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Objective To establish and evaluate a patient-derived orthotopic xenograft ( PDOX ) model of pancreatic cancer. Methods Tissues of patient-derived pancreatic tumor were transplanted into nude mouse pancreas by surgery. The PDOX models were evaluated by the small animal near infrared fluorescence ( NIRF) optical imaging and PET/CT. The traceability of PDOX models was detected by STR technology, and the pathological changes were observed by H&E staining, immunohistochemistry, and serum level of CA19-9 was detected by ELISA. Results Apparent NIRF were observed to be accumulated in pancreatic site by optical imaging system. The location and size of the xenografts tumor were revealed by fluorescence intensity. The PET/CT images with 18F-FDG molecular probe confirmed the tumor's location and size. Ex vivo NIRF imaging of isolated organ further showed the tumor formation. The traceability of PDOX models was 99. 99% with human origin. H&E staining pathology and immunohistochemistry indicated the pancreatic cancer characteristics. The high serum level of ca19-9 confirmed the mice bearing tumor. Conclusions Pancreatic PDOX models are successfully established in this study, and it can be evaluated comprehensively by NIRF optical imaging and PET/CT, providing an appropriate platform for further research of pancreatic cancer.
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Objective To evaluate the therapeutic effect of chemotherapeutic drugs on pancreatic carcinoma based on patient-derived xenograft(PDX)models,and to screen an individualized treatment strategy. Methods Fresh human pancreatic carcinoma tissues were subcutaneously transplanted into nude mice to establish PDX models which could be stab-ly passaged. The traceability of PDX models was determined by STR analysis. The PDX models were treated with three dif-ferent clinical chemotherapeutic drugs oxaliplatin, gemcitabine and irinotecan, respectively, and the tumor volumes were measured at different times. The therapeutic effect of those drugs was assessed by TGD mathematical model and plasma CA19-9 test. Results The traceability of patient-derived xenograft samples was up to 99.99%. Compared with the con-trol group,the treatment with irinotecan and gemcitabine inhibited tumor growth significantly(P=0.001), and gemcit-abine had even better result. The minimum toxic effect in the mice was induced by irinotecan treatment,followed by gem-citabine treatment. Conclusions Pancreatic carcinoma PDX models are successfully established and can be stably pas-saged. Gemcitabine shows the most inhibitory effect on tumor growth based on TGD mathematical model assessment, and deserves to be recommended as the preferred drug for individual treatment of pancreatic carcinoma.
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Objective To study the tumor targeting ability and application of farnesylthiosalicylic Acid (FTS) and heptamethine carbocyanine fluorescent dye-mediated near-infrared imagine in living animals, and confirm the inhibitory effect of this compound on growth of tumor cells.Methods Human breast cancer cell line MCF-7, glioma cell line U251 and prostate cancer cell line PC3 were cultured to logarithmic growth phase, and different concentrations of FTS and FTS-IR783 were added, respectively.We observed the inhibitory effect of those two compounds on the growth of tumor cells.Under fluorescence microscopy, specific accumulation of FTS-IR783 in these tumor cells was observed.The tumor cells (1×106) were transplanted subcutaneously into nude mice.These mice were subjected to intraperitoneal injection of FTS-IR783 (10 nmol/mouse) two weeks later.In the in vivo imaging, near infrared fluorescence signal and tumor volume were measured and their correlation was analyzed.Results Compared with FTS, FTS-IR783 significantly inhibited the growth of MCF-7, U251 and PC3 cells in vitro.FTS-IR783 was specifically uptaken by these three kinds of tumor cells, showing strong near infrared fluorescence in cell agglomerates.After subcutaneous injection of FTS-IR783, the correlation between fluorescence intensity and tumor volume was 0.987, 0.998 and 0.971, respectively.Conclusions The compound of FTS conjugated with near infrared fluorescent dye IR-783 can specifically recognize tumor cells, in both in vitro and in vivo imaging.At the same time, the compound can significantly inhibit the growth of tumor cells, and may be expected to become a new potential targeted drug.
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Objective To study the application of hepatamethine cyanine near-infrared fluorescence (NIRF) dye IR-783 in the mouse models of human liver cancer exenografts, and to analyze the molecular mechanisms of the NIRF dye targeting tumor cells.Methods Luciferase-tagged HepG2 cells were inoculated subcutaneously into the nude mice.We detected the correlation of NIRF intensity and bioluminescence intensity (BIL) in the tumor region.Patient-derived xenograft (PDX) model was established in mouse by subrenal capsular implantation of clinic liver cancer specimen.After injecting the IR-783 dye, the interface between mouse kidney and the xenograft tumors was confirmed by NIRF analysis, and the tumor tissue in kidney was observed by pathology using H&E staining.The expression of CEA, AFP, HIF1α and OATP3A1 in the liver cancer tissue was detected by immunohistochemical staining.The intracellular retention of NIRF dyes was observed under fluorescence microscope after adding Mito Tracker or Lyso Tracker into cultured HepG2 cells.We added IR-783 in a co-culture system of HCCs and normal liver cells to test the specifical identification ability of IR-783 of the liver cancer cells.Results There was a good correlation between NIRF intensity and BIL intensity of the subcutaneous liver cancer xenograft region in nude mice.The margin between the mouse kidney tissue and xenograft tumors was clearly identified by IR-783.Compared with normal kidney tissue, CEA, HIF1α, OATP3A1 and AFP were highly expressed in the tumor region detected by IHC staining.The NIRF dye IR-783 was mainly accumulated in the mitochondria and lysosomes of cancer cells.GFP-tagged HepG2 cells could be recognized directly, whereas red fluorescence was not detected in normal liver cells.Conclusions IR-783 is a novel near-infrared fluorescent dye with tumor targeting and imaging properties.Its targeting ability may be related to the high expression of HIF1α and OATP3A1 in the liver cancer tissue.
