BACKGROUND: As a main gene engineering vector, adeno-associated virus (AAV) is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV-293 cells to meet the need of gene engineering.OBJECTIVE: To establish a culture method of AAV-293 cells in vitro.DESIGN: An opening study.SETTING: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate OMEM (H-DMEM) powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company.METHODS: This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not (observed by fluorescence inverted microscope) after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed.MAIN OUTCOME MEASURES: ① Morphological observation of AAV-293 cells; ② the growth curve; ③ the package of AAV.RESULTS: ① AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei appearances. ②The growth curve showed that the growing adaptive phase was the first day after AAV-293 cells were passaged, the actively growing phase was from the second day to the fifth day, and the growing platform phase was after the sixth day. ③ AAV-293 cells with green fluorescence observed by fluorescence inverted microscope, and cotransfection of the there AAV system plasmids was successful. AAV-293 cells gave out green fluorescence after infected with AAV supernatant, and AAV package succeeded.CONCLUSION: The culture method established by the authors in the experiment is simple and useful, and the cultured AAV-293 cells remain a good function of AAV package.

Objective To explore the method for measuring and calculating both absorbed dose and effective dose received in organ and tissues of occupational workers by using TLDs for the implantation of 125Ⅰ seed sources.Methods The experiments with 60Co γ-rays were carried out for the stability.A group of TLD chips was exposed to 125Ⅰ seed sources to establish standard dose curve for air kerma.During the 125Ⅰ seed implantation, the TLD chips were pasted to 13 locations like thyroid inside and outside the lead aprons worn by occupational workers to measure average absorbed dose and calculate the absorbed doses and effectives to organs and tissues.Results For 3 cases of prostate cancers with implantation of 125Ⅰ seeds, the worker's organs and tissues received the absorbed dose 0.02 -3.80 μ Gy and effective dose 0.06- 1.81 μSv outside lead aprons and the highest absorbed dose 2.35 μ Gy and effective 0.02 μSv inside lead aprons, respectively, with more than 65.9% of rays shielded.For 3 cases of brain cancers with implantation of 125Ⅰ seeds, the workers received the absorbed dose 0.23 - 11.31 μGy and effective dose 0.88 - 4.07 μSv outside lead aprons and the highest absorbed dose 2.22 μ Gy and effective dose 0.09 μSv inside lead aprons, respectively, with more than 54.5% of rays shielded.For 3 cases of lung cancers with implantation of 125Ⅰ seeds, the workers received the absorbed dose 0.03 - 14.78 μGy and effective dose 0.35 -7.59 μSv outside lead aprons and the highest absorbed dose 4.09 μGy and effective 0.22 μSv inside lead aprons, respectively, with more than 58.4% of rays shielded.For 2 cases of mediastinum cancers with implantation of 125Ⅰseeds, the workers received the absorbed dose 0.06 - 74.91 μGy and effective dose 0.83 - 17.96 μSv outside lead aprons and the highest absorbed dose 10.29 μGy and effective 0.5 μSv inside lead aprons, respectively, with more than 85% of rays shielded.For one case of ovary cancer with implantation of 125Ⅰ seeds, the worker received the absorbed dose 0.09 - 14.29 μGy and effective dose 2.40 - 4.50 μSv outside lead aprons and the highest absorbed dose 7.77 μGy and effective 0.12 μSv inside lead aprons, respectively, with more than 34% of rays shielded.For one case of eye cancer with implantation of 125Ⅰ seeds, the workers received the absorbed dose 2.2 -39.84 μGy and effective dose 4.48 - 10.06 μSv outside aprons and the highest absorbed dose 5.19 μGy and effective 0.16 μSv inside aprons, respectively, with more than 54.6 % of rays shielded.Conclusions The method of using TLDs to measure the doses to the occupational workers in the course of the implantation of 125Ⅰ seed sources is simple and easy to operate.It would be an effective approach to protecting medical workers in the case of brachytherapy.

Objective To observe the expression of chemokine (MCP-1) and chemokine receptor (CCR2) in bone marrow cells,bone marrow stromal cells of multiple myeloma (MM) patients.Methods 15 cases were diagnosed by domestic uniform standard for MM patients,7 cases of male,8 cases of female,age range from 38 to 67 years,mean age 53.7 years old.According to the Durie-Salmon staging system,patients were divided into Ⅰ (2 cases),Ⅱ (5 cases) and Ⅲ period(8 cases).Control group were from 10 cases of non-malignant blood disease patients.MCP-1,CCR2 expression were measured by flow cytometry.Results Almost 14 cases of bone marrow cells expressed MCP-1and CCR2 in MM patients,while in the control group,bone marrow cells almost did not express MCP-1and CCR2.Stromal cells had similar MCP-1and CCR2 expression profile (68.17 ％ vs 4.27 ％. P＜0.05).Tumor cells of MCP-1/CCR2 expression rates were 3.25 ％ and 32.76 ％. Compared MCP-1/ CCR2 expression of stromal cells and tumor cells with different stages of disease, the activated stage and the stable stage had similar level (68.71％ and 32.76 ％ vs 70.12 ％ and 53.39 ％. P＞0.05). Conclusion Most patients with MM bone marrow were expressed MCP-1and CCR2.MCP-1and CCR2 are the major MM cell surface expression of chemokine/receptor, which play important roles in the progress of.

