动物实验,血管内皮细胞,索拉非尼,肝细胞癌 z-bo 2020-09-05 521

　　Introduction: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death in the world, and the liver is considered the most common site of blood-borne tumor metastasis. Local treatment (TACE) is considered an effective treatment for advanced liver cancer. These treatments only maintain the short-term stability of the lesion and provide limited survival benefits. Vascular endothelial growth factor (VEGF) is one of the most effective tumor angiogenesis regulators. After local treatment of liver cancer, it significantly increases and is considered to be one of the important factors for tumor survival and recurrence. So far, the anti-angiogenic multikinase inhibitor sorafenib inhibits the activity of Raf serine kinases and receptor tyrosine kinases (such as VEGFR-2, PDGFR-β, c-KIT, FLT-3 and RET). Leads to inhibition of tumor cell growth and angiogenesis. The efficacy of sorafenib has been confirmed in BCLCC stage liver cancer. However, 6.5 months after sorafenib treatment, the local reaction was 3.3%, and drug resistance appeared. In explaining the condition, the BCLC stage C with portal vein tumor thrombosis is not comprehensive enough (PVTT), and at this stage it is not yet possible to clearly distinguish primary or branch portal vein tumor thrombosis. The safety and effectiveness of hepatic artery chemoembolization have been confirmed in the treatment of portal vein cancer. In fact, the two therapies have no clear contraindications to TACE? +? Most patients who receive combined treatments of stage B and C, such as sorafenib, have a good prognosis. Retrospective studies have shown that TACE combined with sorafenib is beneficial to patients with advanced HC. At the same time, it was found that different studies used different methods and time of administration. Obviously, these differences may cause controversy in these trials. In this study, animal studies were used to study the best starting point for sorafenib combination therapy. Methods: Tumor cells and animals: Walker-256 tumor cells, 4-week-old BALB/c nude mice (12-15g, N≥2) were injected with tumor cells into the outer thigh. .. Inoculate liver tumors to adult male SD rats (weight 150-200g; N≥50). Place the animal under SPF conditions at a temperature of 23-±3°C and a relative humidity of 50-±10%. Establishment of subcutaneous tumor model: Use Walker256 tumor cells to establish liver tumor model, and then establish phosphate buffered saline (PBS, 16.5mM phosphate, 137mM sodium chloride and 2.7mM potassium chloride, pH 7.4). )used. The tumor cell suspension was diluted to 107 cells/ml and inoculated subcutaneously on the outer thighs of BALB/c nude mice. About 9 days after tumor cell inoculation, a subcutaneous tumor with a diameter of about 1 cm was seen at the injection site. Establishment of liver cancer model: use the above method for tumor transplantation. Tumors were collected subcutaneously from donor animals, minced, SD rats were opened through a midline incision in the lower abdomen, and intraperitoneally anesthetized with 10 mg chloral hydrate at a dose of 300 mg/kg. The left lobe of the liver extends outside the abdominal cavity. Using ophthalmic forceps, prepare a subcapsular tunnel depth of approximately 2-3 leaf thickness. Insert the solid tumor mass into the tunnel. Cover the wound with a small piece of gelatin sponge. Hemostasis is not necessary, all experimental rats returned to normal after the operation and resumed normal activities the next day. Experimental treatment: Ten days after transplantation, magnetic resonance imaging (MRI) was used to evaluate the establishment of liver tumor models. The experimental animals were anesthetized by intraperitoneal injection of chloral hydrate, and then MRI scans were performed. The scanning parameters are: echo time: T1WI is 82ms, T2WI is 1.5ms, repetition time T1WI is 1500ms, T2WI is 508.7ms, and the reconstruction thickness of T1WI and T2WI is 5mm. Forty qualified rats with liver tumors were randomly divided into four groups. Group A was the control group, and the animals were injected with VEGF (20 mg/L, 1 mL/kg, body weight) only. 72 hours before VEGF injection, group B (group B, n = 10?) was gavaged with sorafenib (100 mg/kg bw). Group C (C, n≥10) received gavage sorafenib and vascular endothelial growth factor injection. Group D (D, N = 10 -4) received sorafenib by gavage 72 hours after vascular endothelial growth factor injection. Sorafenib was taken orally once a day for 100 consecutive days at a dose of 100 mg/kg body weight. Recombinant rat VEGF165 (20 mg per tube) was centrifuged at 1000 pm for 30 seconds, dissolved in 100 ml of deionized water, and diluted with saline of desired concentration. Rats in groups B, C and D were injected with 20 mg/LVEGF. Imaging and pathological studies: Magnetic resonance imaging is used to assess the development of tumors and ascites. 