Objective Mapping the responsible gene for congenital nuclear cataract in a family for five generations in Yantai City,Shandong Province,China.Methods Family history and clinical data were recorded.9 unaffected members and 13 affected members in this family were involved in the study.The genes of all the involved members were amplified by polymerase chain reaction(PCR).28 microsatellite polymorphism in the 15 reported disease loci were used as genetic markers.The PCR products from each DNA sample were separated on a 6% polyacrylamide gel and analyzed.Allele-sharing analysis was carried out for exclusion,and linkage analysis was calculated with the LINKAGE(Version 5.1)package.Direct sequencing was used for GJA3 gene.Results The clinical phenotype in this family was isolated congenital nuclear cataract,the pathogenic nutation of the phenotype of which has not been reported yet.For all the 28 markers around the 15 candidate loci,there was no allele-sharing between the affected family members.At the 0.00 recombination frequency,the LOD score was-∝ in 27 of the 28 microsatellite markers with exception of D11S898.No GJA3 gene mutation was found.It indicated that there was no linkage between these markers and the pathogenic gene in this family.Conclusion The responsible gene for the congenital nuclear cataract in this family is not located on the 15 reported loci,which further indicates the clinically and genetically heterogeneity of inherited cataract,and an important clue is provided for finding more cataract responsible genes.The pathogenic gene in this family should be identified through extensive scanning of genes.

BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.

Objective To investigate the difference of retinal nerve fiber layer thickness(RNFL)in opti-cal coherence tomography(OCT)under low and moderate signal strength. Methods Four hundred eyes of people aged 46~75 with clear fundus image,no obvious fundus diseases,and satisfactory optical coherence tomography were classified according to their ages and signal strength. The peripapillary and 4 quadrants of RNFL were detect-ed with OCT. Results In satisfactory OCT images ,the signal strength that reached 4/10 and 7/10 was 1.5% and 27% respectively. Under low and moderate signal strength ,the maximum thickness of RNFL was at the superior and inferior,and the minimum thickness of RNFL was at the nasal and temporal. There were no significant differ-ences in RNFL thickness under low or moderate signal strength. Conclusion RNFL results are reliable under low and moderate signal strength.

The build-in low-pressure monolithic column combined with hydride generation atomic fluorescence spectrometry ( HG-AFS ) was employed for speciation analysis of fish meat. The sample pretreatment and separation approach could be accomplished within 30 min. The proper amount of fish sample was weighed and smashed into puree. The extraction solution composed of 10% HCl, 1% thiourea, and 0. 15% KCl was added before loaded into the automatic temperature controlled vertex system with 2000 r/min. The sample solution was separated through Merck monolithic column, with 3% ( V/V) acetonitrile, 30 mmol/L amonium acetate and 0. 03%(V/V) 2-mercaptoethanol (2-ME) as the eluent. The after-column eluent was digested by novel UV digestion device with pipeline sintered into the lamp, and then detected by hydrid-generation AFS. The rapid LC separation enabled fast mercury speciation of fish sample within 10 min. The different UV lamp digestion effects, eluent components, carrier gas, shield gas, lamp current, as well as PMT working power was optimized. Under the optimal conditions, the robust system achieved detection limits (DL) of 0. 15 μg/L and 0. 14 μg/L for methylmercury and HgⅡ, respectively. The RSD (n=7) was less than 5%, the linear correlation coefficient was 0 . 999 , and the matrix spiked recovery was in the range of 85%-110% for Hg speciation. This method was used for the determination of Hg speciation in fish and soil samples, and was proofed to be a reliable, easy approach for daily inspection.

Objective To analyse the efficacy of methotrexate combined with mifepristone in treatment of 130 cases heterotopic pregnancy. Methods 130 cases heterotopic pregnancy from January 2014 to December 2014 in obstetrics and gynecology department of First Affiliated Hospital of Liaoning Medical University were divided into observation group and control group, randomly, 65 cases in each group.Patients in control group were treated with 1 mg/kg methotrexate of intramuscular injection, one time per day; patients in observation group were treated with mifepristone of oral, one time per day, 75mg per time on the basis of control group.The serum β-subunit of human chorionic gonadotropin (β-hCG), normal pregnancy rate, size of enclosed mass, rate of treatment success and adverse reactions in two groups were compared.Results The recovery time of serum β-HCG in observation group was shorter than that in control group ( P <0.05 ) , and normal pregnancy rate in observation group was higher than that in control group (P<0.05).After 7 and 14 days’ treatment, size of enclosed mass in observation group was smaller than that in control group (P<0.05).After two months’ treatment, the efficacy in observation group was higher than that in control group (92.31%vs.61.54%;χ2 =17.33,P<0.05).There was no significant difference in adverse reactions rate between two groups (χ2 =0.34,P>0.05).Conclusion Methotrexate combined with mifepristone could improve the clinical efficacy which is a safe therapy with few adverse reactions.

