| Backgroud:Gastric cancer is a high-incidence malignant tumor and it causes huge economic losses to our country every year. Its incidence and mortality remain high. The key to its prevention is ’early detection and early treatment’. But the early detection rate of gastric cancer is very low in our country. When the patients with gastric cancer are in confirmed diagnosis, it is too late to surgery. The emergence of molecular imaging offers a new way for the diagnosis and treatment of gastric cancer. The key to molecular imaging is to find the specific molecules targeting the tumors in vivo. The peptides homing to tumor vasculature are screened out using phage peptide library and identified in vivo. Some peptides has entered the stage of clinical trials, and they maybe have potential to clinical application.We have been committed to the research of gastric cancer angiogenesis, and screened out the GX1 peptide homing to the tumor vasculature. The earlier studies showed that it can specifically bind to tumor vascular endothelial cells and target the tumor in vivo. Wedid a lot of researches and prepared a series of probes and drugs in the diagnosis and treatment of gastric cancer using the GX1 peptide. But the affinity and targeting ability of GX1 are still capable. So in this study, we will modify GX1 and identify modifications in vivo and in vitro. It will provide a new thinking for the diagnosis and treatment of gastric cancer.Aims:1) To synthesize GX1 dimers and identify whether the affinity and targeting properties are superior to the monomer;2) To explore the labeling method of188 Re, to prepare188Re-PEG-(GX1)2, and to apply it to SPECT imaging and treatment of gastric cancer;3) To explore the labeling method of68 Ga, to prepare68Ga-DOTA-KEK-(GX1)2, and to apply it to PET imaging of gastric cancer;Methods:1)188Re-PEG-(GX1)2 was prepared and identified. The labeling yield and stablity were performed. The specificity of188Re-PEG-(GX1)2 to Co-HUVECs was verified using cell-binding assay in vitro and competitive inhibition assay. MTT assay and flow cytometry were carried out to investigate the role of188Re-PEG-(GX1)2 to suppress proliferation of Co-HUVECs and to induce cell apoptosis.2) The targeting ability of188Re-PEG-(GX1)2 was confirmed using SPECT imaging and T/H quantification. The progress of treatments was monitored using BLI.3) The tumor tissues were stained by CD 31, Ki 67 and TUNEL ex vivo to give the functional evidence of188Re-PEG-(GX1)2. The toxicity and side effects were checked using HE staining of liver and kidney and biochemical analysis.4)68Ga-DOTA-KEK-(GX1)2 was prepared and identified. The labeling yield and stablity were performed. The specificity of68Ga-DOTA-KEK-(GX1)2 to Co-HUVECs was verified using cell-binding assay in vitro and competitive inhibition assay. The affinity of68Ga-DOTA-KEK-(GX1)2 was validated using immunofluorescence and uptake and efflux assay.5) The targeting ability of68Ga-DOTA-KEK-(GX1)2 was confirmed using Nano PET/CT imaging, SUV quantification and biodistribution.Results:1)188Re-PEG-(GX1)2 was successfully prepared. Its labeling yield was over 96 % and it was stable in the serum in 72 h. A series of experiments were performed in vitro. The results showed that188Re-PEG-(GX1)2 bound to Co-HUVECs specifically and the affinity was higher than188Re-GX1 and188Re-URP.188Re-PEG-(GX1)2 could inhibit proliferation of Co-HUVECs and induce cell apoptosis.2)188Re-PEG-(GX1)2 could target the tumor tissue in the tumor-bearing model according to SPECT imaging. T/H ratio of188Re-PEG-(GX1)2 group was higher than188Re-GX1 group. BLI showed that188Re-PEG-(GX1)2 could suppress tumor growth effectively.3) The staining of tumor tissues ex vivo showed that188Re-PEG-(GX1)2 could inhibit angiogenesis of tumor, suppress cell proliferation and induce cell apoptosis. And the effects were better than that of188Re-PEG-(GX1)2.4)68Ga-DOTA-KEK-(GX1)2 was successfully prepared. Its labeling yield was over 97 %and it was stable in the serum in 90 min. A series of experiments were performed in vitro. The results showed that68Ga-DOTA-KEK-(GX1)2 bound to Co-HUVECs specifically and the affinity was higher than68Ga-DOTA-GX1 and68Ga-DOTA-URP.The binding of68Ga-DOTA-KEK-(GX1)2 to Co-HUVECs was higher than to HUVECs, BGC 823 and GES cells. The uptake of68Ga-DOTA-KEK-(GX1)2 in Co-HUVECs reach the maximum at 60 min.5)68Ga-DOTA-KEK-(GX1)2 could target the tumor tissue in the tumor-bearing model according to Nano PET/CT imaging. The SUV of68Ga-DOTA-KEK-(GX1)2 group was higher than68Ga-DOTA-GX1 group. The biodistribution showed that68Ga-DOTA-KEK-(GX1)2 was excellently reserved in the tumor tissues.Conclusions:1) GX1 was modified successfully, and the properties of GX1 dimer were superior to GX1.2)188Re-PEG-(GX1)2 was successfully prepared and it could been used in the diagnosis and treatment of gastric cancer.3)68Ga-DOTA-KEK-(GX1)2 was successfully prepared and it could been used in PET imaging of gastric cancer.4)188Re and68 Ga were applied successfully and they could been used in clinical application in the future. |
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