in control was more serious than that in MEIN group.The cytokines were detectable in the mouse tumor and serum of every group, and the concentration of these cytokines in the control group was non-significantly higher than those in MEFN, MEIN+CTX and CTX group (P>0.05). The WBC in control group was significantly higher than that in other groups(P<0.05). The serum CRP in the control group was significantly higher than that in MEIN+CTX group (P<0.05). The result suggested that MEIN maybe inhibit the inflammation in the body.The apoptosis in the tumor tissue was evaluated by the terminal deoxynucleotidyl transferase (TdT)-mediated digoxigeninuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay. The TUNEL results showed that the control group positive rate was+/-, MEIN group and CTX group positive rate was++, and MEIN+CTX group was higher than the other three groups, its positive rate was+++. It suggested that MEIN and cyclophosphamide play a strong inhibition role to the tumor cells, in addition, the inhibition effect has further improved with collaboration of MEIN and cyclophosphamide.VEGF, Ki-67and CD34proteins in tumor tissue were respectively detected.The tumor tissue Ki-67positive rate in the control group and CTX group was++, MEIN group was+/-, MEIN+CTX group was+. It was indicated that the tumor cells were in resting phase in MEIN and MEIN+CTX group, because of MEIN could keep the tumor cells resting phase, and inhibit the reproduction of tumor cells.The tumor tissue VEGF positive rate in the control group was+++, inMEIN+CTX group and CTX group was+, and VEGF detection in MEIN group was extremely low, was+/-. It was demonstrated that, in the interference of MEIN or cyclophosphamide, the tumor angiogenesis was reduced.The tumor tissue CD34positive rate in the control group was+++, other three groups was+. It was showed that tumor angiogenesis was reduced, the tumor growth was also restrained in the interference of MEIN or cyclophosphamide,.The result suggested that MEIN could inhibit the tumor growth in murine Lewis Lung cancer model and C26cancer model. The anti-inflammatory function of MEIN may play a role in the inhibition of tumor growth. Part Ⅱ:Establishment of liver fibrosis model using urokinase-type plasminogen activator gene-deficient miceThe incidence of liver fibrosis and cirrhosis has become an important public health and social issue.To evaluate the feasibility of uPA knock-out (uPA-/-) mice liver fibrosis model by observing the pathologic changes and analysing the expression of a-Smooth Muscle Actin (a-SMA) and Matrix Metalloproteinase-13(MMP13) Adult male BL/6J WT mice and uPA knock-out (uPA-/-) mice were divided into four groups with10mice in each group:control groups (WT, uPA-/-) and liver fibrosis model groups (WT, uPA-/-). Mice were intraperitoneally injected0.15ml10%CCl4(or olive oil as control) twice per week for6weeks.Animal body weight, liver weight, spleen weight and kidney weight were measured, and organ coefficients were calculated. General pathology of livers was observed at sacrifice, histopathology was performed using HE staining and Masson staining, and electron microscopic exam was performed to observe the ultrastructural changes. The mRNA expression of a-SMA and MMP13was detected by real-time polymerase chain reaction (PCR) and the protein expression of a-SMA and MMP13was analyzed by western blot and immunohistochemistry. Liver weight and liver coefficient of uPA-/-model mice increased significantly compared with WT model mice (P<0.05). The enlargement of the liver, pale gray color, and apparent granular nodules were observed in uPA-/-model mice. HE staining, Masson staining and electron microscopy all showed in consistency that liver cells were severely damaged, extensive collagen was deposited and false lobules were formed in uPA-/-model mice. uPA-/-model mice showed more severe liver fibrosis compared with WT mice by observing histopathology. The mRNA and protein expression of a-SMA were significantly higher in the uPA-/-mice. The knocking-out of the uPA gene promoted the hepatic stellate cells transforming into myofibroblasts, thus speeding up the deposition of extracellular matrixin in the liver. Also, the protein expression of MMP13in liver fibrosis model mice was significantly increased.uPA-/-mice are susceptible animal strain to establish liver fibrosis model. |
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