Objective To construct the expression box of fusion gene NT4-p53(N15)-Ant.Methods The gene of p53(N15)-Ant was obtained by PCR of two primers templating with each other and T-vector cloning method.The positive clone was identified and analyzed by the restriction enzymes and sequencing respectively.After digested with restriction enzyme,the interest gene of p53(N15)-Ant was subcloned into the plasmid pBV220/NT4.Results The gene of p53(N15)-Ant was confirmed by the digestion of restriction enzyme and sequencing.The recombinant plasmid pBV220/NT4p53(N15)Ant was identified by the digestion of restriction enzyme and agarose gel electrophoresis and the results conformed theoretical values.Conclusion The plasmid pBV220 containing the expression box of NT4-p53(N15)-Ant was successfully constructed by molecular cloning and recombination techniques in vitro,which will guide further study on gene therapy of cancer.

AIM: To investigate the relationship between NF-?B activation and intimal hyperplasia.METHODS: The protein extract from aorta was used to detect the expression of NF-?B p65,I?B?,ICAM-1 and COX-2 by Western blotting,and detection of threonine phosphorylation of p65 was performed by immunoprecipitation analysis.RESULTS: The level of p65 reached a maximum level at day 7,and then decreased significantly,which was higher than that in the control,in both cellular extract and nuclear extract at day 21 after balloon injury.The p65 level in cytoplasm had no obvious changes at different time points after balloon injury.The threonine phosphorylation of NF-?B p65 was negative correlated with the nuclear translocation of NF-?B.I?B? level showed a 15% decrease at day 1 after balloon injury,compared with control group,then returned at day 14,and reached to normal level at day 21.However,the expression of ICAM-1 and COX-2 was maximal at day 14,and then declined but higher than that in control group at day 21 after balloon injury.CONCLUSION: The activation of NF-?B p65 and the expression of ICAM-1 and COX-2 are parallel to the neointimal thickening after balloon injury.

Objective:To construct a recombinant adenovirus vector harboring fusion gene NT4-p53(N15)-Ant,laying a foundation for gene therapy research of malignant tumors.Methods:The p53(N15)-Ant gene was obtained by T-vector method and was inserted in pBV220/NT4 vector after digested with restriction enzyme.The fusion gene of NT4-p53(N15)-Ant was subcloned into the shuttle plasmid of adenovirus;the products were cotransfered into HEK-293 cell line with helper plasmid PJM17.The recombinant adenovirus was produced by homologous recombination of above 2 plasmids in HEK-293 cells and its titer was measured by plaque-forming.The expression of Ad.NT4p53Ant in transfected 293 cells was confirmed by reverse transcription polymerase chain reaction(RT-PCR)procedure.The effect of Ad.NT4p53Ant on HepG2 cell line was measured by a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Results:The p53(N15)-Ant gene was confirmed by restriction enzyme digestion and DNA sequencing.High titer of recombinant adenovirus was obtained by homologous recombination in HEK-293 cells(1?10 11pfu/ml).The expression of NT4-p53(N15)-Ant gene in 293 cells was confirmed by RT-PCR.Ad.NT4p53Ant had strong killing effect on HepG2 cells.Compared with Ad.GFP,Ad.NT4p53Ant significantly decreased the survival rate of HepG2 cells.Conclusion:The recombinant adenovirus vector encoding gene NT4-p53(N15)-Ant has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques,laying a foundation for further research of gene therapy of cancer.

ObjectiveTo evaluate the inter-and intra-fractional clinical target volume (CTV) positioning errors of patients receiving postoperative simplified intensity-modulated radiotherapy (SIMRT) using cone beam computed tomography (CBCT).MethodsTwelve patients with liver tumors underwent postoperative SIMRT.CBCT images were acquired before and after the treatment.The clipbox volume for registration included the fiducial markers in the tumor bed and excluded the ribs and vertebral bodies.If any translational parameter of setup error before treatment exceeded 3 mm or rotational parameter exceeded 3°,the treatment couch was adjusted and a verification CBCT was acquired to assess residual setup error.Automatic bone match was used.A total of 214 acquisitions of CBCTs in 111 groups were analyzed.Inter-fractional translational CTV positioning errors in left-right (x),superior-inferior (y) and anterior-posterior (z) axis were calculated in 111 groups,and intra-fractional translational CTV positioning errors in 70 groups.Clinical to planning target volume (PTV) margins were calculated according to the formula:margin =2.0 ∑ + 0.7σ ( ∑ is systematic error,σ is random error).ResultsInter-fractional translational CTV positioning errors in x,y and z axis were -0.03 mm,-0.43 mm,1.02 mm,with systematic error ( ∑ ) of 1.50 mm,5.89 mm,1.97 mm,and random error (σ) of 1.76 mm,4.13 mm,2.42 mm,respectively.Intra-fractional translational CTV positioning errors in the x,y,z axis were 0.04 mm,0.86 mm,- 0.46mm,with systematic error (∑) of 0.46 mm,1.14 mm,0.31 mm,and random error (σ) of 0.95 mm,1.38 mm,0.91 mm,respectively.The calculate CTV to PTV margins were 4.5 mm,15.0 mm,5.8 mm in the x,y,z axis,respectively.ConclusionsThe CTV errors were inevitable when patients with liver tumors received SIMRT.Fiducial markers placed in tumor bed during operation were helpful for accurate positioning error analysis.

