[Effect of RNA interference targeting mammalian target of rapamycin on the proliferation of vascular smooth muscle cell and the intimal hyperplasia].

OBJECTIVE: To construct RNA interference (RNAi) vector of mammalian target of rapamycin (mTOR) and study the effect on the proliferation of vascular smooth muscle cell (VSMC) and the intimal hyperplasia (IH). METHODS: The sequence of short hairpin RNA (shRNA) targeting mTOR was designed and synthesized by chemical method and inserted into a retroviral vector pLXIN, then the vector was packaged in PT67 cells. The efficiency of inhibition was verified by Northern blot and Western blot after transfection to VSMC. The changes of p70s 6k and 4E-BP1 were also determined. The proliferation activity of VSMC was determined by flowcytometry and MTT. Autogenous vein graft model were established in 54 rats. Three groups were divided, including gene therapy group, control group and vein graft group. The grafted veins were harvested 7 days, 14 days and 28 days after transplanting respectively. The expression of mTOR was determined by immunohistochemistry stain and Western blot. IH was also observed. The apoptotic VSMC was determined by TUNEL. RESULTS: The shRNA fragments was inserted into pLXIN vector. The shRNA vector was successfully constructed. Additionally, the vector targeting mTOR gene could efficiently decrease the mRNA and protein expression of mTOR and p70s 6k in VSMC, while the expression of 4E-BP1 increased significantly. The cell cycle of VSMC was stunned in phase G(0)/G(1). The protein expression of mTOR in vein graft was inhibited by transfection of pLXIN-shRNA targeting mTOR (P < 0.01) and the IH was also inhibited (P < 0.01), but the apoptosis of VSMC increased significantly (P < 0.01). CONCLUSION: The mTOR RNAi retroviral vector has been constructed successfully, which can significantly inhibit the proliferation of VSMC and the IH, and promote apoptosis after vein grafting.

[The expression of mammalian target of rapamycin and its substrates in autogenous vein graft in rats].

OBJECTIVE: To investigate the expression of mammalian target of rapamycin (mTOR) and its substrates including p70s6k and 4E-BP1 in autogenous vein graft. METHODS: Autogenous vein graft model was established in 64 Wistar rats by transplanting the right common jugular vein to infrarenal abdominal aorta. Vein graft samples were harvested 6 hours, 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks after surgery. The mRNA expression of mTOR, p70s6k and 4E-BP1 were measured by RT-PCR and in situ hybridization. Western blot and immunohistochemistry methods also were used to detect the protein expression of mTOR, p70s6k and 4E-BP1. Proliferating cell nuclear antigen (PCNA) was also detected at the same time. RESULTS: The mRNA expression of mTOR and p70s6k increased soon after vein graft transplanting, rose quickly and reached the peak 3 days to 2 weeks after surgery, which recovered 6 to 8 weeks after surgery. The expression of 4E-BP1 mRNA decreased soon after surgery and reached the lowest at 1 week, then rose to the peak 4 to 6 weeks after transplantation. Protein expression of mTOR and p70s6k reached the peak 2 to 4 weeks and recovered to normal level 8 weeks after surgery, but the expression of 4E-BP1 decreased to the lowest during 1 to 2 weeks and reached the peak 4 to 6 weeks after transplanting. The positive cells mostly located in vascular smooth muscle cell (VSMC) just like PCNA. CONCLUSIONS: The expression of mTOR and its substrates were activated in vein graft soon after transplantation, which means that mTOR and its substrates might become new targets for the prevention and therapy of stenosis or obliteration after vein graft transplanting.

[Cyclic fatigue of Vita mark II machinable ceramics under Hertzian's contact].

OBJECTIVE: To investigate the cyclic fatigue modes of Vita mark II machinable ceramics under Hertzian's contact. METHODS: Hertzian's contact technique (WC spheres r = 3.18 mm) was used to investigate the cyclic fatigue of Vita mark II machinable ceramic. All specimens were fatigued by cyclic loading in moist environment, furthermore, surviving strength was examined by three point test and morphology damage observation. RESULTS: In homogeneous Vita mark II machinable ceramics, two fatigue damage modes existed after cyclic loading with spheres under moist environment, including conventional tensile-driven cone cracking (brittle mode) and shear-driven microdamage accumulation (quasi-plastic mode). The latter generated radial cracks and deeply penetrating secondary cone crack. Initial strength degradation were caused by the cone cracks, subsequent and much more deleterious loss was caused by radial cracks. CONCLUSION: Cyclic fatigue modes of Vita mark II machinable ceramics includes brittle and quasi-plastic mode.

[Inhibition of nuclear factor kappa B attenuates multiple organ injury following ruptured abdominal aortic aneurysm: an experiment with rats].

