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 investigate the variations of the spatial position and overlap ratio for gross tumor volume (respiratory phase 50％) (GTV50) and internal gross tumor volume (IGTV) of primary thoracic esophageal cancer during conventional fractionated radiotherapy based on repeated four-dimensional computed tomography (4DCT) scans.Methods Thirty-three patients with thoracic esophageal cancer underwent contrast-enhanced 4DCT scans before radiotherapy and at the 10th and 20th fractions of radiotherapy.Scans were registered to the baseline 4DCT scan using bony landmarks.The GTV50 was delineated by the same radiotherapist on each 4DCT imaging data set,and the IGTV was constructed accordingly.The target volume,degree of inclusion (DI),and matching index (MI) were compared in different phases.Results The volumes of GTV50 and IGTV decreased along with treatment course.No significant changes in the centroid position were observed for the GTV50 and IGTV.The median DIs of the target volumes at the 10th and 20th fractions in the original target volume were 0.75 and 0.63(P =0.000) for GTV50 and were 0.79 and 0.66(P=0.000) for IGTV,while the median MIs were 0.61 and 0.56(P=0.002) for GTV50 and were 0.68 and 0.58 (P =0.005) for IGTV.A positive correlation between the variation of volume ratio and the variation of DI was found for GTV50 and IGTV (r =0.632,r =0.783),and the variation of volume ratio was also positively correlated with the variation of MI (r =0.387,r =0.483) ;the 3D vector was negatively correlated with the MI (r =-0.455,r =-0.438).Conclusions During conventional fractionated radiotherapy,the variation of spatial position is less than 0.8 cm for GTV50 and IGTV of primary thoracic esophageal cancer,and the decline of the target leads to varying degrees of decreases in DI and the MI.

In order to investigate the neuroprotective effects of cyclin-dependent kinase-5 (cdk-5) inhibition in mice with Niemann-Pick disease type C (NPC) (npc(-/-)), recombinant adeno-associated virus (rAAV) carrying the small interfering RNA (siRNA) specific for cdk-5 gene was injected into 3-day-old npc(-/-) mice intracerebroventricularly. The rAAV-GFP-injected age-matched npc(-/-) mice and non-surgery age-matched npc(-/-) mice were employed as controls (n=6-10/group). From the 4th to 8th week after the treatment, mice were weighed, and evaluated for limb motor activity by using the coat hanger test once a week. Eight-week-old npc(-/-) mice were sacrificed by decapitation, and brains were quickly dissected and halved sagittally. Immunohistochemistry, Western blotting, and HE staining were used to evaluate the neuropathology in npc(-/-) mice. The results showed that rAAV-cdk-5-siRNA-GFP significantly reduced the number of axonal spheroids, delayed the death of Purkinje neurons, ameliorated motor defects in npc(-/-) mice, and significantly attenuated the hyperphosphorylation of tau proteins. These data suggested that inhibition of cdk-5 activity has neuroprotective effect on neurons in NPC mice.

Objective To compare volumetric size, conformity index (CI), degree of inclusion (DI) of internal gross target volumes (IGTV) delineated on 4D-CT-MIP and PET-CT images for primary thoracic esophageal cancer. Methods Fifteen patients with thoracic esophageal cancer sequentially underwent enhanced 3D-CT, 4D-CT and PET-CT simulation scans. IGTVMIP was obtained by contouring on 4D-CT maximum intensity projection ( MIP). The PET contours were determined with nine different threshold methods (SUV≥2?0, 2?5, 3?0, 3?5), the percentages of the SUVmax(≥20%, 25%, 30%, 35%, 40%) and manual contours. The differences in size, conformity index (CI), degree of inclusion ( DI) of different volumes were compared. Results The volume ratios ( VRs) of IGTVPET2. 5 to IGTVMIP , IGTVPET20% to IGTVMIP, IGTVPETMAN to IGTVMIP were 0?86, 0?88, 1?06, respectively, which approached closest to 1. The CIs of IGTVPET2?0,IGTVPET2.5,IGTVPET20%,IGTVPETMAN and IGTVMIP which were 0?55, 0?56, 0?56, 0?54,0?55, respectively, were significantly larger than other CIs of IGTVPET and IGTVMIP (Z= -3?408-2?215,P <0?05). There were no statistical significance in the DIs of IGTVMIP and IGTVPET2.5,IGTVMIP and IGTVPET20%, IGTVMIP and IGTVPETMAN(0?77,0?82,0?71,0?67, 0?68,0?82,P>0?05). Conclusions The targets delineated based on SUV threshold setting of≥2?5, 20% of the SUVmax and manual contours on PET images correspond better with the target delineated on maximum intensity projection of 4D-CT images than other SUV thresholding methods.

