| Part One: Studies on the binding of125I-c(RGD)2on lungadenocarcinoma cell A-549, the biodistribution and imaging of125I-c(RGD)2in nude mice bearing tumorsObjective: To study the biding of125I labeled novel c(RGD)2on lung adenocarcinoma cellA-549and evaluate the biodistribution and the planar SPECT camera imagingcharacteristics of125I-c(RGD)2in tumor-bearing nude mice.Methods:①The novel c(RGD)2was labeled with125I by Ch-T method and theradiochemical purity was determined.②The stability of125I-c(RGD)2was detected bybeing kept in human serum or0.9%normal saline(0.9%NS) in37℃with the ordinarytemperature of (25±2)℃at different time.③The binding rate of125I-c(RGD)2and lungadenocarcinoma cell A-549was tested. The cell suspension of A-549was extracted and thedensity of the cells was modified to1×106/mL. Then106/well were seeded onto6-wellplate.3.7KBq (50μL)125I-c(RGD)2was added in each well. The binding rate wascalculated at1h,2h,4h,8h,16h and24h according to the formula [B/(B+F)]×100%.④Thenude mice model which bearing human lung adenocarcinoma cell A-549were established.⑤The biodistribution of125I-c(RGD)2in tumor-bearing mice was detected.24tumor-bearing mice were divided into6groups by the method of random digits table. Thetumor to non-tumor issues ratios (T/NT) and the percentage of injected dose per gramtissue (%ID/g) of different organs at1h,2h,4h,8h,16h,24h after tail vein injection with125I-c(RGD)2were calculated.⑥15tumor-bearing mice were imaged by SPECT at1h,2h,4h,8h and24h after tail vein injection with125I-c(RGD)2.Results:①The labeling efficiency and the radiochemical purity of125I-c(RGD)2reached 63.92%±6.22%and97.89%±0.23%, respectively. The radiochemical purity of125I-c(RGD)2was stable. The radiochemical purity of125I-c(RGD)2was still high thatreached to97%even at48h.②After adding125I-c(RGD)2for1h,2h,4h,8h,16h and24h,the cell binding rates were (5.89±0.84)%,(5.61±0.60)%,(4.29±0.84)%,(4.04±0.44)%,(3.49±0.99)%and (3.23±0.56)%respectively. Cell binding rates were negatively correlatedwith time(r=－0.813, P<0.05).③The%ID/g of the tumor sites was4.56±1.08at1h.Theuptake of radioactivity of different organs reduced by the time gradually and T/NTincreased as time passed.④The uptake of radioactivity in tumor sites were identified at1hafter injection of125I-c(RGD)2. The background faded with excretion of the radioactivedrugs through kidneys gradually over time. The tumor sites were obvious for2h to4h. Theaccumulation of radioactivity of tumor sites could still be observed at24h.Conclusions:125I-c(RGD)2concentrated in the tumor can be observed at1h. The tumorsites were more obvious during2h to4h. The uptake of tumor could still be observed at24h. The high uptake125I-c(RGD)2suggested it could be a new targeting imaging agent inthe future.Part two: Studies on inhibition effect of131I labeled c(RGD)2on humanlung adenocarcinoma cellsObjective: To study the inhibition effect of131I, novel c(RGD)2and131I labeled c(RGD)2on human lung adenocarcinoma cells.Methods:①The novel c(RGD)2was labeled with131I by Ch-T method and theradiochemical purity was determined.②131I, c(RGD)2and131I-c(RGD)2were added to theexperimental groups (250μCi131I,6μL c(RGD)2in each well).20uL0.9%NS was added tothe control groups to establish the blank control groups. The OD values were measured byusing the method of MTT. The formula of cell proliferation inhibition rate (%) was(ODcontrol groups－ODexperimental groups)/ODcontrol groups×100%.③Each control group wasadded with1.0mL0.9%NS and131I, c(RGD)2and131I-c(RGD)2were added to theexperimental groups (250μCi131I，6μL c(RGD)2). The flow cytometry was used to evaluate the effect of cell apoptosis by different drugs in different time.Results:①Lung adenocarcinoma cells entered the logarithmic phase in the first4daysand reached the platform on the4th day, then decreased on the6th day.②The labelingefficiency and the radiochemical purity of125I-c(RGD)2reached99.06%±0.7%and91.93%±1.7%, respectively.③The OD values of experimental groups with131I, c(RGD)2and131I-c(RGD)2were higher than the OD values of control groups in different time. In131Igroup, the OD values were0.224±0.134,0.524±0.061,0.961±0.013at24h,48h and72hrespectively. In c(RGD)2group, the OD values were0.244±0.310,0.507±0.358,0.968±0.119at24h,48h and72h respectively. In131I-c(RGD)2group, the OD values were0.194±0.279,0.408±0.515,0.539±0.143at24h,48h and72h respectively. The OD valuesof each group were positively correlated with time(P<0.01). The OD values of131I-c(RGD)2group for72h was the lowest.④The inhibition rate of each group wascalculated in different time. In131I group, the inhibition rates of the group were(21.52%±4.71%),(24.71%±8.81%),(48.59%±0.70%) at24h,48h and72h respectively. Inc(RGD)2group, the inhibition rates of the group were (14.99%±10.81%),(27.06±5.15%),(48.23±0.06%) at24h,48h and72h respetively. In131I-c(RGD)2group, the inhibition ratesof the group were (31.93%±9.80%),(41.32%±7.40%),(71.09%±7.65%) at24h,48h and72h. The inhibition rate of each group increased over time and the inhibition rates of thegroups with131I-c(RGD)2were the highest at different time.⑤The early apoptosis rate ofeach experimental group increased with time. The early apoptosis rates of the groups with131I-c(RGD)2were the highest.Conclusions:131I-c(RGD)2had obvious inhibition effect on lung adenocarcinoma A-549cells. The inhibition effect of131I-c(RGD)2was stronger than the single inhibition effect of131I and c(RGD)2on lung adenocarcinoma A-549cells.131I-c(RGD)2may become one ofthe molecular targeting drugs on lung cancer in the future. |
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