Objective To observe the effect of Wnt/β-catenin pathway on proliferation of human umbilical vein endothelial cell (HUVEC) using the glycogen synthase kinase 3β (GSK-3β)-targeting RNAi recombinant adenovirus vector. Methods Homologous recombination and cloning techniques were used to construct RNAi recombinant adenoviral expressive vectors specific to GSK-3β. Then, the adenovirus plasmids was transfected into HEK 293A cells to produce adenovirus and amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock. The GSK-3β and β-catenin protein expressions were detected by Western blot and immunohistochemistry. The proliferation of HUVEC was detected with MTr assay. Results The RNAi adenovirus vectors specific to GSK-3β were successfully produced with high titer. The expression of GSK-3β protein in HUVEC could be down-regulated efficiently by the RNAi adenovirus, along with increased β-catenin protein expression. The proliferation of HUVEC was significantly increased (P < 0.05 or P < 0.01) after infected with GSK-3β RNAi recombinant adenovirus for 3, 5, 7 days. Conclusion RNAi adenovirus is an important tool that can inhibit the expression of GSK-3β efficiently, along with increased β-catenin protein expression. Up-regulating of the Wnt/β-catenin pathway might play an important role in the proliferation of HUVEC.

Objective To prepare polyclactic-co-glycolic acid(PLGA) polymer ultrasound contrast agent loading sudan black dye (SB-PLGA) and study the enhancement effect of it on rabbit lymph nodes imaging and as a sentinel lymph node (SLN) biopsy in the feasibility and value of the indicator.Methods SB-PLGA ultrasound contrast agent was made by double emulsion and freeze-drying methods.Thigh subcutaneous rabbit VX2 tumor model of eight lymph nodes were set up with the foot pad subcutaneous injection of contrast agents to get contrast-enhanced ultrasound imaging of the popliteal lymph node and the second inguinal lymph node stations.Then the blue stained lymph nodes were resected by popliteal lymph node dissection and the second station lymph nodes were also observed after 30 mins respectively,lymph nodes were examined by frozen sections HE staining in parallel.Results SB-PLGA microbubbles had a tight size distribution.Ultrasound imaging can be significantly enhanced lymph node imaging,the second station lymph node imaging was not obvious.Popliteal lymph node dissection for lymph node staining,30minutes later,frozen sections HE staining were seen within the lymph node contrast agent present in the lymphatic sinus and a large entry of macrophages.Inguinal lymph node dissection showed the second station of lymph nodes no blue staining.Conclusions SB-PLGA ultrasound contrast agent is good for sentinel lymph node imaging and biopsy indicator.Contrast agent retention and accumulation in the lymph nodes by the uptake of macrophages may be associated with the mechanisms of lymph node enhanced imaging.

Objective To prepare a kind of superparamagnetic iron oxide (SPIO) and doxorubicin loaded multifunctional polymer microbubbles (MPMBs),and to explore its potential application as an ultrasound(US)/magnetic resonance (MR) imaging contrast agent in vitro.Methods The MPMBs and normal polymer microbubbles (PMBs) were made by double emulsion and freeze-drying methods.The physical property,drug encapsulation efficiency and the drug-loading efficiency of MPMBs were determined,and the release property and US/MR imaging enhancement of MPMBs were observed.Results The MPMBs had a regular shape and narrow size distribution.The drug encapsulation efficiency was (60.20±2.69) ％,and the drug-loading efficiency was (6.02 ± 0.27) ％.The in vitro release experiment showed that ultrasound can promote the release of doxorubicin in MPMBs.US imaging in vitro showed that the enhancement of MPMBs was better than PMBs,and MR imaging in vitro conformed that MPMBs could well enhance MR imaging.Conclusions The MPMBs is a multifunctional contrast agent with the treatment function as well as US/MR dual-mode imaging enhancement effect.

Objective To verify whether the periodic or continuous exposure to high glucose may have different effects on human umbilical vein endothelial cell (HUVEC)apoptosis, and to explore the effect of NF-κB pathway on apoptosis of HUVEC induced by high glucose using the RNAi adenovirus vector. Methods RNAi combinant adenovirus vector which targeted 1566 site of NF-κB p65 mRNA was constructed and the effect of p65 gene knockdown in HUVEC was detected by Western blot analysis. Then, the RNAi adenovirus was transducted to explore the role of NF-κB pathway on the regulation of apoptosis in HUVEC induced by high glucose. The apoptosis of HUVEC was tested by flow cytometry and TUNEL assay. Results High glucose could induce apoptosis of HUVEC. p65 protein expression of nuclear extracts was significantly increased in high glucose culture as compared to control group, but only slightly increased in NF-κB-specific knockdown group, which maintained at basal state. Compared with normal glucose group, the number of TUNEL-positive cells in high glucose group was significantly increased (25.81%±1.77% vs 8.20%±0.63%, P<0.05). The number of TUNEL-positive cells was decreased in 30.5 rmnol/L glucose plus Ad-1566 than that in 30.5 mmol/L glucose plus Ad-DEST (11.49%±0.92% vs 26.10%±0.98%, P<0.01). Flow cytometry and TUNEL assay showed that the apoptosis of human umbilical vein endothelial cells induced by high glucose was inhibited by the RNAi adenovirus. Conclusion High glucose induces apoptosis of HUVEC. Knockdown of NF-κB p65 may protect HUVEC from apoptosis by preventing high glucose-induced NF-κB nuclear translocation.