Objective: To obtain highly purified recombinant CP4-EPSPS antigen with well reserved original antigenicity by a simple and fast protocol,and to study immunoactivity of recombinant CP4 EPSPS antigen.Methods: Recombinant expression vector pET-EPSPS containing CP4-EPSPS gene from transgenetic soy bean was induced by optional concentration of IPTG.After antigen activity was identified by strip-test method,recombinant protein expressed in the supernatant was purified by Ni-NTA-chelated agarose affinity chromatography method.Immunoactivity of purified CP4-EPSPS was further confirmed by ELISA assay,strip-test method,and Western blot analysis.Results: Recombinant CP4-EPSPS antigen was mainly expressed in the form of inclusion bodies in E.Coli system,whereas the strong antigenicity of CP4-EPSPS was detected in the induced supernatant by Colloidal-Gold strip-test,no antigenicity of CP4-EPSPS could be detected by Colloidal-Gold strip test for inclusion bodies or renatured sample from inclusion bodies.CP4-EPSPS protein was successfully purified from induced supernatant via propagating host cell culture and reducing the re-suspension volume of induced culture.ELISA assay indicated the specific antibody titer of recombiant antigen-immunized serum sample could reach 1:625,000 after three months of immunization.Using serum sample derived from recombinant antigen-immunized mice as primary antibody,CP4-EPSPS standard and purified recombinant antigen showed a highly specific and strong reaction with this serum by Western blot analysis.Conclusion: The original antigenicity of CP4-EPSPS cannot be recovered completely during gradual renaturing procedures by simple dilution methods.The epitope related to the rapid detection of CP4-EPSPS cannot be recovered by renaturing the inclusion bodies by the method in this study.However,CP4-EPSPS in induced supernatant is found to have a well-reserved original antigenicity.Colloidal Gold Strip-test to detect recombinantCP4-EPSPS antigen is a highly sensitive,and convenient method to follow through the whole process of recombinant expression and purification.It plays a crucial role in identifying the induced recombinant CP4-EPSPS for purification and further application in the immunization for the monoclonal antibody development.

The progress of modern science and technology stimulates reformation of teaching.PBL(Problem Based Learning) encourages the students to explore and solve the problems in the learning process.PBL can arouse the enthusiasm and creativity of the students through the carefully designed questions,focused discussions and(related) experiments etc.It can create an easy and active learning atmosphere that can improve the quality of teaching.

Adolescent ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Lymphohistiocytosis, Hemophagocytic ; genetics ; Mutation ; Signaling Lymphocytic Activation Molecule Associated Protein

