| 1. BackgroundThe surgical resection is considered to be the most promising first choice therapeutic option for many malignant tumors in human, but, only about 20 percent of the patients with hepatic carcinoma are eligible for surgical resection because of various reasons, such as severe impairment of hepatic functional reserve, extrahepatic metastasis, involvement of the portal vein, severe extrahepatic disease and so on [1-3]. Many patients have to accept other non-surgical treatments. Therefore, some local ablative therapies have been hot spot,for instance, percutaneous ethanol injection (PEI), percutaneous acetic acid injection (PAI), radiofrequency ablation(RFA), cryoablation therapy(CST), microwave coagulation therapy(MCT), interstitial laser photocoagulation (ILP). Among them, RFA has gained the widest acceptance because of its minimal invasion, safety, effectiveness, repeatability in local therapy for primary and secondary liver malignancies [4-8].Radiofrequency ablation (RFA) is a thermal treatment technique that produces localized tumor coagulative necrosis by heating tumor tissue to temperatures exceeding 50℃.High frequency alternating current is passed across the RFA needle electrode arrays deployed directly inside the tumor and is changed heat energy which results in the final destruction of the tumor tissue [8-9]. Although RFA has been widely used to treat various hepatic tumors and has got good effects, Unfortunately, incomplete ablation often are caused, the remnant tumors are produced ,due to the size, number and anatomic distribution of tumor nodules and other factors .According to some reports, the overall complete ablation rate is 50-93% ,and, the complete ablation rate is 77-100%,84-93.5%,41-71% respectively after a single session for the tumor in diameter≤3cm,3-5cm,≥5cm[9-22].The remnant tumor rate may be higher after RFA for the tumor in the special anatomical location. In a study by Pawlik et al [23], the residual tumor was observed in 100% of tumors adjacent to the vena cava, in 57% of the tumors adjacent the portal vein, and in 33% tumors adjacent to the hepatic veins after a single session. The high remnant tumor rate has been one of the limitation factors for RFA developing.In the light of the high remnant tumor rate, more and more researchers have begun the study of remnant tumor characters。Nevertheless, all the study results are different significantly [24-38]. Van Duijnhoven [24] established a secondary liver malignancy model by inoculating the CC531 colon carcinoma tumor cells in rats liver, then, RFA was applied .The result indicated that the outgrowth and metastasis of the residual tumor cells were inhibited. He thought the changes were produced probably because of improving immunopotency. In a study by P. von Breitenbuch [26] et al, a metastasis was established by injection of CT-26 colon carcinoma cells into the right liver lobe of Balb/C mice. Five days later, GFP-transfected CT-26 tumor cells were injected again, and the metastasis was treated by resection or RFA. The result showed RFA significantly promoted the proliferation of neoplastic cells, compared with resection. Some clinical studies have found too that RFA can hasten metastasis of the remnant tumor and promote the proliferation of residual neoplastic cells [26-29]. What can be concluded about the activity of the remnant tumor outgrowth and metastasis after RFA? At present, the answer can not be determined yet. Otherwise, all the study suspected the changes were caused by the microenvironment changes of the whole body and the local organize or the vasa injury, such as improved immunopotency, intratumoral pressure increased, cytokines produced and so on. Whether the RFA can change the proliferation and metastasis activity of the residual tumor cells themselves?2. ObjectivesBased on the animal test, this study is designed to observe the proliferation and metastasis activity of residual hepatic VX2 tumor cells after radiofrequency ablation and study the changes of the proliferation and metastasis activity of tumor cells after RFA3. Methods and Results(1).Establishment and evaluation of the metastasis model of VX2 tumor lines in rabbitsThe VX2 tumor tissue was transplanted into rabbits'spleens. After2,3,4 weeks, 6 rabbits were killed respectively. The volume and weight of the tumor, the tumor pathology information and the tumor metastasis information were observed. Six rabbits with VX2 tumor were food normally and recorded spontaneous survival time. Results :①The occurrence rate of tumor was 100% after being implanted .The volume and weight of the tumor in the spleen was 278.28±55.77mm3,0.31±0.07g respectively on the postoperative week 2.