OBJECTIVE: To investigate the effect of inhibition of nuclear factor kappa B on multiple organ injury following ruptured abdominal aortic aneurysm. METHODS: Forty-five Wistar rats underwent catheterization to observe the intestinal microcirculation blood flow, and were randomly divided into 3 equal groups. Rats of the ruptured abdominal aortic aneurysm (RAAA) group underwent laparotomy and extraction of blood to cause hemorrhage shock for 1 h (hemorrhagic shock phase), by the end of this phase normal saline at the dose of 50 ml/kg was injected intravenously, after that the abdominal aorta and bilateral common iliac arteries were blocked with artery clamps for 45 min so as to cause lower torso ischemia, and then the extracted blood was reperfused. The lungs, small intestine were taken out to undergo histological examination, and examination of lung polymorphonuclear neutrophilic leukocyte (PMN) sequestration, lung wet tissues/dry (W/D) tissues ratio, and myeloperoxidase (MPO) activity. The rats of pyrrolidine dithiocarbamate (PDTC) group were perfused with PDTC, a specific inhibitor of nuclear factor kappa B (NF-kappaB), by the end of the hemorrhagic shock phase. And the rats of the sham operation group were perfused of normal saline. RT-PCR was used to detect the mRNA expression of NF-kappaB p65 and intercellular adhesion molecule (ICAM). Western blotting was used to detect the protein expression of NF-kappaB p65 and ICAM-1. Immunohistochemistry was used to detect the expression of NF-kappaB p65 and ICAM-1 in the lung and small intestine tissues. RESULTS: Histological examination showed that severe damage could be found in the lung and small intestine of the RAAA group, and damages were significantly mild in the PDTC group. Lung PMN sequestration, W/D ratio, MPO activity were significantly increased in the RAAA group and these changes were relatively mild in the PDTC group (all P < 0.01). The intestinal microcirculation blood flow was 0.25 +/- 0.04 mlxmin(-1)xg(-1) in the RAAA group, significantly less than that of the sham operation group (0.71 +/- 0.11 mlxmin(-1)xg(-1), P < 0.01), and was 0.64 +/- 0.06 mlxmin(-1)xg(-1) in the PDTC group, significantly higher than that of the RAAA group (P < 0.01). The mRNA expression of NF-kappaB p65 in the lung of the RAAA group was 0.68 +/- 0.22, significantly higher than that of the sham operation group (0.11 +/- 0.02, P < 0.01) and that of the PDTC group (0.23 +/- 0.07, P < 0.01). The mRNA expression of NF-kappaB p65 in the intestine of the RAAA group was 0.48 +/- 0.10, significantly higher than that of the sham operation group (< 0.20 +/- 0.05, P < 0.01) and that of the PDTC group (0.27 +/- 0.06, P < 0.01). The mRNA expression of ICAM-1 in the lung of the RAAA group was 0.92 +/- 0.31, significantly higher than that of the sham operation group (0.07 +/- 0.02, P < 0.01) and that of the PDTC group (0.21 +/- 0.04, P < 0.01). The mRNA expression of ICAM-1 in the intestine of the RAAA group was 0.74 +/- 0.15, significantly higher than that of the sham operation group (0.14 +/- 0.05, P < 0.01) and that of the PDTC group (0.25 +/- 0.08, P < 0.01). The protein expression of NF-kappaB p65 in the lung of the RAAA group was 1.04 +/- 0.26, significantly higher than that of the PDTC group (0.52 +/- 0.13, P < 0.01). The protein expression of NF-kappaB p65 in the intestine of the RAAA group was 1.20 +/- 0.30, significantly higher than that of the PDTC group (0.64 +/- 0.21, P < 0.01). The protein expression of ICAM-1 in the lung of the RAAA group was 0.40 +/- 0.12, significantly higher than that of the PDTC group (0.18 +/- 0.06, P < 0.01). The protein expression of ICAM-1 in the intestine of the RAAA group was 0.46 +/- 0.15, significantly higher than that of the PDTC group (0.22 +/- 0.05, P < 0.01). Immunohistochemistry showed that NF-kappaB p65 and ICAM-1 positive cells were widely distributed in the lungs and intestine of the RAAA group and were rarely distributed in the sham operation group. CONCLUSION: PDTC attenuates the multi-organ injury by inhibiting the expression of NF-kappaB p65, thus reducing the mRNA and protein expression of its downstream gene ICAM-1 gene.

[The expression and significance of hypoxia-inducible factor-1 alpha and related genes in abdominal aorta aneurysm].

OBJECTIVE: To study the expression of hypoxia-inducible factor (HIF)-1alpha and related genes in abdominal aorta aneurysm (AAA) and explore the underlying pathogenesis. METHODS: Twenty-two AAA specimens were collected and 5 normal abdominal aorta tissue were used as control. Northern blot, western blot and immunohistochemistry were used to evaluated the expression of HIF-1alpha mRNA and protein product. Western blot and immunohistochemistry method were also used to determine the expression of vascular endothelial growth factor (VEGF) and caspase-3. Microvessel density (MVD) was studied by immunohistochemistry stain of CD34. RESULTS: The expression of HIF-1alpha mRNA and protein product were significantly higher in AAA than that in normal abdominal aorta (P < 0.01). The expression of VEGF and caspase-3 were also higher in AAA and both had a significantly positive relationship with HIF-1alpha expression (P < 0.01). Most of the positive cells located in VSMC and adventitia of AAA. The MVD counts were higher in AAA. CONCLUSION: HIF-1alpha may have an important role the development of AAA, which maybe obtained by regulating the expression of VEGF or caspase-3.