BACKGROUND: Parkinson disease(PD) is a series of clinical symptom induced by decreased dopamine (DA) in the striatum due to nigral dopaminergic neuronal degeneration. The intracerebral transplantation of secretory DA can reverse or improve the symptoms to a certain extent, but immunologic rejection is still existed.OBJECTIVE: To probe into cell transplantation with immunoisolation in treatment of in rats without application of immunosuppress and observe its mechanical intensity and the biocompatibility of microcapsule .DESIGN: Randomized controlled experiment was designed.SETTING: Biomedical Material Engineering Group, Dalian Institute of ChemicalPhysics , Chinese Academy of Sciences, and Department of Neurology, Second Hospital of Jilin University.MATERIALS: The experiment was performed in Animal Experimental Center of Second Hospital of Jilin University from August 2003 to February 2004, in which, 40 male Wistar rats were employed. PC12 cell was provided from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences.METHODS: 6-hydroxydopamine solution was infused in the striatum to prepare animal model of Parkinson disease. Twenty-five rats of those had been prepared successfully and were randomized into microencapsulated cell transplantation group (12 rats), in which, 25 μL cell-loading sodium alginate-chitosan-solium alginate(ACA)microencapsul suspension (equal to 2.5×104 cells) was injected stereotaxically on two points of the right (affected side) striatum of animal model; non-microencapsulated cell transplantation group (7 rats), in which, 25 μL PC12 cell suspension (equal to 5×104cells) was injected; and empty microcapsul transplantation group (6 rats),in which, 25 μL empty microcapsules suspension was injected . On the 7th day after transplantation, in every group, apomorphine (APO) prepared with saline solution was injected (0.05 mg/kg) subcutaneously in the neck; afterwards, the revolving behavior was recorded for each rat, once per week,totally for 12 weeks. In the 12th week after operation, the rats were sacrificed with anesthesia. The brain tissue was collected for pathological observation and microcapsule were retrieved to evaluation of biocompatibility and immunoisolation.numbers before and after transplantation of each group.RESULTS:Twenty-five rats entered result analysis and the rest was sule: the retrieved ACA microcapsule was integrative in morphology,munoisolation of microcapsule: microencapsuled PC12 cells were prolifercycles before and after transplantation of each group: the records of lateral revolving of rats in every group before transplantation were not significantly different (P ＞ 0.05). In microencapsuled cell transplantation group, 2weeks later, the average number of revolving was significantly lower than that before the transplantation, or even the revolving stopped; the improved symptoms were maintained till the 12th week after transplantation. In nonmicroencapsulated cell transplantation group, the average revolving number was also significantly lower than that before the transplantation, but that on the 8th and 12th weeks was in tendency of increase, without obvious change compared with that before the transplantation (P ＞ 0.05). The revolving number before and after transplantation in non-microencapsulated transplantation group was similar[(10.5±1.4), (10.5±1.3) cyclos/min, P ＞ 0.05].microcapsule provides immune protection. The grafted encapsulated PC12cells survive for along term in the brain of rats with PD, maintain continuously the normal physiological function and improve the symptoms of PD by synthesizing and releasing DA.

ObjectiveTo investigate the prognostic value of fluid responsiveness in patients with acute respiratory stress syndrome (ARDS).Methods Fifty-nine mechanically ventilated patients with ARDS were enrolled.Their stroke volume variations (SVV) were detected by pulse contour continuous cardiac output (PiCCO) analysis device,and then the patients were subsequently divided into fluid responsive group (SVV≥15％) and fluid non-responsive group (SVV ＜ 15％ ).The differences in 28-day survival,length of ICU stay and duration of mechanical ventilation between the 2 groups were compared.Thereafter,the 28-day survival of patients was analyzed by Kaplain-Meier survival model and the relationship between SVV and mortality within 28 days was analyzed by logistic regression model.Results In comparison with fluid non-responsive group,the 28-day survival of fluid responsive group was significantly increased (85.3％ vs.56.0％,P =0.012),the length of ICU stay was significantly shortened [( 13.1 ±5.2 ) d vs. (21.6±9.0) d,P=0.008],and the duration of mechanical ventilation was significantly shortened as well [( 11.4 ± 5.3 ) d vs.( 18.3 ± 4.9) d,P =0.022] ; Logistic analysis revealed that SVV ＜ 15％ increased the risk of 28-day mortality ( OR =4.82 ; 95％ CI:2.67 ～ 11.71,P =0.009 ).ConclusionsSVV-based fluid responsiveness can be served as a prognostic predictor for ventilated patients with ARDS.

