| ABSTRACT(ONE)Transfection of red fluorescent protein into BxPC3 pancreatic cancer cells and in vitro fluorescence imagingObjective:pDsRed2-N,a red fluorescent protein reporter gene,was prepared to label human pancreatic cancer cell line BxPC3.Tumor cells stably and highly expressing luciferase reporter gene were monitored by in vivo imaging.The pancreatic cancer in situ model was established by in situ planting in nude mice pancreas.Methods:The pDsRed2 gene was cloned into the Lentivirus Expression Vector PT67-pLPCX-EGFP,expression plasmid and packaging plasmid were co-transfected into NUGC-4 cells by Lipofectamine 2000 mediated gene transfection,and the activity titer of mKate 2 lentivirus was determined.After infection of BxPC3 with MOI=6 for 96 hours.the infection efficiency was detected by fluorescence microscopy and flow cytometry(FACS),and 2*106 pDsRed2-BxPC3 cells were collected for fluorescence imaging through optical imaging system to determine the optimal imaging parameters.The cells were implanted in the pancreas of nude mice after stable transmission.The tumorigenesis of the transfected cells in nude mice was detected by in vivo imaging.Results:Sequencing results showed that the pDsRed2 gene sequence was correct without mutation or deletion:the activity titer of pDsRed2 lentivirus was 1.6×106 TU/mlThe emission light(710+28)nm is the best parameter for fluorescence imaging of pDsRed2 BxPC3 cells.A stable monoclonal cell line with high expression of luciferase gene was obtained.The monoclonal cell line was implanted into the pancreas of nude mice.In vivo fluorescence imaging showed high intensity red fluorescence development in nude mice.Conclusions:Lentivirus can mediate the high-efficiency labeling of red fluorescent protein reporter gene pDsRed2 on human pancreatic cancer cell line BxPC3.and pDsRed2-labeled BxPC3 can be used for in vitro cell fluorescence imaging.In vivo imaging of tumor animal model is an ideal model to expand the research of tumor growth,metastasis and treatment in vivo.ABSTRACT(TWO)Establishment of BxPC3 pancreatic tumor in situ animal model and imaging scan in vivoObjective:In this study,we used mKate 2-BxPC3 mouse pancreatic cancer cells to construct an in situ tumor model and explore the method of growth observation of transplanted tumor animals.Methods:Subcutaneous transplantation of human pancreatic cancer cells from mKate 2-BxPC3 mice was made into tumor bodies.The tumor bodies were embedded in situ in the tail capsule of healthy BALB/c female mice.Twenty mice were inoculated successfully.The growth of transplanted tumor in mice was continuously observed by Xenogen.According to the tumor formation in vivo imaging mice,the tumor growth should be 10 mm.The growth of transplanted tumor in mice was observed by 64-slice spiral CT(Siemens),1.5T-MRI(Siemens),7T-MRI(Bruker)and Xenogen.Results:Imaging examination showed that all the orthotopic transplantation tumor models of human pancreatic cancer in mice were successfully established,the success rate was 100%,the best median time of tumor formation was 14 days,and the maximum diameter of tumor was 10 mm.CT imaging has good spatial resolution,soft tissue structure can not be accurately displayed,1.5T-MRI imaging of small animals lack good tissue resolution,7T-MRI combined with animal coils,can clearly show the abdominal cavity structure,T2DWI has good tissue and spatial resolution.In vivo imaging system can display tumor growth in real time and show the size of tumor directly.Conclusions:Subcutaneous in situ tumor embedding and amplification is an ideal method for establishing orthotopic transplantation tumor model of human pancreatic cancer in mice,which is easy to operate and has a high success rate;7T-MRI and in vivo imaging are reliable methods for detecting the growth of transplanted tumor,and can be used for monitoring the animal model of pancreatic cancer,accurately locating the tumor in situ,and for the later test.The study provides a good assessment method for tumor intervention.ABSTRACT(THREE)125 seed brachytherapy combined with CARNK-Robotl immunotherapy for BxPC3 pancreatic carcinoma in situ:imaging evaluationObjective:125 I seed brachytherapy combined with CAR-NK-Robot 1 immunotherapy for BxPC3 pancreatic tumor in situ was evaluated by imaging.Methods:The experimental animals were divided into two groups:G1 group and G2 group,with 12 rats in each group.G1 group was treated with implantation of 125I seeds in tumor and intravenous injection of Robol-specific BiCAR-NK cells.G2 group was treated with implantation of 125 I seeds in BxPC3 tumor model.Robol BiCAR-NK cell infusion was divided into two courses with an interval of 7 days and 2 times.The number of cells per course was 10×108,30×108or 50×108,and the interval of each infusion was 1 day,i.e.1,3,5 days.The tumor volume,body weight and body weight of the subjects were recorded according to the observation points of the experimental time.Survival time,fluorescence imaging value and 7T-MR scanning were analyzed to observe the early morphological changes of the tumor.Non-water-depressed internal reference water located at 4.7 ppm was used as a reference for quantitative analysis of other metabolites.The ratio of peak area