Objective:To investigate the rat retinal DNA damage induced by acute hypobaric hypoxia and the protective effect of Radix Astragali seu Hedysari compound on it. Methods:Seventy-two healthy clean grade male Sprague Dawley (SD) rats were randomly divided into normal oxygen control group, hypoxic model group and Radix Astragali seu Hedysari compound gavage group by computer random number distribution, with 24 rats in each group.The rats in the normal oxygen control group were fed in normal condition, while the rats in the hypoxic model group and Radix Astragali seu Hedysari compound gavage group were fed in a hypobaric oxygen chamber, which simulated an altitude of 5 kilometers, and the rats were administered intragastrically with Radix Astragali seu Hedysari compound (0.1 g/kg) or the same volume of normal saline once a day according to grouping.Retinal tissue was obtained on the 7th day of continuous administration of drugs after the rats were sacrificed by euthanasia.Histopathological staining was performed to observe the pathological morphology of retina in each group.Immunohistochemistry was used to identify the expressions of p53 and histone family 2A variant ( γH2AX). Real time-PCR was used to determine the relative expression levels of 8-oxoguanine nucleoside triphosphatase (MTH1) and 8-hydroxyguanine glycolsylase (OGG1) mRNA.The use and care of the animals followed Regulations of the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results:The retina of hypoxic model group was thicker than that of the normal oxygen control group and Radix Astragali seu Hedysari compound gavage group, especially the nerve fiber layer and the ganglion cell layer.The positive staining intensities of p53 and γH2AX were stronger in the retinas of the hypoxic model group than those in the the normal oxygen control group and Radix Astragali seu Hedysari compound gavage group.The relative expression levels of MTH1 and OGG1 mRNA in the hypoxic model group were 0.573±0.081 and 0.772±0.136, which were significantly lower than 0.846±0.160 and 1.013±0.168 in the normal oxygen control group, respectively (both at P<0.05). The relative expression level of MTH1 mRNA was 0.748±0.114 in the Radix Astragali seu Hedysari compound gavage group, significantly higher than that in the hypoxic model group ( P<0.05). No significant difference was detected in OGG1 mRNA expression level between the Radix Astragali seu Hedysari compound gavage group and the hypoxic model group ( P=0.743). Conclusions:The intervention of Radix Astragali seu Hedysari compound plays a protective role in hypobaric hypoxia-induced retinal DNA damage through modulating the expression of p53, γH2AX, OGG1 and MTH1.

Background Meningothelial cells (MECs) occupy the predominant cell component of barrier between optic nerve and the cerebral spinal fluid,and any change of cerebral fluid components probably affects the MECs function and further impairs the optic nerve.Objective This study was designed to investigate the influence of glutamate,a potentially excitotoxic amino acid,to the functional changes of MECs and provide a theoretical evidence for clarifying the mechanism of optic nerve disorders.Methods Human MECs strains were cultured in vitro and prepared into cell suspension.The cells were inoculated to 96-well plates with the densities of 1 × 104/we11.The glutamate of 100,200,400,600,800 and 1 000 μmol/L was added into medium for 12,24,36,48 and 72 hours,respectively,and the cultured cells without glutamate were used as normal control group.MTS assay was employed to measure the proliferative rate (absorbency) of the cells.The regularly cultured MECs were divided into 600 μmol/L glutamate-treated group and normal control group and the cells were treated for 12 and 24 hours respectively,and the expression of superoxide dismutase (SOD) mRNA and heat shock protein 90 (HSP90) mRNA in the cells was detected by real-time PCR;the level of total anti-oxidative capacity (T-AOC) of the cells was processed by enzyme linked immunosorbent assay (ELISA),and the reactive oxygen species (ROS) production was determined by DCFH-DA probe.Results Cultured MECs grew well and formed 80％ confluence after 72 hours culture.The proliferative rate of the cells were gradually decreased with the increase of glutamate dose and the lapse of affected time,with significant differences among different concentrations of glutamate and various time points (F tration =52.501,P＜0.001;Ftime =8.505,P＜0.001).The relative expression level of SOD mRNA was significantly reduced in the glutamate-treated group compared with the normal control group in both 24 hours and 48 hours after culture (t =20.278,t =16.724,both at P＜0.001),and the expression of HSP90 mRNA in the cells was significantly lower in the glutamate-treated group than that in the normal control group in 24 hours after culture (t =5.065,P =0.002).No significant difference was found in T-AOC activity between glutamate-treated group and normal control group in 24 hours after culture ([30.835±2.094] nmol/(min · L) vs.[32.873±2.317] nmol/(min · L)) (t=1.599,P =1.414).In 48 hours after culture,T-AOC activity was (29.561 ± 1.831) nmol/(min · L) in the glutamate-treated group,which was significantly lower in comparison with normal control group (33.680±2.039) nmol/(min · L)(t =3.682,P =0.004).Fluorescence staining showed that the intensity of green fluorescence of ROS in MECs in the normal control group was weaker than that in the glutamate-treated group under the immunofluorescense microscope.The ROS level was 48.110± 1.712 and 40.982± 1.853 at 24 hours and 48 hours in the glutamate-treated cells,and which was significantly elevated in comparison with 36.608± 1.009 and 37.153 ± 1.424 in the normal control group (t=14.178,P＜0.001;t=4.012,P=0.002).Conclusions Glutamate inhibits the proliferation of MECs in vitro,and excitatory toxicity of glutamate on MECs probably is associated with oxidative stress response.

