| Purposeliver cancer, in particular hepatocellular carcinoma, is one of the most common fatal cancers in China or in the world and soon may reach epidemic levels because of increased viral - induced hepatitis. Although some advances have been achieved in the diagnosis and treatment of HCC, the long - term outcome for patients with HCC is still very poor. The prognosis for HCC depends mainly on the clinicopathological characteristics regarding invasion and metastasis. The major obstacle to the improvement of the prognosis for HCC is the high incidence of recurrence or metastasis after routine surgical treatment or the transcatheter arterial chemoembolization ( TACE ). TACE has been one of the most common used method to treat liver cancer in middle and late phase, unfortunately routine TACE is limited by poor response rates, severe toxicities and high recurrence rates resulting in a lower mean survival time. To circumvent these multiple shortcomings, we develop a new chemotherapy agent arsenic trioxide( As2O3) in TACE. As2O3 the main component of a traditional Chinese drug " Pishuang" , has obviously selective anti - tumor effect on human HCC in both in vitro and in vivo studies, and some studies has illustrated the antiangiogenic effect of As2O3 in treatment of leukemia. While angiogenesis is the most important step in the process of tumor recurrenc and metastasis after TACE, we think that As2O3 used in embolization should be a better select for liver cancer because of its double therapeutic effectiveness: selectively inducing tumor cell apoptosis and antian-giogensis. As a result, we made an experimental study in an animal model, toinvestigate the antiangiogenic effect of As2O3in TACE and to provide experimental basis for clinical application in suppressing postembolization recurrence and metastasis.Materials and Methods1. Tumor implantationAdult Japanese White rabbits (40 total) weighing 2. 5 ~3kg were used. The rabbit VX2 tumor was selected for implantation in the liver because of its blood supply to that of human hepatomas. Other characters of this tumor include rapid tumor growth, development of a sizable tumor that can be readily identified by X - ray imaging (fluoroscopy and angiography) , in addition, the rabbit is large enough that selective manipulation of catheters in the hepatic artery for delivery of agents is possible. For successful implantation of the VX2 tumor into the liver, the tumor was first grown for 2 weeks on the hind leg of a carrier rabbit. Each carrier rabbit was used to supply tumor cells for implantation into the left or right central lobe of the liver of ten separate rabbits.All of the animals, carriers and recipients, were anesthetized with " sumi-anxin" administered i. m. . The VX2 tumor was then excised from the carrier rabbit and placed in hanks'solution. The tumor tissue were minced into small cubes as 1 mm size. Then the abdomens of the recipient rabbits were shaved and layed on the bed, after which a midline subxyphoid incision was made. The anterior surface of the liver was exposed and tumor cubes were directly implanted into the left or right central lobe of the liver. This method allows the growth of a single solitary, well demarcated tumor in the liver of each recipient rabbit. The abdomen was closed in two layers. Proper aseptic technique was rigorously observed during each implantation. After surgery, animals were returned to their cages, and monitored in the animal laboratory. The tumors were allowed to grow for another 2 weeks, at which time they received enhanced CT examination and commonly reached an ellipsoidal shape with dimensions of 1. 5 x2 x2.5cm, recorded the maximum and minimum diameter.2. Animals group and preparation of embolic agentsForty rabbits with implanted VX2 tumor were randomly divided into 5 groups: control group ( without any treatment) ; groups of UFLP alone; UFLP combined with ADM; UFLP combined with As2 03; and UFLP combined with ADM and As2O3. Each rabbit with TACE used 0.8ml UFLP, 2mg ADM or 2mg As2O3 or their mixture.3. TACE procedureAfter 2 weeks of tumor implantment and CT examinations, the animals with liver tumor were treated with different methods. Administration of anesthesia, i. m. access as described above. Transcatheter hepatic artery injection of chemo-embolic agents was performed under fluoroscopy. The animals were brought to the angiography room and subxyphoid incision was made to expose the liver hilar structure, then separate the hepatic artery from common hepatic artery to the distal end of proper hepatic artery. Puncture needle of 24G(Braun, German) was used to puncture the common hepatic artery, after which a hepatic arterio-gram was performed to confinn the location of tumor. The tumor staining could easily be visualized on the left or right central lobe of liver. The nutrition artery, which ususlly provides most of the blood flow to the tumor, was selectively catherterized. After having adequately positioned the catheter within the nutrition artery, the chemoembolic agents were infused