【Abstract】 Objective prepared rabbit cirrhosis of liver cancer model, and the injection of VX2 model of metastatic carcinoma of the liver cell preparation the contrast observation liver cancer after irradiation pathomorphological changes. 16 cirrhosis of liver cancer in rabbits (experimental group) were randomly divided into two groups, each group of eight were given 20,30 Gy single dose stereotactic irradiation; the eight simple vaccination hepatoma rabbit (experimental group 2), random into two groups of four, according to the method described above to give the same irradiation. Experimental group 1 and experimental group 2, the the HE staining compare before and after irradiation experimental group 1 and experimental group 2 liver pathomorphological changes were sacrificed 3 weeks after irradiation; reticular fiber staining, Masson trichrome compare and determine the experimental group before and after irradiation with experimental group 2 liver fibrosis. Large tumor cells of the tumor area after irradiation necrosis, interstitial fibrous tissue swelling, part fibrous tissue rupture. The experimental group 1 tumor tissue surrounding hepatocellular necrosis obvious; experimental group 2, tumor tissue surrounding liver cells mainly degeneration. Reticular fibers and Masson trichrome before and after irradiation: experimental group 1 irradiation and before irradiation compared to fibrous tissue around the fracture; 2 before and after irradiation fibrous tissue of the experimental group did not change significantly. Cirrhotic liver cancer radiotherapy Conclusion liver state compared with the normal state hepatoma liver tolerated dose is significantly reduced.
Key words cirrhosis HE staining of liver cancer stereotactic irradiation reticular fiber staining Masson trichrome
Primary liver cancer is one of the common malignant tumors in China, the high degree of malignancy and poor prognosis. China die each year from primary liver cancer by more than 11 million people, accounting for 45% of the world’s primary liver cancer deaths . Early treatment of liver cancer is still the preferred surgery. With the development of radiation physics, conformal radiotherapy technology implementation, tumor target by the exact conformal high-dose irradiation, while protecting the surrounding normal liver tissue , radiation therapy has gradually become the main non-surgical treatment of liver cancer One of the ways [3,4]. This study rabbit liver cirrhosis liver cancer foundation uplink single different dose stereotactic irradiation, the pathological changes of liver cancer after irradiation application histopathology, histochemistry observed liver disease status, in order to guide the HCC dimensional conformal radiotherapy, improve the clinical therapeutic effect .
1 Materials and methods
1.1.1 rabbit cirrhosis of liver cancer alone HCC model preparation of 25 male New Zealand white rabbits, intragastric administration of carbon tetrachloride (CCl4, 1.598g/ml) method, once / week, starting dose of 50 mg / kg, adding 2ml corn oil were mixed into the oil solution of 5%, and to gradually increase the amount according to the experimental group 1 tolerated, the maximum dose amount can be added to CCl4160mg/kg 3ml corn oil was added in the synthesis of a 10% oil solution. Administration of 6 months, made a rabbit model of hepatic cirrhosis. 16 successfully made cirrhosis rabbits (experimental group), surgery VX2 tumor cells in situ vaccination in rabbit right lobe of the liver, made rabbit cirrhosis of liver cancer models. Eight normal male New Zealand white rabbits (experimental group 2), according to the above surgical methods inoculated VX2 tumor cells, made of rabbit the simple liver cancer model.
16 1.1.2 Animals and packet processing have been successfully prepared cirrhosis of liver cancer rabbit (experimental group 1) and simple hepatoma rabbit 8 (experimental group 2) were randomly divided into A and B groups, 12 in each group (experimental group 1 to 8, the experimental group 2 to 4) were given 20Gy and 30Gy stereotactic irradiation were sacrificed 3 weeks after irradiation, the liver tissue, the conventional preparation of paraffin sections alternate. All experimental animals were male New Zealand white rabbits, the average weight of 3.5kg.
1.2 Reagents and instruments
1.2.1 Reagent reticular fiber staining reagents: (1) 0.25% potassium permanganate solution; (2) 2% oxalic acid solution; (3) 2% aqueous solution of ammonium ferric sulfate; (4) Gomori’s silver ammonia solution; ( 5) 10% neutral buffered formalin solution; (6) nuclear fast red solution.
The Masson trichrome reagents: (1) Weigert’s iron hematoxylin; (2) Beginning of Spring the red acidic magenta liquid; (3) 1% aqueous solution of phosphomolybdic acid; (4) aniline blue liquid.
