ABSTRACT
In this study, the recombinant adenovirus (Ad) vector containing dual reporter gene [i.e. human transferrin receptor gene (TFRC) and firefly luciferase reporter gene] was constructed to provide a novel experimental tool for magnetic resonance (MR) and bioluminescence dual-modality molecular imaging. The cDNA of TFRC was amplified by polymerase chain reaction (PCR) and cloned into the multiple cloning site of pShuttle-CMV-CMV-Luciferase vector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luciferase vector and the adenoviral backbone vector (pAdeno) were subjected to homologous recombination. The correct recombinant plasmid was then transfected into 293 packaging cells to produce adenoviral particles and confirmed by PCR. After infection of human colorectal cancer LOVO cells with Ad-TFRC-Luciferase, the expressions of transferrin receptor (TfR) and luciferase protein were detected respectively by Western blotting and bioluminescence imaging in vitro. The results showed that TFRC gene was successfully inserted into the adenoviral shuttle vector carrying luciferase gene. DNA sequence analysis indicated that the TFRC gene sequence in the shuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmid was identified correct by restriction digestion. Ad-TFRC-Luciferase recombinant adenovirus was constructed successfully, and the virus titer was 1.6×10(10) pfu/mL. Forty-eight h after dual reporter gene transfection, the expressions of TfR and luciferase protein were increased significantly (P<0.01). It was concluded that the recombinant adenovirus vector with dual reporter gene was successfully established, which may be used for in vivo tracing target cells in multimodality imaging.
Subject(s)
Adenoviridae , Genetics , Genes, Reporter , Genetics , Genetic Engineering , Methods , Genetic Vectors , Genetics , Molecular Imaging , MethodsABSTRACT
<p><b>BACKGROUND</b>Metformin is the most widely used anti-diabetic drug in the world. An increasing body of evidence shows metformin also blocks cell cycle progression and selectively induces apoptosis via caspase activation in some breast tumor cells. Diffusion-weighted imaging (DWI) and bioluminescence imaging (BLI) have great potential in the evaluation of the early response to cancer therapies. We used DWI and BLI in evaluating the response of breast cancer to metformin.</p><p><b>METHODS</b>The luciferase-engineered human breast cancer cell line MDA-MB-231 was inoculated into the mammary fat pad of nude mice. Twelve female nude mice bearing tumors were divided into two groups. The mice in the treatment group received metformin (2 mg/ml in drinking water daily) after tumor inoculation, and the mice in the control group were offered drinking water without any drug added. We performed 7T magnetic resonance imaging and optical imaging every week. Imaging included T1- and T2-weighted imaging, DWI, and BLI. After imaging. The tumors were collected and subjected to histological analysis.</p><p><b>RESULTS</b>The mean photons/second of tumors in the treatment group was (3.00 ± 0.43)× 10(6) at day one, (1.01 ± 0.14)× 10(7) at 2 weeks, (5.79 ± 1.42)× 10(7) at 4 weeks, and (2.33 ± 0.70)× 10(7) at 8 weeks. The mean photons/second of tumors in the control group was (3.29 ± 0.59)× 10(6) at day one, (3.59 ± 0.63)× 10(7) at 2 weeks, (3.87 ± 0.56)× 10(8) at 4 weeks, and (4.12 ± 1.72)× 10(8) at 8 weeks. Compared to the control group, the treatment group showed an obvious decrease in the mean bioluminescence (photons/s) of the tumors and fewer metastases. Histological examination confirmed the presence of fewer metastases. DWI showed the apparent diffusion coefficient (ADC) value of the tumors; the mean ADC value was (0.9287 ± 0.04346)× 10(-3) mm(2)/s in the treated tumors and (0.7553 ± 0.01804)× 10(-3) mm(2)/s in the untreated tumors. The ADC value of tumors in the treatment group was significantly higher than the control tumors (P = 0.0013).</p><p><b>CONCLUSIONS</b>The growth and metastasis of MDA-MB-231 breast cancer may be inhibited by metformin. DWI and BLI have great potentials in the evaluation of the early response to metformin treatment. BLI has a high degree of sensitivity and is able to detect micrometastasis, thus can be used for identifying tumor metastasis in vivo.</p>
Subject(s)
Animals , Female , Mice , Breast Neoplasms , Drug Therapy , Pathology , Cell Line, Tumor , Cell Proliferation , Diffusion Magnetic Resonance Imaging , Luminescent Measurements , Metformin , Therapeutic Uses , Mice, Nude , Multimodal Imaging , Neoplasm MetastasisABSTRACT
In this study, the recombinant adenovirus (Ad) vector containing dual reporter gene [i.e. human transferrin receptor gene (TFRC) and firefly luciferase reporter gene] was constructed to provide a novel experimental tool for magnetic resonance (MR) and bioluminescence dual-modality molecular imaging. The cDNA of TFRC was amplified by polymerase chain reaction (PCR) and cloned into the multiple cloning site of pShuttle-CMV-CMV-Luciferase vector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luciferase vector and the adenoviral backbone vector (pAdeno) were subjected to homologous recombination. The correct recombinant plasmid was then transfected into 293 packaging cells to produce adenoviral particles and confirmed by PCR. After infection of human colorectal cancer LOVO cells with Ad-TFRC-Luciferase, the expressions of transferrin receptor (TfR) and luciferase protein were detected respectively by Western blotting and bioluminescence imaging in vitro. The results showed that TFRC gene was successfully inserted into the adenoviral shuttle vector carrying luciferase gene. DNA sequence analysis indicated that the TFRC gene sequence in the shuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmid was identified correct by restriction digestion. Ad-TFRC-Luciferase recombinant adenovirus was constructed successfully, and the virus titer was 1.6×10(10) pfu/mL. Forty-eight h after dual reporter gene transfection, the expressions of TfR and luciferase protein were increased significantly (P<0.01). It was concluded that the recombinant adenovirus vector with dual reporter gene was successfully established, which may be used for in vivo tracing target cells in multimodality imaging.
ABSTRACT
Objective To dynamically analyze the evolutionary process of cerebral edema absorption and the level of local iron in rats with intra-cerebral hematoma by high-field strength 7 Tesla MRI and explore the characteristics and mechanism of secondary injury after intra-cerebral hematoma.Methods Sixteen adult SD rats (about 150 g) were randomly divided into experimental group (n=10) and control group (n=6).Rat models in the experimental group were established by performing injection of 50 μL their own venous blood into their right caudate nucleus accurately. Rats in the control group were used normal saline,instead.After that,head MRI (T2 and T2-star scans) was performed 1,2,3,7 and 14 d after the injection; their imaging features were compared. Results Nine rats in the experimental group survived and 1 died after the operation; in the early days (within 3 d), the T2 weighing imaging showed that the time of relaxation surrounding the hematoma was longer than that in control group,suggesting that the zone of the edema surrounding the hematoma became more clearly.In the early days (within 3 d),T2-weighted imaging was clear,and the time of relaxation surrounding the hematoma increased rapidly,steadily improved 3 d after the operation and reached its peak level 7 dafter the operation; the damage area absorption decreased steadily but turned widening 3 d later and reached the peak 7 d later.T2-star value reached the peak rapidly 3 d after the operation,and then,moderated the downturn.The rats in the control group showed no obvious signal changes under MRI,except those with needle tract injury. Conclusion Secondary injury after intra-cerebral hemorrhage shows a rapidly injury progress in the short terrn at first,and then,has intensify again after a stable period; the local iron diffusion trend is synchronized to the secondary injury,suggesting that iron may play a key role in the mechanism of secondary brain edema.
