| BackgroundLiver cancer is one of the most common malignant tumors and has rank in thirdin our country. Operation is the most effective treatment liver cancer in early stage.Due to its occult onset, the late visit to doctors combined with cirrhosis leading to badliver function, the rate of the surgical operation is only 20%-30%. For unsecetablepatients with medium and latter stage liver cancer, microinvasive treatment still canget the good curative effect. Therefore, microinvsive therapy has become anindispensable and important method in the treatments of the later period liver cancer.Frequently the microinvasive treatment to the liver cancer have two main type: one isangio-microinvasive treatment, including the transcatheter arterial infusion(TAI),transcatheter artery embolization(TAE) and transcatheter arterialchemoembolization(TACE);Another is none angio-microinvasive treatment,including percutaneous chemo-ablation and percutaneous physico-ablation, theformer includes percutaneous alcohol injection (PEI) and percutaneous acetic acidinjection (PAI), and latter includes the cryocareTM cryoablation, radiofrequencyablation (RFA), microwave coagulation therapy (MCT), laser mesenchymalthermotherapy and high intensity focused ultrasound. Among them, cryocareTM cryoablation, radio frequency ablation and microwave thermotherapy are wiedlyapplied in the clinical. The three kinds of microinvasive therapy belong topercutenousphysico-ablation techniques, which can destroy or kill tumor cell by cold or hoteffect and attain" destruction in one time "therapeutic outcome." destruction in onetime " is defined that technique can destroy and kill tumor cell in the treatment scopein one time. Therefore, using these three kinds of microinvasive therapy in thetreatment of the liver cancer has strong comparability in its clinical curative effect.But for the three kinds of microinvasive therapy, which is the most excellent inclinical curative effect is not clear, and currently there is no the related experimentcontrast or the clinical contrast research in domestic and intemational. The model ofrabbit VX2 liver cancer which was established on the big animal is suitable for theexperimental study of liver cancer microinvasive treatment. VX2 tumor cell strain isoriginated from the squamous carcinoma that is derived from the rabbit's papillomainduceing by Shope virus. Through seventy-two times transplantion and passage,VX2 tumor cells formally produce strain. At present, it is one of the best ideal livercancer experiment models. It is widespread applied to empirical study such as livercancer oncology, tumor therapeutics,radiobiology, pharmacokinetics of anticancerdrugs and tumor imageology. The purpose of this study is comparing respectivelygood and bad therapeutic efficacy by observing the size of tumor ablation target area,tumor cell necrosis and residue, tumor metastasis, and the change of immunologicalfunction as well as rabbit's life span through putting the VX2 liver cancer model ofrabbit into group with above-mentioned three kinds of microinvasive treatmenttechnique. To explore the mechanisms and clinical therapy of three micrioinvasivetechniques and provide theoretical and expermental bases for properly choosing themin liver cancer treatment in clinical.Chapter one The establishment of rabbitVN2 liver cancer modelObjectivesIt has been proofed that microinvasive treatment is an important part in middle and advanced stage of the liver cancer, it is the first choice of nonoperative treatmentto the liver cancer. But now it still exits many problems, which include furtherimprovement of blood-supply theories, the prostecdtive efficacy and recurrence ofmicroinvasive treatment, the relationship between microinvasive treatment andapoptosis and drug resistant and so on. Moreover nonvascular treatment of eachsection remains to be studied such as the refrigeration, radio frequency, microwaveetc. A stable bigger animal model needs to be established. The ideal liver cancermodel should be the same or similar to liver's biology behavior of human beings,which has the high achievement ratio of the model, lowmortality, good repeatability,convenient and animal size or figure suitable for the research of the etiopathogenisis,diagnosis and treatment. The method must be harmless or little harm to human bodyand little pollute the environment. The former tumor model establishes mostly in