1.2.2 Instrument PICKER PQS 2000 (AMERICA) whole body spiral CT; Medrad CT pressure syringe; Leibinger STP3 (GERMANY) stereotactic conformal radiotherapy treatment planning system; Varian 600C / D linear accelerator; paraffin slicing machine; electric oven; microwave .
The the 1.3.1 experimental group and experimental group 2 stereotactic irradiation (stereotactic irradiation, SRT) scheme anesthesia fixed: rabbit from the ear vein injection anesthesia with 3% sodium pentobarbital 0.5ml/kg dose is fixed in a vacuum mat; CT scan positioning: PICKER spiral CT scan across the top to the right lobe, 3mm thick positioning, the first level sweep, then pressure syringe injection 0.3ml / s iodine Pierer 7ml (Italy MAKE) enhanced scan and CT the positioning scan information transmission to FISHER3DTPS (three-dimensional treatment planning system) workstation; the target draw: GTV (measurable tumor area) is determined in accordance with the requirements of the International Radiation Dose Units and Measurements Committee, CTV (clinical target volume), PTV ( planning target volume), the TV (treatment zone), IV (irradiated area); stereotactic irradiation: at said target to distinguish between an angle of 5 to 6 stereotactic irradiation, were given a single dose of experimental group 1 and the group A, the experimental group 2 20Gy group B single dose of 30Gy. With Varian600C / D linear accelerator implementation of the plan, with a 6MV the X-ray irradiation, dose rate 5Gy/min.
1.3.2 reticular fiber staining (modified Gomori’s method)  slices flat on a staining rack, dropping 0.25% potassium permanganate solution (to be overshadowed by the slice) oxidation 5min; slightly washed; 2% oxalic acid solution bleach 1 ~ 2min; slightly washed; 2% aqueous solution of ammonium ferric sulfate mordant 5min; slightly washed; trickle Gomori’s silver liquid ammonia role for 3 ~~ minutes; slightly washed distilled water; 10% neutral buffered formalin solution restore 1min; running water for 5 ~ 10min stained; liquid with nuclear fast red 5 ~ 10min; slightly washed; conventional dehydration transparent, neutral gum cementing; reference to the results of the light microscope, collagen fibers brownish yellow to brownish black reticular fibers.
Masson trichrome : Weigert’s iron hematoxylin Dyed 5 ~ 10min, wash water slightly; 1% hydrochloric acid alcohol differentiation, running water for several minutes; beginning of spring the red acidic magenta liquid dyed ~~ 10min, slightly rinse of distilled water; 1% phosphomolybdic acid aqueous solution is about 5min; without washing, the direct liquid stained with aniline blue 5min; 1% glacial acetic acid for 1min; 95% alcohol dehydration times; anhydrous alcohol dehydration, xylene, neutral gum cementing; under the light microscope observations, collagen fibers were blue, cytoplasmic, red muscle fibers.
The 2.1 experimental group 1 and experimental group 2 before and after irradiation the pathomorphological changes shown in Figure 1.
2.1.1 experimental group 1 and experimental group 2 before irradiation HE staining irradiation prior to infection deaths experimental group and the experimental group 1 rabbit anatomy parallel routine HE staining, the experimental group of normal liver tissue, hepatic lobule a polygonal structure, liver cell cords arranged radially around the central vein, hepatic sinusoid gap between the liver cell cord. Cirrhosis 1 organization in the experimental group, the normal liver structure damage, cell disorder, portal area more fibrous tissue hyperplasia, split hepatic lobule to pseudolobules structure. Inoculation of VX2 cell mass in the liver tissue, a small number of cancer cell apoptosis and necrosis, and a small amount of connective tissue hyperplasia.
After the the HE staining experimental group 2 cancer tissue surrounding normal liver tissue in the the 2.1.2 experimental group 1 and experimental group 2 different doses of irradiation to accept irradiated with a single dose of 20Gy, only visible degenerated liver cells, as shown in Figure 1a; irradiated with a single dose of 30Gy visible liver cells degenerated, and visible focal necrosis, as shown in Figure 1d.