BACKGROUND: Formerly, it was thought that the damaged hair cells could not have the repair ability. Recent studies demonstrate that mammal vestibule hair cells also possess certain repair ability after being destroyed.Then, whether mammalia animal cochlea hair cells possess regenerative ability after being destroyed is disputed.OBJECTIVE: To observe cochlear hair cells condition and threshold value change of auditory brainstem response (ABR) at different time following gentamicin ototoxicity by using scanning electron microscope (SEM) technique combined with ABR test, so as to investigate whether cochlear hair cells of mammals can be regenerated after being injured.DESIGN: A randomized and controlled animal experiment.SETTING: Department of Physiology, Shenyang Medical College.MATERIALS: This experiment was carried out at the Hearing Research Room of China Medical University from November 2001 to May 2002. Totally 60 healthy adult white Guinea pigs, with red eyes and sensitive auricle reflex, of clean degree, were used and randomly divided into gentamicin group and normal control group with 30 guinea pigs in each one.METHODS: 100 mg/kg gentamicin was intraperitoneally daily injected into the guinea pigs, serving as gentamicin group. Same volume of normal saline (2.5 mL/kg) was intraperitoneally daily injected into the guinea pigs,serving as normal control group. All the guinea pigs were given medication for 10 successive days. Threshold value of ABR was detected respectively pre-operatively and at the 1st, 3rd, 30th days postoperatively; after withdrawal and execution, scanning electron microscope was used to observe cochlear hair cells of guinea pigs in each group.MAIN OUTCOME MEASURES: ① Threshold value of ABR. ②Cochlear hair cell change of guinea pigs at different time following gentamicin ototoxicity.RESULTS: All the 60 experimental animals entered the stage of result analysis with no loss in the midway. ①At 1,3 and 30 days after withdrawal of gentamicin, threshold value of ABR was significantly higher as compared with normal control group, with significant difference [(38.00±3.75), (2.22 ±3.63) dB nHL,t=30.651, P ＜ 0.001];[(39.09±4.22), (2.50±3.54) dB nHL, t=29.708, P ＜ 0.001];[(14.50±3.69), (1.50±2.42) dB nHL,t =13.175, P ＜ 0.001]. Threshold value of ABR recovered obviously on day 30, but did not reach the normal level. ②On the first day after withdrawal of gentamicin , stereocilium of hair cells in second turn of cochlea of guinea pigs presented fusion, distortion, lodging, loss or incompetence and other pathological changes , especially severe in the third turn , also cystic form protrutions appeared outside the stereocilium of inner hair cell; On day 3 after withdrawal of gentamicin, stereocilium of outer hair cells in the second turn of cochlea of guinea pigs still presented fusion, loss, lodging and other pathological changes. Stereocilium of inner hair cell still showed lodging,but the outside cystic form protrutions decreased; On day 30 after withdrawal of gentamicin, stereocilium of outer hair cells in the second turn of cochlea presented fusion, loss , lodging and other pathological change ,which were obviously weaker than those on the 1st day and 3rd day after withdrawal of gentamicin , and at the same time , new born stereocilium appeared in the third turn of cochlea.CONCLUSION: Cochlear hair cell morphology recovery appears in those which survive for 30 days after cochlear hair cells of guinea pigs are damaged following gentamicin ototoxicity, and threshold value of ABR also recovers to some extent, suggesting that cochlear hair cells possess regenerative and repair ability following gentamicin ototoxicity. Hair cells after gentamicin-induced cochlear damage possess regenerative ability.

Objective To evaluate the clinical value of the afterloading brachytherapy inverse intensity-modulated radiotherapy of gynecological tumor.Methods Twenty patients with cervical cancer,were randomly divided into A and B groups,10 cases for each group.Group A received the afterloading brachytherapy inverse intensity-modulated radiotherapy.Group B received the three-dimensional comformal afterloading brachytherapy.The target volume dose distribution,organs at risk (rectum,bladder),shortterm curative effect and radioactive complications were analyzed on both groups.Results The dose homogeneity index of the target volume of group A was 52.43-± 0.45,better than that of group B (46.37 ± 1.45) (t =0.92,P ＜ 0.05).The maximum dose of rectum and bladder of group A were about 37％,35％,less than that of group B (t =1.34,1.39,P ＜ 0.05).The 75％ prescription dose irradiated volume of rectum and bladder of group A were about only 1/2 of group B (t =1.23,1.13,P ＜ 0.05).The local control rate of 96％ for group A was better than 93％ for group B (t =1.25,P ＜ 0.05).Conclusions Afterloading brachytherapy inverse intensity-modulated radiotherapy technique could be better than the three-dimensional comformal afterloading brachytherapy.It should be recommended for gynecological tumor.

Objective To investigate the effects of orexin-A on learning and memory of pentyleneterazol (PTZ)-kindled epileptic rats.Methods Adult Wistar rats were randomly divided into control group(normal saline,NS)and PTZ group.The PTZ-kindled rats were randomly divided into,orexin-A group and NS group administrated by intracerebroventricular(i.c.v.)injection of 10μl orexin-A(1.4 nmol/μl)or 10μl NS.Using Morris water msze experiment,the ability of learning and memory was measured in all rats.Results Eighty percent of rats in PIZ group were kindled successfully after intraperitoneal injection of 35mg/kg PTZ every day for 30 days.Compared to control group,the escape latency in the PTZ-kindled rats was significantly increased in place navigation test(PNT)(F=200.956,P<0.01),whereas a remarkable reduction of time spent in the target quadrant and number of pool circlings in 120 seconds Was observed during probe trials.Following injection of orexin-A,the latency of escape platform was significantly declined in both PTZ-kindled((39.73±2.03)8,(33.76±2.96)s)and NS rats,increased the number of crossing the platform(10.83±1.80)vs(4.67±3.34).In addition,the treatment with orexin-A markedly increased swim velocity and number of pool circlings in beth groups(P<0.01),particularly to the PTZ-kindled rats.Conclusion Spatial learning and memory in the PIZ-kindled rats can be improved by treatment with orexin-A.

