| Objective: Cervical cancer is one of the most common gynecological malignancy. The morbidity of cervical cancer in developing country showed a trend of getting younger in recent years. The treatments such as surgery, radiotherapy and chemotherapy could improve the prognosis of patients at the early stage and it was unsatisfactory for recurrent and metastatic cancer which was the chief causes of the death of patients. The invasive surgery may be less willing for people and after long-term of chemotherapy, toxic effect could be accumulated and drug-resistance could be formed. Therefore it was topic to find a new anticancer durg with high-effencient, nontoxic and safty.It had been proved that Artesunate had anti-tumor effects on central nervous system tumors, lung cancer, colon cancer, leukemia, melanoma, renal cell carcinoma. And the side-effect was fewer. Vascular endothelial growth factor(VEGF) and cyclooxygenase-2(COX-2) had a close relations-hip with angiogenesis, which was closely linked to the occurrence and develo-pment of tumor. To explore the expressions of VEGF and COX-2 and the relation between MVD,VEGF and COX-2 by the effect of artesunate on cervical transplanted tumor, and analysed the changes in different time. To explore the anti-tumor effect of artesunate by restrained angiogenesis.Methods:1 Establishment of animal model and experimental groups:Mouse uterine cervical cancer cell line U14 on logarithmic phase was collected and inoculate into mice abdominal cavity by the concentration of 1×10~7/ml. Drawn and centrifuged the ascites after 5 days when the ascites was formation,Diluted to 5×10~7/ml and inoculated subcutaneously to right lower limb of the experienmental animal, KM mice, 5-6week-year-old, 15-17 g, femal, 0.2ml each mice. It was time to go on the further study when maximum length of tumor reach to 5mm. The model group animals were randomly divided into 4 groups,without any tumor injected as blank control group(Normol), tumor model was established, mice which were injected saline as tumor Control group(Control),and ART as ART group,and injected cisplatin into the abdominal cavity as positive control group(DDP),and all of cured was lasted to 7 days. Each group was randomly divided into 4 groups, the mice were sacrificed in 10,20,30 and 40 days. Each groups had 20 mice.2 Analysis of expression of VEGF and COX-2 and the count of MVD on tumor issue.After mice in each group was killed, tumor was prepared for immunohistochemical staining and image analysis. The gray scale(GS) was measured under imagine analysis system to show the expression of VEGF and COX-2, and the negative relationship between value of GS and intensity of expression, and the percentages of positive cells were calculated under light microscope with rectangular track.The positively stained cells of VEGF are tan, and the method of weidner was used to the quantitative analysis of MVD. The slides were examined under 100× magnication to identify the highest vascular density area within the tumor, and six areas of highest MVD were selected for counting. The average of the six areas was recorded as the MVD level of this case. Any brown-staining endothelial cell or endothelial cell cluster that was clearly separate from adjacent microvessels, tumor cells, and other connective tissue elements was considered as a single countable microvessel. Even those distinct clusters of stained endothelial cells which might be from the same vessel snaking its way in and out of the section were considered distinct and countable as separate microvessels. Vessel lumina did not have to be present. Macrovessels, characterized by thick muscular walls or with lumina greater than eight red blood cells in diameter were excluded from the count.3 Satistical analysis: SPSS13.0 software was used. after analysis of normality and variance. Meet the normal distribution, One-way ANOVA was used to analyze the expressions of VEGF and COX-2 and the count of MVD in tumor, and K-independent samples rank-sum test was used otherwise. SNK was used in compareing every two data.Results: 1 The count of