Objective To analyze the clinical results and early complications of corpectomy and reconstruction with titanium mesh cage implantation and pedicle screw fixation via a single posterior approach for severe thoracic and lumbar fractures.Methods Forty-four patients treated by reconstruction with titanium mesh cage implantation and pedicle screw fixation via a single posterior were studied retrospectively.There were 35 males and 9 females,with an average age of 37.3 years(range,19-66 years).The injury segments include 1 case at T11,5 cases at T12,20 cases at L1,11 cases at L2,5 cases at L3 and 2 cases at L4.According to AO classification,there were 24 cases of A3,17 cases of B1 and B2,and 3 cases of C1.According to ASIA,there were 10 cases of grade A,17 cases of grade B,10 cases of grade C and 7 cases of grade D.The neurologic function and effectiveness of correction of preoperative,immediate postoperative and 2years follow-up were compared,and the clinical outcome and early complications were analyzed.Results The follow-up time was 24 to 58 months,mean 38.9 months.At the time of 2 years postoperation,43 cases of incomplete neurologic deficit had improved 1 or 2 ASIA grades except 1 case of grade A.The results of decompression and reduction were satisfactory from the postoperative radiographic examinations.The correction maintained well and the implant loosening was not seen in 43 cases(97.7%)at the last follow-up.The com plications include:excessive blood loss(＞1500 ml)in 9 cases,transient nerve root injury in 4,cerebrospinal fluid leakage in 3,instrumentation failure in 1,mesh cage malposition in 3,iatrogenic leaving of free bone granula into the canal in 2,and superficial infection in 1.Conclusion This technique is effective for decompression and fusion,less invasive than combined anteroposterior procedure,and may be another good alternative for the treatment of severe thoracic and lumbar fractures.The early complications are not rare,but most of them are not serious and are relative to techniques.

ObjectiveTo evaluate the role of nitric oxide (NO) in ischemia reperfusion injury (IRI) and acute rejection (AR) of intestinal transplantation in rats.MethodsThe rat orthotopic intestinal transplantation was performed. Animals were assigned to the following 4 groups with random methods:transplant control group,L-arginine (L-Arg) group,NG-Nitro-L-arginine methyl ester (L-NAME) Ⅰ group (group Ⅰ ) and L-NAME Ⅱ group (group Ⅱ ).The rats in different group were given saline,L-Arg (150 mg· kg-1 · d-1 ),L-NAME (4 and 8 mg· kg-1 · d-1 ) injection respectively from the operative day.The recipient survival time was observed.The pathologic changes were observed by HE staining.The activity of nitric oxide synthases (NOS) was measured by using immunohistochemistry.The abilities of glucose absorption and serum NO levels were tested.Results The recipient survival timein transplant control group,L-Arg group,group Ⅰ and group Ⅱ were (11.7 ± 1.2),(10.2 ± 1.0),( 12.3 ± 1.5) and ( 17.3 ± 1.9) days respectively,and the survival in group Ⅱ was prolonged significantly (P＜0.01).As compared with control group,the Park scores in L-Arg group and group Ⅰ were reduced,and IRI were attenuated; the Park score in group Ⅱ was increased (P＜0.01),the IRI was aggravated,but the AR was attenuated.As compared with control group,during the IRI period,the iNOS staining in group Ⅰ was decreased,and both iNOS and nNOS staining in group Ⅱ was decreased; during the AR period,the iNOS staining in group Ⅱ was decreased obviously.The serum NO levels were increased gradually in all groups.As compared with control group,the increase of serum NO level in group Ⅱ was delayed.As compared with control group,the glucose absorption levels in L-Arg group were increased significantly from 30 min after reperfusion to POD-3 (P＜0.01),and the postoperative glucose absorption levels in groups Ⅰ and Ⅱ maintained the low levels.ConclusionNO may play a dual role as both cytotoxic and cytoprotective effects in IRI,and aggravate mucosal damage in AR in rats intestinal transplantation.The glucose absorptive capacity of graft is promoted by supplementation of LArg at early postoperative period.