Background BackgroundMeningothelial cells (MECs) are major cell type in the meningeal sheath around optic nerve,which form a fluid barrier between optic nerve and the cerebral spinal fluid.The impairment of the cerebral fluid-optic nerve barrier probably affects the balance of cerebral fluid components.Currently,the investigation on the role of MECs in neuropathy is less performed.Objective This study attempted to explore hypoxia-induced function changes of MECs,and to shed a new clus for the future research of optic nerve disorders.Methods Human MECs strains were cultured in vitro and cell suspension was prepared with the cell densities of 2.5 ×103/hole,5.0× 103/hole and 1 x 104 /hole,respectively.The suspensions of 100 μl were separately collected to incubate in 96-well plates and cultivated for 2 days in 21％ O2(normoxia group) or 1％ O2(hypoxia group).MTS was used to detect and compare the proliferative value (A490) of MECs between the normoxia group and the hypoxia group.The changes of MECs diameter and volume were measured by CASY1 assay.ATP product in the cells after MECs exposed to different oxygen environments with or without substrate (100 mmol/L pyruvate and 100 mmol/L malate) for 1,2 days were assayed by Luminometer method.The expression and distribution of cytochrome C in the cells of the normoxia group and the hypoxia group were determined by immunofluorescence.Results A490 of MECs in the 2.5× 103/hole,5.0× 103/hole and 1 × 104/hole were 0.399±0.009,0.393±0.009 and 0.496±0.026 in the hypoxia group,which were lower than 0.424±0.131,0.413±0.111 and 0.537±0.021 in the normoxia group (t =3.777,P =0.004 ; t =3.251,P =0.009 ; t =3.037,P =0.013).Compared with the normoxia group,the diameter and volume were significantly increased in the hypoxia group ([20.970 ±0.127] μm vs.[21.198 ±0.048] μm,t =-3.762,P=0.006; [5805±73] fl vs.[6026±106] fl,t=-4.124,P=0.002).ATP products were (0.900±0.225)mmol/(L· g) and (0.952± 0.075) mmol/(L · g) in the hypoxia group and the hypoxia+substrate group,which were significantly lower than (1.389±0.145) mmol/(L · g) and (1.401±0.122) mmol/(L · g) in the normoxia group and the normoxia +substrate group (P =0.001,0.002,0.001).Immunofluorescense staining showed that the green fluorescence of cytochrome C located at mitochondria of MECs in the normoxia group,but in the hypoxia group,cytochrome C distributed in the cytoplasm extensively.Conclusions Hypoxia induces malfunction of MECs,which might impact the intact of the cerebral spinal fluid-optic nerve barrier and therefore influence the microenvironment of the subarachnoid space and neuronal function.