②. Macroscopic observation showed the tumors were round, there was obvious necrosis in the center of the tumor in diameter exceeding 2 cm.Observed with light microscope , the tumor cells were arranged irregularly with invasive growth potential .③Many tumors metastasized could be observed in the liver, lung and enterocoelia lymphonodes on the postoperative week 2-3.④Animals'mean spontaneous survival time was 56±6.07 days. All the animals were died of multiple system organ failure resulted from tumors'growth and metastases.(2). Observation of the metastasis activity of the residual VX2 tumor cells after radio frequency ablationRabbit hepatic tumor models were established with VX2 tumor cell lines by the transplantation .Two weeks later, RFA was applied to the tumor and the remnant tumor was intended to bring about by controlling the temperature of the marginal tumor tissue at 43-45℃level, the shock- temperature, and maintaining the temperature for 30 min [38]. The experimental animals were divided into control group and treatment group. The treatment group (18 animals) and the control group (18animals) were established by transplanting remnant tumors tissues and VX2 tumor tissues non-treated by RFA respectively in the rabbit spleens. The number of the tumor in the liver, the positive index of the E-cad and MT1-MMP by immunohistochemistry staining in the tumor were compared between the two groups by killing 6 animals respectively on postoperative week 2, 3, 4. Six animals with VX2 tumor in each group were food normally and recorded spontaneous survival times, then, the animal spontaneous survival times were compared too. Results:①. Mean tumor number in the liver had no significant differences between the groups on postoperative week 2,3,4.②Mean survival span had no significant differences between the treatment group and the control group (50±13.4days vs,56±6.07days,P=0.40).③The difference of the positive index of the E-cad in tumors was not significant between two groups on postoperative week 2, 3, 4.④. Positive index of the MT1-MMP in tumors was significant higher in the control group than in the treatment group on postoperative week 2 (91.5±5.65%, 82.2±4.79% respectively, p=0.01) and had no significant differences between the groups on postoperative week 3, 4.(3). Observation of the proliferation activity of remnant VX2 tumor after radio frequency ablationThe remnant tumor was got by the same methods as the first test .The experimental animals were divided into control group and treatment group. The treatment group (18 animals) and the control group (18animals) were established by transplanting remnant tumors tissues and VX2 tumor tissues non-treated by RFA respectively in the rabbit spleens. The tumor volume, the tumor weight and the proliferation index (PI)of the tumor cells were compared between the two groups after killing 6 rabbits respectively on postoperative week 2,3,4. Results:①Mean tumor volume and the mean tumor weight were significantly greater in the control group than in treatment group at postoperative 2 week (278.27±55.77mm3 vs 100.31±25.51mm3 P=0.02;0.3049±0.071g vs 0.1191±0.095g P=0.04. )②. Tumor volume and tumor weight had no differences between two groups on postoperative week 3, 4.③PI showed no significant differences between the groups on postoperative week 2, 3, 44. Main Conclusions(1) The metastasis model of VX2 tumor lines established in rabbits has such characteristics as easy reproduction, short period of tumor occurrence and metastasis, high success rate, high stability. It is a perfect metastasis model in vivo established in big animal with normal immune system.(2) The rabbit spleen is a good carrier for the propagation and proliferation. After VX2 tumor is implanted the rabbit spleen. In short time, the model is a perfect spleen tumor.(3) In the fist 4 weeks after treatment,RFA did not change significantly the proliferation and metastasis activity of residual VX2 tumor cells.(4) The period needed for the remnant tumor after RFA getting completely activity is 2-3 weeks. It may be essential and effective to find out remnant tumor and treat it in first two weeks after RFA for improving the therapy effectiveness... |
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