Objective To explore the effect of minimally invasive pedicle screw fixation for the treatment of thoracolumbar spine fracture.Methods Totally 80 patients with thoracolumbar spinal fracture accepted pedicle screw internal fixation in our hospital from January 2012 to December 2015 were selected as the observation object.According to the operation mode,they were equally divided into minimally invasive surgery group and open surgery group.The operation effect,quality of life and the incidence of complications of the two groups were compared.Results The operation time of the two groups had no significant difference.The amount of blood loss and postoperative drainage volume in minimally invasive surgery group were less than those in open surgery group(P ＜ 0.05).The anterior and posterior Cobb's angles of the two groups had no significant difference.The anterior and posterior Cobb's angles of the two groups both decreased 3 months after operation,and it decreased more significantly in the minimally invasive surgery group compared with the open surgery group with statistically significant difference(P ＜0.05).The VAS and ODI scores between the two groups had no significant difference before operation.And the scores of the two groups all decreased 3 months after operation,but the reduction in the minimally invasive surgery group was more significant (P ＜ 0.05).The incidence rate of complications of the two groups had no significant difference (P ＞ 0.05).The quality of life of the two groups had no difference before surgery,and it increased 3 months after the operation both in the two groups,and the minimally invasive surgery group increased more significantly (P ＜ 0.05).Conclusion The minimally invasive pedicle screw internal fixation for thoracolumbar spine fracture has a better therapeutic effect,which can significantly improve the patients clinical symptoms,signs,and their quality of life.

Objective To study the effect of electroacupuncture (EA) combined with repetitive transcranial magnetic stimulation (rTMS) on endogenous neural atem cells, as well as learning and memory in rats with cerebral ischemia. Methods One hundred and twenty male Wistar rats were randomly divided into 5 groups: a normal group, a model group, an EA group, a rTMS group and an EA plus rTMS group. After establishing acute middle cer-ebral artery occlusion, the rats in the various experimental groups were treated with EA, rTMS or EA plus rTMS. The rats' ability to avoid an electric shock was observed using a jumping stand, Immunohistochemical techniques were used to measure the infarct area and the number of Brdu-positive cells. Results A small quantity of Brdu labelled cells was observed in the subgranular zone (SGZ) and the subventricular zone (SVZ) in rats in the normal group. In the EA, rTMS and EA plus rTMS groups, the numbers of Brdu labelled cells in the SVZ and SGZ was significantly higher than in the model group after both 7 and 14 days. The expression of Brdu around the SVZ and SGZ of the 3 treatment groups increased, especially in the EA plus rTMS group. At 7, 14 and 28 d there was significant improve-ment in the neural function and electric shock avoidance test scores in the EA, rTMS and EA plus rTMS groups com-pared with the model group,with the EA plus rTMS animals scoring best. Conclusion EA plus rTMS can promote the proliferation of nerve stem cells, facilitate neurological recovery and improve learning and memory in rats after cerebral ischemia.

BACKGROUND:Intravertebral bone graft to rebuild anterior and middle column structure and to recover vertebral morphology has been re-understood, and a suitable bone graft material can promote bone healing and be conducive to rebuild the long-term stability of the spine. OBJECTIVE:To discuss the differences in clinical efficacy of three kinds of bone graft materials through unilateral pedicle to treat thoracolumbar burst fractures. METHODS:Total y 102 thoracolumbar burst fracture patients were randomized into three groups:autologous bone, autologous bone combined with al ogeneic bone and al ogeneic bone were implanted via the unilateral pedicle, respectively, in the three groups. We measured the percentage of height of the anterior edge of vertebral body and Cobb angle by X-Ray before and after bone grafting, and used CT to observe bone graft healing, and used Mimics to measure the defect area of vertebral body at the last fol ow-up. RESULTS AND CONCLUSION:Al the 102 patients were fol owed-up for 24-36 months. The percentage of height of the anterior edge of vertebral body and Cobb angle of three groups were restored after bone grafting (P<0.05), but there was no difference in the percentage of height of the anterior edge of vertebral body of three groups at different time point after bone grafting. The Cobb angle in the al ogeneic bone group was bigger than that in the autologous bone group and autologous bone combined with al ogeneic bone group at 9, 12 and 24 months after bone grafting (P<0.05). The fracture healing rate of the al ogeneic bone group at different time points was lower than that of the autologous bone group and autologous bone combined with al ogeneic bone group (P<0.05), and the area of bone defect was bigger than that in the autologous bone group and autologous bone combined with al ogeneic bone group (P<0.05). These findings indicate that these three bone graft materials can rebuild the vertebral body via the unilateral pedicle to treat thoracolumbar burst fracture, reduce the loss of vertebral height and Cobb angle, and decrease defect area of the vertebral body. The clinical efficacy of autologous bone combined with al ogeneic bone to heal bone graft and reduce bone defect is similar to autologous bone, both of which are better than al ogeneic bone alone.