Objective To investigate the variations of the spatial position and overlap ratio of the internal target volume (ITV) and planning target volume (PTV) of primary thoracic esophagus carcinoma using repeated four-dimensional computed tomography (4D-CT) scanning during conventional fractionated radiotherapy.Methods Thirty patients with thoracic esophageal carcinoma were included whose 4D-CT scans were conducted before radiotherapy and between every ten fractions.The gross tumor volumes (GTVs) were delineated by the same radiation oncologist on each 4D-CT image phase,and the ITV and PTV were constructed afterwards.Results No significant difference of the isocenters was observed for the ITV and PTV during the treatment course,yet both the volumes of the ITV and PTV decreased.The median DI (the degree of inclusion) of the target acquired subsequently in the original target were 0.85,0.77 (Z=-3.10,P ＜0.05) for ITV and 0.86、0.82(Z =-2.49,P ＜0.05)for PTV respectively during entire treatment.The variation of volume ratio correlated strongly with the variation of DI (the DI of the target acquired subsequent in the original target) (rITV =0.71,rPTV =0.77,P ＜0.05).The variation of volume ratio and the variation of the matching index (MI) were positively correlated (rITV =0.47,rPTV =0.59,P ＜ 0.05).The 3D vectors of ITV and PTV motions were negatively correlated with the corresponding MI (rITV =--0.52,rPTV =-0.36,P ＜ 0.05).If the initial PTV was used for treatment planning,8.80％ and 6.37％ of the target volume would be missed at the tenth and twentieth fraction (Z =-0.55,P ＞ 0.05),respectively.In the meanwhile,11.45％ and 18.49％ of the normal tissues would be wrongly irradiated at the corresponding time points (Z =-2.49,P ＜ 0.05).Conclusions The variations of the spatial position of all targets were all less than 0.6 cm.The DI and the MI of the target decrease by various degrees during the treatment course,which lead to target mispositioning and normal tissue irradiation at different levels.

Objective To analyze the volume and position of the gross tumor volumes (GTV) in primary esophageal cancer based on contrast-enhanced three-dimensional (3D),four-dimensional (4D) and cone beam (CB) computed tomography (CT).Methods A total of thirty-four patients underwent 3D-CT and 4D-CT simulation scans for computer treatment plan and contrast-enhanced CBCT scans were conducted prior to the first treatment.GTV3D,GTV4D50,internal GTVMIP (IGTVMIP) and internal GTVCBCT (IGTVCBCT) were delineated on 3D-CT,4D-CT50 (the end expiratory phase),4D-CTMIP (the maximum intensity projection),and CBCT datasets,respectively.The IGTV10 was defined as 10 respiratory phases GTVs in 4D-CT.To evaluate the difference in position,volume and the volumes encompassed characteristic.Results The significant difference was observed in the volumes [IGTV10 ＞ (IGTVCBCT or IGTVMIP) ＞ (GTV3D or GTV4D50)] regardless of the tumor location.Regarding IGTV10 as the standard volume,the underestimations or overestimations between IGTV10 and IGTVCBCT were larger than that of between IGTV10 and JGTVMIP (t =-8.294--3.192,P ＜ 0.05).However,there was no significant difference between the areas of IGTV10 which excluded in IGTVCBCT and IGTV3D (P ＞ 0.05).The GTV4D50/ IGTVCBCT ratio for upper esophageal tumors was negatively correlated to motion vector (r =-0.756,P ＜ 0.05).The centroid coordinates of IGTVCBCT in AP direction were significantly different from the test volumes (GTV3D,GTV4D50,IGTVMIP and IGTV10) (t =-3.559--2.435,P ＜ 0.05).The IGTV10/IGTVCBCT ratio was positively correlated to motion vector (r =0.695,P ＜ 0.05) for middle esophageal tumors.The centroid coordinates of IGTVCBCT were significantly different IGTV10 (t =2.201,P ＜0.05) in AP direction.For distal esophageal tumors,the significant difference was observed in the centroid coordinate between IGTVcBcT and IGTVMIP (t =-2.365,P ＜ 0.05) in LR direction.The percentage of IGTV10 excluded the IGTVcBcT were significantly correlated to the motion vector (r =0.540,0.678,P ＜ 0.05) for both middle and distal esophageal tumors.The mean MI value of IGTVCBCT to the other four test volumes ranged from 0.65 to 0.72.Conclusions CBCT has much motion information than 3D-CT but less than IGTV10.CBCT was similar to MIP images based on respiration motion.However,the target motion information encompassed in CBCT and MIP images cannot be exchanged to each other.