therat,due to it's body is small and blood vessel is thin, the tumor is small, this methodis not suitable for using as the treatment research of.the interventional therapy of thetumor. The other animal is few used, such as pig and dog, because the lure livercancer of these two kinds of animals is different, the individual is difficult to compare;the achievement ratio of transplanting the liver cancer is low because of rejection andhard to live. But the figure of rabbit is bigger than rat, which is suitable for livercancer microinvasive treatment experimental research. The rabbit VX2 liver cancermodel is suitable for the experimental study of liver cancer microinvasive treatment.Strain of VX2 tumor cell is originated form the squamous carcinoma that is derivedfrom the rabbit's papilloma inducing by Shope virus. Through seventy-two timestransplantion and passage, VX2 tumor cells formally produce strain.The model ofrabbit transplantation VX2 liver cancer has many advantages: (1)The model is thebiggest liver cancer model in china at present; (2)It has been demonstrated that tumorisrich in vascular, whose blood is provided by liver artery. The tumor is the similar to human primary hepatic carcinoma; (3) In pathology the tumor is similar to hugetype cancer of liver with huge lump solid tumor, infiltrate growth and plentiful bloodprovides; (4) Generally, the time needs three to four weeks for the rabbit VX2 livercancer diameter grows to 3cm. So the rabbit VX2 liver cancer model has been one ofthe most ideal liver cancer experimental models. It is widely applied to empiricalstudy such as liver cancer oncology, tumor therapeutics, radiobiology,pharmacokinetics of anticancer drugs and tumor imageology. The rabbit liver VX2cancer model in this study was established via coeliotomy-inoculation method.MethodsLeft hepatic lobe is usually choosed for plantting cell line in rabbit VX2 livercancer model.The methods include :(1) cut the belly open to embed; (2) cut the bellyopen to puncture; (3) direct puncture per cutem; (4) cell suspension innoculation. 80New Zealand big and white rabbits were established the model of liver tumors by cutthe belly open to puncture.This study ensured the inoculated location and the depthaccurate as far as possible, to increase the experiment comparability throughestablishing the rabbit liver VX2 cancer model by the coeliotomy. This method wassimple, economic, high achievement ratio and accurate inoculation location, whichwas good for playing the microinvasive target ablation technique through planting thetumor to 1～3cm in 2～3 weeks.Results76 among 80 New Zealand rabbit models were built successfully and confirmedby CT and pathological examination .Inoculation rate was 95% in line with literaturereport.ConclusionsThe rabbit VX2 liver cancer model is a ideal model for microinvasive treatmentstudy, whose inoculation method is simple, economic, high achievement and accurate inoculation location through planting the tumor to 1～3cm with 2～3 weeks.Chapter Two Comparison of ablation effect cryocareTMcryoablation and radiofrequency ablation, microwavecoagulation therapy in rabbit with VX2 liver cancerObjectivesThere are two kinds of microinvasive treatment in the clinical. One is an angio-microinvasive treatment, including the transcatheter arterial infusion chemotherapy(TAI), transcatheter arterial embolzatization(TAE) and transcatheter hepatic arterialchemoembolization(TACE). Another is non-angio-microinvasive treatment,including percutaneous chemo-ablation and percutaneous physico-ablation, theformer includes percutaneous alcohol injection (PEI) and percutaneous acetic acidinjection (PAl), the latter includes the cryocareTM cryoablation, radio frequencyablation (RFA), the microwave coagulation therapy (MCT),laser mesenchymalthermotherapy and high intensity focused ultrasound. Three kinds of the mostcommon microinvasive treatments in liver cancer include the cryocareTMcryoablation,radio frequency ablation (RFA), the microwave coagulation therapy(MCT). They kill the tumor cell through cold or hot effect. The main biology curativeeffect of the cryocareTM cryoablation treatment is the refrigeration that argongenerates. But the hot effective that argon created effects as well, because in a shorttime refrigeration and heat can create the ice crystal inside and outside the cell,damaging the tumor cell thoroughly. The mechanism by which cancer cell