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The Effects of Autophagy on Vascular Smooth Muscle Cells Proliferation

Author: ZhangPeiPei
Tutor: WangChen
School: Suzhou University
Course: Human Anatomy,Histology and Embryology
Keywords: vascular smooth muscle cells autophagy proliferation rapamycin 3-MA
CLC: R363
Type: Master's thesis
Year: 2011
Downloads: 114
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Abstract


Objective:To investigate the effects of autophagy on vascular smooth muscle cell of rat in vitro and provide a new compelling evidence for prevention of the proliferation of vascular smooth muscle cells.Methods: Abdominal aorta of healthy SD rat were used for primary VSMCs culture in 10% FBS Deulbecco’s Modified Eagle Medium( DMEM) at 37℃in a humidified atmosphere containing 5% CO2. Cells crawl out at 3 to 5 days, and could receive the first passage at 7 to 9 days.According to the different detection methods, cells were used as follows:①The experimental cells of each group used for Immunofluorescence staining,were plated in 24-well culture plates with a piece of glass placed horizontally at the bottom of each well.Every well contains 1 ml cell suspension (cell density:5×104/ml).②The VSMCs of each group,which were collected to detect the expression of PCNA and LC3 by western blot,were culutred in a 50ml culture flask.Every culture flask contains 3ml cell suspension(cell density:5×105ml). VSMCs used were those that have underpassed 3 to 6 passages . After the passaged VSMCs sticking to the wall of the culture flask about 16 h,Cells were incubated in 0.5% serum for 24 hours.Then VSMCS were divided into 7 groups:①the control group;②rapamycin group (10ng/ml);③3 - methyl adenine (3-MA) group (10ng/ml);④thrombin group (1U/ml);⑤rapamycin (10ng/ml) plus thrombin (1U/ml) group;⑥3-MA (10ng/ml) plus thrombin (1U/ml) group.⑦rapamycin plus pepstatinA(0.01μmol/ml)group .After dosing Cultured for 48h , Immunofluorescence,western blot analysis were used to detect the expression levels of the proliferation related protein PCNA and autophagy related protein LC3.Results: 1. Effect of Autophagy on proliferation of VSMCsThe expression levels of PCNA and LC3 in VSMCs were detected with Immunocytochemistry and Western Blot methods after rapamycin and 3-MA treatment,the results showed that : rapamycin significantly inhibited the proliferation of VSMCs when compared with tthe control group ( P <0.01), while 3-MA promoted the proliferation (P <0.01). With western Blot analysis,the results further revealed that F2 can promote smooth muscle cell proliferation, but when combined with rapamycin, the promotion effect of F2 was greatly reduced (P <0.01). these results suggest that autophagy can inhibit proliferation of VSMCs, and may has regulatory effect.2. Effect of proliferative factor (F2) on the proliferation and autophagy of VSMC Thrombin (F2) can promote the proliferation of VSMCs. F2 treatment significantly can decreased the expression of LC3 while significantly upregulated the expression of PCNA. However, combination use of F2 with rapamycin significantly upregulated the expression of LC3 while significantly decreased the expression of PCNA ; combination use of F2 with 3-MA inhibited autophagy and significantly increased the expression of PCNA. These results suggest that: F2 have a certain role in suppressing VSMC autophagy, but the combination therapy of F2 and rapamycin appeared to promote autophagy (P < 0.01),that mean F2 is not have great effect in inhibiting autophagy.3.The mechanism of inhibition proliferation of VSMC induces by autophagy After inhibition of lysosomal signaling pathway with pepstatin A, the expression levels of LC3 and PCNA were detected. Combination use of pepstatin A with rapamycin significantly upregulated LC3, while significantly decreased the expression of PCNA when campared with that treated with rapamycin alone, indicating that the inhibition of autophagy in VSMCs has relation with lysosomal pathway.Conclusion:1. The inhibition role of rapamycin on VSMCs are related the activation of autophagy.Autophagy can inhibit proliferation of VSMC.2. Proliferation factor (F2) had inhibitory effect on autophagy, although the effect was not significant. 3. Inhibitory effect of autophagy in VSMCs may has correlation with lysosomal pathway.

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