Objective To verify the reliability of radiotherapy dosimetric parameters in reference and non-reference conditions using thermoluminescent dosimeters (TLDs).Methods Using the established TLD method,the dose variations with different radiation field sizes and 45 ° wedge plate were verified for 10 photon beams of 6 MV,together with dosimetric parameters at the point of maximum axial dose for 4 electron beams of 9 MeV under reference and non-reference conditions.Comparisons were made between TLD results and finger ionization chamber results.Results The average relative deviation,for 6 MV photon beams,between TLD results and finger ionization chamber measurements was 4.7％,within ± 7％ as required by the IAEA.The average relative deviation,for 9 MeV electron beam,between TLD results and plane parallel ionization chamber measurements was 2.4 ％,not beyond ± 5％ permitted by IAEA.Conclusions Using TLD method to verify the radiotherapy dosimetric parameters in reference and non-reference conditions was reliable,simple and feasible.

Objective To verify the methodology for auditing dosimetric parameters in reference and non-reference conditions with thermoluminescent dosimeters (TLDs).Methods Under reference and non-reference conditions,the established TLD methods were used to observe the absorbed dose variations with depth,SSD,field size and 45 wedges for 10 photon beams at 5 hospitals.Dosimetric parameters,including doses at Dmax points in axis,on 5 electron beams of 9 MeV were measured.The measurement results were compared between the TLDs and plane parallel ionization chambers.Results For 6 MV photon beams,the relative deviation of between finger ionization chamber method and TLD chips was in the range of-1.7％ to 5.4％ under on-axis non-reference conditions,and-6.3％ to-0.6％ under off-axis non-reference conditions,respectively,all within the range of ≤ ± 7％ as required by the IAEA.The relative deviation between plane parallel chamber and TLD method was-2.3％ to 3.7％,within ± 5％ as required by the IAEA.Conclusions It is convenient and feasible to use TLD method for quality audits of dosimetric parameters in radiotherapy.

The damage degree of neurons in perilesion at different time points was observed in order to explore the optimal operation occasion. Piglet lobar hematomas were produced by pressure-controlled infusions of 2.5 mL autonomous blood into the right frontal hemispheric white matter over 15 min, and the metabolic changes were ambulatorily detected with MRS at 3rd, 12th, 24th and 48th h after hematoma induction. Brain tissues of perihematoma were also obtained at different time points. The transcription level of Bax gene was detected by in situ hybridization and apoptosis by TUNEL technique, and the pathologic change of neurons was observed under an electron microscope. The results showed that the number of Bax positive cells reached the peak at 24 h (79.00 +/- 4.243/5 fields). There was no significant difference in A values between 3 h and 6 h, 12 h (P > 0.05), but there significant difference between 24 h and 3 h, 6 h, 12 h (P < 0.05). The number of apoptotic cells reached the peak at 24 h (P < 0.001), and there was no significant difference between 3 h and 6 h (P = 0.999). The area of the apoptotic cells showed no significant difference between 3 h and 6 h or among 3 h, 6 h and 6 h (P > 0.05). Lac peak mainly occurred at 24 h and 48 h, while on the healthy side, no Lac peak was detectable. The ratio of NAA/Cr presented a descent tendency, but there was no significant difference among the groups before 12 h (P > 0.05), there was very significant difference between 3, 6 and 24, 48 h (P < 0.01). Under electronic microscopy, the neuronal damage surrounding hematoma in 3 to 6 h was milder than in 24 h to 48 h. It was concluded that the secondary apoptosis, damage and metabolic disturbance of the neurons surrounding hematoma was milder in 3-6 h in acute intracerebral hemorrhage, while obviously aggravated in 24-48 h. An effective intervention is needed to reduce secondary damage as soon as possible.
Brain/*pathology ; Cerebral Hemorrhage/*pathology ; Hematoma/*pathology ; Magnetic Resonance Imaging ; Neurons/pathology ; Random Allocation ; Swine ; Swine, Miniature ; Time Factors