death thatdue to the super low temperature refrigeration is : 1. the cell dehydration andcrenulation; 2. the cell electrolyte toxicity condense and PH value change; 3.degeneration in the cell membrane lipoprotein composition; 4. mechanical damage of the formation and ice crystal in cells; 5. the stasis of blood stream andmicro-thrombosis; 6. the immunity response. Radio frequency ablation (RFA) andmicrowave coagulation therapy (MCT) belongs to scope of the hot ablation, whichkill the cancer cell using super heat, and the mechanisms of action are : 1.the tumororganizes congestion and hypoxia; 2. PH of the tumor reduces,the acidityincreases,lysosome increases,lysosome enzymelization; 3. the DNA and RNA proteinsynthesis of lump cell suffers from repression; 4. the organism immune reactionstrengthen .In the progress of using the three kinds of physico-ablation mircoinvasive,the size of the target area and the ability to kill the tumor cancer affect the therapeuticefficacy directly. The purpose of this experiment was to observe and compare thesize of ablation target area, cell death and residual situation via cryocareTMcryoablation, radio frequency ablation and microwave coagulation therapy to meltthe rabbit VX2 liver tumor.MethodsThe model of liver tumors were established in 27 New Zealand big and whiterabbits (inoculated VX2 cell stub empresses successfully 3 weeks ago).They wererandomizedly divided into 3 groups with 9 rabbits in each group, after using spiral CTto measure the perpendicular diameter and transverse diameter. CryocareTMcryoablation was A, the group of radio frequency ablation was B, microwavecoagulation was C. The rabbits were fixed on the operation table which wereanesthetized generally by 3% Pentobarbital (1mg/Kg). The rabbits were cut the bellywith 2-3cm long cut through the abdomen median incision, then the tumor weremeasured through exposing the liver and extruding left hepatic lobe outside withtweezers. After that, the tumors were melted individually with the cryocareTMcryoablation system of United States Endocare company, HGCF-3000 cool-tip radiofrequency ablation treatment machine of HeJia medical treatment equipments company in Zhuhai and MTC-3C microwave coagulation treatment appearance ofNanjing Qinghai mircowave electron institute. The each melt time is 1/3 the clinicaltreatment time patient with liver cancer. All the rabbits were killed after 3 days, thetumors were taken and fixed with Formaldehyde, then send for pathologicexaminination. All data was analyzed by SPSS software. Quantitative data wasexpressed by x±s, before statistics the test of homogeneity of variance were applied,ANOVA and S-N-Ktest or Welch and Games-Howell test in the comparison of threegroups were used. P＜0.05 were defined as statistical significance.Results1. The traverse diameter of ablation target area:that is 2.28+0.12cm,1.96±0.09cm and 1.93±0.10 cm in group A,BandCrespectively. There was significantdifference in the comparison of the three groups (F=31.138,P=0.000)and between Aand B,A and C(P=0.000). There was no significant difference between B and C(P=0.653).2. The average area in the ablation target area:that of A was 5.79±1.34 cm2, Bwas 4.40±0.43 cm2, C was 4.31±0.59 cm2. The differences between Aand B,A and C(P=0.003,P=0.002 )and differences among groups(F=8.047,P=0.002) are significant.No significant difference was found between B and C (P=0.836).3. The rate of tumor complete ablation in Awas 88.9 %, 44.44 % in B, 33.44 %in C.Significant differences were seen in the comparisons of 3 groups(x2=6.300,P=0.043) and between A and B,A and C(P＜0.05).There was no significantdifference between B and C (P＞0.05).4. The tumor cell residual rate were 22.22 %, 77.78%,77.78% in A, B and Crespectively. There was significant difference in the comparison of the 3groups(x2=7.670,P=0.022); There was significant difference in the comparison of thetumor cell residual rate between A and B, A and C. But there was no significant difference between B and C (P＞0.05).5. The complete necrosis rate in A (66.67%) is higher than that of B and C(50.00%).ConclusionsIn the three kinds of microinvasive treatment, cryocareTM cryoablation wasbetter than RFA and MCT, no matter in the ablation target area and transversediameter, the rate of complete tumor ablation, or in the residual tumor rate. Howeverthe RFA equaled with the result of MCT. The study showed the effect of