Objective To observe the efficacy and safety of preoperative concurrent chemoradiotherapy or radiotherapy alone in patients with T3,T4 or lymph node-positive rectal cancer.Methods 141 rectal cancer patients with locally advanced or node-positive based on imaging from 2000 to 2009 were retrospective analyzed.Ninety-seven patients received preoperative concurrent chemoradiotherapy and 44 received preoperative radiotherapy alone.Two-dimensional or three-dimensional radiation technique and four types of chemotherapy regimens were used.Results The following-up rate was 91.5％.106 patients were followed up for at least 3 years and 68 patients for at least 5 years.The 3-and 5-year overall survival rates were 85.8％ and 65.7％,respectively.The 3-and 5-year local recurrence rates were 9.2％ and 14.1％,respectively.The 3-and 5-year metastasis rates were 33.8％ and 45.8％,respectively.The downstaging rate was up to 59.0％ (82/139) and the rate of sphincter preservation was 65.5％ (91/139).The median disease-free survival in patients treated with preoperative concurrent chemoradiotherapy was superior to radiotherapy alone (51 months vs 31 months,x2 =12.88,P =0.000).The time to metastasis in patients with downstaging was significantly delayed than that in patients without downstaging (60 months vs 29 months,x2 =14.65,P =0.000).Most acute toxicity was grade 1 and grade 2.The incidence of delayed wound healing and anastomotic leakage was very low.Conclusions Preoperative concurrent chemoradiotherapy or radiotherapy alone has excellent tumor downstaging effect and helps in sphincter preservation,with tolerable side effects.

Objective To investigate the treatment outcomes of 41 patients with suprasellar and pineal germinoma who are treated in our department in recent 18 years,and to explore related therapeutic strategies.Methods A total of 41 patients with concurrent suprasellar and pineal germinoma who were treated in our department from January 1996 to August 2013 were enrolled.There were 35 male patients and 6 female patients,and the median age was 16 years (range 5-39 years).Five patients had pathologically confirmed germinoma and 36 patients had clinically diagnosed germinoma.Conventional radiotherapy was performed for 33 patients, and intensity-modulated radiotherapy was performed for 8 patients.Combined-modality chemoradiotherapy was performed for the 5 patients with pathologically diagnosed germinoma.The 6 MV X-ray was applied for radiotherapy;6 patients received whole ventricular irradiation and a boost in tumor region, 16 received whole-brain radiotherapy and a boost in tumor region,and 19 received craniospinal irradiation and a boost in tumor region.The median radiation dose for tumor region was 45.0 Gy (37.8-50.0 Gy),and the median dose for prophylactic irradiation was 25.0 Gy (17.8-35.0 Gy).The survival was caculated using Kaplan-Meier method.Results The number of patients followed were 26 at 5-years.The 5-year overall survival rate and relapse-free survival rate were 95% and 85%,respectively.Relapse and metastasis were noted in 8 patients,and 3 of them died.There were 4 patients with spinal cord metastasis,1 patient with ventricular dissemination,and 3 patients with periventricular relapse.Among the 22 patients who did not undergo spinal irradiation,4(18.2%) experienced spinal cord metastasis,and all the other 19 patients who underwent spinal irradiation did not experience spinal cord metastasis.The 8 patients undergoing intensity-modulated radiotherapy and the 5 patients undergoing combined-modality chemoradiotherapy did not experience treatment failure during follow-up.Conclusions Radiotherapy for intracranial germinoma has a good therapeutic effect,and the patients with suprasellar and pineal germinoma who do not undergo spinal cord irradiation have a high failure rate.It is suggested to perform spinal cord irradiation for patients with suprasellar and pineal lesions.

