ABSTRACT
The translation of gene therapy from bench to bedside depends on efficient intracellular gene delivery. The macromolecular biologics such as gene combined with vectors tend to enter into the cells by means of endocytosis, where the biologics may encounter the risk of degradation in endolysosome. Recently, photochemical internalization (PCI) has emerged as a promising technique to overcome endo-lysosomal sequestration, which utilizes photosensitizer and light resulting in reactive oxygen species at sub-lethal level to destruct biofilm and facilitate intracellular drug delivery. In this article, the mechanism of PCI technology and its development for gene delivery were reviewed, which can provide the scientific basis for the possible utilization of PCI to solve the problem of endo-lysosomal escape in gene delivery.
ABSTRACT
The translation of gene therapy from bench to bedside depends on efficient intracellular gene delivery. The macromolecular biologics such as gene combined with vectors tend to enter into the cells by means of endocytosis, where the biologics may encounter the risk of degradation in endo-lysosome. Recently, photochemical internalization (PCI) has emerged as a promising technique to overcome endo-lysosomal sequestration, which utilizes photosensitizer and light resulting in reactive oxygen species at sub-lethal level to destruct biofilm and facilitate intracellular drug delivery. In this article, the mechanism of PCI technology and its development for gene delivery were reviewed, which can provide the scientific basis for the possible utilization of PCI to solve the problem of endo-lysosomal escape in gene delivery.
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Objective To investigate radionuclide imaging and routine CT in diagnosing hepatic focal nodular hyperplasia (FNH) and the combined diagnostic value of the two modalities. Methods Thirty-two patients with hepatic FNH were retrospectively studied. All patients underwent routine CT scan. Twenty-four patients were examined by 99Tcm-sulfur colloid (SC) hepatic planar scintigraphy and SPECT/CT imaging, and then patients who had abnormal foci underwent 99Tcm-diethyl iminodiacetic acid (EHIDA) triple-phase hepatobiliary imaging. x2 -test of four-table or Fisher exact probabilities in 2 × 2 table was applied for statistical analysis. Results Of all 32 patients pathologically diagnosed as FNH with single solitary nodule, 25 were classified as classic type and the rest 7 as non-classic type. Although routine CT found all hepatic lesions, only 15 cases were diagnosed pathologically as FNH classic type but the rest were either misdiagnosed or left as indeterminate. On radionuclide imaging (hepatic colloid scintigraphy plus triple-phase hepatobiliary images), 11 patients with big foci (with maximal diameter ＞3 cm) out of 24 patients were correctly diagnosed as FNH, with 7 diagnosed as classic type FNH and 4 as non-classic. Other 13 patients were either misdiagnosed or simply missed. The diagnosing rates of routine CT and radionuclide imaging were60.0% (15/25) and 38.9% (7/18) for FNH classic type, 0/7 and 4/6 for non-classic type,50.0% (10/20) and 73.3% (11/15) for big foci, 41.7% (5/12) and 0/9 forsmall foci (with maximal diameter≤3 cm), respectively. The total diagnosing rate of radionuclide imaging combined with routine CT was significantly higher than that of routine CT or radionuclide imaging alone ( x2 = 4. 48, P ＜ 0. 05;x2 =4.27, P ＜0.05 ). Conclusion Radionuclide imaging in combination with routine CT may improve the diagnostic accuracy for hepatic FNH patients.
