| Objective: To choose CDCRGDCKC polypeptide (RGD-4CK) labeled 99mTc as an imaging agent, which was used for imaging and biodistribution on mice bearing H22 hepatic carcinoma that already treated by antiangiogenic agents (endostar), and discusss whether endostar would compete with 99mTc-RGD-4CK, and estimated the value of imaging agent (99mTc-RGD- 4CK) on monitoring endostar's value on H22 cancer. Method:1 Experimental groupsPart one: If the endostar inhibited the uptake of the imaging agent (99mTc-RGD-4CK) by the tumor tissue12 well-grown mice with no significant differences in their tumor size were selected and then divided randomly into a (injected with high dose endostar), b (injected with medium dose endostar) c (injected with low dose endostar) d (injected with sodium chloride) four groups, The same dose of imaging agent was injected in a tail vein of respectively 3 each group. Two hours following tracer injection, SPECT imaging were perfomed using regions of interest technique (ROI) to calculate the ratio of tissue to non-tissue (T/NT). After the acquisition the blood was obtained from retrobulbar venous plexus and then the animals were euthanized by cervical dislocation and samples from tumor were obtained. Then we weighed the tissue and assessed activities of the tracers using a gamma-well counter. All tissue counts were corrected for background and decay during the time of counting. Tissue activities of the tracers were expressed as percent of the injected dose per gram of wet weight (%ID/g). Statistical analysis was carried out on the ration of tumor to non-tumor and %ID/g value.Part two: Monitoring therapeutic effect 36 well-grown H22-bearing KM mice with no significant differences in their tumor size were selected and then divided randomly into A, B, C, D four groups with 9 in each group. The mice in each group were intraperitoneally injected high dose, medium dose, low dose endostar and sodium chloride respectively. The injection was performed every day. And every group in four was randomly divided into three groups, which have 3 mices in each, then monitered the therapeutic effect respectively after 3, 5, 7 days after the injection.1 SPECT imaging: 99mTc-RGD-4CK was injected (18.5MBq-200μl) in a tail vein of the mice. Two hours following tracer injection, the animals were placed under the pin-hole collimator of single photon emission computed tomography (SPECT) and a 200k-counts whole-body planar image acquisition was performed. Using regions of interest (ROI) technology to measure the same size of the tumor regions of interest (T) and the contralateral limb muscle area (NT) of radioactive counts to calculate the ratio (T/NT).2 Biological Distribution: extracted blood from eyeballs after the acquisition, euthanized them by cervical dislocation removed the tumor tissue, measured its maximum diameter (L) and shortest diameter (W), then calculated the volume V which equal to LW2/2. Then samples from the heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle and tumor were obtained. Organs were quickly rinsed and activities of the tracers were assessed using a gamma-well counter. Tissue activities of the tracers were expressed as percent of the injected dose per gram of wet weight (%ID/g). calculate the various organs and tumor organizations of the radioactivity per gram of uptake (%ID/g).3 Immunohistochemical staining of microvessel densityObtained appropriate tumor tissues, fixed the tissues by 10% formalin, and immunohistochemical staining was performed to evaluate the expression of the antibody of CD34, BCIP/NBT color. CD34 expression in vascular endothelial cells, according the colored of the endothelial cells to determine microvessel density (MVD). The T/NT values, tumor size, %ID/g values and microvessel density were analyzed statistically.Results:Part one: The results of detecting whether a tumor uptake of imaging agents was inhibited by endostatin demonstrated that there were not significant differences (a=0.05) between the a, b, c, d four groups on tumor tissue's %ID/g values and T/NT value. It was proved that tumor tissue uptake of imaging agents was not inhibited by the endostar injection. Part two: Results in monitering the therapeutic effects of the endostar for H22 hepatic carcinoma1 The results tested for the first time1.1 Results of SPECT imagingAfter three-days treatment, the analysis results showed that the T/NT ratios of four groups