Objective To investigate the correlations between the displacements of surgical clips,skin markers and the gross tumor volume (GTV) delineated by surgical clip and seroma based on fourdimensional computed tomography (4DCT) during normal breathing.Methods On each of the 10 respiratory phases of 4DCT with 15 patients of breast cancer,the surgical cavity,which was defined as GTV and formed by both surgical clips and seroma,all surgical clips in the cavity,and skin markers on the skin were delineated by the same radiation oncologist.The center displacements of GTV,selected boundary surgical clips and skin markers in the left-right (LR),anterior-posterior (AP),and superior-inferior (SI) directions were recorded and analyzed by Pearson method.Results In the AP direction,there was a positive correlation between the displacements of the GTV and the inner clip (r =0.643,P =O.013).In the SI direction,the displacement of GTV was positively correlated with those of the lower clip and the skin marker at anterior body midline (r =0.857 and 0.643,P =0.002 and 0.013).In the LR and AP directions,there was a positive correlation between the displacements of the outer clip and the ipsilateral skin marker (r =0.757 and 0.697,P =0.001 and 0.025),but they were negatively correlated with each other in the SI direction (r =-0.647,P =0.043).In the AP direction,there was a positive correlation between the displacements of the inner clip and the skin marker at the anterior body midline (r =0.738,P =0.015).Conclusions The correlations between the displacement of GTV delineated by surgical clips and seroma and the displacements of surgical slips and skin markers vary under different conditions,and so does the correlation between the displacements of surgical clips and skin markers.

BACKGROUND: The blood levels of specific markers of platelet activation, such as platelet granule membrane protein (GMP-140) and tumor necrosis factor alpha (TNF-α) are very low in healthy individuals, while the plasma levels of them in patients with cerebral infarction increase. Is the protection of brain hydrolysate injection correlated with the phenomenon?OBJECTIVE: In this study, the plasma levels of GMP-140 and TNF-α in patients with acute stroke were measured, the brain protection of brain protein hydrolysate injection on patients with ischemic stroke were investigated, and were compared with the therapeutic effect of compound danshen injection.DESIGN: It was designed for case study.SETTING: This study was conducted at the Medical Department of the General Hospital of Pingdingshan Coal Co. Ltd and the Neurology Department of the Second Hospital Affiliated to Zhengzhou University.PARTICIPANTS: From January 2001 to October 2003, 144 inpatients with hypertension and acute stroke in the Medicine Department of the General Hospital of Pingdingshan Coal Co. Ltd were selected and divided into 2 groups, as experiment group containing 72 cases, 47 males and 25females, with an age from 42 to 90 and in average of (69±11) years old and control group containing 72 cases, 49 males and 23 females, with an age from 37 to 85 and in average of (68±10) years old.METHODS: All the patients in these two groups underwent oxygen inhalation therapy, antihypeetensive therapy, dehydration therapy and anticoagulation therapy. Patients in control group were coadministered 500 mL compound salvia miltiorrhiza injection QD once a day, with a 14-day course of therapy. Patients in experiment group were treated with 500 mL compound salvia miltiorrhiza injection QD and 20 mL protein hydrolysate European stroke scale (ESS), from 0 (worst possible health status) to 100(best possible health status), were used to evaluate the recovery status of (from 24 hours to 72 hours) and 3 weeks post-treatment, 5 mL blood samples were obtained from antecubital veins, then plasma levels of GMP-140and TNF-α were quantified using an RIA (radioimmunoassay) and the changes in neural function before and after brain protein hydrolysate injection were evaluated.MAIN OUTCOME MEASURES: Before treatment and at 3 weeks postwere quantified using an RIA.RESULTS: All the 144 patients entered the statistical analysis procedure.ESS were significantly higher than the pre-treatment scores [Experiment groups: 79.95±18.64 and 59.65±19.87; Control group: (74.66±15.88) and (61.25±18.68), (t=2.678-4.351, P ＜ 0.01). The post-treatment scores of ESS in experiment group were higher than those in control group (t=2.016-2.158,groups, the post-treatment outcomes were significantly lower than the pre-treatment outcomes [Experiment group: (22.12±9.52) μg/L and (50.41±22.35) μg/L, (1.05±0.24) μg/L and (1.62±0.50) μg/L; Control group: (26.66±8.22) μg/L and (48.63±21.54) μg/L, (1.35±0.44) μg/L and (1.66±0.48) μg/L; (t=2.678-4.351, P ＜ 0.001)]. And the post-treatment levels of the two markers were lower in experiment group than those in control group (t=2.016-2.158, P ＜ 0.05).CONCLUSION: Brain protein hydrolysate injection can significantly decrease the plasma levels of GMP-140 and TNF-α in patients with acute stroke, and it is capable of increasing the ESS scores and improving the impaired neural functions greatly.

