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.

Background The hyperplasia of human Tenon capsule fibroblasts (HTFs) is a common cause of filtering surgery failure in glaucomous eye.Researches demonstrated that hydroxycamptothecin is a cell cycle arresting drug and induce apoptosis of cancer and fibroblasts.However,its mechanism is currently less understood.Objective This study was to investigate whether hydroxycamptothecin induce the apoptosis of HTFs and explore the possible mechanism.Methods Human Tenon capsule tissue was obtained from EyeBank of Jiangsu Province Hospital.HTFs were cultured using explant method in vivo and passaged in DMEM containing 10％ FBS.The cells were identified using vimentin and keratin by immunochemistry,and the cells of generation 3-6 in the logarithmic growth phase were used in the experiment.The cells were incubated with 0.01,0.05 or 0.10 g/L hydroxycamptothecin for 5 minutes respectively,and the cells without any hydroxycamptothecin were served as the control group.Cell viability then was assessed by cell counting kit-8 (CCK8) for the optimal inhibition concentration.The cells were treated by 0.10 g/L hydroxycamptothecin for 24 hours,and the apoptotic rate of the cells were assayed with annexin V/PI double-staining.Mitochondrial membrane potential of HTFs was assessed using JC-1 staining.The expressions of caspase-3,caspase-9 and cytochrome C (cyt C) in mitochondria and cytoplasm of HTFs were detected by Western blot.Results The proliferative value (A450) of the HTFs 0,0.01,0.05,0.10 g/L was 0.9716±0.0608,0.8035 ± 0.0346,0.7048 ±0.0446,0.6265 ±0.0286,with a significant difference (F =26.372,P =0.002).A450 of HTFs in the 0.01,0.05,0.10 g/L groups was significantly lower than the control group (P＜0.05),with the lowest A450 value in the 0.10 g/L group.The apoptotic percentage of HTFs was (18.72±1.41)％,in the 0.10 g/L hydroxycamptothecin group and that of the control group was (3.67 ±0.36)％,showing a significant difference between them (t =-10.374,P=0.001).The expression intensity of caspase-3 and caspase-9 protein in HTFs was higher in the 0.10 g/L hydroxycamptothecin group than that in the control group.JC-1 staining showed that the green fluorescence of the monomer JC-1 in cytoplasm was stronger in the 0.10 g/L hydroxycamptothecin group than that in the control group,but the red fluorescence of the polymer JC-1 in the 0.10 g/L hydroxycamptothecin group was weaker than that in the control group.The grey scale of cyt C protein in HTFs in mitochondrion was 0.0605±0.0022 in the 0.10 g/L hydroxycamptothecin group,showing a significant increase in comparison with 0.0301 ±0.0016 of the control group (t=4.865,P=0.014).However,the grey scale of cyt C protein in cytoplasm was declined in the 0.10 g/L hydroxycamptothecin group than that in the control group (0.0605 ±0.0022 vs.0.0301 ±0.0016) (t =-11.177,P =0.001).Conclusions Hydroxycamptothecin can induce the apoptosis of HTFs through activating the mitochondrial apoptosis pathway.

OBJECTIVETo observe the effect of Guilingji Capsule (GC) on the fertility, liver functions, and serum lactate dehydrogenase (LDH) of adult male SD rats exposed by 900 MHz cell phone.

METHODSTotally 18 adult male SD rats and 36 adult female rats in child-bearing period were selected and randomly divided into three groups according to weight equilibrium principle, i.e., the normal group, the radiated group, and the GC group, 6 males and 12 females in each group. Male rats in the normal group and all female rats were not radiated. Male rats in the radiated group and the GC group received radiation for 4 h per day, lasting for 18 successive days. Rats in the GC group received GC suspension at the daily dose of 0. 15 g/kg by gastrogavage at the same time. Equal volume of normal saline was administrated to other male rats. Then male rats were mated with corresponding female rats from the 14th radiation night to the 18th radiation night in the ratio of 1:2. Male rats were killed following on the next morning of ending the radiation. Female rats were normally fed and then killed before delivery. The pregnant outcomes of female rats in responding groups (the rates of pregnancy and the number of death fetus, birth weight, body length, and tail length) were observed and compared. Serum alanine aminotransferase (ALT), aspartate transferase (AST), AST/ALT, and LDH levels of the male rats were detected by colorimetry. Histological and morphological changes of liver were observed by HE staining.