The experimental group 1 cancer tissue surrounding liver tissue visible multifocal necrosis obvious near the tumor tissue surrounding hepatocellular necrosis, fibrous tissue around the fracture. Cirrhosis tissues received radiotherapy 20Gy irradiation, visible necrosis in the liver tissue, as shown in Figure 1b; After and cirrhosis tissues radiotherapy 30Gy showed large patchy necrosis in the liver tissue, only a small amount of residual cirrhotic liver tissue, as shown in Figure 1e.
Three weeks after liver VX2 tumor SRT: tumor area were seen large areas of necrosis, cancer cell shrinkage, nuclear fragmentation, disappeared surrounding visible residues little cancer tissue, interstitial fibrous tissue swelling, some breaks. Given liver cancer radiotherapy 20Gy visible within the cancer tissue necrosis, as shown in Figure 1c; given liver cancer radiotherapy 30Gy large flake visible cancer tissue necrosis, hemorrhage, see Figure 1f.
Experimental group and the experimental group showed no hepatic vein occlusion or thrombosis.
The 2.2 experimental group and the experimental group the pathological chemistry reticular fiber staining, Masson tri-color change before and after irradiation, as shown in Figure 3. Before and after irradiation line reticular fiber staining and Masson trichrome, reticulin staining fibrous tissue dyed black, Masson trichrome stained blue fibrous tissue.
Fibrous tissue in the experimental group 2 normal liver tissue after irradiation had no significant changes, as shown in Figure 2a, 2d, Figure 3a, 3d.
Experimental group cirrhotic liver tissue irradiation significantly before the fibrous tissue hyperplasia, thickness ranging single irradiation 20Gy visible part of the collagen fiber fracture, as shown in Figure 2b, Figure 3b; complete rupture of collagen fibers is visible after a single irradiation 30Gy see Figure 2e , 3e.
Liver VX2 tumor before irradiation with visible coarse collagen fibers, single irradiation 20Gy visible after the collagen fiber portions broken, was intermittent connection, see Fig. 2C, Fig 3c; the single illumination 30Gy, complete rupture of collagen fibers within the vision, and no connection, see Figure 2f, 3f.
Reticular fiber staining the special staining , reticular fibers of extracellular matrix components present in the tissues and organs of the human body, is the product of the combination of type III collagen fibers and argyrophilic proteoglycan by fibroblasts cells, fibroblasts, vascular adventitia cells of mesenchymal cells and mesenchymal tumor cells, the reticular fibers dyed fibrous tissue dyed black. Masson trichrome connective tissue is the most commonly used special staining method , mainly used to distinguish between the collagen fibers and myofibrils.
In this experiment, we use the reticular fiber staining and Masson trichrome special staining fiber expression of cirrhosis of liver cancer organization received a single high-dose irradiation. Reticular fiber staining collagen fibers brownish yellow, the reticular fibers brownish black , Masson color collagen fibers dyed blue, the cytoplasmic red muscle fibers . Before irradiation in normal liver tissue, only seen in the portal area a small amount of fibrous tissue. In cirrhotic liver tissue, the visible obvious pseudolobules structure in hepatocellular carcinoma and fibrous tissue obvious proliferation. Given after irradiation, the context of normal liver the HE staining saw liver cell degeneration, network stained and Masson trichrome visible fibrous tissue did not change significantly, and cirrhosis of the liver tissue to give 20Gy irradiation HE staining visible liver tissue necrosis , giving 30Gy irradiation, the liver tissue, showed large patchy necrosis, only a small amount of residual cirrhotic liver tissue. Network stained and Masson trichrome visible part of the collagen fiber breakage after 20Gy dose irradiation, the 30Gy dose irradiation after complete rupture of collagen fibers within the field of view is visible. Given two doses of irradiation, fibrous tissue in the liver disease state, there are varying degrees of fracture, surrounded by a small amount of fibrous tissue hyperplasia, cirrhosis of liver cancer tissue necrosis after irradiation, after a certain period of time showed necrotic hyperplasia, that clinical said radioactive liver injury early liver fibrosis, the general performance of 4 to 12 weeks after irradiation, the experiment because of the observation period of only three weeks, so the performance of liver fibrosis is not obvious, and also showed no radioactive liver damage typical hepatic vein occlusion or thrombosis.
Radiation therapy of liver cancer nearly two years has made significant progress in liver disease state , prolong the survival of patients with advanced hepatocellular carcinoma, and improve quality of life. With the continuous improvement of radiologic technology and equipment , the continuous development of radiobiology, radiation treatment of cirrhosis of liver cancer will become a safe and effective means of