Objective To examine the interfractional dosimetric variations among inverse three-dimensional (3D) plan, forward 3D plan, and two-dimensional (2D) plan of intracavitary brachytherapy for cervical cancer, and to discuss the risk of implementing the interval plan on different implantation applicators at short time intervals.Methods Twenty-five groups of CT-guided intracavitary brachytherapy (two consecutive radiations at ≤4 d apart) plans from 11 cervical cancer patients who received radical radiation therapy in our hospital were reviewed and compared.The dwelling location and time of the first intracavitary brachytherapy plan (Plan-1) were simulated on the CT image of the second intracavitary brachytherapy to form Plan-1-S.The target coverage indices and D 2 cc of organs at risk (OARs) of Plan-1-S and Plan 2(actual plan of the second intracavitary brachytherapy) under the three planning modes were recorded and compared using the paired t-test, Wilcoxon signed rank test, and ANOVA.Results The D90, D100, and V100 of high-risk CTV were significantly lower in Plan-1-S created under the inverse mode in the actual plan (-9.11±13.46%,-13.16±18.79%, and-7.80±13.34%, P=0.002, 0.002, and 0.005, respectively).D90, D100, and V100 of the interval plan had the greatest reduction under the inverse mode (76%, 80%, and 76%, respectively).The maximum reductions in D90, D100, and V100 were 332.14 cGy (2D), 244.12 cGy (forward), and 41.76%(inverse).OAR overdose occurred most frequently under the forward mode;the rates of D90, D100, and V100 reductions accompanied by one OAR overdose were 29.41%, 37.50%, and 25.00%, and the rates of D90, D100, and V100 reductions by two OAR overdoses were 5.88%, 12.50%, and 6.25%,respectively.Overdose occurred most frequently in the small intestine (36%).Comparison of the three planning modes showed that the inverse plan had a greater reduction in each target coverage index than the 2D plan.Conclusions The simulated interval plan can significantly reduce target coverage and increase the risk of OAR overdose regardless of the planning mode and the short time intervals, and is therefore not recommended for clinical application.

Objective To investigate the inhibitory effects of silencing expression ofEZH2 gene on the cell proliferation of human QBC939 cells and its mechanisms. Methods The targeting siRNA was designed and trans-fected into QBC939cells. The expressions of EZH2 mRNA and protein were detected by real-time qPCR and west-ern blotting,respectively. The ability of cellproliferationwas analyzed by MTT assay and plate clone formation assay. Cell apoptosis and cycle percentage weremeasured by flow cytometry. Cell senescence was assessed byβ-galactosi-dase dyeing.The expressions of H3K27me3,P14ARF,P16INK4a,P53,P21 and E2F1 proteinwere determined by West-ern blotting.Results Compared with the control group ,the expressions of mRNAand protein were significantly elevat-ed in experimental group. The ability of cellproliferation in the experiment group was significantly down regulated , which could also cause a rise of G1/S phase ,but not a marked variation of apoptosis rate. Silencing EZH2 would induce a obvious senescence phenotype in QBC939 cells. EZH2-siRNA transferredcould also down-regulate the expressions of H3K27me3 and E2F1 protein,while up-regulating the expressions of P14ARF,P16INK4a,P53 and P21 protein in QBC939 cells.Conclusions Silencing EZH2 could induce a significant inhibition on cell proliferation of QBC939 cells,the mechanism of which may be associated with the senescence pathway regulation.

Objective To analyze the outcomes of radiotherapy for 74 patients with intracranial ger-minoma. Methods Between 1990 and 2007,74 patients with intracranial germinoma(9 pathologically diag-nosed and 65 clinically diagnosed) were treated with radiotherapy in our hospital. The median age at diagno-sis was 15 (range 5-45) years. Radiation treatment fields varied among patients, including craniospinal irra-diation(CSI), whole brain irradiation, whole ventricular irradiation with primary tumor boost, and involved-field irradiation only to the primary tumor plus margin. The dose was 38.5 -50.0 Gy to the tumor,18-25 Gy to the whole brain/ventricular,and 21-25 Gy to the whole spinal cord in fractions of 1.6-2.0 Gy per day,5 fractions per week. Results The median follow-up time was 80(range 12-168) months and the fol-low-up rate was 97%. Fourteen patients had been followed up for over 10 years. The 1-,5- and 10-year o-verall survival rates were 99% ,96% and 93%. The corresponding disease free survival rates were 97%, 90% and 83%, respectively. Relapses occurred in 9 patients. For the 6 patients with in-field relapse, the dose to the tumor was 38.5-40.0 Gy in 3 patients, 41-45 Gy in 2 and 46-50 Gy in 1. Relapse in the spinal cord was found in 3 patients and none of them received spinal irradiation. Twenty-one patients re-quired hormonal replacement therapy because of radiation induced hypofunction of prehypophysis. Conclu-sions Radiotherapy alone is a curative treatment for intracranial germinoma. The proper dose should be de-termined by tumor numbers and the examination of cerebrospinal fluid.