RESULTSCompared with the normal group, the pregnancy rates of female rats decreased and the number of death fetus increased, the serum LDH level obviously increased in the radiated group (P < 0.05). Serum levels of ALT, AST, and AST/ALT were no significantly changed in the radiated group. The hepatocyte nuclear atrophy and cytoplasm vacuolar degeneration appeared. Compared with the radiated group, the pregnancy rates increased, the number of death fetus dropped, and the serum level of LDH decreased in the GC group (P < 0.05). There was no obvious change in serum levels of ALT, AST, or AST/ALT. The hepatocyte nuclear atrophy and cytoplasm vacuolar degeneration were significantly attenuated. The histomorphological structures recovered to normal basically in the GC group.

CONCLUSIONSThe pregnancy rates could be decreased, the number of death fetus increased, histomorphological structures abnormal, and serum LDH level increased by exposure toy GSM 900 MHz cell phone. GC could prevent and treat the aforesaid lesion. But there was no statistical difference in serum ALT or AST levels.

Animals ; Cell Phone ; Drugs, Chinese Herbal ; pharmacology ; Female ; Fertility ; Lactate Dehydrogenases ; blood ; Liver ; drug effects ; Male ; Pregnancy ; Radiation ; Rats ; Rats, Sprague-Dawley

The traditional Chinese medicine of Radix Hedysari plays an important role in invigorating gas for as-cending, benefiting blood for promoting production of fluid, and promoting circulation for removing obstruction in collaterals, which is consistent with the principle of treatment for osteoporosis. This study is designed to investigate the bioactive components on increasing peak bone mass (PBM) by exploring the spectrum-effect relationship between chromatography fingerprints and effect. Multiple indicators are selected to evaluate the pharmacological activity. In fingerprints, 21 common peaks are obtained, five of which are identified. Furthermore, gray relational analysis (GRA) is a quantitative method of gray system theory and is used to describe the correlation degree of common peaks and pharmacological activities with relational value. 21 components are then divided into three different regions, of which ononin and calycosin play an extremely significant role in increasing PBM. In addition, factor analysis and hierarchical cluster analysis (HCA) are used to screen the optimal producing area for Radix Hedysari. This provides a comprehensive and efficient method to improve the quality evaluation of Radix Hedysari, confirming the bioactive components for PBM-enhancement and further develop its medicinal value.

