The Experimental Study Of Cryotherapy In Combination With Differentiation-inducing Agent Against C6 Rat Intracerebral Glioma

Objective To determine the synergistic differentiation-inducing effects of cry-otherapy in combination with all-trans retinoic acid against C6 rat intracerebral glioma and to elucidate its changes of biologic characters and potential molecular mechanism for the purpose of making the basis for further clinical trials.Methods 1 C6 cell culture:The C6 rat glioma cell line used in this study was maintained in DMEM supplemented with 10% newborn calf serum,100u/ml Penicillin and 100μg/ml streptomycine under the condition of 5% Carbon dioxide and 37℃. 2 model of Wistar rat cerebral glioma was established:Rats were anesthetized with 10% chloral hydrate at a dose of 3ml/kg.With the help of a stereotactic frame and Hamilton syringe, 125 male wistar rats,200g-250g,were respectively injected C6 glioma cells suspension containing 1 × 10⁶/10μl cells in the exponential phase of growth into their cortex S₁ area in the right.When the tumor was 7 days old,5 rats were used to evaluate the established models by MRI and HE stain.3 grouping and performace of treatment: 120 tumor-bearing rats were averagly and randomly divided into four groups, when the tumor diameter was 4mm approximately on the seventh day after implantation.(1)the control group:one milliliter solution containting 10% ethyl alcohol and 90% PBS were delivered via syringe injection into the rat peritoneal cavity each day for two weeks.(2)the cryotherapy group: A 2mm diameter cryoprobe cooled was applied to the core of the exposed tumor. When the iceball was reached the rim of tumor, then allowed to thaw naturally.freeze thawing was operated for the twice.(3) the ATRA treatment group: ATRA was diluted in 10% ethyl alcohol and 90% PBS to yield a 1mg/ml suspension,One milliliter doses were delivered via syringe injection into the rat peritoneal cavity each day for two weeks.(4)the combination treatment group:ATRA was applied to rats after cryotherapy.20 rats of each group were randomly drawed to sacrifice for different study purpose on the 22nd day following inoculation,The rest of each group were used for MRI scaning and the observation of lifetime.4 The expressionof different antigen P27> GFAP were detected by immunohistochemical staining. 5 The different phases of cell cycle of tumour cell proliferation were analyzed by flow cytometry. 6 The tumour size was measured by MRI. 7 The rat survival time of each group was directly observed.Results 1 The expression levels of P27% GFAP protein were increased in thetreatment groups as compared with that of the control group,especially in thecombination treatment group.2 In the treatment groups,particularly in the combinationtreatment group,ratio of Gi phase was increased and S phase was decreased.Gi phasearrest was brought about.3 The tumour size was reduced and the rat survival time wasprolonged in the treatment groups ,extraordinarily in the combination treatment group,in contrast with that of the control group.4 There was a positive correlation between theexpression of P27 protein and the expression of GFAP protein, ratio of Gi phase, lifespan;There was a negative correlation between the expression of P27 protein andtumour size.Conclusions the combined use of cryotherapy and ATRA can induce differentiation in the C6 rat brain glioma more significantly than that of them alone,its mechanism is concerned with the upregulation of P27 protein.