Background Protein kinase B (Akt) is the center of multiple cellular signaling pathways,and it participates in the regulation of cell function,such as proliferation,migration,and metabolism of cells.Phosphoinositide 3-kinase (PI3K) promotes activation of Akt and therefore triggers many signal pathways.PI3K inhibitor can silent Akt,but whether it can affect the biological behavior of lens epithelial cells (LECs) during the posterior capsular opacity (PCO) is worthy of investigation.Objective This study was to explore the effect of LY294002,a PI3K inhibitor,on Akt activation in human LECs.Methods Human LECs strain,HLEC-B3 cells,were cultured and passaged.The cells were incubated to 96-well plate for 24 hours,then LY294002 was added with the final concentration at 0,10,20,30,40,50,60,70,80 μmol/L,respectively.After incubation for 24 hours,cell counting kit-8 (CCK-8) was used to detect the inhibitory rate of cell proliferation.Transforming growth factor-β2 (TGF-β2) of 10 μg/L was added in the medium of cells as TGF-β2 group,TGF-β2 + LY294002 (20 μmol/L) was used to coculture the other group of cells,and the cells without TGF-β2 and LY294002 served as the control group.Phosphorylation of Akt (p-Akt) in the cells was detected by laser scan confocal immunofluorescence microscope.The expression level of p-Akt was evaluated using Western blot.Results CCK-8 assay showed that the A value of the cells was gradually reduced with the increase of LY294002 concentration (Fgroup =9.72,P =0.00),but the A value was significant raised along with the lapse of time (Ftime =1737.54,P=0.00).Confocal immunofluorescence revealed that a little of p-Akt was expressed in the control cells.After induced by TGF-β2,lots of red fluorescence of p-Akt was seen in cells around the cell membranes.But in the TGF-β2+LY294002 co-culture cells,the fluorescence of Akt was much weaker.Western blot showed that the expression level of p-Akt was 0.91±0.08,1.48±0.13 and 0.95±0.19 in the control group,TGF-β2 group and TGF-β2 +LY294002 group,respectively,with a significant difference among the three groups (F =15.04,P =0.00).Conclusions LY294002 can inhibit the activation of Akt induced by TGF-β2.LY294002 may have utility in the prevention and treatment of PCO.

Background The pathogenesis of age-related cataract is associated with the apoptosis of lens epithelial cells (LECs) caused by oxidative stress.Previous studies showed that intracellular focal adhesion kinase (FAK) pathway can be activated by H2O2 in vitro,which induced apoptosis of cells.To investigate the effect of oxidative on FAK expression in LECs is one of important studies in the prevention of age-related cataract.Objective This study was to investigate the expression and function of FAK in human LECs treated by H2O2.Methods Human LECs strain (HLECs-B3) were cultured in vitro in the low glucose DMEM with 10％ fetal bovine serum.Different concentrations (0,30,50,70,100,300,500,700,1000 μmol/L) of H2O2 were added into the culture medium for 24 hours.The survival rate of the cells was detected by Cell Counting Kit-8 (CCK-8) assay.Cell morphology as well as the expression and distribution of FAK in the cells were observed by immunofluorescent staining under the laser confocal microscope.Apoptosis was observed by hoechst33258 staining,and Western blot assay was used to quantitatively detect the expression and phosphorylation of FAK.Results The survival rate of the cells was (1.00±0.03) ％,(1.24±0.03)％,(1.36±0.24) ％,(0.93±0.02)％,(1.75±0.19)％,(1.37±0.18) ％,(0.64±0.01)％,(0.59±0.11)％,(0.14±0.05)％ in 0,30,50,70,100,300,500,700,1000 μmol/L H2O2 groups,with a significant difference among them (F =95.30,P =0.00).The survival rates of the cells in the below 300 μmol/L H2O2 groups were significantly higher than those in the 0 μmol/L H2O2 group,and survival rates of the cells in the above 500 μmol/L H2O2 groups were significantly lower than those in the 0 μmol/L H2O2 group(all at P＜0.05).After H2 O2 treatment for 24 hours,HLECs-B3 cells transformed from polygon shape to spindle shape and extended pseudopodiums,meanwhile the green fluorescence for FAK exhibited in the cytoplasm.Cell apoptosis was found in the 1000 μ mol/L H2O2 group.Western blot assay revealed that the expressing levels (grey scale) were significantly different among the various groups (F=28.08,P=0.00),and FAK expressing levels in the below 300 μmol/L H2O2 groups were significantly higher than those of the 0 μmol/L H2O2 group; while the expressing levels in the above 500 μmol/L H2O2 groups were lower than those of the control 0 μmol/L H202 group (all at P＜0.05).After treated by different concentrations of H2O2,the phosphorylation level of intracellular FAK (p-FAK) was significantly higher in 3 hours group than that in 30 minutes group (all at P＜0.05).Conclusions H2 O2 can affect the survival,proliferation and morphology of human LECs by activating the intracellular FAK pathway,indicating that FAK may play roles in the regulation process of cell biological behavior.

