| Primary bronchogenic carcinoma is one of the malignant tumor which cause serious damage to the human health.Integrin receptor αvβ3 is highly expressed in vascular endothelial cell and the surface of the tumor.The peptide of Arg-Gly-Asp can recognize integrin receptor αvβ3 specifically.This research compound a cyclic peptide CDCRGDCKC(cRGD)which has two disulfide bonds.The Structure is c(Cys-Arg-Gly-Asp-dSer-Cys)-dSer-Lys(Gly-(D)-Ala-Gly-Gly-Aba)-Lys-dSer-c(Cys-Arg-Gly-Asp-dSer-Cys).Using 99Tcm and 188Re mark this micromolecule peptide to research the imaging and treatment of the non-small cell lung cancer.1.The experimental research of imaging in nude mice model of non-small cell lung cancer which contains c(RGD)2 marking by 99Tcm1.1 The experimental research of c(RGD)2 marking by 99Tcm in the nude mice of non-small cell lung cancerObjective Study the method of marking c(RGD)2 with 99Tcm which can reach a high marking rate and is also stable.Methods Adopting the pretinning procedure marks c(RGD)2 with 99Tcm,marking method:using a bottle of penicillin lOmL,adding 100μ1 tartaric acid buffer,20 μL RGD stoste and 5 μl Stannous chloride solution,adjusting pH to 3-4 by mixing 1M sodium hydroxide,reacting at room temperature filled with nitrogen overnight,and then adding 5 μL Stannous chloride solution,adjusting pH to 3-4 by mixing 1M sodium hydroxide repeatedly,adding 100 μl 99TcmO4-solution,adjusting pH to 3-4 by mixing little 1M sodium hydroxide,reacting at the temperature of 95 ℃ filled with nitrogen for 45 minutes.Results The marking condition as follows:Marking reaction time is 45 minutes,mass concentration of SnCl2·2H2O is 10g/L,20μl c(RGD)2(2 g/L),volume of eluant is 100μl,marking rate of 99Tcm-c(RGD)2 can reach(96.02±3.47)%.After 24 hours,radiochemical purity of 99Tcm-c(RGD)2 is still greater than 90%in Fresh human serum.Conclusion Adopting the pretinning procedure marks c(RGD)2 with 99Tcm which can reach a high marking rate and is also stable.1.2 The experimental research of the distribution of 99Tcm-c(RGD)2 in the normal miceObjective Study the distribution of 99Tcm-c(RGD)2 in the normal mice.Methods Dividing 36 normal ICR mice into 6 groups randomly,six mice in each group.Each mouse is injected with 0.1ml 99Tcm-c(RGD)2(about 370 KBq)which is freshly prepared through audal vein.Killing one group of the mice after 30min,1h,2h,4h,8h and 24h.collecting the tissue of brain,heart,liver,spleen,lungs,Kidneys,stomach,intestines,pancreas,muscle,thyroid,bone and 100μl blood from Carotid artery.Calculating the percentage of each gram of organ or tissue in the total radioactive count(%ID/g)by counting the radioactive count using y-ray counter after weighting.Results The kidney has the highest radioactive count after injecting 99Tcm-c(RGD)2,which shows the marker metabolising through the kidney.Conclusion The 99Tcm-c(RGD)2 which is injected through audal vein is metabolized through the kidney.1.3 The experimental research of 99Tcm-c(RGD)2 in the tumor-bearing nude miceObjective Study the 99Tcm-c(RGD)2 in the tumor-bearing nude mice.Methods Preparing 36 tumor-bearing BALB/c nude mice with cell A549 of non-small cell lung cancer.Each one is injected with 0.1ml 99Tcm-c(RGD)2(about 370 KBq).Killing six mice after 30min,1h,2h,4h,8h and 24h.Collecting the blood and the tissue of brain,heart,liver,spleen,lungs,stomach,pancreas,intestines,Kidneys,muscle,thyroid,bone and the tumor tissue.Weighting and counting the radioactive uptake to calculate the ID%/g.Results The radioactive uptake of the tumor tissue reaches a peak after I h,then declines gradually.Meanwhile,there is a high radioactive uptake in the abdominal organs like kidneys,liver,spleen and so on.99Tcm-c(RGD)2 eliminates faster in the blood.The radioactive count of tumor to muscle ratio is 3.09±0.27 