The Effect Of Creatine Phosphate Sodium On Angiogenesis In Mice S₁₈₀ Sarcoma And Its Mechanism

Objective: To explore the effect of creatine phosphate sodium on angiogenesis in mice S180 sarcoma and to study the related mechanism.Methods: Sixty kunming mice were inoculated with S180 sarcoma cell by right armpit to estabilish carcinoma model.The next day ,The model mice were divided into 4 groups: Sodium Chloride control group, low dose CP group, middle dose CP group and high dose CP group , fifteen mice in each group. The four groups were injected drug by intraperitoneal way. Low dose group was injected with CP of 200mg/kg/d, Middle dose CP group was injected with CP of 400mg/kg/d, High dose CP group was injected with CP of 800mg/kg/d, The mice in control group was injected with Sodium Chloride.The four groups were injected for 7 days and once a day. All mice were killed at 10 days later and the tumor tissues were taked and weighed. Then the inhibition rate of tumor growth were calculated. Flow cytometry was used to detect the expression of MMP-2 protein, TIMP-2 protein in the S180 transplanted sarcoma tissue. Immunohistochemistry (IHC) image analysis was performed for detecting vascular enthothelial growth factor,tissue inhibitors of metalloproteinases and microvessel density. Semiquantitative RT-PCR was used to detect the expression of VEGF mRNA and bFGF mRNA in the transplanted tumor.Results: 1Tumor weight and inhibition rate: The tumor weight of control group was (3.65±0.22)g , the tumor weight of low dose CP groups was (3.48±0.20)g, the inhibition rate was 4.66%; The tumor weight of middle dose CP group was (3.43±0.23)g,the inhibition rate was 6.03%;The tumor weight of high dose CP group was (2.97±0.10)g ,the inhibition rate was 18.63%; It was no statistical significance of the monitoring singnal among control group,CP low dose group and CP middle dose group(P>0.05).High dose group was compared with control group, CP low dose group and CP middle dose group, The weight of transplanted tumor was low, the difference has statistical significane(P<0.01).2 Flow cytometry was used to detect the expression of TIMP-2 protein, MMP-2 protein in the transplanted tumor: the expression level of TIMP-2 protein in control group, low dose CP groups ,middle dose CP groups and high dose groups was 276.59±7.96,284.45±3.76,286.32±10.41,308.90±14.50. the expression level of MMP-2 protein in control group, low dose CP groups ,middle dose CP groups and high dose groups was 386.47±2.25,382.41±5.08,378.52±8.89,323.10±9.27;It was no statistical significance among control group, CP low dose group and CP middle dose group(P>0.05). There was statistical significance of the control group,low and middle dose CP group comparing with high dose CP group (P<0.01).3 Immunohistochemistry image analysis was performed for detecting the VEGF and bFGF protein and microvasvular density: The expression level of VEGF protein in control group, low dose CP group ,middle dose CP group and high dose group was 4.67±0.48,4.47±0.64,4.33±0.72,3.07±0.59. The expression level of bFGF protein in control group, low dose CP group ,middle dose CP group and high dose group was 4.00±0.76,3.80±0.77,3.60±0.74,3.00±0.53;the value of MVD control group, low dose CP group ,middle dose CP group and high dose group was 15.20±1.08,14.80±0.94,14.53±0.92,11.73±1.38;It was no statistical significance among control group, CP low dose group and CP middle dose group(P>0.05). There was statistical significance of the control group,low and middle dose CP group comparing to high dose CP group(P<0.05). 4 The expression level of VEGF and bFGF mRNA in tumor tissue were detected by reverse polymerase chain reaction.the expression level of VEGF mRNA in control group, low dose CP group ,middle dose CP group and high dose group was 0.1602±0.005,0.1556±0.0038,0.1548±0.0041,0.1393±0.0029. The expression level of bFGF mRNA in control group, low dose CP group ,middle dose CP group and high dose group was 0.1602±0.0059,0.1556±0.0038,0.1548±0.0041,0.1393±0.0029. It was no statistical significance among control group,CP low dose group and CP middle dose group(P>0.05).. There was statistical significance of the control group,low and middle dose CP group comparing to high dose CP group(P<0.01).Conclusions: 1 High dose CP can inhibit the proliferation of S180 sarcoma in mice. Low,middle dose CP is no effect on the proliferation of S180 sarcoma in mice.2 High dose CP can reduce the expression of MMP-2 protein and promote the expression of TIMP-2 protein. Low,middle dose CP is no effect on the expression of the MMP-2 and TIMP-2 protein.3 High dose CP can reduce the expression of the VEGF mRNA,bFGF mRNA and protein. Low,middle dose CP is no effect on the expression of the VEGF mRNA,bFGF mRNA and protein.4 High dose CP can inhibit the angiogenesis of S180 sarcoma in mice. Low,middle dose CP is no effect on the angiogenesis of S180 sarcoma in mice.5 Our research proved that CP could not promote the angiogenesis of S180 sarcoma in mice. This provides a theoretic reference for the application of CP on the myocardial protection in chemotherapy.