AIM： To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS： Human pancreatic tissue samples （malignant and normal） were o...AIM： To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS： Human pancreatic tissue samples （malignant and normal） were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% 02. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection.RESULTS： With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants. CONCLUSION： The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.展开更多
Objective: We explored the expression of coxsackie and adenovirus receptor (CAR) in small cell lung cancer (SCLC) tissue. Methods: CAR expression in 31 SCLC was assessed in formaldehyde-fixed, paraffin-embedded tissue...Objective: We explored the expression of coxsackie and adenovirus receptor (CAR) in small cell lung cancer (SCLC) tissue. Methods: CAR expression in 31 SCLC was assessed in formaldehyde-fixed, paraffin-embedded tissue according to the EnVision immunohistochemistry procedure, while 3 samples of surgical specimens of non-malignant lung disease were taken as the negative control. Results: We observed that the expression of CAR was detectable positive in all the 31 cases from the small cell lung cancer tissue, in contrasting that non-malignant lung tissue control. Conclusion: The high expression of CAR appeared in SCLC tissue indicates that it play an important role in of adenovirus vector-based gene therapy in SCLC.展开更多
OBJECTIVE: To study whether recombinant adeno-associated virus (rAAV) mediated foreign gene, LacZ, could pass the blood brain barrier by intra-carotid artery delivery and express in vivo in ischemic brain of the foc...OBJECTIVE: To study whether recombinant adeno-associated virus (rAAV) mediated foreign gene, LacZ, could pass the blood brain barrier by intra-carotid artery delivery and express in vivo in ischemic brain of the focal embolic stroke rats to investigate a possibility of delivering foreign gene through carotid artery to treat acute ischemic stroke. METHODS: The carotid artery territory in 41 rats was embolized with or without arterial-like fibrin rich clots to make a model of focal embolic stroke rat. rAAV containing LacZ gene (rAAV-LacZ) was constructed in 293 cells by calcium phosphate cotransfection. The rats were assigned to one of the following treatments: 1 control (without embolism) groups, including PBS treated (n = 6), pLacZ treated (n = 6 ) and rAAV-LacZ treated (n = 6): 2 embolic groups, including embolism + PBS (n =7),embolism + pLacZ (n = 8) and embolism + rAAV-LacZ (n = 8). Brains were cryosectioned and kappa-Gal stain was performed at 2, 4, and 8 weeks, respectively, after transfection, and then infarct volume was measured and the percentage of LacZ staining-positive cells was calculated. RESULTS: In all the control groups and embolism + PBS treated animal, no kappa-Gal staining-positive cells were found, but in embolism + pLacZ (n = 8) and embolism+rAAV-LacZ groups a lot of kappa-Gal staining-positive cells were found. The expression cells were in the tissues around the infarction. The gene expression persisted only nearly four weeks in embolic group with pLacZ. In the embolic group with rAAV-LacZ the expression was very stable during the experiment course (eight weeks) and the percentage of the expressed cells was significantly higher than that of its contralateral areas at the same time points, respectively. CONCLUSIONS: The plasmid vector and rAAV could enter the brain through the ischemia-damaged blood barrier and foreign gene can be expressed in brain. The positive gene expression is mainly in the peripheral areas of the infarction. rAAV as a permanent expression vector may ultimately be used for gene therapy of human ischemia cerebravascular diseases.展开更多
基金Supported by The Dutch Cancer Society(grant.UvA2002-2604)
文摘AIM： To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS： Human pancreatic tissue samples （malignant and normal） were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% 02. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection.RESULTS： With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants. CONCLUSION： The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.
基金Supported by a grant from the Society Development Foundation of Jiangsu (No. BS2007025)
文摘Objective: We explored the expression of coxsackie and adenovirus receptor (CAR) in small cell lung cancer (SCLC) tissue. Methods: CAR expression in 31 SCLC was assessed in formaldehyde-fixed, paraffin-embedded tissue according to the EnVision immunohistochemistry procedure, while 3 samples of surgical specimens of non-malignant lung disease were taken as the negative control. Results: We observed that the expression of CAR was detectable positive in all the 31 cases from the small cell lung cancer tissue, in contrasting that non-malignant lung tissue control. Conclusion: The high expression of CAR appeared in SCLC tissue indicates that it play an important role in of adenovirus vector-based gene therapy in SCLC.
基金ThisstudywassupportedbythegrantforKeyProjectofNationalNaturalScienceFoundationinChina (No39730170)andthegrantforGeneralProjectofNSFC (No 3 9770 810 )
文摘OBJECTIVE: To study whether recombinant adeno-associated virus (rAAV) mediated foreign gene, LacZ, could pass the blood brain barrier by intra-carotid artery delivery and express in vivo in ischemic brain of the focal embolic stroke rats to investigate a possibility of delivering foreign gene through carotid artery to treat acute ischemic stroke. METHODS: The carotid artery territory in 41 rats was embolized with or without arterial-like fibrin rich clots to make a model of focal embolic stroke rat. rAAV containing LacZ gene (rAAV-LacZ) was constructed in 293 cells by calcium phosphate cotransfection. The rats were assigned to one of the following treatments: 1 control (without embolism) groups, including PBS treated (n = 6), pLacZ treated (n = 6 ) and rAAV-LacZ treated (n = 6): 2 embolic groups, including embolism + PBS (n =7),embolism + pLacZ (n = 8) and embolism + rAAV-LacZ (n = 8). Brains were cryosectioned and kappa-Gal stain was performed at 2, 4, and 8 weeks, respectively, after transfection, and then infarct volume was measured and the percentage of LacZ staining-positive cells was calculated. RESULTS: In all the control groups and embolism + PBS treated animal, no kappa-Gal staining-positive cells were found, but in embolism + pLacZ (n = 8) and embolism+rAAV-LacZ groups a lot of kappa-Gal staining-positive cells were found. The expression cells were in the tissues around the infarction. The gene expression persisted only nearly four weeks in embolic group with pLacZ. In the embolic group with rAAV-LacZ the expression was very stable during the experiment course (eight weeks) and the percentage of the expressed cells was significantly higher than that of its contralateral areas at the same time points, respectively. CONCLUSIONS: The plasmid vector and rAAV could enter the brain through the ischemia-damaged blood barrier and foreign gene can be expressed in brain. The positive gene expression is mainly in the peripheral areas of the infarction. rAAV as a permanent expression vector may ultimately be used for gene therapy of human ischemia cerebravascular diseases.
