Objective To explore the possibility of the inner vertical outer spiral complex tubular urethra scaffold vascularization in repairing long segment of urethral defect.Methods From August 2014 to October 2015,27 clean male New Zealand white rabbits were divided into 3 groups,S1 group was transfected recombinant vascular endothelial growth factor(VEGF) gene lentiviral vector group.S2 group was vascular pedicle transfer tube group.C group was simple stent group.A 3.0 cm inner vertical outer spiral complex scaffold was constructed by using the small intestine acellular matrix (SIS) and polylactic acid copolymer (PLGA) modified by type Ⅰ collagen surface,and adipose-derived mesenchymal stem cells (ADSC) and smooth muscle cells after transformation from New Zealand white rabbits.In S1 group,the seed cells were transfected by recombinant vascular endothelial growth factor (VEGF) gene lentivirus,which express VEGF protein.The complex scaffold was used to repair 3.0 cm rabbit urethral defect In S2 group,the untransfected cells were seeded into the scaffold and embedded in the skin near the groin artery 3 weeks for repairing urethral defect with vascular pedicle transfer tube.In group C,the unseeded scaffold was used to repair the urethral defect alone.Postoperative observation and urethrography were followed 4,8 and 24 weeks after implantation.The HE staining,fluorescence tracing,immunohistochemical and scanning electron microscopy were evaluated at the same phase.Results In S1 group,there were one urinary fistula and one urethral stricture-related death,respectively.The urethra was smooth and patent,histological examination showed active hyperplasia of urethral capillary.In S2 group,there were one urinary fistula and two urethral stricture-related deaths,respectively.The urethral was rough,local thinning or dilated.Fat accumulation and mucosal contraction were found in the urethral submucosal,respctively.In C group,there were one urinary fistula,three hypospadias,and three urethral stricture-related deaths.The thickness of the urethra was uneven and stricture bending.The urethral mucosa was poorly repaired and the scar was narrow.HE and CD31 staining showed that S1 and S2 groups were active in the proliferation of urethral capillaries,and the angiogenesis was abundant.VEGF staining showed that the cytoplasm of endothelial cell layer,smooth muscle layer of vascular wall and the urothelial epithelial cell layer were fully expressed at 24 weeks,especially in epithelial cell layer.CKpan staining showed that the epithelium of S1 and S2 group developed to stratified epithelium,and the morphology of urethra was similar to normal urethra at 24 weeks.The urethral epithelial in C group of grew poor as single-level,irregular arrangement,24 weeks is still a lack of effective stratified epithelium.HE and oα-SMA staining showed that the smooth muscle and actin gradually increased in group S1 and S2,α-SMA staining in group C was scarce and increased at 24 weeks.PLGA was encapsulated by the surrounding tissue and the structure of electrospinning was clear after 4 weeks,absorbed and degraded after 8 weeks and absorbed after 24 weeks.Conclusions The inner vertical outer spiral comnplex tubular urethra scaffold maybe a reasonable method in repairing long segment urethral defects,and the methods of tubular urethra scaffold vascularization by transfected VEGF gene recombinant lentiviral vector and vascular flap deserve more research.

Objective To investigate the feasibility of measuring femoral neck torsion angle and anteversion angle by laser projection method .Methods The femoral neck torsion angle and anteversion were observed and described .An angle measuring device was designed and produced .With the device , the femoral torsion angle and anteversion angle were measured by laser projection method two times .Statistical analysis was performed on the measured value , and sides difference .Results The differences between femoral neck torsion angle and anteversion angle were observed .There was no significant difference ( P >0.05, power =100%) between the two measurements by laser projection method . Measurements of the femoral anteversion were 13.58 °±6.55 °on the left side , and 12.15 °±5.83 °on the right side . Measurements of the femoral neck torsion angle were 18.50 °±7.38 °on the left and 19.08 °±8.59 °on the right .There was no significant difference between left and right side ( P >0.05 ) .Conclusion The laser projection method is the effective method in measuring femoral neck torsion angle and anteversion angle , and has excellent repeatability .

AIM: To construct a recombinant lentivirus RNAi vector carrying cytochrome C oxidase gene to obtain the titer of the lentiviral stock for investigation of the expression in the eukaryotic cell and the affection of the COX gene silencing in the eukaryotic cells. METHODS: According to the DNA of the cytochrome C oxidase gene, we designed and synthesized complementary single-strand DNA oligos, annealed the single-stranded oligos to generate a ds oligo, cloned the ds oligo into pENTR/U6 to obtain an entry clone; An LR recombination reaction was performed between the pENTR/U6 entry construct and pLenti6/BLOCK-iT-Dest to generate expression construct, the 293FT cell line was cotransfected with pLenti6/BLOCK-iT expression construct, and the viral packaging mix, viral supernatant was harvested to determine the titer. RESULTS: The DNA sequence of interest clone to the vector was constructd to generate an entry clone and an expression clone successfully, which were proved by sequence determination. A vector producing cell line 293FT was established, and the titer for transfection was obtained. Western blotting analysis demonstrated that COX shRNA expression construction could suppress the expression of MTCOX-I. CONCLUSION: A lentivirus RNAi vector containing cytochrome C oxidase gene was successfully constructed.

Objective To study the correlationship between type Ⅰcollagenα1 (COL1A1) gene polymorphism and the occurrence of osteosarcoma. Methods Peripheral blood from 54 patients with osteosarcoma and 126 normal ones were collected, rs1061970 genotype of COL1A1 gene was amplified with PCR and products were analyzed by pyrosequencing among the samples. Results The allele frequency of TT (13.0%) and CT (48.1%) was significantly higher in pathologi-cal group than that in the normal control group, which manifested a allele frequency of TT(11.9%) and CT(30.2%) (P<0.05). Additionally, allele frequency of T in patients with osteosarcoma was 37.0%, higher than the control group (27.0%), with OR of 1.59 and 0.99-2.57 of 95%CI, with no difference of statistically significant (P>0.05), but the risk was still on the rise of osteosarcoma. Conclusion COL1A1 gene polymorphism may be related with the incidence of osteosarcoma, patients who carry the T allele of gene of COL1A1 may increase the risk of osteosarcoma occurrence.