Objective:To explore the anti-tumor and hypoxia regulatory effect of HIF-1?siRNA in tumor tissues. Methods:The HIF-1?siRNA adenovirus vectors targeting HIF-1?were constructed and the rabbit VX2 tumor model was established.The constructed vectors were injected into rabbits through ear vein;noninterference sequence was injected as control.PET(positron emission computed tomography)was used to observe the changes of hypoxia and necrosis in tumor tissues.The tumors were removed and the tumor volumes were measured.H-E staining was used to observe the degree of the necrosis;RT-PCR was used to detect the change of HIF-1?expression in tumor tissues.Results:The recombinant HIF-1?siRNA adenovirus vectors were successfully constructed,with a titre of 1.1?10~(10)pfu/mL.PET imaging showed that the SUV of ~(18)F-FDG was significantly decreased in HIF-1?siRNA injected group than that in the control group ([3.51?0.36]%vs[8.73?0.83]%,P

Objective To study the proliferation and differentiation patterns of hepatic stem cells from mice cultured in vitro isolated identify their characteristics.Methods Mice were divided into 5 groups according to the pregnant (embryo days,ED13,ED15 and ED18) or born age (day0 and 3).Hepatic stem cells were isolated and cultured in vitro.The amount of the stem cells as well as their growing situation and differentiation pattern were observed and compared among different groups and markers of stem cells (CD117,CD90.1,CD49f,c-Met),hepatic cells(AFP) were used to identify the cultures.Resulls The cells with best situation of growing as colonies were obtained from ED15 group.Their expression of specific markers suggested that they were hepatic stem cells.The stem cells isolated from ED15 mice in subculturing proliferated in line pattern and differentiated in reverse line pattern.The expression of AFP varied as normal distribution as cell differentiation development.Conclusion Most cells have characteristics of hepatic stem cells isolated from fetal liver and the number of these cells decreases gradually as embryo duration prolongs.The hepatic stem cells proliferate in line pattern and differentiate into hepatic cells after in vitro culture.

Objective To construct a sequence tag library of HepG2 cells by serial analysis of gene expression (SAGE). Methods Total RNA of HepG2 cells was extracted using a Trizol agent. The double strand cDNA were reverse-translated from mRNA which was isolated from total RNA. According to the protocol of SAGE, a sequence tag library of HepG2 cells was constructed. The transcript, which was represented by each tag, was analyzed by consulting the GenBank and UniGene, and the abundance of the tags was analyzed by SAGE analysis software. Results A total of 14 181 SAGE tags out of 15 586 tags were steadily and effectively amplified and the lengths of the tags were between 200-3000 bp. After sequence analysis of the tags, 2023 specific genes were revealed. Conclusions The use of improved reverse transcription agent kit may guarantee obtaining the full length cDNA, which provides materials for the construction of SAGE library. SAGE is a powerful method to investigate the gene expression profile, and it establishes a foundation for gene researches.

Objective To evaluate the efficacy of prevention of recurrent femur fracture in children with osteogenesis imperfecta.Methods Data of 35 patients (19 boys,16 girls) who were treated for femur deformity due to osteogenesis imperfecta at our hospital from Jun 2009 to Jun 2013 were retrospectively analyzed.The average age of the patients was 9 years 3 months,ranging from 3 years 6 months to 13 years 1 month.All the patients had suffered from recurrent femur fractures before which led to femoral deformity.The mean angulation angle was 55° (range,10°-90°).10 patients were operated at one side;the other 25 patients were treated bilaterally.None of them received operation before for fractures.The average incidence of femur fracture before operation was 2.4/year (range,2-4).According to the modified Sillence classification system,9 were type Ⅲ OI,14 type Ⅳ,and 12 type I.The curved deformity of the femurs were corrected by osteotomy and stabilized by telescopic intramedullary rods in 35 patients.Results All the 35 patients were followed up for a mean period of 62 months (range,36-72).The average healing time of the osteotomy site of the femur was 8.5 weeks (range,7-12).The patient was encouraged to begin weightbearing and walking when the Xray film showed healing of the osteotomy site.By the latest follow up,80％ of the patients could stand and walk independently.The incidence of femur fracture decreased significantly to the level of 0.3±0.1/year,compared with the level of 2.4± 1.3/year before operation.All the parents of the children were satisfied with the result of deformity correction.At the latest follow up,the Barthel index score increased from 71.82 (range,51-92) before operation to 92.32 (range,82-100) postoperatively,while the WeeFIM score increased from 53.32 (range,42-72) before operation to 78 (range,70-86) postoperatively.The difference of Barthel index score and WeeFIM score between that before and post operation was significant.The children's selfcare and motion ability improved obviously after operation.During follow up,22 patients suffered from recurrent fracture of the femur by various degree,7 of them were treated by open reduction and telescopic rodding surgery,while the other 15 patients were treated conservatively because the fracture displaced or angulated minimally and the fracture healed uneventfully.Conclusion The telescopic intramedullary rod can maintain the correction of the femur deformity and improve the quality of the bone,thus prevent the recurrent fracture of the femur in children with osteogenesis imperfecta effectively.