under different metabolites between normal pancreas and pancreatic cancer was analyzed by statistics.Results:The tumor volume of G1 group was smaller than that of G2 group(P=0.009),the weight of G1 group was smaller than that of G2 group(P=0.004),the survival time of G1 group was 39.000(+2.449)days,the survival time of G2 group was 40.000(+0.797),there was no significant difference between the two groups(P=0.156);the fluorescence attenuation of G1 group was significantly higher than that of G2 group(t=3.0).The ratio of lipid-water,fatty acid-water and choline-water in pancreatic head of normal nude mice was higher than that of G1 and G2 groups,and the ratio of lipid-water and choline-water was significantly different between normal pancreatic head and pancreatic head cancer(P 0.038 and 0.00015 respectively).There was no significant difference(P value 0.152)in the ratio of subapical area between normal pancreatic head and pancreatic head cancer(P value 0.346).The ratio of subapical area between normal pancreatic head and pancreatic head cancer was statistically lower than that of pancreatic head cancer(P value 0.346).0.028).On the 14th day of MRI,there was no significant difference between G1 and G2 groups and normal nude mice pancreas(P=0.832,0.135);on the 7TMR T1 and T2 enhanced scan.there was no significant difference between G1 group and G2 group(t=2.753;P=0.0512),G0,G1,G2 groups were significantly different(F=16.85;P=0.0003).Conclusions:BxPC3 pancreatic tumor in situ is an ideal model of tumor in situ.In vivo fluorescence imaging can judge the curative effect according to the fluorescence intensity.lipid/water,fatty acids/lipid and choline/internal water can be used as early diagnostic basis for tumor,and the 13th day can be used as the observation point of tumor metabolism.7T-MR could detect the changes of tumor cells on the 7th day after tumor intervention,but there was no significant difference in imaging between the two ways.ABSTRACT(FOUR)Study on the mechanism of fine invasion and metastasis of pancreatic cancer by radioactive 125I seed irradiationObjective:By irradiating pancreatic cancer cells with 125I seeds and taking the changes of extracellular matrix as the research platform,the biological factors causing the invasive changes of pancreatic cancer were studied.Methods:Fibroblasts overexpressing FAP induced by tetracycline were arranged in a simulated 3D extracellular matrix system in vivo.Extracellular matrix(ECM)was irradiated with 125I seeds.Angle changes of fibroblasts in ECM were measured and ECM components were analyzed by Western blot.To evaluate the effect of FAP+on the biological behavior of Panc-1 tumor cells,the migration of tumor cells in different components of extracellular matrix was analyzed by Western blot.Results:The percentage of fibroblasts in ECMs with FAP+,FAP+and FAP+inhibitors was 27%.45%and 29%.respectively.The expression of alpha-SMA,fibronectin and type I collagen increased significantly(p=0.009,p=0.002.p=0.001)and the expression of tenascin C decreased significantly(p=0.001)in FAP+ECM compared with FAP-ECM.FAP+inhibitors increased the level of type I collagen(p=0.007).and the expression of fibronectin and alpha-SMA was only significantly higher than that of FAP(p=0.04 in both cases),but not significantly higher than that of FAP+(p=0.32 and P=0.6)(P<0.05).In FAP+cell matrix,the invasion and migration abilities of AsPC-1(AV=5.1 ± 0.7,D=9.5±2.9,T=54.8 ± 5.0)and HPAF-II(AV=5.5 ± 1.7,D=16.6 ± 4.8,T=58.1 ± 18.1)cells were weaker than those of Capan-1(AV=11.3 ± 0.7,D=32.2 ± 0.7,T=120.8 ± 7.9)and Panc-1(AV=12.0 ± 1.4,D=94.1±13.1,T=128.2 ± 15.0).Panc-1 cells were inoculated into three different matrices produced by FAP-,FAP+or FAP+inhibitor fibroblasts.Compared with FAP-stromal cells(AV=7.8±0.8,D/T=0.36±0.04),Panc-1 cells exhibited greater speed and directivity of movement(AV=12.0±1.4,D/T=0.72±0.06)(p=0.05 and P<0.001).In addition,the orientation of the matrix of FAP+inhibitor was significantly weakened(D/T=0.52±0.06,p=0.01),and the velocity was slightly inhibited(AV=9.1± 1.6,p=0.19).Inhibition of integrin beta 1 significantly decreased the speed(67%inhibition,P<0.001)and directivity(70%inhibition,P<0.001)of Panc-1 cells in FAP+,but not in FAP-matrix(p=0.07 and P=0.06).In addition,the specific inhibition of integrin alpha 5 beta 1 decreased the speed(43%inhibition,P=0.002)and directivity(53%inhibition,P<0.001)of Panc-1 cells in FAP+matrix.Conclusions:FAP can restructure ECM by regulating the expression of protein,mainly increasing the composition of fibronectin and collagen fibrin.The changes of FAPrelated structure and composition promote the invasion of tumor cells by altering the arrangement of parallel fibroblasts.After 125 I internal irradiation,the structure of extracellular matrix was changed,but in FAP+extracellular matrix,FAP+extracellular matrix can be significantly changed.Accelerate the movement of tumor cells,while blocking the FAP enzymatic reaction leads to the disordered arrangement of extracellular matrix,can inhibit the activity of tumor cells.FAP+ECM-mediated tumor invasion is mediated by the beta-integrin(beta-integrin)/FAK(focal adhesion kinase)signaling pathway. |
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