Background Researches showed that wild-type p53(wtp53)and Rb94 genes inhibit the growth of retinoblastoma(RB),and these genes are involved in signal pathway in the induction and maintenance of cellular senescence.Thus the combination of two genes to inhibit growth of RB is concerned.Objective This study was to observe the inhibitory effect of the co-transfection of Rb94 and wtp53 gcnc into subretina on RB with ultrasound microbubble.Methods HXO-Rb44 suspension was subretinally injected to establish the RB model in 40 SPF female Balb/c nude mice.The RB models were randomized into model control group,wtp53 group,Rb94 group and wtp53+Rb94 group,and 0.1 ml relevant gene microvesicle suspension was injected via caudal vein in the different groups,but no any gene was used in the model control group.Seven days after modeling,followed by 0.5 W/cm2ultrasonic wave irradiated the eyeballs immediately for 4 seconds ×2 times and interrupted for 24 seconds.Eyeballs were extracted 7 days after gene transfection,and the expressions of wtp53 mRNA and Rb94 mRNA in tumor tissuc were detected by RT-PCR,and wtp53 and Rb94 protein in tumor tissue were assayed using Western blot.Immunochemistry was used to exam the VEGF expression,and TUNEL was used to evaluate the apoptosis of the tumor cells.Results The model successful rate after HXO-Rb44 suspension was 80％ (32/40)and obvious malformation cells were seen under the light microscope.In 7 days after gene transfection,no response band for wtp53 mRNA and Rb94 mRNA were found.The relative expression valuc of wtp53 mRNA was 0.65±0.07 in the wtp53 group,and that in wtp53+Rb94 group was 0.32±0.02,showing a significant difference between them (t =11.743,P =0.000).Rb94mRNA relative value was 0.42 ±0.03 in Rb94 group,and that in the wtp53 + Rb94 group was 0.23 ± 0.03,with a significant difference(t=5.041,P=0.001).The response bands of wtp53 and Rb94 proteins were seen in wtp53group,Rb94 group and wtp53+Rb94 group.Immunochemistry showed that the positive reactive intensity for VEGF in tumor tissue was obviously weaker in wtp53+Rb94 group than that in the wtp53 group,Rb94 group and model control group.Apoptotic index(Al) was 37.35±2.14 in the wtp53+Rb94 group,showing a significant increase in comparison with model control group (0.46 ± 0.05),wtp53 group (5.05 ± 0.80) and Rb94 group (6.43 ± 1.02) (t =-34.395,-28.206,-26.006,P＜0.01).Conclusions Ultrasound microvesicle enable double gene transfecting into RB tumor tissue,and Rb94 gene cooperation with wtp53 gene enhance the inhibitory effect on RB by promoting the apoptosis of RB cells.

Animals ; Cadmium Chloride ; toxicity ; Dose-Response Relationship, Drug ; Estradiol ; toxicity ; Female ; Organ Size ; drug effects ; Rats ; Rats, Sprague-Dawley ; Uterus ; drug effects ; pathology

To study the in vitro anti-angiogenesis effect of three curcumin pigments (curcumin, demethoxycurcumin, bisdemethoxycurcumin). In the study, the inhibitory effect of the three curcumin pigments on proliferation of HUVEC cells induced by OX-LDL and the effect on migration of HUVEC cells were detected. The effect on neovascularization was observed by chorioallantoic membrane (CAM) test. The effect on cell adhesion factors ICAM-1 and VCAM-1 of HUVECs were tested by Real-time RT-PCR. It was found that the three curcumins could inhibit the proliferation of HUVEC cells induced by OX-LDL within the dosage range 4, 8, 16 mg x L(-1), with a dose-dependence. The proliferative effect of curcumins on HUVECs was greater than the other two derivatives (P < 0.01). All of the three curcumin pigments inhibited the migration of HUVEC cells and the angiogenesis of chick chorioallantoic membrane (CAM). The migration inhibition rate of curcumins at middle and high concentrations was greater than the other two (P < 0.01). All of the three curcumin could down-regulate the expression of VEGF and ICAM-1, and curcumins showed more obvious effect in down-regulating VEGF than demethoxycurcumin and bisdemethoxycurcumin(P < 0.01); Bisdemethoxycurcumin showed the most significant effect in down-regulating ICAM-1 (P < 0.01). All of the three showed no remarkable effect on expression of VCAM-1, and only bisdemethoxycurcumin showed the down-regulating effect (P < 0.05). According to the findings, all of the three curcumin pigments could resist angiogenesis by inhibiting proliferation and migration of endothelial cells and down-regulating the expression of VEGF and adhesion molecules ICAM-1.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Cell Movement ; drug effects ; Cells, Cultured ; Chorioallantoic Membrane ; drug effects ; Curcumin ; analogs & derivatives ; pharmacology ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; RNA, Messenger ; analysis ; Vascular Cell Adhesion Molecule-1 ; genetics ; Vascular Endothelial Growth Factor A ; genetics

