| Part one: Establishment of breast cancer mouse model with “human microenviroment”Purpose: To establish the novel human breast tissue-derived orthotopic mouse model of breast cancer--“breast-breast” mouse model.Materials and Methods: Twenty-two female Severe Combined Immunodeficient(SCID) mice of four to six weeks old were enrolled and randomly separated into two groups. In the test group, 3 pieces of normal human breast tissue(4×4×4mm 3) were implanted subcutaneously into SCID mouse on the both sides, while in the control group, only on the right side. One week after the transplantation, the human breast cancer cells(MDA-MB-231) were inoculated into the implants of left side with the dose of 5 × 105 in the test group, while the breast cancer cells were injected subcutaneously into the left side with the dose of 5 × 105 in the control group. The orthotopic tumors and metastasis of SCID mice were observed by the whole-body optical imaging system per week after tumor cells injection. Eight to twelve weeks after tumor cells injection, the SCID mice were killed by cervical vertebra dislocation. The orthotopic tumors and metastasis of SCID mice were examined by pathologist.Results: Two mice were dead after transplantation in the control group, which were excluded. It was showed that the rate of the orthotopic tumors 90.9%(10/11) and the rate of the metastasis 36.4%(4/11) in the test group, while 44.4%(4/9) and 88.9%(8/9) in the control group by the whole-body optical imaging system. And there was no tumor in the right side of human breast tissue transplantation not only in the test group but also in the control group by the whole-body optical imaging system. It was showed that the rate of the orthotopic tumors 100%(11/11), the rate of the metastasis in the mouse tissue and right human transplantation 27.3%(3/11), the rate of the metastasis only in the right human transplantation 45.5%(5/11), the rate of the metastasis only in the mouse tissue 9.1%(1/11) and the rate of no metastasis 18.2%(2/11) in the test group, while 44.4%(4/9), 33.3%(3/9), 0%(0/9), 55.6%(5/9) and 11.1%(1/9) in the control group by pathology. Compared by the control group, the rate of the orthotopic tumors and the rate of the metastasis only in the right human transplantation was obviously increased(P<0.05), while the rate of the metastasis only in the mouse tissue was obviously decreased(P<0.05) in the test group. There were living mammary ducts and lobules in the human breast tissue transplantation. The human anti-CD34 and anti-CD31 were positive in the human breast tissue transplantation, while the human ER and PR of orthotopic tumor cell(GFP-MDA-MB-231) were negative.Conclusion: “Human microenviroment” was provided for the human breast cancer cells by the “breast-breast” mouse model, in which proliferative breast cancer cells would prefer to shift into the human breast tissue. The good stability and repeatability of the “breast-breast” mouse model were guaranteed by the homogeneity of breast cancer cells.Part Two: In vivo MR imaging of bevacizumab-targeted magnetic nanoparticles in the “human microenviroment” breast cancer mouse modelPurpose: 1.To design bevacizumab-targeted magnetic nanoparticles as contrast media; 2.To monitor the distribution and effect of bevacizumab-targeted magnetic nanoparticles by MRI in the “human microenviroment” breast cancer mouse model.Materials and Methods: 1. Twenty-one female Severe Combined Immunodeficient(SCID) mice of four to six weeks old were enrolled and randomly separated into three groups. Three pieces of normal human breast tissue(4×4×4mm 3) were implanted subcutaneously into SCID mouse on the left side. One week after the transplantation, the human breast cancer cells(MDA-MB-231) were inoculated into the implants of left side with the dose of 5 × 105 in all SCID mice. The orthotopic tumors of SCID mice were observed by the whole-body optical imaging system after tumor cells injection. 2. Through covalent coupling, the antibody(Avastin) was conjugated onto the surface of Fe3O4-DMSA, which was acquired subsequently after synthesis of Fe3O4@OA and stored at 4 °C. Morphology and size of the prepared Fe3O4 nanoparticles were characterized by transmission electronic microscopy. Electronic diffraction was used to determine the crystalline structure of the nanoparticles. Hydrodynamic size and zeta potential were measured by Dynamic Light Scattering. 3. The drug(Fe-Avastin) would be injected into peritoneal cavity with the dose of 0.5mg-1mg/ 400?l(0.4ml) per 4 days for 3 times in the test group. The drug(Fe) would be injected into peritoneal cavity with the dose of 0.5mg/ 400?l(0.4ml) per 4 days for 3 times in the control group1. The drug(Avastin) would be injected into peritoneal cavity with the dose of 1mg/ 400?l(0.4ml) per 4 days for 3 times in the control group2. 4. Series of in vivo MR images were obtained before injection, 4th day,8th day and 12 th day after injection. The SCID mice were killed by cervical vertebra dislocation 12 th day after injection. MRI findings were compared with pathological results(H-E stain, Berlin's blue stain and transmission electronic microscopy).Results: 1.Four mice were dead. There were fifteen mice with orthotopic tumors and two mice without orthotopic tumors observed by the whole-body optical imaging system. Fifteen mice were enrolled and randomly separated into three groups. 2. The transmission electronic microscopy image indicated that the average size of Fe3O4 nanoparticles was 12.2±4.3 nm. The structure of Fe3O4 nanoparticles was cubic spinel. Hydrodynamic size and zeta potential of Fe3O4-DMSA nanoparticles was 30.2±0.7 nm and-42.7±10.8 m V separately. The solution of bevacizumab-targeted magnetic nanoparticles was stable and fully water-soluble. The average size of bevacizumab-targeted magnetic nanoparticles was from 10 nm to 20 nm, equal to the size of ultra-small superparamagnetic iron oxide(USPIO). 3. There emerged low signal region in the peripheral zone of orthotopic tumor in test group from the 4th day to the 8th day on the T2 weighted image. As the time went by, the size of the low signal region increased gradually. There was no signal change in the orthotopic tumor of control group1 and control group2. It was showed that bevacizumab-targeted magnetic nanoparticles distributed in the peripheral zone of orthotopic tumor of test group, while no bevacizumab-targeted magnetic nanoparticles in the peripheral zone of orthotopic tumor of control group1 by H-E stain and Berlin's blue stain. There was necrosis in the central zone of orthotopic tumor of test group, while no necrosis in the central zone of orthotopic tumor of control group2. In the form of pinocytotic vesicle, magnetic nanoparticles distributed in the cytoplasm of orthotopic tumor cells of test group, while no magnetic nanoparticles distributed in the cytoplasm of orthotopic tumor cells of control group1 by transmission electronic microscopy. There were no magnetic nanoparticles distributed in the interstitial substance of orthotopic tumor oftest group or control group1.Conclusion: Through the cooperation of positive target and negative target, bevacizumab-targeted magnetic nanoparticles distributed in the peripheral zone of human breast cancer, which led to necrosis in the central zone of breast cancer. In the form of pinocytotic vesicle, bevacizumab-targeted magnetic nanoparticles distributed in the cytoplasm of human breast cancer cells by transmission electronic microscopy. |
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