Objective To investigate the clinical significance of CD55,CD59 and Aeromonas hydrophila toxin variant (FLAER) in the diagnosis of anemia and paroxysmal nocturnal hemoglobinuria (PNH).Methods Collected 30 healthy controls,22 cases of PNH,33 cases of aplastic anemia (AA),37 cases of iron deficiency anemia (IDA),45 cases of megaloblastic anemia (MA),30 cases of hemolytic anemia (HA) and 31 cases of myelodysplastic syndrome (MDS) from January 2009 to March 2017,CD55,CD59 and FLAER negative cell ratio of peripheral blood neutrophil of them were detected by multipa rameter flow cytometry.Results The detection rates of FLAER in PNH,AA and MDS groups were higher than those of CD55 and CD59,but there was significant difference in AA (x2 =7.759,5.518,P=0.005,0.019＜0.05).The average CD55,CD59 and FLAER deletion rate in PNH and AA group were significantly higher than those in normal control group and other groups (t=2.163～17.890,P=0.000～0.038＜0.05).The number of FLAER in PNH group was higher than CD59 and CD59 was higher than CD55 with the statistically significant difference (t=2.503 ～ 6.308,P=0.000 ～0.016＜ 0.05).Conclusion CD55,CD59 and FLAER have important value in the diagnosis of PNH and differential diagnosis with other anemia diseases,and can also be used to detect the presence of MDS and AA in patients with PNH.FLAER outperforms CD59,CD59 outperforms CD55.

Background Surgical induced astigmatism induced by a 5.5mm of superior scleral tunnel incision is a key cause of influencing vision outcome in the high myopic eyes after implantation of Artisan phakic intraocular lens(IOL).Objective This study was to evaluate the surgical induced astigmatism through a 5.5mm superior scleral tunnel incision during the implantation of Artisan phakic IOL.Methods This was a case observational study.The clinical data of 202 eyes of 111 patients with Artisan IOL implantation for high myopia from October 2004 through October 2008 was collected.The patients were followed-up for 12 months.The uncorrected visual acuity(UCVA),best spectacle-corrected visual acuity(BCVA),spherical equivalent(SE),cylinder equivalent(CE) and cylinder axis were examined before surgery and postoperative 1,3,6,12 months.The patients were divided into with rule group and against rule group according to the cylinder axis.Surgery-induced astigmatism value was calculated using Holladay vector analysis formula.This clinical trial was approved by the Ethics Committee of Guangdong General Hospital.Written informed consent was obtained from each patient prior to the surgery.Results The UCVA of 195 eyes(94.1%) was 0.5 or better in postoperative 12 months,and that of 172 eyes(85.1%) showed the improvement of BCVA during the follow-up duration.The mean CE values were reduced in 1,3,6,12 months after surgery in comparison with before surgery(t=-4.30,P=0.00;t=-2.27,P=0.01;t=-2.04,P=0.04;t=-2.79,P=0.01).Vector analysis of total surgically induced astigmatism revealed that the mean incision-induced astigmatism value was +1.94D,+2.26D,+2.29D,+2.25D at 171°,170°,181°,175° in 1,3,6,12 months after surgery.The surgery-induced astigmatism was(+1.97±1.84)D,(+2.25±1.75)D,(+2.27±1.76)D,(+2.24±1.75)D in with rule group at postoperative 1,3,6,12 months respectively;while those in against rule group was(+1.75±1.88)D,(+2.35±1.74)D,(+2.38±1.76)D,(+2.34±1.74)D respectively.No significant differences were found between the two groups(t=0.54,-0.29,-0.27,-0.29;P=0.59,0.78,0.79,0.78).Conclusion Implantation of Artisan IOL is an effective approach for correction of high myopia.A 5.5mm superior scleral tunnel incision in operation induces a +2.25D astigmatism at 175° meridian.