cryocareTMcryoablation might be better than RFA and MCT in curing the rabbit VX2 liver cancer.Chapter Three Comparison of therapeutic effect ofcryocareTM cryoablation and radiorequency Ablation,microwave coagulation therapy in rabbits with VX2 LivercancerObjectivesLiver cancer is one of the most common malignant tumors and has rank in thirdin our country. Operation is the most effective treatment liver cancer in early stage.Due to its occult onset, the late visit to doctors combined with cirrhosis leading to badliver function, the rate of the surgical operation is only 20%～30%. For unsecetablepatients with medium and latter stage liver cancer, microinvasive treatment still canget the good curative effect. Therefore, microinvsive therapy has become anindispensable and important method in the treatments of the later period liver cancer.Frequently the microinvasive treatment to the liver cancer have two main type: one isangio-microinvasive treatment, including the transcatheter arterial infusion(TAI),transcatheter artery embolization(TAE) and transcatheter arterialchemoembolization(TACE);Another is none angio-microinvasive treatment,including percutaneous chemo-ablation and percutaneous physico-ablation, the former includes percutaneous alcohol injection (PEI) and percutaneous acetic acidinjection (PAI), and latter includes the cryocareTM cryoablation, radiofrequencyablation (RFA), microwave coagulation therapy (MCT), laser mesenchymalthermotherapy and high intensity focused ultrasound. Among them, cryocareTMcryoablation,radio frequency ablation and microwave thermotherapy are wiedlyapplied in the clinical. The three kinds of microinvasive therapy belong topercutenousphysico-ablation techniques, which can destroy or kill tumor cell by cold or hoteffect and attain" destruction in one time "therapeutic outcome." destruction in onetime" is defined that technique can destroy and kill tumor cell in the treatment scopein one time. Therefore, using these three kinds of microinvasive therapy in thetreatment of the liver cancer has strong comparability in its clinicai curative effect.Three kinds of microinvasive therapy, which is the most excellent in clinicalcurative effect is not clear, and currently there is no the related experiment contrast orclinical contrast research in domestic and worldwide. In this experiment we createdindependently the rabbits VX2 liver cancer with three kinds of microinvasive therapy,cryocareTM cryoablation,radio frequency ablation,comparing with operational groupand the control group, then we told the result good from bad by the observationaltumor remained, tumor metabasis, the immunity function variety and the rabbitexisting period. We approached the function mechanism and clinical curative effectsof these three kinds of microinvasive therapies. In this way we can provide thetheories experiment accordance for the accuracy chance of the three kinds ofmicroinvasive therapies in the clinic treatment of the liver cancer.Methods45 New Zealand big and white rabbit( inoculated VX2 cell lines successfully 3weeks ago) were randomly divided into five groups (each group contains 9), namelythe cryocareTM cryoablation group(A group) radio frequency ablation group (B group),microwave coagulation group (C group), surgical operation group (D group) and thematched group (E group),through using the spiral strengthened CT to scan check thetumor size and the transfusion in liver,lung and in the lymphoid node of the belly cavity. The rabbits were fixed on the operation table which were anesthetizedgenerally by 3% Pentobarbital (1mg/Kg). The rabbits were resected from the bellywith 2-3cm long cut through the abdomen median incision, then the tumor weremeasured through exposing the liver and extruding left hepatic lobe outside withtweezers. After that, the tumors were melted respectively with the EndocareCryocareTM Surgical System of United States Endocare company, HGCF-3000cool-tip radio frequency ablation treatment machine of HeJia medical treatmentequipments company in Zhuhai and MTC-3C microwave coagulation treatmentmachine of Nanjing Qinghai mircowave electron institute. The each melt time is 1/3the clinical treatment time in patient with liver cancer. Used the surgical operation tocuts off the tumor of the D group, and the incisal edge exceeded tumor edge 1.0 cm.Opened the stomach of the E group to look into it but did not interfere. Aftercompleting the five