after 4h.Conclusion The radioactive uptake of the tumor tissue reaches a peak after 1h,and then declines gradually.This could be a targeted imaging agent for non-small cell lung cancer.1.4 The SPECT imaging of 99Tcm-c(RGD)2 in the tumor-bearing nude mice with cell A549Objective Study the SPECT imaging of 99Tcm-c(RGD)2 in the tumor-bearing nude mice with cell A549.Methods Five tumor-bearing nude mice are injected 0.1ml 3.70～5.55MBq 99Tcm-c(RGD)2 through audal vein.After 30min,1h,2h,4h,and 6h,take the imaging of the nude mice in prone position one by one by using the SPECT which contains low energy high resolution parallel hole collimator.The radioactivity concentration can be observed in the tumor location one hour later.With time passing,the radioactivity concentration of the tumor location disappears gradually.There is a radioactivity concentration in liver,spleen,Kidney and bladder.Other organs like brain,lungs,small intestine and muscle have no radioactivity concentration.Results The radioactive uptake of the tumor tissue reaches a peak after 1h,and then declines gradually after 2 to 6hours.Conclusion 99Tcm-c(RGD)2 could be a targeted imaging agent for non-small cell lung cancer.2.the experimental research of imaging in nude mice model of non-small cell lung cancer which contains c(RGD)2 marking by 188Re2.1.The experimental research of c(RGD)2 marking by 188ReObjective Study the research of c(RGD)2 marking by 188Re.Methods Adopting the pretinning procedure marks c(RGD)2 with 188Re-c(RGD)2,marking method:using 20μl RGD,adding 100μl tartaric acid buffer,shaking so that it is dissolved.Then adding 15μl(lOg/L)SnCl2,adjusting pH to 3-4 by mixing 7μl NaOH,adding 100mL 188ReO40 Fresh eluent.reacting at 95℃ in the oil bath pan for 30min.Results The marking condition as follows:Marking reaction time is 30 minutes,mass concentration of SnCl2·2H2O is lOg/L,10 μl c(RGD)2(2 g/L),volume of eluant is 100μl.The best marking rate of 188Re-c(RGD)2 can reach(95.39±3.07)%.After 24 hours,radiochemical purity of 188Re-c(RGD)2 is still greater than 90%in Fresh human serum.Conclusion Adopting the pretinning procedure marks c(RGD)2 with 188Re which can reach a high marking rate and is also stable.2.2 The experimental research of the distribution of 188Re-c(RGD)2 in the normal miceObjective Study the distribution of 188Re-c(RGD)2 in the normal mice.Methods Dividing 36 normal ICR mice into 6 groups randomly,six mice in each group.Each mouse is injected with 0.1ml 99Tcm-c(RGD)2(about 370 KBq)which is freshly prepared through audal vein.Killing one group of the mice after 30min,1h,2h,4h,8h and 24h.collecting the tissue of brain,heart,liver,spleen,lungs,kidneys s stomach,intestines,pancreas,muscle,thyroid,bone and 100μL blood from Carotid artery.Calculating the percentage of each gram of organ or tissue in the total radioactive count(%ID/g)by counting the radioactive count using γ-ray counter after weighting.Results The liver,spleen,and kidneys have the highest radioactive count after injecting 188Re-c(RGD)2.Conclusion The 188Re-c(RGD)2 which is injected through audal vein is metabolized through the liver and kidneys which is different from the 99Tcm-c(RGD)2.2.3 The experimental research of the distribution and the SPECT imaging of 188Re-c(RGD)2 in the tumor-bearing nude miceObjective Study the distribution and the SPECT imaging of 188Re-c(RGD)2 in the tumor-bearing nude mice.Methods Preparing 36 tumor-bearing BALB/c nude mice with cell A549 of non-small cell lung cancer.Each one is injected with 0.1ml 188Re-c(RGD)2(about 370 KBq).Killing six mice after 30min,lh,2h,4h,8h and 24h,collecting the blood and the tissue of brain,heart,liver,spleen,lungs,stomach,pancreas,intestines,kidneys,muscle,thyroid,bone and the tumor tissue.Weighting and counting the radioactive uptake to