OBJECTIVETo explore the clinical effects of Dynesys system for the treatment of multiple segment lumbar degenerative disease.

METHODSA total of 28 patients with lumbar degenerative disc disease treated with Dynesys system from December 2008 to May 2011 were retrospectively reviewed. There were 16 males and 12 females, aged from 27 to 75 years old with an average of 49.1 years. Thirteen patients with multiple segmental lumbar intervertebral disc protrusion, including L3-L5 in 7 cases, L2-L4 in 1 case and L4-S1 in 5 cases. Fifteen patients with multiple segmental lumbar spinal stenosis, including L3-L5 in 10 cases, L4-L5 in 4 cases and L2-S1 in 1 case. The symptoms of lumbago and (or) intermittent claudication in all patients were treated with conservative treatments for more than 6 months and these methods did not work. Visual analogue scale (VAS) was used to analyze the lumbar and leg pain, imaging data were used to measure the intervertebral space height and intervertebral motion of fixed segment and upper adjacent segment, Oswestry Disability Index (ODI) was used to evaluate the clinical effect.

RESULTSAll operations were successful and the patients were followed up from 38 to 65 months with an average 50.6 months. At final follow-up, ODI and VAS of the low back pain and leg pain were (25.10±6.52)%, (1.25±0.70) points and (1.29±0.89) points, respectively and were decreased compared with preoperative (P<0.05). Postoperative intervertebral space heights were increased and intervertebral motions were decreased in fixed segment compared with preoperative (P<0.05). There were no significant differences in intervertebral space heights and intervertebral motions of upper adjacent segment between preoperative and postoperative (P>0.05).

CONCLUSIONDynesys system may obtain long-term clinical curative effect in treating multiple lumbar degenerative disease. It can partially preserve the intervertebral motions of the fixed segments, have little effect on adjacent segments. The long-term clinical effect of Dynesys still need longer time follow-up observation.

Adult ; Aged ; Female ; Follow-Up Studies ; Humans ; Intervertebral Disc Degeneration ; pathology ; surgery ; Joint Instability ; Lumbar Vertebrae ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Visual Analog Scale

Background Some aboard studies showed that polyatomic pathway play an important role in the microsvasculopathy of type 2 diabetes,and sorbitol dehydrogenase (SDH) gene is involved in the pathogenesis of diabetic retinopathy(DR).There is no relevant research at home up to now.Objective This study was to investigate the correlation of sorbitol dehydrogenase (SDH) G-888C gene polymorphism with diabetic retinopathy (DR) in type 2 diabetes mellitus.Methods The SDH genotypes were determined by polymerase chain reactionreaction fragment length polymorphism (PCR-RFLP) in 187 patients with diabetes and 123 normal contrels.Patients were divided into two groups depending on the presence or absence of DR(DR:n=118;NDR:n=69).The frequencies of SDH genotype and allele were assayed and compared among these groups.This study was approved by Ethic Committee of Guangdong General Hospital.Written informed consent was obtained from each individual prior to any relevant medical procedure.Results The disease course in the DR group was significantly longer than that in NDR group(t=2.070,P=0.042),and other clinical features in both groups were non-significant (all P>0.05).The genotype frequencies in the DR group,NDR group and normal control group were 24.6%,8.7% and 8.1%,respectively,and frequencies of the G allele were 42.4%,25.4% and 29.7%,showing statistically significant differences among these three groups.The GG genotype and G allele frequencies were significantly higher in the DR group than those in the NDR group and normal control group (GG:P=0.007,P=0.001;G:P=0.001,P=0.004).There were no significant differences in the frequencies of the GG genotype and G allele between the NDB group and normal control group ( P>0. 05) as well as the proliferative DR group and non-proliferative DR group (P>0.05).Conclusion SDH G-888C gene polymorphism is associated with the development of diabetic retinopathy in southern Chinese.