Calcium hydroxide (CH) is applied to improve disinfection of root canals in most root canal retreatment. This study aimed to analyze the CH removal efficacy using 7 different root preparing files (K file, pre-curved K file, EndoActivator, Ultrasonic file, pre-curved ultrasonic file, F file and needle irrigation alone) with apical transportation. Standardized models of curved canal with such apical transportation or not were set up before applying CH to root canal for 7 days. Seven techniques described above were used for its removal. Then the roots were disassembled and digital photos were taken. The ratio of residual CH in the overall canal surface was calculated using the image analyzer image pro plus 6.0. The data were analyzed using one-way ANOVA with post hoc Tukey test. Results revealed that CH was effectively removed (P<0.05) by using all 6 mechanical methods except irrigation alone. In curved root canals with apical transportation, EndoActivator, pre-curved ultrasonic file and F file were found to be more effective in removing CH than the other four file (P<0.001), while there was no significant difference among EndoActivator, pre-curved ultrasonic file and F file groups (P>0.05). The percentage of residual CH in the canal with apical transportation was higher than that in the canal without apical transportation (P<0.05). In conclusion, CH can be hardly removed completely. Canal with apical transportation will result in insufficient CH removal. EndoActivator, pre-curved ultrasonic file and F file are more effective in the curved root canal with apical transportation.

AIM :To investigate the association of primary open-glaucoma (POAG), intraocular pressure (IOP) and systemic blood pressure in a rural population aged 50 years old or above in Shaanxi Province, China.METHODS: In the population-based, cross-sectional study, 1 775 (83.53%) residents, aged 50 years old or above, from 3 counties of Shaanxi Province, China, undertook an interview with a standard questionnaire and a detailed eye examination, including logarithm of the minimum angle of resolution visual acuity, slit-lamp biomicroscopy, applanation tonometry and dilated fundus examination. Two blood pressure reading in the sitting position were taken. Gonioscopy was performed if a narrower peripheral anterior chamber (less than one fourth of the corneal thickness) was indicated. Automated visual field testing was performed by participants assessed to have suspicion of glaucomatous disc damage or if IOP was 22mmHg or higher.RESULTS: IOP significantly correlated with systemic blood pressure, and both IOP and systolic blood pressure increased significantly with increasing age. No association between POAG and hypertension was found. The frequency of POAG increased significantly with lower diastolic perfusion pressure.CONCLUSION: Data in our study are accordance with those reported in other population-based studies, and confirm that lower diastolic perfusion pressure is a significant risk factor for primary open angle glaucoma.

AIM: To assess the prevalence and risk factors for age-related macular degeneration (AMD) in a rural population in Shaanxi Province of China.METHODS: A total of 2 835 (81.00%) people aged 40 years old or more, from Fuping county, Jingbian county and Yang county of Shaanxi Province, China, underwent a comprehensive interview and a relative eye examination. The present of AMD was classified into neovascular AMD (NV) and pure geographic atrophy (GA) by using direct ophthalmoscopy for fundus examination according to International Classification System.RESULTS: The prevalence (95% CI) of AMD was 3.00% (2.42, 3.71) in this population, of which NV accounted for 1.45% (1.05, 1.98) and 1.55% (1.14, 2.10)for GA. The prevalence of AMD increased significantly with increasing age (P ＜0.001). AMD was present in 0.47% of participants aged 40 to 49 years, rising to 11.90% of participants older than 80 years, of which the corresponding data increased from 0.28% to 4.76% for NV and from 0.19% to 7.14% for GA. No significant difference was found in the prevalence of NA and GA between genders in this population. With multiple logistic analyses, apart from advancing age, only smoking was found to have a strong association with any type of AMD.CONCLUSION: The prevalence of AMD in the rural population of Shaanxi Province of China is lower than that reported from other population-based studies in different provinces of China, less than that reported in whites, more than that reported in blacks. Except increasing age, smoking is also a significant well-known risk factor for AMD.