groups with different methods, retumed the left leaf liver into thebelly cavity, and sutured stomach. The rabbits were intramuscularly injected 20,000ugentamicin in left back legs, then send them back to the animal building for feeding.Observed them one. Made autopsy in each died rabbit to observed the remainedtumor and tumors transfusion, including the liver inside transfusion, the lungtransfusion, the implantation and the metastasis in lymphoid node in belly cavityand so on. All rabbits were taken the blood before inoculated, before treatment andtreated 10 days later, then used the ELASA bi-anti-sandwich method to examinesIL-2R and ALT. All the data was analyzed by SPSS software. Quantitative data wasexpressed by x±s, before statistics the test of homogeneity of variance were applied;ANOVA and S-N-K test in the comparison of three groups were applied. P＜0.05 weredefined as statistical significance.Results1. The tumor remained and metabasis: (1) The tumor remained inside the liver:2, 4, 5, 0 and 9 had tumor remainedinside the liver from the group A, B, C, D, E respectively. The residual rate was groupE＞group C＞group B＞group A＞group D.There was significant difference in thecomparison of the five groups (x2=20.700,P=0.000).(2) The metabasis in liver: 1, 3, 4, 6 and 9 had metabasis in liver whose rate wasgroup E＞group D＞group C＞group B＞group A.There was significant difference in thecomparison of the five groups(x2=15.652,P=0.004).(3) The metabasis in the lung and lymphonode of abdominal cavity:There were9 found metabasis in the lung and lymphonode of abdominal cavity in each group.(4) The carcinomatosis in abdominal cavity:The 2, 5, 6, 0 and 1 had thecarcinomatosis in abdominal cavity whose rate was group C＞group B＞groupA＞group E＞group D. There was significant difference in the comparison of the fivegroups (x2=13.894,P=0.008). The high carcinomatosis rate of group B and C wererelated to the "boiling effect",that is during RFA and MCT treatment, boilinginterstitial fluid (and possibly viable tumor cells) emanated from the probe path intothe peritoneal cavity.The above results demonstrated: From the remained tumor and the metabasiswe concluded that the surgical operation is the most valid method, the next is thecryocareTM cryoablation, the last are the RFA and MCT in the treatment of rabbit VX2liver cancer.2. The immunity change: There was no significant difference in sIL-2R among thosegroups before treatment, it had a descent in the cryocareTM cryoablation group, noobvious variety in the RFA and MCT group, a raise in the surgical operation group,and it showed significant advance in the matched control group, sIL-2R can reflectthe immunity change indirectly. Although above four kinds of treatment methodscouldn't completely converse the anti- tumor immunity of the cancer rabbits, prevented the anti-tumor immunity from continuously descending, such as thematched control group and promoted the normal immunologic function partiallyrecovery. In the function of promoting immunity, the cryocareTM cryoablation groupwas the best, the next was the RFA and MCT group, the surgical operation group wasthe third.3. The change of the live function:There was no significant difference in the ALT .SocryocareTM cryoablation, RFA, MCT and surgical operation had no significanteffect on the live function of the rabbits.4. The mean survival time:That of the cryocareTM cryoablation group, the RFA andMCT group and the surgical operation was notably higher that of matched controlgroup. There was significant difference in the comparison of the fivegroups(F=73.084,P=0.000). The CryocareTM cryoablation group and the surgicaloperation group were higher than the RFA and MCT group; There was no significantdifference in the mean survival time between the cryocareTM cryoablation and thesurgical operation group, this result can be seen on the RFA and MCT group.ConclusionsIn the treatment of the rabbit VX2 liver cancer, the effect of the cryocareTMcryoablation was superior than the RFA and MCT in the aspects of reducing tumorremains and metabasis, strengthening the immunity and prolonging the mean survivaltime. However the RFA equaled with the effect of MCT. During RFA and MCTtreatment, because occurrence of "boiling effect", the carcinomatosis in abdominalcavity is produceed easily. This result deserves to cause the clinical concerns.
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