Objective To study the efficacy,late complications and prognostic factors of postopera-tive radiotherapy for bladder cancer: Methods Between July 1995 and January 21307,109 patients with primary bladder cancer who had received adjuvant pelvic radiation therapy were retrospectively analyzed. The age ranged from 41 to 94 (median 68) years. There were 29 patients with T1 disease,60 with T2,17 with T3, 3 with T4, and 7 with positive lymph node. Three-dimensional radiotherapy was given with a median total dose of 49.2 Gy(39.3 -62.2 Gy) in conventional fractionation after surgery. Field-in-field intensity modu-lated radiotherapy was given to 57 patients. Results The total follow-up rate was 98% ,with a median fol-low up time of 36(2-144) months. The 1-,3- and 5-year local control rate was 63% ,47% and 42% ,respec-tively. The 1-,3- and 5-year overall survival rate was 80% ,48% and 37% ,respectively. Among the 109 pa-tients,33 died of tumor progression or metastasis,42 showed local recurrence,and 22 had lymph node metas-tasis. According to the RTOG criteria,grade 1,2,3 and 4 radiation related early urological side effects were 22% ,59% ,12% and 1% ,respectively;The corresponding late urological side effects were 29% ,28% ,2% and 1%, respectively. Two patients underwent whole bladder cystectomy due to the radiation related side effects. Conclusions Postoperative three-dimensional radiotherapy, achieving high response rate with tol-erable side effects, can be offered as an alternative option to the radical cystectomy in the bladder conserving treatment.

Objective To evaluate treatment results and prognostic factors of 47 patients withprimary urethral transitional cell carcinoma treated with post-operative adjuvant radiotherapy. Methods From October 1998 to October 2008, 47 patients with primary urethral transitional cell carcinoma received postoperative adjuvant radiotherapy. Thirty-one patients had stage T_3/T_4 disease, 7 had lymph node metastasis. Thirty-nine patients had G3 tumor, 13 had stump-positive. The median radiotherapy dose was 60 Gy (36-64 Gy). 81% patients (38/47) were treated with regional irradiation. Results The median follow-up time was 21 months (6 -88 months). The follow-up rate was 92%. The median overall survival time was 35 months (5 -88 months). The 2-and 5-year overall survival rates were 57% and 49%, respectively. In univariate analysis, the median overall survival time was better in patients with stage T_1 or T_2 compared with stage T_3 or T_4 tumor (42 months vs. 19 months,Χ~2 =7. 28,P=0. 007), with age of ≤65 years compared with >65 years (28 mouths vs 18 months,Χ~2 =8.23 ,P =0. 004). There was no significantdifference in the long term survival in patients with non-radical surgery compared with radical mastectomy (21 months vs. 20 months, Χ~2 = 0. 90, P = 0. 344). In multivariate analysis, the stage T_3 or T_4 (Χ~2 = 7. 89, P =0. 005), >65 years old (Χ~2 = 4.85, P = 0. 028), renal pelvis involvement (Χ~2= 5.65, P = 0. 018), and tumor located in the mid or inferior segment (Χ~2=6. 08 ,P =0. 014) were factors associated with poorer prognosis. Conclusions Postoperative adjuvant radiotherapy can improve the efficacy of patients with locally advanced urethral transitional cell carcinoma. Advanced T stage and > 65 years age are associated with poorer prognosis.