Objective To identify high-risk group among gastric cancer patients treated with curative resection and more than D1 dissection, and investigate the indications for proper adjuvant therapy.Methods 297 patients who met the following enrolled criteria were retrospectively analyzed:treated between January 2002 and December 2004, primary gastric or gastroesophageal cancer, underwent curative gastrectomy and more than D1 lymphadenectomy, pathologically staged as T3-4N0-1M0,or TxN2-3M0.The overall survival (OS), disease-free survival (DFS), local-regional recurrence-free survival (LRFS) and distant metastasis-free survival (DMFS) were calculated, and possible prognostic factors were analyzed.Results The median follow-up time was 61 months.The follow-up rate was 92.3%.The 5-year OS, DFS, LRFS and DMFS were 57.9%, 52.2%, 70.6% and 71.7%, respectively.Four independent prognostic variables identified for OS, DFS, LRFS and DMFS using multivariate analysis were Borrmann type (Ⅰ+Ⅱ/Ⅲ+Ⅳ), total number of dissected lymph nodes (>18/≤18), number of positive lymph nodes (0-3/≥4), and 6th AJCC TNM stage (Ⅱ+Ⅲ a/Ⅲ b+ⅣM0)(χ2=3.94-16.34,P<0.05).If one unfavorable prognostic factor was scored as 1, according to the total scores of the four prognostic factors, four risk groups were generated as low (score:0), low-intermediate (score:1), high-intermediate (score:2) and high risk group (score:3 or 4).The 5-year OS, DFS, LRFS and DMFS were 85.7%, 61.0%, 58.6% and 38.6%(χ2=31.20,P<0.01) in low risk group, 85.2%, 61.3%, 48.1% and 31.8%(χ2=31.88,P<0.01) in low-intermediate risk group, 94.4%, 77.8%, 64.4% and 57.2%(χ2=18.36,P<0.01) in high-intermediate risk group and 87.9%, 75.0%, 74.2% and 55.5%(χ2=19.30,P<0.01) in high risk group.Conclusions Even with R0 resection and more than D1 lymphadenectomy, the outcome was poor for gastric cancer patients with two or more unfavorable prognostic factors.Prospective study is warranted to evaluate the efficacy of adjuvant concurrent chemoradiotherapy for this group of patients.

BACKGROUND: The transplantation of microencapsulated cell is becoming a hotspot modality in the therapy of Parkinson disease (PD). The application of Alginate-polysysine-alginate (APA) is currently limited due to fragility and pericystic fibrosis although it has been used in clinic. In this study, the native Alginate-chitosan-alginate(ACA)microencapsulated pheochromocytoma cells (PC12 cells) are transplanted into the region of corpus striatum in the injured side of the brain of the PD rat model, the functional recovery of rotational behavior and pathological changes are also observed in the control, sham and treated groups.OBJECTIVE: To observe whether the transplantation of ACA microencapsulated PC12 cells into the brain can improve the rotational behavior in the rat model of PD.DESIGN: Randomized controlled experiment.SETTING: Dalian Research Institute of Physiochemistry, Chinese Academy of Sciences.MATERIALS: Totally 40 adult male Wistar rats with body mass of(220±10) g, ACA microcapsule and PC12 cells were used in this study.METHODS: The experiment was carried out in the animal experimental laboratory of Second Hospital, Jilin University and Dalian Research Institute of Physicochemistry, Chinese Academy of Sciences between May and December 2002. Native ACA were used to microencapsulate the PC12cells. These rats were randomly divided into the following three groups,treated group (10 rats received microencapsulated PC12 cell transplantation), control group (7 rats received unencapsulated PC12 cell transplantation) and sham group (6 rats received empty microencapsule transplantation). The transplantation site was the region of corpus striatum in the injured side of brain. The difference of rotational behavior included by apomorphine was compared before and after the transplantation in these rats,the morphological changes of the transplanted microcapsules and activity of the microencapsulated cells were also detected.MAIN OUTCOME MEASURES: ①Rotational behavior of the rats before and after transplantation. ②Pathological change in the regions of substantia nigra and corpus striatum. ③ The integrality of retrieved microencapsule and the bioactivity of retrieved PC12 cells.RESULTS: ① At the 4th week of transplantation, rotational behavior was significantly decreased in the encapsulated PC12 cells treated group compared with that of the groups received empty microencapsules transplantation [(6.9±2.8),(10.5±1.6) r/min, P ＜ 0.05].Tbis behavioral improvement could last at least three months. Although the unencapsulated PC12 cells also can improve the rotational behavior compared with before transplantation[(5.6±l.1 ), (9.5±1.5) r/min, P ＜ 0.05], which only lasted two months and fetal tumor formed in the skull of some rats. There was no significant difference in rotational behavior of the rats before and after transplantation in the empty microencapsule transplantation group. PC12 cells of retrieved microencapsulate grew well after re-culture, and have bioactivity.CONCLUSION: Transplantation of ACA microencapsulated PC12 cells into the brain can improve can improve the rotational behavior of rat PD model induced by apomorphine. ACA microcapsule can both isolate the host's immune system effectively and prevent the formation of tumor, and have a promising application in clinic.