Objective:To investigate the renal injury of portal hypertensive rat after one hour of occlusion of portal vein and inferior vena cava.Methods:Healthy male Wistar rats were taken randomly as normal control、portal hypertensive control and trial group.The recoverable portal hypertensive model was induced firstly.Three weeks later ,15 model rats were taken randomly as portal hypertensive control group,others had another operation and were divided randomly into 0,6h,12h,24h,48h,72h,7d group according to different reperfusion time after 1 hour of occlusion of portal vein and inferior vena cava.At the corresponding time points after reperfusion,the examinations below were done:serum ALT,TBIL,BUN,Cr concentrations;mor-phological changes of liver and kidney,the ultrastructure of renal tissue.Results:Serum and Cr in trail group reached their peak value 12~24 hours after reperfusion,then decreased gradually,and returned to normal 72 hours after reperfusion.The main injury of kidney was located in proximal tubular epithelial cell ,it peaked at 12 hours and 24 hours after reperfusion,the sporadic karyopyknosis and karyorrhexis could be seen,but the basement membrane preserved well.48 hours later,the restoration could be seen.7 days later it restored obviously.Conclusion:There are obvious injury in tubular epithelial cell in the portal hypertensive rat after 1 hour of occlusion of portal vein and inferior vena cava.But the injury of kidney is reversible.

Objective To evaluate the therapeutic efficacy of soft contact lenses on corneal diseases. Methods A total of 70 cases (82 eyes) of corneal diseases in the treatment group were treated with therapeutic soft contact lenses combined with topical antibiotic eye drops and artificial tears, but 64 cases (81 eyes) suffering from corneal diseases in the control group were treated with eye drops and eye ointments. ? 2 test was used to compare the cure rate of corneal epithelial defects and corneal ulcers caused by chemical and thermal burns between the two groups. Double sample t test was used to compare the average treatment time between the two groups. Results The cure rates of the corneal epithelial defects and corneal ulcers caused by chemical injury and thermal burn in the treatment group and the control group were 83% and 57%, respectively. In the treatment group, 100% patients with bullous keratopathy and 90% patients with dry eye disease were free from symptoms after wearing the lenses, but in the control group, the percentages were 50% and 63.6%, respectively. In the treatment group, 3 cases (4 eyes) of corneal epithelial defects caused by explosive injury and 3 cases of corneal epithelial defects after lameller keratoplasty were all cured, and 4 cases(4 eyes) of corneal perforations were healed. In the control group, 1 out of the 2 cases of corneal epithelial defects caused by explosive injury was cured, and 4 cases of corneal epithelial defects after lamellar keratoplasty surgery were cured, but 2 cases (2 eyes) of corneal perforation failed in the therapy, and corneal surgery was performed. In the treatment group, the cure rates of corneal epithelial defects or corneal ulcers caused by chemical injury and thermal burn were significantly higher than those in the control group ( P

