Targeting of angiopoietin 2-small interfering RNA plasmid/chitosan magnetic nanoparticles in a mouse model of malignant melanoma in vivo.

The aim of the present study was to observe the in vivo targeting characteristic of angiopoietin 2-small interfering RNA (Ang2-siRNA) plasmid/chitosan magnetic nanoparticles in an established nude mouse model of malignant melanoma (MM) under an external magnetic field. The nude mouse MM model was first established, then divided into 3 groups, including the control group, the non-targeting group and the target group, the control group was given normal saline and the non-targeting and targeting groups were administrated particles through the tail vein; the non-targeting group was not under external magnetic field and the control group and the targeting group were under external magnetic field for 60 min. The mice were then sacrificed and the tumor tissues were stained with hematoxylin and eosin and Prussian blue in order to verify the particle distributions in the tumor tissues. The control group exhibited negative Prussian blue staining in the tumor tissues, the non-targeting group demonstrated weakly positive Prussian blue staining in tumor tissues and the targeting group revealed strongly positive Prussian blue staining in tumor tissues. Ang2-siRNA plasmid vector/chitosan magnetic nanoparticles directly moved towards tumor tissues under the action of external magnetic field, thus it demonstrated good targeting characteristic.

Construction of Ang2-siRNA chitosan magnetic nanoparticles and the effect on Ang2 gene expression in human malignant melanoma cells.

The aim of the present study was to construct angiopoietin-2 (Ang2)-small interfering (si)RNA chitosan magnetic nanoparticles and to observe the interference effects of the nanoparticles on the expression of the Ang2 gene in human malignant melanoma cells. Ang2-siRNA chitosan magnetic nanoparticles were constructed and transfected into human malignant melanoma cells in vitro. Red fluorescent protein expression was observed, and the transfection efficiency was analyzed. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess the inhibition efficiency of Ang2 gene expression. Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed, and at a mass ratio of plasmid to magnetic chitosan nanoparticles of 1:100, the transfection efficiency into human malignant melanoma cells was the highest of the ratios assessed, reaching 61.17%. RT-qPCR analysis showed that the magnetic chitosan nanoparticles effectively inhibited Ang2 gene expression in cells, and the inhibition efficiency reached 59.56% (P<0.05). Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed. The in vitro studies showed that the nanoparticles inhibited Ang2 gene expression in human malignant melanoma tumor cells, which laid the foundation and provided experimental evidence for additional future in vivo studies of intervention targeting malignant melanoma tumor growth in nude mice.

[Anatomic study and application of TRAM flap with partial preservation of abdominal rectus muscle in the breast reconstruction].

OBJECTIVE: To investigate the feasibility and effectiveness of transverse rectus abdominis musculocutaneous (TRAM) flap with partial preservation of abdominal rectus muscle based on the anatomic study in cadavers. METHODS: 5 adult female cadavers which provided by department of anatomy of Fujian Medical University were dissected after injection with medical red latex from the starting point of the inferior epigastric artery and superior epigastric artery. The TRAM flap with partial preservation of lateral abdominal rectus muscle were dissected for breast reconstruction. The location, route, branches and anastomosis of inferior and superior epigastric arteries were observed. Based on the anatomic study, breast reconstruction were performed in 8 cases with muscle-sparing TRAM flaps. RESULTS: The inferior epigastric artery arises from external iliac artery (9/10, 90%) or femoral artery (1/10, 10%) at the joint point between the internal third and lateral two third. There are extensive anastomoses between superior and inferior epigastric arteries above the umbilicus, mostly between the 2cm below the first tendinous intersection and umbilical level. From Sept. 2009 to Sept. 2010, 8 cases received breast reconstruction with muscle-sparing TRAM flap. The patients were followed up for 3 months to one year. Fibrosis happened in subcutaneous fat at flap IV zone in 2 cases, borderline necrosis and subcutaneous fat liquefaction occurred in some areas of flap IV zone in 2 cases, which healed after debridement. The other 4 cases healed with no complication. Except for unsatisfied shape in one case, good result achieved in 7 cases. There was no abdominal weakness, hemia or other complication. CONCLUSIONS: It is an effective and safe method in breast reconstruction with muscle-sparing TRAM flap. It is practical with comparatively short operation time and less morbidity in donor site.

[Auricular reconstruction for concha-type microtia].

OBJECTIVE: To investigate the method of auricular reconstruction for concha-type microtia. METHODS: Two-staged auricular reconstruction was applied in 13 cases (14 ears) with concha-type microtia. The cartilage auricular framework was fabricated and implanted in the first stage, followed by ear elevation and cranio-auricle angle formation at the second stage. RESULTS: The patients were followed up for 2 months to 2 years with satisfactory aesthetic result. The reconstructed ears had a good appearance and position, and were symmetric to the healthy ears. CONCLUSIONS: The two-staged auricular reconstruction with autologous cartilage framework is ideal for concha-type microtia.

[Construction of recombinant lentiviral vector of Tie2-RNAi and its influence on malignant melanoma cells in vitro].

OBJECTIVE: To construct lentivector carrying Tie2-Small interfering RNA (SiRNA), so as to study its influence on malignant melanoma cells. METHODS: Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with XbaI, ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-I or pNL-EGFP-U6-Tie2-II, and then the electrophoresis clones was sequenced. Plasmids of pNL-EGFP-U6-Tie2-I and pNL-EGFP-U6-Tie2-II were constructed and combined with pVSVG and pHelper, respectively, to constitute lentiviral vector system of three plasmids. The Lentiviral vector system was transfected into 293T cell to produce pNL-EGFP-U6-Tie2- I and pNL-EGFP-U6-Tie2-II lentivirus. Then the supernatant was collected to determine the titer. Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. RESULTS: The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing. And the titer of lentiviral vector was 8.8 x 10(3)/ml, which was determined by 293T cell. The results of Realtime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells (P<0.01). There was no significant difference in the expression level (P>0.05) between the two lentiviral vectors of Tie2-RNAi. CONCLUSIONS: Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly. The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.