Objective To evaluate the role of postmastectomy radiotherapy in four subgroups of high-risk breast cancer patients, who were grouped by the status of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 (Her-2). Methods A total of 437 invasive breast cancer patients with T3-4N1 or N2-3 and available immunohistochemistry results of ER, PR and Her-2 were retrospectively analyzed. Patients were classified into 4 subgroups according to hormone receptors (ER or PR, Rec) and Her-2 status:Rec-/Her-2-(triple negative), Rec-/Her-2 +, Rec +/Her-2 + and Rec +/Her-2-. Rec-was defined as ER-and PR-. Rec + was defined as ER + and/or PR +. Her-2 positive was defined as Her-2 + + or Her-2 + + +. End points were isolated locoregional recurrence (LRR), distant metastasis (DM), disease-free survival (DFS) and overall survival (OS). Results The median follow up time was 48 months. Sixty-nine (15. 8%) patients were Rec-/Her-2-, 62 (14. 2%) Rec-/Her-2 +, 89 (20.4%) Rec +/Her-2 + and 217 (49.7%) Rec +/Her-2-. 480(93.4%) patients received chemotherapy and 352(80. 5%) received radiotherapy. Radiotherapy significantly reduced the 5-year LRR rates of all the four subgroups (Rec-/Her-2-: 13.1% vs. 33. 3%, Rec-/Her-2 + :9. 3% vs. 21.2%, Rec + /Her-2 + :9. 7% vs. 47.0%, Rec +/Her-2-:3. 2% vs. 15.4%). Radiotherapy significantly lowered the 5-year DM rates (26. 7% vs. 49.4%, 27.6% vs. 67. 5%, 18.4% vs. 100%) and improved the 5-year DFS rate (66. 7% vs. 33. 3% , 67.7% vs. 33. 3% , 72. 6% vs. 0%) as well as OS (73.9% vs. 25.2% ,69. 8% vs.41.5%, 91.0% vs. 32. 8%) of patients with Rec-/Her-2-, Rec-/Her-2 + and Rec +/Her-2 +. Conclusions In high-risk breast cancer patients, all subgroups of patients grouped by ER, PR and Her-2 status can benefit from postmastectomy radiotherapy.

Objective To observe the serum levels of calcium, phosphorus, alkaline phosphatase (ALP), parathy-roid hormone (PTH) and 25-hydroxyvitamin D, and the influence of biochemical markers of bone turnover in Graves’dis-ease. Methods Sixty-two patients with Graves’disease were enrolled into the Graves’disease group and 91 healthy indi-viduals as a control group. Electrochemical luminescence was used to evaluate the plasma levels of PTH and 25-hydroxyvita-min D in two groups. The serum levels of calcium, phosphorus and ALP were measured with biochemistry methods in two groups. Results The serum levels of calcium, phosphorus, ALP and 25-hydroxyvitamin D were significantly higher in the Graves’disease group compared with those in control group (P<0.01). The serum levels of calcium, phosphorus, ALP and 25-hydroxyvitamin D were significantly higher in female patients than those of control group, and the level of PTH was lower than that of control group. For male patients, the levels of ALP and 25-hydroxyvitamin D were higher than those of control group, and the level of PTH was lower than that of control group. In Graves’disease group, patients with vitamin D deficien-cy were 17 cases (27.4%), insufficiency 20 cases (32.3%) and sufficiency 25 cases (40.3%), respectively. In control group, there were 54 cases with vitamin D deficiency (59.3%), 31 cases with insufficiency vitamin D (34.1%) and 6 cases with suffi-ciency vitamin D (6.6%), respectively. There was no correlation in plasma levels of PTH, 25-hydroxyvitamin D, serum calci-um and serum phosphorus in Graves’disease group. Conclusion The bone turnover is accelerated in Graves’disease. The increased plasma level of 25-hydroxyvitamin D is related with increased calcium level and decreased PTH level in Graves’ disease. The increased serum phosphorus reduces 1-α-hydroxylase activity. Vitamin D deficiency plays a minor role in bone metabolism of Graves’disease.