Objective To investigate the serum concentrations of protein-bound uremic toxins of hippuric acid ( HA) , indoxyl sulfate ( IS ) , p-cresyl sulfate ( PCS ) and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid ( CMPF ) in patients with chronic kidney disease(CKD) 3-5 stages(CKD3-5) and to assess the correlation between renal function and pro-tein-bound uremic toxin concentrations in CKD3-5 patients.Methods Serum concentrations of HA, IS, PCS, and CMPF from 60 healthy volunteers and 112 CKD3 -5 patients were measured by liquid chromatography mass spectrometry/mass spectrometry ( HPLC-MS/MS ) .Correlation analysis was conducted between the levels of HA, IS, PCS, CMPF and the estimated glomerular filtration rate( eGFR) .Results Compared with healthy subjects, serum concentrations of these four solutes were significantly increased in CKD3-5 patients (all P<0.01).The serum levels of HA,IS and PCS in CKD3-5 patients were significantly increased (all P<0.05),while those of CMPF did not significantly change (P>0.05).Linear correlation analysis showed that HA, IS, PCS and CMPF were in significantly negative correlation with eGFR.The curve regression analysis showed that the curvilinear regression fitting equation was Y=-46.171lnX+209.464(R2 =0.601,P<0.01)for HA and eGFR, Y=-62.570 lnX+279.537(R2 =0.633,P<0.01)for IS and eGFR, Y=-84.297 lnX+383.172(R2 =0.529,P<0.01)for PCS and eGFR, and was Y=-7.648 lnX+53.546(R2 =0.172,P<0.01)for CMPF and eGFR .Conclusion The levels of the four types of protein-bound toxins in CKD3-5 patients increase significantly compared to healthy subjects.The serum levels of HA,IS and PCS are increased when the renal function decreases, but the level of CMPF changes little.Renal dysfunction can lead to significantly elevated levels of HA,IS and PCS in CKD3-5 patients, but has little effect on CMPF.

The two pabAB genes encoding aminodeoxychorismate synthase(ADC synthase)from a L-serine producing strain Corynebacterium glutamicum SYPS-062 and model strain Corynebacterium glutamicum ATCC 13032 were ampilified by PCR.The result of nucleotide sequence analysis showed that both pabAB fragments were 1863bp,encoding 620 amino acids.16 bases differences that resulted in the changes of 7 amino acids were found in the pabAB of SYPS-062.The two pabAB were inserted into pET-28a to yield the recombinant expression vector pET-28a-pabAB and then transfromed into BL21(DE3).Upon IPTG induction,soluble ADC synthase was over-produced by E.coli BL21(DE3)harboring the expression construct.Recombinant ADC synthase purified by Ni-NTA affinity chromatography showed a single band about 67kDa on SDS-PAGE gel,and activity of aminodeoxychorismate synthase analysis show that the enzyme specific activity of SYPS-062 is 46.6% lower than ATCC 13032.

Objective To explore the possible relationship between alteration of cell cycle and JAK/STAT3 signal transduction pathway inhibition induced by arsenic trioxide (As_2O_3,) in myeloma cell lines U266 and RPMI8226 in vitro. Methods Multiple myeloma cell lines U266 and RPMI8226 were used as in vitro models. The influence of AS_2O_3 on myeloma cells were evaluated by MTT assay and flow cytometry. Meanwhile, methylation specific PCR and Western blotting were employed to detect the methylation status of gene SOCS-1 and protein expression level of P-STAT3 in these cells after AS_2O_3 treatment. Results AS_2O_3 significantly inhibited the growth of U266 and RPMI8226 cells in a dose-dependent manner. Furthermore, cell cycle was arrested at G0/G1 phase with inhibition of protein expression level of P-STAT3 and SOCS-1 gene demethylation after exposure to As_2O_3 for 72 h( r = 0. 85, P < 0.05). Conclusion AS_2O_3 could induce the alteration of cell cycle which might be related to JAK/STAT3 signal transduction pathway inhibition and SOCS-1 demethylation in myeloma cell lines. The study puts forward a new idea of AS_2 O_3 treatment in multiple myeloma.

Objective To develop an ideal cultural method to amplify embryonic hepatic stem cells and inhibit their differentiation in vitro. Methods Suspension of ED 14 Fischer (F) 344 rat em-bryonic hepatic stem cells was prepared by collagenase digestion and mechanical disaggregation. Then cells were divided into two groups randomly. The cells in group 1 were seeded into type I collagen-coated plates by adherent culture while those in group 2 were seeded into soft agar medium by suspen-sion culture. After culture for 2 weeks, the morphology and ultrastructure of cells in both groups were observed and compared by inverted microscope and transmission electron microscope, respectivley.The expression of CD90. 1 and CD49F, the two specific stem cell surface markers, was tested by flow cytometry to manifest the establishment of embryonic hepatic stem cells. Alkaline phosphatase stai-ning was used to detect stem cell differentiation. Result Embryonic hepatic stem cells in group 2 were characterized by higher nucleus-cytoplasm ratio and less cell organelles, higher expression of CD90. 1 and CD49F, and stronger positive reaction for alkaline phosphatase staining compared with those in group 1. Moreover, the cells in group 1 showed significant differentiation features. Conclusion Em-bryonic hepatic stem cells cultured suspendedly in soft agar medium experience less differentiation than those adherently cultured in serum-added culture medium, and can proliferate and form clone ball with a specific stem cell feature.