Background Cell immunologic therapy for retinoblastoma(RB)is becoming a hot research topic.Cancer-testis antigen is a human immunogenic protein and is used to treat some tumors.However,its effect on RB has not been investigated.Objective The present study was to discuss the antigen specific anti-tumor effect of cytotoxic T lymphocytes(CTL)induced by the cancer-testis antigen,NY-ESO-1-sensitized dendritic cells(DCs),on human RB.Methods PCR was performed to amplify target gene fragments from the NY-ESO-1 plasmids,and then the target gene fragments were digested with the restriction enzymes SalI and EcoRI.Harvested fragments were inserted into the pDC316 plasmid to construct the recombinant plasmid pDC316/NY-ESO-1.The expression of NY-ESO-1 protein in human RB cells strain,HXO-RB44,was detected by immunofluorescence and Western blot.Monocytes were isolated from 60 ml of peripheral blood from a healthy donor using Ficoll density-gradient centrifugation with a cell density of 1 × 107/ml.DCs isolated from blood were stimulated with recombinant human granulocyte-macrophage colony stimulating factor(rhGM-CSF)and recombinant human interleukin-4(rhIL-4).The recombinant plasmid pDC316/NY-ESO-1 was transfected into DCs and the DCs were co-cultured with T lymphocytes.The resultant CTL were used as effector cells.The growth of the CTL was detected by MTT assay.The CTL were then added into the growth medium used for culturing HXO-RB44 cells and the vitality of the HXO-RB44 cells was assayed by MTT assay.Results The sequence of the cloned DNA fragment of the recombinant plasmid pDC316/NY-ESO-1 was conforms with the sequence of the NY-ESO-1 gene.The expression of the NY-ESO-1 protein in HXO-RB44 cells was tested by immunofluorescence and Western blot.DCs were successfully induced with rhIL-4 and rhGM-CSF from PBMC.The recombinant expression plasmid pDC316/NY-ESO-1 was successfully transferred into DCs.These DCs had high expression of surface molecules such as HLA-DR(42.1％),CD80(54.2％),CD83(39.7％)and CD86 (94.8％).The CTL that was induced by DCs-sensitized with NY-ESO-1 specifically killed HXO-RB44 cells.CTL induced by the sensitized DCs had a stronger cytotoxic effect against HXO-RB44 cells compared with un-sensitized DCs and CTL un-induced with DCs,as shown by MTT asssay(P＜0.05).The anti-tumor activity was highest when the ratio of effector to target was 75∶1(P＜0.05).Conclusions DCs transfected by the recombinant plasmid pDC316/NY-ESO-1 can induce the proliferation of allogenic CTLs,which showed a specific anti-tumor effect against HXO-RB44 cells.These results present a new type of immunotherapy for the treatment of RB.