Background The pathogenesis and development of cataract is associated with oxidative stress-induced apoptosis of human lens epithelial cells(LECs).BH3-only protein is a factor that can initiate apoptosis,and thus the apoptotic process is probably related to the activation of the c-Jun N-terminal kinase(JNK).However,the relationship between oxidative stress-induced apoptosis of human LECs and the JNK pathway remains to be illuminated.Objective This study was to investigate the effects of the JNK/c-Jun pathway and its target gene,Bim (Bcl-2 interacting mediator of cell death)and PU M A(p53 up-regulated modulator of apoptosis),on oxidative stressinduced apoptosis of human LECs.Methods The human LECs cell line(HLEC-B3)was cultured and passaged in DMEM with 10％ fetal bovine serum in vitro.Confluent cells were incubated in 24 well plates and divided into 4 groups.Hydrogen peroxide(H2O2)(50 μmol/L)was used to treat the cells for 4,8 or 12 hours,and cells without H2O2 treatment served as the control group.Apoptosis was detected using Hoechst 33258 staining and quantified by counting the number of cells with pyknotic nuclei.In addition,confluent cells were seeded in 6 well plates,and Western blot and RT-PCR were used to detect the expression of the caspase-3,c-Jun,Bim and PUMA proteins and their mRNA in HLEC-B3,respectively.The JNK/c-Jun pathway inhibitors,CEP11004 or SP600125,were added into cultured media with H2O2,and cells treated with DMSO or H2O2 only served as negative and positive control groups.The expression of the p-JNK,JNK,p-c-Jun,c-Jun,Bim,PUMA proteins was detected by Western blot and apoptosis was assayed using Hoechst 33258 staining.200 pmoL/L of Bim or PUMA small interference RNA(siBim or siPUMA)fragments were transfected into the cells for 24 hours,respectively,and H2O2 was then used to treat the cells for 8 hours.The expression of the Bim and PUMA protein and their mRNA in the cells was detected by Western blot and RT-PCR,respectively.Results After H2O2 treatment in HLEC-B3 cells for 4,8,or 12 hours,the rates of apoptosis were 4.30％±1.15％,27.08％±0.74％ and 46.59％±0.91％,showing a significant difference among them (F=1909.433,P=0.000),and those of the 4,8,12 hour groups were significantly increased in comparison to the control group(P =0.049,0.000,0.000).Compared to untreated cells,the levels of expression of the JNK,Bim,PUMA proteins and their mRNA in HLEC-B3 cells were significantly elevated.After the addition of CEP11004 or SP600125,the expression of these protein and mRNA in HLEC-B3 cells in the presence of H2O2 was significantly weaker than that in the DMSO control group(P =0.000,0.000).After the tranfection of siBim or siPUMA,the apoptosis rates of the H2O2 treated groups were significantly higher than those in the Bim-/-or PIMA-/-group (P＜0.05).Conclusions H2O2 can activate the JNK/c-Jun pathway and up-regulate the expression of its target genes Bim and PUMA in human LECs in a time-dependent manner.Inhibiting the JNK/c-Jun pathway and interfering with the expression of Bim and PUMA can protect human LECs against oxidative stress-induced apoptosis.