had no significant differences (P=0.3191>0.05). The highest T/NT ratio was sodium chloride group (3.26±0.37), while the lowest one was medium-dose group (2.92±0.30).1.2 Results of detection tumor volumeAfter three days treatment, A, B, C, D four groups of tumor volume data, the middle dose group (B group) is the maximum (0.94±0.37cm3), the low-dose group (C group) is the minimum (0.49±0.6cm3), but the result of the statistical analysis (SNK-q) between the four groups was no significant difference (P=0.1163>0.05).1.3 Results of detection bio-distributionAfter three days treatment, The mean of percent of the injected dose per gram of tumor tissue (%ID/g) in the A, B, C, D four groups were: 5.12±0.35, 5.29±0.36, 5.59±0.25 and 5.70±0.26, Statistical analysis between the four groups had no significant difference (P=0.1582>0.05).1.4 Results of microvessel densityAfter three days treatment, the mean of the tumor's microvessel density in the A, B, C, D four groups were: 14.6±2.07, 16.2±1.92, 17.2±2.59 and 18.0±3.87, There are no significant difference between four groups (P=0.27>0.05). 2 The effect of the test results in the second times2.1 Results of SPECT imaging After five days treatment, There are no significant difference between the four groups in the tumor's T/NT value (P=0.4836>0.05).2.2 Results of detection tumor volume After five days treatment, There are no significant difference between the four groups in tumor's volume (P=0.88>0.05).2.3 Results of bio-distribution After five days treatment, the mean of percent of the injected dose per gram of tumor tissue (%ID/g) in four groups were(%ID/g): 4.02±0.08, 5.54±0.27, 5.37±0.25, 5.63±0.35. Through the statistical analysis, there are significant difference (P=0.0002 <0.05) between A group and B, C, D three groups, And there are no significant difference between B, C, and D groups.2.4 Results of microvessel density After five days treatment, the mean of the tumor's MVD value in the four groups, A group is the minimum (11.2±1.5), and D group is the maximum (19.4±2.7), B, C groups were 13.4±2.3 and 15.8±1.6 respectively. The result of the statistical analysis (SNK-q), between A group and B, C, D group have significantly difference (p<0.0001).3 Results of the effect in the third times3.1 results of SPECT imaging After seven days treatment,there are no significant difference between A, B, C, D four groups of tumor tissues's T/NT value (P=0.2291>0.05).3.2 Results of tumor volumeAfter seven days treatment, There are no significant difference between A, B, C, D groups tumor's volume (P=0.8039>0.05).3.3 Results of bio-distributionAfter seven days treatment, The mean of A group of The percent of the injected dose per gram (%ID/g) is minimum (3.69±0.26), the following is the B groups (3.91±0.44) the largest is the D group (5.4±0.10). After statistical analysis, there are significant difference between A, B group and D group (P=0.0007<0.05), and there are no significant difference between C and D.3.4 Results of microvessel densityAfter seven days treatment, the mean of the tumor's MVD value in the A, B, C, D four groups were: 9.4±2.6, 11.4±2.7, 13.2±1.48 and 19.6±3.36. Through statistical analysis, there are significant difference between A, B, C groups and D group (P<0.0001), there are no significant difference in A, B and C groups.4 Related Research4.1 There were positive correlation between Tumor tissue's MVD value and %ID/g values (r=0.8501, P=0.0005<0.05).4.2 There was no correlation between tumor tissue's MVD value and T/NT values (r=0.8039, P=0.0016<0.05).4.3 The T/NT value from the bio-distribution tumor has no correlation with that from SPECT imaging (r=0.5384, P=0.0709>0.05).Conclusion:1 The first part of the experiment demonstrated the absorption of imaging agent by tumor doesn't be inhibited by endostar within 0.84mg dosage.2 The second Part2.1 The uptake of the imaging agent (99mTc-RGD-4CK) by tumor can be used to evaluate the inhibition of endostar's therapeutic effect. The uptake of the imaging agent (99mTc-RGD-4CK) by tumor was positively correlated with the blood vessel density.2.2 The T/NT acquired from planar imaging has large error, has no correlation with that from distribution measurements and can't be accurately reflect the uptake of imaging agents by tumor tissue.2.3 Measurement of tumor volume can not be used to evaluate the early efficacy of Endostar.
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