Objective:To differentitate the myogenic from arthrogenic component of knee contracture resulting from being immobilized in extension for 4 weeks. Method:Ten rabbits were used as experimental animals. The right hindlimbs were immobilized with knee in extension for 4 weeks, while the left severed as controls. The range of motion ( ROM ) of both knees were measured before and after myotomy. Result:In immobilized and control hindlimbs, the mean ROM of the knee were 79.5°and 138.5°, respectively. The gain of ROM after myotomy in immobilized and control hindlimbs were 8.7° ±0. 45°, 11.6° +0.58°, respectively. Only 14.75% of the limitation of motion in ROM were accounted for by the myotomy component. Conclusion:After immobilizing for 4 weeks, the ROM of knee was significantly limited. However, the myogenic part was small and not different from normals.

Objective To differentiate the myogenic and arthrogenic components of knee contracture resulting from being immobilized in extension for 4 weeks.Method Ten rabbits were used as experimental animals. The right hindlimbs were immobilized with the knee in extension for four weeks, while the left served as controls. The range of motion (ROM) of both knees was measured before and after myotomy.Result In immobilized and control hindlimbs, knee mean ROM were 79.5° and 138.5° respectively. The gain of ROM after myotomy in immobilized and control hindlimbs were (8.7± 0.45)° , (11.6± 0.58)° respectively. Only 14.75％ of the limitation of motion in ROM was accounted for by the myotomy component. Conclusions After immobilized for 4 weeks, the ROM of knee was limited.However,the myogenic part was small and not different from normals.Because this study reports on only one time point,further investigations are needed.

Objective:To study the effect of corticotrophin-releasing hormone(CRH) on estrogen production in human trophoblasts and its mechanisms. Methods: Cytotrophoblasts were isolated from term human placentas and cultured for 72 h. The cultured cells were then treated with various concentrations of CRH and CRH receptor antagonist,?-Helical CRH9-41 for 24 h; estradiol was measured by radioimmunoassay in the culture media. Expression of aromatase(P450arom),the key enzyme for estrogen synthesis,was analyzed by Northern blot. Results: CRH significantly stimulated estradiol production and the expression of P450arom mRNA in placental cells. It was found that ?-Helical CRH9-41 blocked the effect of CRH and inhibited estradiol production and P450arom mRNA expression(P

Objective To modify the posterior procedures for severe thoracolumar fractures. Methods Twenty-one patients of thoracolumbar fractures with over 40% of anterior compression were studied. After reduction and fixation with short-segment pedicle instrumentation was performed, the transpedicular vertebroplasty of the fractured vertebrae with injectable self-setting calcium phosphate cement was carried out under fluoroscopic guidance. All patients were checked with X-ray and CT scanning before and after operation. They were followed up for 4-23 months (average 13 months). Results All patients obtained successful surgery without neurological complications or foreign body response. On CT scanning, the fractured vertebrae were well augmented in 13 cases and insufficiently augmented in 4 cases. Slight extrusion of cement into paravertebral tissue occurred in 4 cases without clinical symptoms. All patients were perfectly recovered without notable correction loss or implant failure during the follow-up. Conclusions Reduction and fixation with short-segment pedicle instrumentation followed by vertebroplasty with injectable self-setting calcium phosphate cement is a promising procedure for severe thoracolumbar fractures.