Biofilms

Objective To observe the effect of long-term intensive glucose control therapy on diabetic retinopathy in outpatients with type 2 diabetes mellitus.Methods Forty-nine patients with type 2 diabetes mellitus were randomly assigned to participate in the trial from 2002 to 2007,receiving either intensive (24 cases) or standard glucose control (25 cases).The patients were examined by the same ophthalmologists to identify any new diabetic retinopathy (DR).After 5 years of intensive glucose control,all of the patients were asked to attend our clinic every 6 months,but no attempts were made to maintain their previously assigned therapies.Hemoglobin A1c (HbA1 c) was measured regularly.In November of 2009,a retinal examination was carried out by the same ophthalmologist who worked in the trial.The visual acuity,lens,vitreous body and fundus were examined after pupil dilation to identify diabetic retinopathy (DR).Fundus fluorescein angiography and retinal laser photocoagulation were carried out when necessary.Results In the second year after enrollment in the trail,the median HbA1c level of the intensive-therapy group was significantly lower than that of the standard-therapy group [(6.3 ± 0.6) ％ vs.(7.2 ± 1.2) ％,t =2.09,P ＜ 0.05],and was maintained in a controlled level throughout the following 4 years.During the post-trial monitoring,no new case of of macula edema or diabetic associated blindness occurred in either intensive or standard-therapy group,the whole occurrence of micro-aneurysms,fundus hemorrhage,as well as those who needed retinal laser photocoagnlation and lowering in visual acuity in intensive-therapy group was lower than that in the standard-therapy group (3 vs.14,1 vs.7,2 vs.4,3 vs.11,respectively ;9 vs.36,totally,x2 =4.719,P ＜ 0.05).During the first post-trial monitoring,there was no difference in median HbAlc level between intensive-therapy group and standard-therapy group [(7.2 ± 1.1) ％ vs.(7.3 ± 1.3) ％,t =0.25,P =0.806],which was sustained in the following years.In the trail,no new case of fundus hemorrhage or diabetic associated blindness occurred in intensive-therapy group during the five-year period of therapy.Number of new episodes of micro-aneurysm,macula edema were less in intensive-therapy group than that in standard-therapy group,number of new episodes of lowering in visual acuity,and those who needed retinal laser photocoagnlation,were significantly less in intensive-therapy group than that in standardtherapy group(15 vs.25,4 vs.23,Z =-4.459,P ＜ 0.05) during five-year follow-up.Conclusions The benefit of reduced incidence of diabetic retinopathy in intensive glucose can be maintained because of the legacy effect.

Objective To investigate the effect of sevoflurane on blood pressure in patients with different ages and the optimal concen-tration of sevoflurane. Methods 60 patients underwent selective LC operation with conventional induction and sevoflurane maintenance were divided into three groups:the youth group (34 patients), the middle age group (20 patients) and the older age group (6 patients). The SBP, DBP, MAP, CETsev, number of patients, age of patients before induction and 10 min(T1), 20 min(T2), 30 min (T3), 60 min (T4) after induction were recorded. Results Fluctuate of blood pressure were in the normal range after anesthesia maintenance, and MAP were fluctuated within the range of ± 20% before induction. The age under different CETsev were of no significant difference in each time point (P>0. 05). The age composition ratio in different CETsev were of no significant difference in each time point (P>0. 05). As the time of anesthesia extended, the number of patients under 0% ~1%CETsev decreased from 35 to 11; the number of patients under 1% ~2%CETsev increased from 10 to 20;and the number of patients under 2% ~3%CETsev maintened in 14 approximately. Conclusion The effect of sevoflurane on blood pressure of different ages is approximate. 2% ~3% CETsev is the most optimal concentration during the main-tenance of anesthesia.

Objective: To study the optimum extraction conditions of alkaloid of Rhizoma pinelliae processecd with ginger.Methods:The alkaloid content was used as a marker.The percolation process of Rhizoma Pinellia(processed with ginger) was studied by orthogonal design with 3 factors and 3 levels(L 9(3 4)) .Results:The alcohol concentration was remarkable factor in the test.Conclusion:Considering the demand of manufacture,the best extraction condition is A 3B 1C 1。