Objective To observe senile plaque and iron deposition in cortex and hippocampus of the Alzheimer's disease ( AD ) transgenic mice and investigate their influence on T2 relaxation time.Method All AD transgeic mice were divided into three groups: young group(2,4 months), adult group (6,8,10 months), old group (12,14,16 months), and C57BL/6J mice were as control and were scanned in order by using 4.7 T MR system.Regions of interest (ROI) corresponding to cortex, hippocampus,thalamus, striatum were manually drawn on MR images and T2 MR relaxation times of each ROI were calculated.After MR scan, these mice were decapitated and stained for iron and senile palques.The number of plaque and iron, plaque burden, iron load in cortex and hippocampus were acquired using image pro plus software.Result T2 relaxation times of each group were as following: wild type ( cortex (49.5 ± 2.1 ) ms,hippocampus (51.6 ± 1.1 ) ms ); young ( cortex ( 49.7 ± 0.5 ) ms, hippocampus ( 50.7 ± 0.7 ) ms ); adult (cortex(47.2 ±0.8) ms, hippocampus(47.7 ±0.9) ms) and old (cortex(44.6 ±0.8) ms, hippocampus (45.3 ±0.4)ms).T2 relaxation times in cortex and hippocampus of each group had statistical differences ( cortex F = 18.620, P ＜ 0.01; hippocampus F = 67.925, P ＜ 0.01 ); Compared with young group and wild type mice, T2 relaxation times in corex and hippocampus of adult group mice were decreased significantly.At the same time, T2 relaxation times in old group mice were reduced compared with adult group ( Adult vs young: cortex q =4.284, P ＜0.01, hippocampus q =7.902, P ＜0.01; adult vs wild type: cortex q =4.424, P＜0.05, hippocampus q = 11.450, P ＜0.01; old w adult: cortex q =4.812, P ＜0.01,hippocampus q = 7.034, P ＜ 0.01 ).Histochemical staining for senile plaques found that senile plaques was deposited as early as 4 month.Iron deposition in hippocampus and cortex were detected by perl-DAB as early as 6 months of age, and there was an overall increase in number and load of plaques and iron with age.A positive correlation was observed between plaque burden and iron load ( r = 0.931, P ＜ 0.01 ).At the same time, plaque burden and iron load were negatively correlated with T2 relaxation times ( plaque burden and T2 relaxation times r = - 0.884, P ＜ 0.01; iron load and T2 relaxation times r = - 0.827, P ＜ 0.01 ).Conclusion The changes of T2 relaxation time in AD transgenic mice are attributed to iron and senile plaques.MR T2 relaxation time is a sensitive marker to diagnosis for AD and screen antidementia drugs.

Objective To investigate the perihematomal changes of hyperacute parenchymal hematomas and the clinical value by MRI. Methods Multi-sequence MRI was performed on 4 hematomas in vitro and on 15 pigs with lobar intracerebral hemorrhage (ICH) for about 30～60 min and 3 h respectively. The integrity of blood-brain barrier (BBB) in ICH pigs was assessed by electron microscopy and Evan's blue dye technique. MR scanning was performed on 2 ICH patients proved by CT for 4 and 9 h after onsets. Results FLAIR and T 2-weighted images showed hyperintensity signal around the hematomas in vitro and in pigs with ICH within 1 h, and more obviously at 3 h. When the gelose cavity was cut, plasma was seen around the clot. The perihematomal ADC values of the pigs increased both within 1 and at 3 h after ICH. However, the BBB was intact at 3 h, which was proved by electron microscopy and Evan's blue dye technique. Water-like intensity signal was observed around the hematomas in two patients with acute ICH. Conclusion The perihematomal changes of hyperacute ICH observed on MRI are resulted from the blood clot contraction and the serum formation and extravasation, but not real cerebral edema.

Objective To develop specific targeted magnetic biomarkers which can selectively mark the senile plaques in Alzheimer' s disease (AD) and verify its feasibility and validity.Methods Aβ1-40 peptide and Tat-PTD ( Tat-protein transduction domain) was binded with dextran-coated ultrasmall superparamagnetic iron oxide ( USPIO) particles.Visualization of plaques in vivo in Alzheimer transgenic mice was investigated at 7.0 Tesla using T2 sequences after intravenous administration of the targeted nanoiron contrast agent and verified by histological staining.Results The targeted nano-iron contrast agent could enter the cultured neural stem cells,and was able to accelerate T2 relaxation rates of water protons in the cells and negatively reinforce the T2 signal intensity in the labeled cells.Plaques were specifically detected in vivo by magnetic resonance imaging ( MRI) and correlated well with histological staining after injection of nano-iron contrast agent into the APP/PS1 mice.Conclusion The targeted nano-iron contrast agent has the ability of selectively labeling the senile plaques in AD brain tissues in vivo,which might enable the early detection of plaques by MRI and can be further applied in the studies of early diagnosis of AD.

Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegenerative diseases, we packed recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-odc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful, rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.