calculate the ID%/g.calculating The radioactive count ratio of tumor(T)to other tissue(NT).Results The radioactive uptake of the tumor tissue reaches a peak after 2h,which is 3.64±0.71(%ID/g),then declines gradually.Meanwhile,there is a high radioactive uptake in the abdominal organs like liver,spleen and kidneys.188Re-c(RGD)2 eliminates faster in the blood.The radioactive count of tumor to muscle ratio is 3.07±0.71 after 24h.Conclusion There is a radioactivity concentration in the liver,spleen and bladder after Tumor-bearing mice is injected 188Re-c(RGD)2 through audal vein 1-4h later.The tumor location has no radioactivity concentration.2.4 The experimental research of the cell-killing effects of 188Re-c(RGD)2 by MTTObjective Methods MTT was used to examine the effects of cell growth inhibition,to show the 188Re-c(RGD)2 has an obvious cell killing effect for A549 cell.Methods A549 cell were seeded onto two piece of 98-well paste,initial cell quantity is 1×104 for each hole,the supernatant was removed after 24h when cell attachment.10μL(74kBq)188Re-c(RGD)2 was addede into the experimental group.The first control group added 10μL 188ReO4-,the second contol group added 100μL c(RGD)2,the third control group added 10μL normal saline,and we have a blank group.We used PBS washing 3 times after 4h,each hole injected 100μL 10%RPVI 1640 after supernatant was removed.After 24h and 48h we added 20μL MTT(5mg/mL)and 80μL serum-free RPMI 1640 for each palte.150μL DMSO were added into the plate after 4h,the actuation duration was 15min.The enzyme mark analyzer in 492 nm wavelength was used to detect the optical density(OD)value,the inhibition rate =(control group OD value-group OD value)/control group OD value,calculate the inhibition rate.Results After joined different drugs within 24 h,the OD values of all the groups are falling.The difference between other groups compared with normal saline group had statistical significance(P<0.01).OD value of all the groups were increased after 48h,compared 48h with 24 h the difference was statistically significant(P<0.01).There was no statistically significant difference(P>0.05)between the 188Re group with c(RGD)2 group in 24 h and 48 h.In 188Re-c(RGD)2 group,the cell growth inhibition rate is reduced with extended time,the difference was statistically significant between 24 h with 48 h(P<0.05).At 48h,the growth inhibition rate among 188Re-c(RGD)2 group 188Re group and c(RGD)2 group had statistically significant(P<0.01);There was no statistically significant difference between the other two groups(P>0.05).Conclusion Both the 188Re and c(RGD)2 can inhibit the growth of A549 cell,but 188Re-c(RGD)2 on A549 cell growth inhibition is more than 188 Re and c(RGD)2 separate function.Conclusion:1.The pretinning procedure marks 99Tcmc(RGD)2 is easy and fast.The marking rate can reach 95%and do not need to domore purification.The radiochemical purity of marker is stable,which can reach 90%in 24 hours.2.The 99Tcm-c(RGD)2 which is injected through audal vein is metabolized through the kidney.3.The radioactive uptake of the tumor tissue reaches a peak after 1h.This could be a targeted imaging agent for non-small cell lung cancer.4.Adopting the pretinning procedure marks c(RGD)2 with 189Re which can reach a high marking rate and is also stable.The percentage of each gram of tumor in the total radioactive count is higher.There is no statistic difference between the radioactive count of tumor and muscle in 24h.There is a radioactivity concentration in the liver and spleen.The tumor location has no radioactivity concentration which needing a further study.5.Both the 188Re and c(RGD)2 can inhibit the growth of A549 cell,but 188Re-c(RGD)2 on A549 cell growth inhibition is more than 188 Re and c(RGD)2 separate function. |
PDF Full Text Request