Experimental Studies Of Induced Differentiation Interventional Treatment For Rats Transplanted Hepatocarcinoma

Part one Experimental study in Vivo Test 1 Experimental studies of all trans-retinoitc acid treatment for Walker-256 hepatocarcinoma cell in cell level.objective To study the role of differentiation and apoptosis of Walker-256 hepatocarcinoma cell induced by ATRA ,as well as to explore its mechanism. Method: treated Walker-256 hepatocarcinoma cell with ATRA for 48h, observe the morphologic change of cells by microscope ; the multiplication restrain effect of ATRA on Walker-256 cells was determined by the MTT assay,translocated phosphatidylserine was labeled with PI, the apoptosis incidence was measured by FCM; The changes of fas mRNA and bcl-2 mRNA were determined by RT-PCR;The changes of the expression ofCaspase3,Caspase8 proteins were determined by western blotting. Results Morphological observation revealed the feature of differentiation and apoptosis of Walker-256 hepatocarcinoma cell induced by ATRA with the incease of ATRA dose. ATRA inhibited the growth of hepatocarcinoma cell significantly and the rate of growth lies on the does and treated time. A apoptosis peak of sub-duplation body before G1 phase was measured by FCM and the percentage of the apoptosis cell inceased with the incease of the dose of ATRA. The study revealed that the expression of fas was up-regulated and bcl-2 was down- regulated when treated with ATRA. Conclusion ATRA have the effect of inducing Walker-256 hepatocarcinoma cell differentiation and apoptosis. Part 2 Experimental study in Vitro Test 1 Establishing rat hepatocarcinoma Walker-256 modelobjective To investigate the feasibility of implanted hepatocarcinoma from walker- 256 hepatocarcinoma cell acting as hepatocarcinoma model and study the method of establishing rat Walker-256 hepatoma model,imaging examination and the influence of tumor size on the effect of transarterial chemoembolization。Materials and methods Twenty Wistar hepatocarcinoma rats model were established by walker-256 tumor implantation。The tumor volume (V1) was measured by magnetic resonance imaging 11 days after implantation, then TACE was performed after laparotomy and retrograde placement of catheter into the gastroduodenal artery。The tumor volume (V2) was measured by MRI 14 days after operation。Observe the variation of the tumor volume。Some model rats were performed CT scan before TACE and after 14 days of TACE,DSA during TACE。Results Successful implantation was obtained in all 8 rats,the mean tumor size of pre-operation was 87.3128±7.0225 mm3,post-operation 14 days was 608.9831±108.8147 mm3,unequal variances were assumed after test for equality of variances。Conclusion Rat Walker-256 hepatoma model is suitable for interventional experiment study on hepatocarcinoma。Conventional TACE can inhibit the growth of hepatocarcinoma.Test 2 Treatment effect of ATRAcombined with transarterial chemoembolization forWalker-256 transplanted hepatocarcinoma in wistar ratsobjective To observe the inhibiting effect of ATRA combined with transarterial chemoembolization on rat Walker-256 transplanted hepatocarcinoma。Materials and methods 27 Wistar rats hepatoma model were established by walker-256 tumor implantation。The tumor volume (V1) was measured by magnetic resonance imaging 10 days after implantation, then the rats were splited randomizedly into three group。The therapy scheme performed as below:A group ATRA0.1mg+MMC0.05mg + mitomycin 0.05mg+ultrofluid lipiodol 0.05ml+0.025mg gelfoam spong; B group mitomycin 0.05mg+ultrofluid lipiodol 0.05ml+0.025mg gelfoam spong; C group 0.9%NaCl 0.2ml. After another 11days MRI were performed once again to determine the tumor volume. Tumors tissue sections stain of factorⅧand Ki67 were performed by immunohistochemical method, HE stain were performed for detecting microscopic metastasis foci of paracancer livers tissue. Significant difference existed in growth rates among 3 groups (p<0.01) and between either groups(p<0.05). Significant difference existed in surface metastasis foci of livers among 3 groups (p<0.01), but not between B and C (p>0.05); Results Average tumor volume of the three group rat before operation were 88.26±3.47mm3,87.91±5.63 mm3,87.08±6.51 mm3 ,while after operation, average tumor volume of the three group were 389.77±54.13mm3,609.05±106.91 mm3,1116.64±120.96 mm3,Significant difference existed in growth rates among 3 groups (p<0.01) and between either groups(p<0.05); Significant difference existed in surface metastasis foci of livers among 3 groups (p<0.01), but not between B and C (p>0.05). Significant difference existed in microscopic metastasis foci of paracancer livers tissue among 3 groups (p<0.01), but not between B and C (p>0.05). Significant difference existed in Ki67 expression among 3 groups (p<0.01) and between either groups (p<0.05). Significant difference existed in MVD among 3 groups (p<0.01), but not between B and C (p>0.05) significant difference existed in microscopic metastasis foci of paracancer livers tissue among 3 groups (p<0.01), but not between B and C (p>0.05). Significant difference existed in Ki67 expression among 3 groups (p<0.01) and between either groups (p<0.05). Significant difference existed in MVD among 3 groups (p<0.01), but not between B and C (p>0.05). Conclusion ATRA have the effect of anti-angiogenesis and inhibiting growth and metastasis as well as inducing differentiation and apoptosis on Walker-256 hepatocarcinoma.