10 days after sorafenib treatment, ensure liver transplantation of tumor tissue and monitor the development of tumor and ascites. In the case of rapid tumor development, animals are usually evaluated daily based on fur, nutritional intake and behavior. After the animal died, the rat liver tissue samples were analyzed and compared with MVD and VEGF. We used rabbit anti-mouse VEGF polyclonal antibody and rabbit anti-mouse CD34 antibody. Detection and counting of vascular endothelial growth factor and microvessels: Two senior pathologists will determine the vascular endothelial growth factor protein expression and MVD level on each slide. In each case, count 100 epithelial cells, the epithelial cells are positive, count 5 random spots and count the stained epithelial cells. The average CD34 positive microvessel count was used to detect tumor blood vessels. Under an optical microscope, the five areas with the largest number of capillary staining were identified as "hot spots". In order to quantify the microvessels, the five "hot spots" were counted and averaged using a 200x magnification field of view. It is not counted in areas of necrosis or inflammation. Further analysis excluded five unrecognized "hot spots". If there is a significant difference between the two pathologists, please check again until a consensus is reached. Results: Detection of tumor growth in the liver: Ten days after tumor tissue transplantation, MR routine scanning was used to determine whether the liver of SD rats had liver tumors. Record the size and location of the tumor. Except for tumors in the abdominal wall or in the abdominal cavity, the discovered tumors were subjected to the following experiment: 10 days after the administration of sorafenib, MR scanning was used to measure the tumor size. The largest tumor diameter is used as an evaluation index.

　　Average survival time: The average survival time of A, B, C and D is 19.6±1.78, 31.2±6.99, 27.4±4.9 and 26.5±4.6 days. The results showed that there was a significant difference in survival time between the sorafenib group and the control group. There are significant differences between group B and group D. However, there was no statistical difference between groups B and C and groups C and D. Pathological examination: After the experimental animals died spontaneously, samples of liver tumors were taken for HE staining. As shown in the picture, the normal lobular structure has disappeared, and the tumor cells are arranged in sheets or nests. The frequency of tumor cell nuclear mitosis is very high. The atypical cells of the tumor are obvious, and they are dual or even multinucleated. It can be seen that the large eosinophilic plaque is the area of tumor necrosis. There was no significant difference in tumor morphology between the 4 groups. Compared with the sorafenib group, the tumor cells in the control group were smaller, more compact, and had greater changes in size and shape. Immunohistochemistry: The expression of VEGF in liver cancer is brown and mainly distributed in the cytoplasm. In the control group, a large number of VEGF-positive vascular endothelial cells were detected by immunohistochemistry. The number of VEGF-positive cell samples in the control group was 88.3±13.61, which was 42.8±7.96, 71.9±15.73, and 73.6±13.73 higher than those in groups B, C and D sorafenib, respectively. The expression of VEGF between groups B and D gradually Recovery, indicating that the early administration of sorafenib has a more obvious inhibitory effect. CD34-positive cells stained brown or dark brown. The trends in the four MVD groups were similar to VEGF. There are statistical differences between the control group and the sorafenib group. The relationship between average survival time and tumor: VEGF and MVD are usually used to evaluate the angiogenic activity of tumors. These are consistent, the correlation coefficient is 0.875, which is positive. Using Pearson's continuous variable correlation analysis, the product-moment correlation of the Pearson coefficient between VEGF and average survival time is 0.78, which is a negative value. MVD was negatively correlated with average survival time (0.794). Conclusion: The most appropriate solution to administer sorafenib is to control tumor growth by inhibiting VEGF and VEGF receptor expression before increasing VEGF expression, thereby prolonging the survival rate of experimental animals and tumors.