An Experimental Study Of Radiation Therapy Combined With Hyperihermia On Angiogenesis Of Hepatocellular Carcinoma In Mice

ObjectiveSolid tumors have a large number of neovascularization in their occurrence and development.Neovascularization is the basis of growth,invasion and metastasis of tumor cells. Primary liver cancer is one of the most common malignant tumors characterized by neovascularization. The incidence of primary hepatocellular carcinoma is high. The outcome of treatment on hepatocellular carcinoma is poor.Using H22-bearing Kun-ming mice, we studied the anti-angiogenesis effects of radiotherapy radiation therapy combined with hyperthermia by observing the changes of intra-tumor microvessel density (MVD) and expression rate of vascular endothelial growth factor (VEGF). With hematoxylin-eosin staining (HE) staining in each group of pathological changes,cellular ultra-structure changes using electron microscopy, we study the effects of angiogenesis of radiation therapy combined with hyperthermia on tumor-bearing mice. At the same time,this research will provide a theoretical basis and new treatments for clinical treatment for liver cancer.MethodsCollected ascites of H22-bearing mouse under sterile conditions, in order to 1500r/min under the conditions of centrifugal 20min, abandoned to the supernatant to sterile PBS adjusted to 2×10'/ml.0.2ml cell suspension injected to the right hind limb subcutaneous of Kunming mice.Subcutaneous tumor volume growing to 1cm diameter spherical start to therapy in setting group. The setting groups in tumor size no significant difference.Forty tumor-bearing mice, using random number table is divided into radiotherapy+hyperthermia (combined group, group A), radiotherapy group (group B),. hyperthermia group (group C) and control group (group D),10 in each group. Radiation therapy combined with hyperthermia group were produced by Xinhua Medical Instrument Factory drilled Co-60 long-distance radiotherapy radiotherapy machines. Treatment of good narcotic tumor-bearing mice at the same time fixed in plexiglass plate, Tumor-bearing leg stretch, and fixed on the radiation field, radiation field on the edge of tumor beyond the edge of at least 5mm, the thickness of the tumor surface coverage 5mm compensation film to improve the surface dose, radiation field 15cm×15cm, the source skin distance 60cm, dose rate of 81.05 cGy/min, a single radiation dose of 3Gy, treatment of 1 day, for 10 days with a total radiation dose of 30Gy. After radiotherapy, hyperthermia was given within half an hour. Constant temperature water bath with the right part of the tumor-bearing mice given 41.5℃+0.2℃hyperthermia, every 50 minutes. Treatment two times a week, a total of four times. Radiotherapy group and the hyperthermia group were given a single treatment. Control group, blank control. Every other day during treatment with vernier caliper measuring tumor size, Measurement of body weight with the balance. Five days after treatment mice were sacrificed by detachable neck act. Complete anatomical tumor tissue in mice, electron microscope specimen drawn and measuring tumor weight balance by Analytical Balance. Immunohistochemical detection of tumor tissue MVD value and positive expression rate of VEGF. Pathological changes in tumor tissue by HE staining observed with an optical microscope; ultrastructure of tumor cells by electron microscopy.Statistical significance was determined using SAS8.1 software package.ResultsRadiation therapy combined with hyperthermia (group A), hyperthermia group and control group, tumor growth rate, respectively (9.39 mm3/d 12.02mm3/d,15.91mm3/d, vs.21.18mm3/d(F=3.23, P=0.037).Inhibition rates of tumor were (74.8%,58% and 41.8%,P<0.05), combined group of anti-tumor effect the largest. Body weight of mice in each group no significant difference(F=0.57,P=0.639).Combined group, radiotherapy group,hyperthermia group and control group, the mean MVD values were 35.2±5.1,48.5±4.6,58.1±7.4 vs 69.7±9.6 (F=44.24, P<0.05), the difference was statistically signif-icant.Positive expression rate of VEGF in combined group, radiotherapy group, hyperthermia group and control group,was 11.4%±3.8%,16.9%±3.9%, 20.8%；昌±3.1% vs 25.5%±6.4%(F=17.42 P<0.05), the difference was statistically significant.HE staining Pathology:Radiotherapy combined with hyperthermia group (combined group):A large number of condensed nuclei of tumor cells seen.Tumor cells were arranged loosely, and the neighboring cell separation. Less cytoplasm was significantly eosinophilic.Sinusoids significantly reduced; Radiotherapy group of tumor cells can be seen swelling, nuclear pyknosis. Some of tumor cells the smaller nuclei, sinusoids of tumor tissue less. Hyperthermia group point or Chip ischemic/necrosis can be seen in focal tumor tissue. Some nuclear of tumor cell shrinkage.Cell arrangement looser, blood flow around tumor the rich. Control group:tumor cell cord-like beam in close order in tumor tissue, tumor cells was growing strong. Nuclear larger and visible mitoses can be seen.Marginal sinus enriched, oncology centers due to ischemic necrosis of tumor growth strong and clear.Visible ischemic necrosis in tumor centers due to tumor cell growing strong.Under the inspection of transmission electron microscopy (TEM):Radiotherapy combined with hyperthermia group see a large number of nuclear fragmentation or nuclear pyknosis. cell shape and organelle morphology blurred or disappeared. Tumor cells of radiotherapy group loosely arranged. Intracellular nucleus fragmentation and formation of necrotic material (red arrow) also seen. Chromatin,cytoplasmic and organelles less in tumor cell.Most tumor less shaped, showing that tumor cell less. Loosely arranged tumor cells in hyperthermia group, nucleus larger, nucleus fragmentation can be seen in tumor cells, organelles are the obvious. Control group:tumor tissues are rich in tumor cells, tumor cells closely arranged. Nucleus larger, visible mitoses,chromatin and organelles abundant in the cytoplasm in tumor cell.ConclusionBy radiotherapy combined with hyperthermia on tumor angiogenesis of tumor-bearing mice in experimental research, using immunohistochemical staining found that radiotherapy combined with hyperthermia can significantly inhibit tumor MVD values and the expression of VEGF-positive rate; Pairs of tumor-bearing mice in each group tumor volume and weight in the treatment of different time measurement and found that the greatest inhibition rate of radiotherapy combined with hyperthermia; no significant difference in body weight sho no significant increase in toxicity in combined Group.Pathological tumor tissue with HE staining and electron microscopy ultrastructure of the Joint Group significantly inhibit tumor angiogenesis but can also increase the killing of tumor cells or the promotion of tumor cell apoptosis. Confirm the synergy of radiotherapy combined with hyperthermia in the growth of anti-hepatoma effect, and provide a theoretical basis for comprehensive treatment strategies choosen of hepatocellular carcinoma for some experiments...