Study On The Efficacy Of Ozonated Saline In The Treatment Of VX2 Tumor And Its Effect On Tumor Infiltrating Lymphocytes

Part ? Study on the efficacy and safety of ozonated saline in the treatment of VX2 tumor of rabbitsObjective: To investigate the efficacy and safety of intratumoral injection of ozonated saline in the treatment of VX2 tumor.Methods:A total of 72 New Zealand white rabbits bearing VX2 tumor on the left hind were randomly divided into four groups with18 rabbits in each group.After anesthesia with 3% pentobarbital sodium sulfate(0.8-1ml/Kg)injected via auricular vein,3ml blood was drawn directly from the heart of rabbit.The longitudinal(a)and transverse diameter(b)on the tumor's maximum section was detected by ultrasound to calculate the tumor volume(V=1/2ab2).Group A(control group)was given saline once on the first day,Group B was given ozonated saline once on the first day,Group C given ozonated saline twice on the first and second days and Group D given ozonated saline three times on the first to third days under ultrasound guidance(injection dosage: two times the tumor volume).The tumor size were detected by ultrasound and3 ml blood were collected from the heart on 4th,8thand 12 thafter the first injection.6rabbits randomly selected from each group were killed through auricular venous injection of 15-20 ml air at each time point,tumors were removed and soaked in 10% formalin and the tumor growth rates were calculated using ratio of tumor size before and post operation,white blood cell count,red blood cell count,platelet count,hemoglobin,serum alanine aminotransferase,aspartate aminotransferase,creatinine and urea were examined.Tumor apoptosis was detected with TUNEL.Results:1.The volume of tumor in each group increased in varying degrees at each time point,but the tumor growth rate in the ozonated saline group(group B,group C,group D)was significantly lower than that in saline group(group A),with lowest in group B(group B vs.group A: P=0.000,0.003 and 0.009 on 4th,8th and 12 th day respectively).There was no statistical significance in the tumor growth rates among the ozonated saline groups(P>0.05);2.There was no statistical significance inthe white blood cell count,red blood cell count,platelet count,hemoglobin,alanine aminotransferase,aspartate aminotransferase,creatinine and urea between each group at each time point(P>0.05);3.There was no statistical difference in the tumor apoptosis between each group at each time point(P>0.05).Conclusion: Intratumoral injection of ozonated saline can significantly slow down the growth rate of tumor,and it seems that single injection of ozonated saline is more effective than multiple injection.Intratumoral injection of ozonated saline has no obvious affect on tumor apoptosis,so its mechanism of slowing down the growth of tumor might not be inducing the cell apoptosis in the tumor.Meanwhile,intratumoral injection of ozonated saline dose not damage the liver and kidney function or cause any change to blood routine,so it is a safe and effective antitumor method.Part ? The Effect of intratumoral injection of ozonated saline on tumor infiltrating lymphocytesObjective: To investigate the effect of ozonated saline directly injected intoVX2 tumor on tumor infiltrating lymphocytes.Methods: Immunohistochemistry was used to detect the infiltration of CD3+T lymphocytes,CD4+T lymphocytes and CD8+T lymphocytes in tumor specimens after treatment with ozonated saline or saline.Image pro plus 6.0 biomedical imaging analysis system was used to detect the corresponding staining intensity(i.e.,integrated optical density),and the value of integrated optical density per unit area was recorded.Results: 1.The infiltration of CD3+T lymphocyte within the tumor of group C and group D was more obvious than that of group A and B at any time points but only showing statistical significance with group B on 4th day(P=0.009 and 0.03).There was no statistical significance among the other groups at any time points;2.On 4thday,the infiltration of CD4+T cells in group C was more than that in group A and B with statistical significance(P < 0.05).The infiltration of CD4+T cells in group D was more than that in group A and B but showing significant difference only with group A(P=0.010).There was no statistical difference among other groups.On 8th day,the infiltration of CD4+T cells in group C was more than that in group A,B and D,but there was significant difference only with group D(P=0.036),and there was no statistical difference among other groups.On 12 th day,the infiltration of CD4+T cells in group D was more than that in group A,B and C,but no statistical difference was found,and the CD4+T lymphocytes in the group D were more than that on 8thday(P=0.012),there was no statistically significant difference among the rest groups;3.The CD8+T lymphocyte infiltration in C group was more than that in groups A,B and D at any time points showing statistical significance with group A on 8thday(P=0.030),and there was statistical significance between group A and group B on 12 th day(P < 0.05).The CD8+T cell infiltration in group D was more than that in A and B groups at any time points but only demonstrated significant difference on12th day(P < 0.05),there was no significant difference among other groups or at other time points.Conclusion: Intratumoral injection of 2 times the tumor volume of ozonated saline does not obviously affect the distribution of tumor infiltrating CD3+T lymphocytes,CD4+T lymphocytes and CD8+ T lymphocytes.Twice and three times injection of ozonated saline on sequential 2~3 days can increase the infiltration of CD3+T lymphocyte,CD4+T lymphocyte and CD8+T lymphocyte within the tumor at some time points,and twice intratumoral injection of ozonated saline revealed relatively more obvious effect.