| Background and purposeIn this study, we intend to find out the relationship between mesothelin proteinexpression in gallbladder cancer tissues with patient’s clinicopathological data andprognosis outcome. And by building mesothelin knocked down lentiviral vectors,gallbladder cancer cell’s proliferation and invasion ability was studied after themesothelin gene silencing. Finally, we synthesized a nano-MRI contrast probe targetinggallbladder cancer using mesothelin monoclonal antibody as a guiding marker. It’scytotoxicity and ability to identify GBC cells were verified in vitro.Method1. Study the relationship between mesothelin expression levels in gallbladder cancertissue and patient’s clinical and pathological data and prognosis information.1) Collect115gallbladder cancer patient’s clinical-pathological data and prognosisinformation through follow-up;2) Verify the mesothelin expression amount at mRNA and protein level in gallbladdercarcinoma by realtime-PCR and immunohistochemistry;3) Use statistical analysis software to study the relationships between mesothelinexpression and patient’s clinical pathology data and survival time.2. Study the effects of mesothelin on cell gallbladder cance proliferation, invasion andmetastasis1) Synthesis siRNA sequences according to the mesothelin gene information and insertedit into the lentivirus vector to construct mesothelin gene knock-down vector. Sequencedto verify correct insertion sequence and determining the titer of lentivirus;2) Validation mesothelin lentiviral gene knock-down by using western blot and realtime-PCR 3) Using the MTT assay and colony formation assay to evaluate mesothelin’s effect onthe gallbladder cancer cell proliferation, using transwell chamber experiments andscratches heal assay to study mesothelin’s effect on the role of cancer cell invasion andmetastasis;4) Changes in metastasis related protein’s expression before and after the mesothelin’sknock down was evaluated by western blot.3. Preparation of targeted magnetic nanoprobes for gallbladder cancer active targetingMRI contrast scan1) Preparation7nm monodisperse nano-magnetic particles by solvothermal method, anduse DSL, TEM, VSM to verify its characterization. Use MTT assay to assess particle’scytotoxicity;2)Use polyethylene glycol (PEG) to modify the surface of material, couple mesothelinmonoclonal antibody to the carboxyl of magnetic particles;3) Co-culture the targeted nanoprobes with gallbladder cancer cells and measureintracellular iron content,after that the cell suspension was MR scanned.ResultThe expression levels of mesothelin in cancer tissue specimens was significantlyhigher than the normal tissue and is related to the degree of tissue differentiation. Deepinfiltration gallbladder cancer patients with lymph node metastasis, a low degree ofdifferentiation and a later TNM staging sample had high mesothelin express level.Prognosis analysis showed mesothelin protein expression was independently associatedwith poor prognosis in patients with gallbladder cancer.After mesothelin knock down, theproliferation and metastasis ability of GBC cells were significantly decreased, deeperresearch indicated EMT and secretion of MMP9maybe the possible mechanism.Thenano MRI contrast probe based on mesothelin mono-antibody has a good waterdispersion and low cytotoxicity. It can successfully identify and enter the GBC cell andthus increased the MR scan T2*signal of the cell suspension. It’s a potential gallbladdercancer targeted MR probe to be used in clinic.ConclusionMesothelin is an important bio-marker to access the prognosis of gallbladderpatients. It can significantly impact the proliferation and metastasis ability of GBC cells. A nano-magnetic iron oxide particle use mesothelin antibody as target molecule canbecome a new MRI probe for gallbladder cancer diagnosis. Background and purposeIn this study, we intend to find out the relationship of mesothelin protein expressionin gallbladder cancer tissues with patient’s clinicopathological data and prognosisoutcome. And by building mesothelin knocked down lentiviral vectors, gallbladdercancer cell’s proliferation and invasion ability was studied after the mesothelin genesilencing. Finally, we synthesized a nano-MRI contrast probe targeting gallbladdercancer using mesothelin monoclonal antibody as a guiding marker. It’s cytotoxicity andability to identify GBC cells were verified in vitro.Method1. Study the relationship between mesothelin expression levels in gallbladder cancertissue and patient’s clinical and pathological data and prognosis information.1) Collect115gallbladder cancer patient’s clinical-pathological data and prognosisinformation through follow-up;2) Verify the mesothelin expression amount at mRNA and protein level in gallbladdercarcinoma by realtime-PCR and immunohistochemistry;3) Use statistical analysis software to study the relationships between mesothelinexpression and patient’s clinical pathology data and survival time.2. Study the effects of mesothelin on cell gallbladder cance proliferation, invasion andmetastasis1) Synthesis siRNA sequences according to the mesothelin gene information and insertedit into the lentivirus vector to construct mesothelin gene knock-down vector. Sequencedto verify correct insertion sequence and determining the titer of lentivirus; 2) Validation mesothelin lentiviral gene knock-down by using western blot and realtime-PCR3) Using the MTT assay and colony formation assay to evaluate mesothelin’s effect onthe gallbladder cancer cell proliferation, using transwell chamber experiments andscratches heal assay to study mesothelin’s effect on the role of cancer cell invasion andmetastasis;4) Changes in metastasis related protein’s expression before and after the mesothelin’sknock down was evaluated by western blot.3. Preparation of targeted magnetic nanoprobes for gallbladder cancer active targetingMRI contrast scan1) Preparation7nm monodisperse nano-magnetic particles by solvothermal method, anduse DSL, TEM, VSM to verify its characterization. Use MTT assay to assess particle’scytotoxicity;2)Use polyethylene glycol (PEG) to modify the surface of material, couple mesothelinmonoclonal antibody to the carboxyl of magnetic particles;3) Co-culture the targeted nanoprobes with gallbladder cancer cells and measureintracellular iron content,after that the cell suspension was MR scanned.ResultThe expression levels of mesothelin in cancer tissue specimens was significantlyhigher than the normal tissue and is related to the degree of tissue differentiation. Deepinfiltration gallbladder cancer patients with lymph node metastasis, a low degree ofdifferentiation and a later TNM staging sample had high mesothelin express level.Prognosis analysis showed mesothelin protein expression was independently associatedwith poor prognosis in patients with gallbladder cancer.After mesothelin knock down, theproliferation and metastasis ability of GBC cells were significantly decreased, deeperresearch indicated EMT and secretion of MMP9maybe the possible mechanism.Thenano MRI contrast probe based on mesothelin mono-antibody has a good waterdispersion and low cytotoxicity. It can successfully identify and enter the GBC cell andthus increased the MR scan T2*signal of the cell suspension. It’s a potential gallbladdercancer targeted MR probe to be used in clinic. ConclusionMesothelin is an important bio-marker to access the prognosis of gallbladderpatients. It can significantly impact the proliferation and metastasis ability of GBC cells.A nano-magnetic iron oxide particle use mesothelin antibody as target molecule canbecome a new MRI probe for gallbladder cancer diagnosis. |
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