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Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank .sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C 57BL/6 mice.After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile , real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice .Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas 9.After microinjection, miRNA-29b1 gene-mutated mice were obtained.The sequencing results showed that there were two types of genotype for the mutated mice , one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion.Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly .Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR /Cas9 technology.
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Objective To knockout Rag2 and IL2rg genes and construct severe combined immunodeficiency mice based on CRISPR/Cas9 technology. Method Design and synthesis of 25 bp sgRNA were made according to the Rag2 and IL2rg sequences in Genbank. After annealing, sgRNA was cloned into pX330 vector. Recombination plasmid Rag2?sgRNA, IL2rg?sgRN and Cas9 were then transcribed into RNA, these RNA were microinjected into zygotes and the zygotes were transplanted into recipient ICR mice. F0 founders were born and mutated F0 founders mated with wild type mice to obtain F1 generation heterozygous mice. Mutated F1 mice were crossed and got F2 generation homozygous mice. Genotype and phenotype of the knockout mice were identified by sequencing, flow cytometry and xenograft model. Results Rag2?sgRNA and IL2rg?sgRNA recombination plasmids were constructed and transcribed into RNA. After microinjection and mat? ing, F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2rg gene, 10 bp or 11 bp deletion;however, there was only one genotype in Rag2 gene, which was 8 bp deletion. Compared with wild?type BALB/c mice, the number of CD3 +, B220 + and NKp46 + cells in peripheral blood of the knockout mice was reduced significantly. After inoculation of human breast cancer cell line SKBR?2HL cells, tumor size in the xenograft mouse model was increased gradually along with time extension. Conclusion CRISPR/Cas9 is an efficient way to mutate Rag2 and IL2rg gene in mice in vivo, leading to aberrant T cells, B cells and NK cells.
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According to relevant national laws and regulations, practitioner training was included into laboratory animal science teaching reform.By adjusting the training content and teaching method and use of animal models of typical human diseases, the transformation of training mode was realized and improved.By the assessment of basic theory in combination with practical operation, the thinking ability and hands-on skill of the practitioners are much improved. Through classroom instruction, experimental teaching, quality assessment and tracking survey, the evaluating process of the training quality of training teaching is performed.Therefore, the teaching reform of the laboratory animal science based on the training of practitioners is established.
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Objective To establish a patient-derived gastric cancer xenograft( PDX) model in nude mice and to in-vestigate the application of near infrared fluorescent ( NIRF) dye IR-783 in in vivo imaging of gastric cancer xenograft mod-els.Methods Fresh human gastric cancer tissue was taken and transplanted into the subrenal capsule of nude mice to es-tablish the xenograft model.When the transplanted tumors grew,took part of the tumor tissue to do HE staining and compare the structural characteristics with the primary tumor.Another portion of the tumor was xenografted into nude mice subcutane-ously.Twenty days later,the tumor-bearing mice were injected intraperitoneally with IR-783 dye (10μM) in a dose of 100 mg/20 g.The intensity of the tumor image was monitored by optical NIRF imaging.The correction between tumor volume and fluorescence intensity was analyzed.Finally,the expression of OATP1B3 and HIF1αin the xenografted tumor tissue was detected by immunohistochemistry.Results We successfully established three patient-derived xenograft ( PDH) models of human gastric cancer.The transplanted tumor tissues maintained the histological characteristics of the primary tumor well.NIRF signal can be detec ted in subrenal capsule of the xenografted nude mice.The correlation between tumor size and fluorescence intensity in the PDX models reached higher than 98%.Strong positive expressions of HIF1αand OATP1B3 in the tumor tissues were detected.Conclusions NIRF dye IR-783 can be specifically accumulated at the tumor site,therefore, can be used to detect PDX in vivo early.The tumor targeting property may be related to the expression of OATP1B3 and HIF1α.
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Objective To evaluate the effect of near infrared heptamethine cyanine dye IR-783-mediated specific tumor imaging in spontaneous tumor of dogs .Methods IR-783 was intraperitoneally injected to nude mice models of transplanted tumor in a dose of 5μmol/kg.The metabolic time course of IR-783 was detected by in vivo imager .Based on the results of above observation , IR-783 was injected to dogs with spontaneous tumor in a dose of 1.5μmol/kg.The site of intravenous injection was the hind leg .Tumor and peri-tumoral tissues were removed at 5 days after IR-783-injection for fluorescence imaging , pathology and frozen section fluorescence examinations .Results After i.p.IR-783 injection to nude mice models of transplanted tumor , the transplanted tumor tissues of nude mice had stronger specific fluorescence than normal tissues by imaging at 8 days after injection .After i.v.IR-783 injection to four dogs with spontaneous tumor , the fluorescence signal in the tumor tissues was stronger than that in the normal tissues at 5 days after injection .Conclusions Near infrared fluorescent dye IR-783 could be specifically taken up by tumor tissues , and can be used for specific diagnosis of tumor.It has an important clinical application prospect .