MVD on transplanted tumorCompared with Tumor control group in count of MVD, DDP groups and ART groups were significant decline(P<0.05). The total trend of MVD in transplanted tumor was Tumor control group>ART group>DDP group. Besides in 20 days,ART gourp was significantly higher than DDP group(21.23±2.71 v.s16.69±2.44, P<0.05). The count of MVD in the total trend of 10d<20d<30d<40d, and in Tumor Control group, there was no significant difference between 10 d and 20 d(20.59±2.52 v.s22.72±2.97, P>0.05). There was no significant difference in 20 d and 30 d in ART group(21.23±2.71 v.s20.30±5.06, P>0.05). The difference between other groups is striking. 2 The expression of VEGF and COX-2 on transplanted tumor 2.1 The expression of VEGF on transplanted tumorCompared with the percentage of positive cells of VEGF on transplant tumor in 10 days in Control group(20.58%±3.28%), the DDP group decreased significantly(14.89%±2.70%, P<0.05), ART group had no obvious change(17.95%±2.02%, P>0.05). In 20,30,and 40 days, the trend of percentage of positive cells of VEGF was Control group>ART group>DDP group. Besides in 30 day, ART group and DDP group have no obvious difference(38.54%±7.10%v.s45.76%±11.83%, P>0.05). The difference between others group were striking(P<0.05). The percentage of positive cells of VEGF in ART, DDP and Tumor control group in trend as 10d<20d<30d<40d. Besides in 10 days and 20 days there was no obvious change(16.92%±6.11%v.s19.32%±6.03%, P>0.05). The differences between others groups of were striking(P<0.05).In 10 and 40 day, the GS of VEGF on transplant tumor had no obvious change(P>0.05). In 20,30 days, compared with Tumor control group the GS of VEGF(124.55±1.44,121.67±5.39), ART group was significant decreased(118.14±5.33, 111.24±6.42, P<0.05), and DDP group was significant incrdased in 20 days(132.98±3.49, P<0.05),and slightly decreased in 30 days(120.40±3.89, P<0.05). 2.2 The expression of COX-2 on transplanted tumorCompared with the percentage of positive cells of COX-2 on transplant tumor in 10 days in Tumor control group(34.10%±6.79%), DDP group was decreased significantly(16.92%±6.11%, P<0.05), and ART group had no significant change(30.06%±5.28%, P>0.05). In 20, 30 and 40 days, the overall trend of positive cells percentage of COX-2 on the transplant tumor was Tumor Control group>ART group>DDP group, and the DDP group and ART group had no obvious change(53.83%±5.54%v.s45.76%±11.83%, P>0.05). The differences between those groups of were striking(P<0.05). The overall trend of percentage of positive cells of COX-2 on transplanted as DDP group<ART group<Tumor control group(P<0.05). Besides the ART group and DDP group in 10 days and 20 days have no obvious change(30.06±5.28 v.s31.88±6.72, 16.92±6.11 v.s19.32±6.03, P>0.05).Compared with Tumor control group of GS of COX-2 on transplant tumor cell in 10 days(109.36±5.37), DDP group was significant increased(118.00±3.82, P<0.05), and ART group had no obvious change(109.33±6.40, P<0.05). In20, 30, 40 days, there was no significant difference in different treatment(P>0.05). 3 Correlational analyses between the expression of VEGF and COX-2 and count of MVD on transplantation tumorThere was significant positive correlation on VEGF and COX-2 of positive cell percentage in different time(10d: r=0.526, P<0.05; 20d: r=0.599, P<0.05; 30d: r=0.859, P<0.05; 40d: r=0.951, P<0.05).There was positive correlation of positive cell expression of VEGF and count of MVD(10d: r=0.773, P<0.05; 20d: r=0.558, P<0.05; 30d: r=0.804, P<0.05; 40d: r=0.565, P<0.05). Besides in 10 days, there was positive correlation on count of MVD and percentage positive cell of COX-2(10d: r=0.339, P>0.05; 20d: r=0.615, P<0.05; 30d: r=0.595, P<0.05; 40d:r=0.563, P<0.05).Conclusions:1 Artesunate could inhibit the expression of VEGF and COX-2 on mice transplanted tumor and inhibit tumor cell growth.2 Artesunate could restrain the growth of transplanted tumor by inhibition of angiogenesis.3 There was positive correlation between MVD,VEGF and COX-2 on transplantation of tumor... |
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