Objective To explore the clinical characteristics and risk factors of re-fracture in patients suffering from osteoporosis-related fractures as well as effective interventions.Methods From January 2006 to January 2008,a total of 273 patients with osteoporosis-related fracture were entered in the study,including out-patients and in-patients who were over 50 years old.The patients were divided into fracture group(n=225)and re-fracture group(n=48).The re-fracture rate was followed up for 2 years,during which 11 patients developed re-fracture.General data including age and sex,fracture types,femoral neck bone mineral density(BMD)T-scores tested by dual-energy X-rays absorptiometry(DEXA),Charlson index,timeinterval between two fractures as well as mobility skill assessment were collected and analyzed.Results The average age at the first fracture was 67.7±8.5 years vs.72.7±9.5 years for the re-fracture cases.Female accounted for 70.2% of the fracture group and 77.1% of the re-fracture group.The most common re-fracture type was vertebral fracture for the first time and femoral neck fracture for the second time during the followup.Risk factors for a second fracture in osteoporotic fractures patients include age(＞75 years,HR=1.23; ＞85years,HR=1.68),female sex(HR=1.36),prior vertebral fractures(HR=1.62),prior hip fractures(HR=1.27),BMD T-score＜-3.5(HR=1.38)and weakened motor skills(HR=1.27).The refracture rate in osteoporosis-related fractures was 4.9% followed up for 2 years.The second fracture happened 3.7 years after the first one on average.Conclusion The risks of second fracture among patients with initial brittle fracture are substantial.Mobility skill assessment is an important risk factor for osteoporosis fractures recurrence.There is adequate time between fracture and re-fracture for effective interventions to prevent or reduce the risks of refracture,especially for the old women with a vertebral or hip fracture.Medication,motor function rehabilitation and fall-down prevention training would be helpful.

ObjectiveTo observe and analyze the synapses developing process of newly generated connections of autologus activated Schwann cells (AASCs) in combination with fetal spinal cord cell suspension(FSCS) in the surrounding area of the spinal cord injury site.MethodsA total of 42 Wistar rats underwent unilateral ligation of the saphenous nerve.The portion of nerve tissues distal to the ligation site were harvested 1 week after operation.AASCs were isolated,cultured and purified.Spinal cord injury model produced in 42 Wistar rats on T7 by modified Allen impact method.Three days after injury,20 μl FSCS with a density of 1×105/μl prepared from pregnant rats (El4) in combination with AASCs were injected into the epicenter of the traumatized cavity.Animals were sacrificed at 2,4,6,8,10,12 weeks post transplantation.Light and electronmicroscopic studies as well as immunohistochemical assay were carried out to evaluate the graft survival,its differentation and integration with the host.ResultsIn the transplantation area,AASCs showed good growth and differentiation,and glial scarring surrounding the lesions was less.The neuroblast stretched out the terminal endings 4 weeks after implantation,followed by the presenting of the pre- and post-synaptic membrane.Eight weeks post transplantation,the dense or developed projections were observed in the pre- and post-synaptic membrane,the high electron dense substance full filled the synaptic cleft.All the spherical cleat vesicles,granular vesicles,elliptical vesicles and flattened-f type vesicles were discovered under the electron microscope.Ten weeks after injury,the axosomatic,dendrosomatic,dendro-dendritic,axoaxonic,and dendro-axonic synapses coexisted.Light microscopy showed that the graft cell grew gradually.Immunohistochemical assay showed that NF,5-HT,CGRP and GFAP positive fibers were in the graft.Synapses,glia fibers and blood brain barrier integrated each other.Conclusion1) The transplanted FSCS combined with AASCs can develop mature synapses with miscellaneous synaptic vesicles in the acute injured spinal cord.2) Co-existing indicate the possibility of synaptic connection between FSCS and host.

Objective: Epi-LASIK appeared to be effective and safe in the treatment of high myopia,but some patients complain about visual symptoms postoperation.The purpose of this study was to evaluate the high order aberrations(HOAs) of the vision-decreased eyes treated by Epi-LASIK,and to analyze the causes of postoperative glare,scieropia and monocular diplopia.Methods: We evaluated 76 eyes of 49 patients treated by Epi-LASIK with a questionnaire three months after surgery,and from them selected 34 eyes of 27 patients who scored less than 80 points and complained of postoperative visual symptoms,including glare(25 eyes in 20 patients),scieropia(7 eyes in 5 patients)and monocular diplopia(2 eyes in 2 patients).Another 42 symptom-free eyes of 22 patients treated by Epi-LASIK were included in the control group.The root mean square(RMS) of total HOAs,coma and SA were determined with the iTrace Visual Function Analyzer from the central 6.0 mm zone,and the modulation transfer function(MTF) was simulated with the iTrace 3.1 software.Results: Significant differences were found in the RMS of total HOAs,coma and SA between the eyes with visual symptoms and those without,and the values of ablation zone decentration were significantly higher in the former than in the latter(P