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Objective To evaluate the diagnostic value of ~(99)Tc~m-methoxyisobutylisonitrile (MIBI) SPECT scintigraphy combined Iocalizable CT in the localization of ectopic parathyroid glands in hyperparathyroidism.Methods Retrospective data of surgery,pathology and imaging were collected from 28 patients with hyperfunctioning ectopic parathyroid glands.All cases underwent CT studies.Twenty-five patients had ~(99)Tc~m-MIBI planar imaging first:SPECT scintigraphy combined localizable CT was performed for the patients with abnormal radionuclide foci immediately.The fusion images obtained after reconstruction showed the exact location of the ectopic foci.Operative histopathologic results were regarded as "gold standards".Presuming 4 parathyroid glands as normal findings,findings confirmed by operation and pathology were regarded as positive,otherwise negative.The results of CT and radionuclide imaging were compared by X~2-test of four-foId table.Results Twenty-eight ectopic parathyroid glands were found in 28 patients,all pathologically confirmed as adenomss.CT found 22 foci,of which 17 were true positive,5 false positive,11 false negative,and 79 true negative.~(99)Tc~m-MIBI SPECT scintigraphy combined localizable CT found 23 foci,no false positive,2 false negative,and 75 true negative.The results showed that the sensitivities were 61% (17/28),92%(23/25),specificities 94%(79/84),100%(75/75),accuracies 86%(96/112),98% (98/100),positive predictive values 77%(17/22),100%(23/23),and negative predictive values 88% (79190),97%(75/77),respectively,for CT and radionuclide imaging.~(99)Tc~m-MIBI SPECT scintigraphy combined localizable CT was therefore significantly higher than CT in sensitivity(X~2=6.98,P＜0.01),specificity (X~2=4.61,P＜0.05),accuracy (X~2=10.30,P＜0.01),positive predictive value(X~2=5.88,P＜0.05) and negative predictive value (X~2=5.36,P＜0.05).Conclusion ~(99)Tc~m-MIBI SPECT scintigraphy combined localizable CT is superior to CT alone in the localization of ectopic parathyroid glands in hyperparathyroidism,but false negative can be found in some patients.
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<p><b>OBJECTIVE</b>To investigate bone defect healing by true bone ceramic complex carrying core binding factor a1 (Cbfa1) gene modified rabbit skin fibroblasts.</p><p><b>METHODS</b>Transfect rabbit skin fibroblasts (RSF) with both eukaryotic expression vector pSG5 which could express Cbfa1 gene and pSG5. After being cultured for 48 h, the transfected RSF were seeded into true bone ceramic (TBC) of 2 cm in length and 4 mm in diameter to construct pSG5-Cbfa1/RSF/TBC complex and pSG5/RSF/TBC complex. Forty-eight bone defect model rabbits were randomized into four groups, each has 6 rabbits (12 radius), due to different treatment. group I: with pSG5-Cbfa1/RSF/TBC complex, group II: with pSG5/RSF/TBC complex, group III: with TBC, Group IV: empty control. After being seeded and cultured for about 24 h the complexes were implanted into 2 cm long bone defects in the middle of bilateral radius of rabbits. The radius were inspected by X-ray and then the specimens were collected at the end of the fourth and twelfth weeks after operation. Then, the specimens were decalcified and histologically investigated with Hematoxylin eosin staining and Masson staining methods. Newly synthesized trabecular bone was inspected by image analysis system and the strength of bone defect area treated with graft-implantation was tested with biomechanical method-three point bending test.</p><p><b>RESULTS</b>In group I, trabecular bone was actively synthesized to generate a great amount of trabecular bone and osteon. Preliminary union and bone defect healing were completed with good biomechanical characteristics. There were no newly synthesized trabecular in the other three groups, and bone defect healing were not discovered. The amount of newly synthesized trabecular bone and the results of biomechanical testing differed significantly between group I and the other three (P < 0.01). The efficacy of group I was significantly better than that of the other three groups.</p><p><b>CONCLUSION</b>True bone ceramic complex composed with Cbfa1 gene modified rabbit skin fibroblasts can effectively heal bone defect in rabbits.</p>
Subject(s)
Animals , Rabbits , Bone Regeneration , Bone Substitutes , Bone Transplantation , Cells, Cultured , Core Binding Factor Alpha 1 Subunit , Genetics , Disease Models, Animal , Fibroblasts , Cell Biology , Metabolism , Plasmids , Genetics , Radius , Wounds and Injuries , General Surgery , Random Allocation , Skin , Cell Biology , Tissue Engineering , Methods , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2).</p><p><b>METHODS</b>Cbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope.</p><p><b>RESULTS</b>Cbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells.</p><p><b>CONCLUSIONS</b>New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.</p>