directly into the artery. The procedure was considered successful when forward flow was no longer demonstrated within the nutrition artery. In addition, an intense tumor stain was identified in each case, which suggested a successful embolization procedure. After bringing out the puncture needle, the arterial puncture site was pressed until bleeding stopped, then the abdomen was closed in two layers.4. HistopathologyThe animals were examined with CT scaning after 3 weeks of TACE procedure. Then the animals were killed and the tumor isolated and measured, measure its maximum and minimum diameter to evaluate the tumor volume and the ratio of necrosis area. Then isolated the liver and lung, sliced at 5 mm intervals for gross examination; half of the tissue fixed in 10% formalin solution, observe the abdominal cavity and nearby organ to look for intrahepatic and distal metastasis ; the other half placed in fluid nitrogen for RT - PCR.For each rabbit, target tumor tisse, tissue around tumor and normal liver tissue were selected and embedded completely in paraffin, after which 4jxm section were stained with HE and immuno - histochemical staining, including VEGF, VEGFR/fltl, and CD34. The positive cells were appeared as brown and yellow staining in cytoplasm. Then calculated the positive cells of VEGF, VEG-FR/Fltl, and CD34 according to the methods of Park and Weidner, respective-ly-5. VEGFmRNA measurementThe frozen tumor tissue were minced into small pieces and the total RNA were extracted with RNAout. RT - PCR was carried by one - step method, fj -actin as the internal control. The sense primer for VEGF: 5 ' - TG-GCAGAAGAAGGAGACAATAAAC - 3 '. Antisense primer for VEGF: 5 ' -CTCATCTCCCCTATGTGCTGG - 3 '. Sense primer for internal control |3 - actin : 5 * - ACCGGCATCGTGATGGAC - 3 \ Antisense primer for ï¿¡ - actin: 5'-AGGAAGGAGGGCTGGAACA - 3' All primers synthesized by Dalian Takara biotechnology company. Cycle conditions were set as the manuscript of the RT -PCR. The PCR products were added in 1.2% agarose gel, after electrophore-sis, the gel was placed under ultraviolet ray to analyze the results. The results were expressed by IOD value of VEGFmRNA/p - actin.6. The TUNEL detecting apoptosis of tumor cellsThe section were stained through the methods illustrated in the technical manual. The positive apoptosis cells were appeared as brown to yellow staining in nucleus.Experimentl; At the end of 5 weeks, excising the implanted tumor along the longitudinal diameter, observe the necrosis area and the residual tumor tissue , then measure the longest and shorted diameter of the tumor and necrosis area to calculate the volume size and necrosis ratio of the tumor in five groups. Inspect the liver, lung and other nearby organs carefully to find out intrahepatic and distal metastasis foci, then sum the number together.Experiment: The sample of three groups of control, UFLP and UFLP + ADM were evaluated by immuno - histochemical reaction to illustrate the VRGF/VEGFR expression level and microvessels angiogenesis. Measure the a-mount of VEGFmRNA by RT - PCR in these three groups. The VEGFmRNA and MVD value were analyzed to evaluate the relations with postembolic metastasis.Experiment3: Samples of UFLP, UFLP + ADM, UFLP + As2O3 and UFLP + ADM + As2O3were evaluated by immuno - histochemical staining to illustrate the VRGF/VEGFR expression level and calculate the MVD value. Count the a-mount of apoptosis tumor cells in TUNEL staining. Measure the amount of VEGFmRNA by RT - PCR in these four groups. The VEGFmRNA and MVD value were analyzed to evaluate the relations with postembolic metastasis.ResultsExperimentl: The implanted VX2 tumor grew in round or oval shape in the left or right central lobe. There is no significant difference in the volume size at the end of 2 weeks (p >0. 05). While after the different treatments of the five groups, the tumor growth and metastasis appeared very different(p < 0. 01). Comparing with the control group, the volume size of experimental groups were suppressed obviously ( p <0.01) and larger necrosis area was seen( p <0. 01) ; especially in the 2 As2O3 groups, but there was no significant difference(p >0. 05). In the combination group of ADM and As2O3, there were less intrahepatic and distal metastasis foci comparing with the UFLP or UFLP + ADM group. And the using of As2O3 could reduce the distal metastasis foci apparently comparing with the UFLP group( p <0.01).Experiment2: The amount of VEGFmRNA and MVD value raised apparently in UFLP group comparing with the control and UFLP + ADM group ( p <0. 01). Routine chemoembolization as UFLP + ADM could reduce the VEGFmRNA amount and the MVD value, while there was no effect on the intrahepatic and distal metastasis.Experiment3: In the 2 groups using As2O3, there were more apoptosis appeared, less VEGFmRNA or MVD comparing with the other two without As2O3 (p <0. 01). Comparing with UFLP group, the application of As2O3 could reduce distal metastasis number apparently (p <0.01). The combination using of...
|
PDF Full Text Request