Objective To investigate the protective effects and molecular mechanism of peroxisome proliferate-activated receptor α(PPARα) activation on acute myocardial damage induced by isoproterenol (Iso) in rats. Methods Thirty male Wistar rats, weighting 160～180 g, were randomly divided into control group, Iso group, fenafibrate(FF) group(each n=10) according to physique quantity. Acute myocardial injury caused by Iso abdomen cavity injection induced ischemia was established and the protective effects of peroxisome proliferate-activated receptor α activation were accessed by the level of ereatine kinase(CK), lactic dehydrogenase(LDH) in serum as well as the activities of myoperoxidase(MPO) in myocardium, and the protein expressions of PPABα in myocardium by Western blot. Results The level of serum CK in control group, lso group and FF group, was (62.41±9.47),(101.71±11.05),(75.64±11.73)kU/L, respectively(F= 34.34, P<0.01). Whereas the level of serum CK in Iso group and FF group was higher than that in control group(P<0.01 or<0.05), the level of serum CK in FF group was lower than that in Iso group(P<0.01). The levels of LDH in these three groups were (5912.20±204.44), (6365.78±137.10), (6089.76±169.60) U/L, respectively(F= 17.54, P<0.01). Compared with the control group, the levels of LDH in Iso and Fir groups were significantly increased(P<0.01 or<0.05). But the level of LDH in FIr group was decreased compared with that in Iso group(P<0.01). The activities of myocardial MPO in these three groups were (1.95±0.10),(3.89±0.17),(2.49±0.19)U/g, espectively(F=391.68,P< 0.01). The activities of myocardial MPO in Iso and FF groups were higher than that in the control group (all P< 0.01), while the activities of myocardial MPO in FIr group were lower than that in lso group(P<0.01). The protein expressions of PPARα in myocardium of these three groups were 251.57±10.95,191.97±10.74,215.08±9.61, respectively(F=82.69, P<0.01). Conclusion PPARα activation by its actor FF can exert protective effects on the acute myocardial ischemia injury induced by lso in rats through inhibiting the release of inflammatory cell factors.

Objective To investigate the effect of ultrasound-guided subcostal transverses abdominis plane (TAP) block with dexmedetomidine after laparoscopic radical operation. Methods 40 patients underwent laparoscopic radical operation for colorectal cancer were randomized into dexmedetomidine group (group DEX) and control group (group CON). All the patients received ultrasound-guided subcostal TAP block after operation, Group DEX with dexmedetomidine 1 μg/kg and 0.25% ropivacaine to 20 ml, and group CON with 0.25% ropivacaine 20 ml. All the patients were assessed with Ramsay scores and the pain at rest and on coughing were assessed with Visual Analogue Scale (VAS), 2, 6, 12, 24 and 24 hours after operation. The highest level and the duration of sensory blockade, the first time and the total times of pressing the analgesia pump in the first day after operation, and the requirements of sufentanil were recorded. First flatus time, first diet time and the length of hospital stay were compared. Results The scores of VAS were significantly less (P<0.001), and the Ramsay scores were more in the group DEX than in the group CON (P<0.01) 2, 6 and 12 hours after operation; with the longer time of sensory blockade (P<0.001), the later to first press the analgesia pump (P<0.001), the less frequence of pressing the analgesia pump (P<0.001), and less dosage of sufentanil (P<0.001). The first flatus time, first diet time were significantly earlier in the group DEX than in the group CON (P<0.001), with the less length of total hospital stay (P<0.001). Conclusion Dexmedetomidine can promote the anaesthesia of ultrasound-guided subcostal TAP block with ropivacaine and improve the recovery after laparoscopic radical operation.