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 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 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.

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.

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 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 discuss the clinical application of reverse dorsal metatarsal artery perforator flap for reconstruction of forefoot soft tissue defects.Methods Since January,2008 to March,2016,43 patients with forefoot soft tissue defect due to various causes were reconstructed with the flap based on dorsal metatarsal artery perforator.Dorsal pedis was used as a donor site with dorsal metatarsal artery perforator as the donor artery.The flap size varied from 2.0 cm × 4.0 cm to 6.0 cm × 8.0 cm.Among them,27,8,4 and 4 cases were reconstructed with flap based on first,second,third and fourth dorsal metatarsal artery perforator flap respectively.Results All the patients were followed-up which ranged from 3 months to 24 months.Six flaps suffered from post operative venous congestion.Out of 6,4 survived with early emergency management while the remaining 2 suffered epidermal necrosis which survived with regular dressing change.Eventually,all the flaps survived.They had good texture and they were elastic,good-looking and very wear-resisting.Walking function was normal.Conclusion Reverse dorsal metatarsal artery perforator flap is an ideal choice in reconstruction of small to medium sized soft tissue defects of forefoot.

Objective To detect the changes of CD4 + CD25 + CD127low/-regulatory T (Treg) cells in peripheral blood in patients with portal hypertension and hepatitis B virus infection before and after splenectomy,and to study the effects of splenectomy on the immune function of patients with portal hypertension.Methods The clinical data of 20 patients with portal hypertension,hepatitis B virus infection and hypersplenism who were admitted to the Third Hospital of Hebei Medical University from May 2012 to May 2013 were retrospectively analyzed.The dynamic levels of CD4 + CD25 + CD127low/ Treg cells in the peripheral blood of the 20 patients (portal hypertension group) at 1 day before splenectomy and at postoperative week 1,month 1 and month 3 were detected by flow cytometry,and the dynamic levels of CD4 + CD25 + CD127low/ Treg cells in the peripheral blood of 10healthy individuals (control group) from the same hospital were also detected by flow cytometry.The effects of changes of Treg cells on the immune function were analyzed.All data were analyzed using the t test or repeated measures analysis of variance.Results The proportions of CD4 + CD25 + CD127low/-Treg cells before operation were 5.1％ ± 3.5％ in the portal hypertension group and 1.4％ ± 0.2％ in the control group,with significant difference between the 2 groups (t =2.573,P ＜ 0.05).The proportions of CD4 + CD25 + CD127low/ Treg cells in the portal hypertension group at postoperative week 1,month 1 and month 3 were 9.2％ ±2.7％,5.6％ ± 1.7％and 2.5％± 2.1％,respectively.There was significant difference in the proportion of CD4+ CD25 + CD127low/-Treg cells between postoperative week 1 and that before operation (F =9.814,P ＜ 0.05),while there was no significant difference in the proportion of CD4 + CD25 + CD127low/-Treg cells between postoperative month 3 and that before operation (F =2.364,P ＞ 0.05).Conclusion The proportion of Treg cells increases in a short period after splenectomy,and then it decreases as time passed by,which indicates that splenectomy has slight influence on the immune system from the perspective of Treg cells.