Introduction to the theories on improving coordination level of the new rural cooperative medical system,including the risk theory,great number rule,fair theory,demand theory and supply theory which are cornerstones of enhancing the NRCMS improving the pooling level.The paper also probed into the practices of improving the pooling level of the NRCMS,including the models of high level,middle level and low level pooling.These theories and practices can help the localities better design and manage the pooling level of the NRCMS.

Objective To compare the normal thickness of the esophageal wall measured by contrast?enhanced three?dimensional ( 3DCT ) , four?dimensional ( 4DCT ) , and cone beam computed tomography ( CBCT) ,and to provide a basis for target volume delineation in esophageal cancer. Methods From 2009 to 2016,thoracic contrast?enhanced 3DCT and 4DCT simulations were performed in 50 patients with lung cancer or metastatic lung cancer. Contrast?enhanced CBCT scans were acquired during the first three?dimensional conformal radiotherapy. The normal esophageal wall was contoured on 3DCT images, the end?exhalation phase of 4DCT images ( 4DCT50 ) , the maximum intensity projection of 4DCT images (4DCTMIP),and CBCT images. The wall thickness was measured on each segment and the average thickness of esophageal wall was obtained. Comparison of the thickness of a fixed segment of esophageal wall between different CT images was made by paired t test. Comparison of thickness on the same type of CT images between different segments of esophageal wall was made by one?way analysis of variance. Results For the thoracic and intra?abdominal segments,there was no significant difference in the thickness of esophageal wall between 3DCT and 4DCT50 images ( P= 0?056?0?550 );however, the thickness of esophageal wall was significantly smaller on 3DCT images than on 4DCTMIP or CBCT images (P=0?000?0?004).For the upper and middle thoracic segments,the thickness of esophageal wall was significantly larger on CBCT images than on 4DCTMIP images ( P= 0?008, P= 0?001 ) . On 3DCT, 4DCT50 , and 4DCTMIP images, the thickness of esophageal wall was significantly larger in the lower thoracic segment than in the upper or middle thoracic segments ( P=0.008~0?041);the intra?abdominal segment had a significantly larger thickness of esophageal wall than the thoracic segments ( all P=0?000 ) . There was no significant difference in wall thickness on CBCT images between three thoracic segments ( P=0.088~0?945) . Conclusions A uniform criterion can be adopted to judge the normal thickness of esophageal wall in gross tumor volume ( GTV ) delineation on 3DCT and 4DCT50 images for thoracic esophageal cancer. However,caution should be taken when 5 mm is used as a criterion for normal thickness of esophageal wall in GTV delineation on 4DCTMIP and CBCT images.