Objective To study the mutagenic action of antiepileptic drugs(AEDs), and find an effective way to prevent the mutagenesis induced by AEDs,by observing the effects of AEDs on serum folic acid(FA) level and sister chromatid exchange (SCE) frequency in epileptic children.Methods Ninty epileptic children were divided into different groups on the basis of the different drugs they had taken, then detected the two indexes at different time points.Results The serum FA level and SCE frequency of the patients significantly decreased and increased after they took carbamazepine (CBZ) and valproic acid (VPA)respectively. The two indexes went back respectively when supplied with FA.Conclusions Both CBZ and VPA possess mutagenic action, yet nitrazepam does not.FA may help repair the chromosome damage and reduce the mutagenesis effects.

Objective To study the clinical features,diagnosis and treatment of eosinophilic granuloma of bone(EGB).Methods Twelve cases(8 male and 4 female)of EGB and their medical record,imaging examinations,follow-up data were reviewed,and the lesion sites,symptom and clinical features were analyzed with reference to relative literature.Results Nine unifocal cases were found in the cervical spine(3 cases),the thoracic spine(1 case),the lumber spine(1 case),the femur(1 case),the metacarpal bones(1 case),and the ilium(2 cases).Three multifocal cases were found in the thoracic and lumber spine(1 case),in the femur,the multiple ribs,the skull,the scapula(1 case)and in the skull,the femur,the ilium(1 case).The most common presenting symptom was pain at the lesion site and restricted motion was obviously in the cervical lesion.The case of the thoracic lesions was found neurologic deficit.Osteolytic destruction was found in the radiologic examination in EGB and the periosteal reaction was found in the long bone lesions,and vertebra plana was considered as typical cha-racteristics in the spine lesion.The lesions not in the spine were diagnosed by biopsy,exclusive methods and close following-up were performed in the spine lesions.Two unifocal were healed spontaneously and three were performed with curettage with one bone grafting.Multifocal and the lesions in the spine underwent chemotherapy.All cases were followed up and no recurrence was found.Conclusions EGB is commonly solitary and not seldom in the spine.Topical pain may be chief complaint.Osteolytic image was found in the radiologic examination.Biopsy and exclusive methods combining closely follow-up were used for diagnosis.EGB is self-limited observation,so curettage or chemotherapy can be used in treatment,and prognosis is good.

Objective To discuss the distribution laws of TCM syndromes of early threatened abortion; To analyze the related factors of pregnancy outcome of early threatened abortion. Methods The study included 1010 hospital patients who were diagnosed as early threatened abortion. Excel2010 was used to establish database in order to collect general information such as the age, menstrual history, times of abortion and adverse pregnancy, incidence of solar term and pregnancy week, the type of TCM syndromes and the pregnancy outcome (at least 3 months after being out of hospital) of patients. The distribution laws of TCM syndromes, and related factors of pregnancy outcome were analyzed. Results Among the 1010 cases, 762 cases were with kidney deficiency syndrome (75.4%); followed by blood heat syndrome, a total of 178 cases, accounting for 17.6%; qi and blood deficiency syndrome, a total of 40 cases, accounting for 4%; blood stasis syndrome, a total of 15 cases, accounting for 1.5%; liver stagnation syndrome, a total of 15 cases, accounting for 1.5%. The distribution of TCM syndromes was statistical significance in the different age groups and the incidence of pregnancy week (P<0.01). But there was no statistical significance in menstrual history, times of abortion, times of adverse pregnancy, and the incidence of solar term (P>0.05). Among the 1010 cases, follow-up to 698 cases, 567 cases were successful pregnancies, accounting for 81.2% of the follow-up of patients; 131 cases were failed pregnancies, accounting for 18.8% of the follow-up of patients. In the study, it found that there was statistical significance between different age groups and pregnancy outcomes (P=0.026). The pregnancy outcomes were related to the age groups (P=0.012, OR=1.063), and it was not related to TCM syndromes, times of abortion, times of adverse pregnancy, menstruation, pregnancy week. Conclusion Kidney deficiency syndrome is the main syndrome of early threatened abortion. The distribution of TCM syndromes is related to the age group and the incidence of pregnancy week of patients. Age of patients is the related factor affecting the pregnancy outcome.