celecoxib对血管平滑肌培养细胞的增殖抑制、凋亡诱导作用及分子机制的研究

发布时间：2018-03-26 08:46

本文选题：celecoxib　切入点：环氧合酶　出处：《南京医科大学》2007年硕士论文

【摘要】： 研究背景：经皮冠状动脉内血管成形术(percutaneous transluminal coronary angioplasty，PTCA)已被广泛应用于冠心病的治疗，但术后6个月内扩张部位再狭窄的发生率仍高达33％。关于再狭窄的机制主要认为和血管中膜平滑肌细胞增生有关。环氧化酶-2(cyclooxygenase-2，COX-2)是诱导型环氧化酶，其终产物是前列腺素E_2(PGE_2)。COX-2和PGE_2与炎症时的炎细胞反应和肿瘤的发生发展密切相关。有学者认为PTCA术后的血管平滑肌增生是一种炎症反应。celecoxib是第一个进入临床应用的选择性COX-2抑制剂，已被FDA批准用于关节炎的治疗和家族性多发性肠息肉病的防治，但celecoxib能否抑制血管平滑肌的增生并通过局部用药达到防治PTCA术后再狭窄的发生尚不清楚。本课题通过celecoxib对血管平滑肌细胞(vascular smooth muscle cell，VSMC)体外作用的实验研究，探讨了celecoxib抑制培养动脉血管平滑肌细胞增生的作用及其机制。研究目的： 1．观察COX-2在血管平滑肌细胞中的表达情况。 2．观察celecoxib在体外对血管平滑肌细胞抑制增殖、诱导凋亡的生物学效应。 3．初步探讨celecoxib抑制血管平滑肌细胞增殖、诱导凋亡可能的分子机制。 4．制备PLGA-celecoxib缓释微球。研究方法： 1．细胞培养：取大鼠胸主动脉做血管平滑肌细胞原代培养，传代后按常规方法培养。 2．Western blot实验检测血管平滑肌培养细胞中COX-2蛋白表达水平。 3．倒置显微镜观察celecoxib对细胞生长的影响。 4．细胞生长和活性测定(WST-1)实验检测celecoxib处理后血管平滑肌培养细胞增殖的变化。 5．流式细胞仪检测药物处理后血管平滑肌培养细胞的凋亡指数。 6．Western blot实验检测celecoxib用药前后caspase-3的激活片段、Akt(Thr~(308))磷酸化水平的改变。 7．乳剂溶解蒸发法制备celecoxib-PLGA缓释微球，倒置显微镜下观察微球形态。研究结果： 1．Western blot实验结果显示，培养的血管平滑肌细胞中能够检测到COX-2蛋白的表达。 2．倒置显微镜下观察，celecoxib处理后，随着时间和浓度的增加，培养平滑肌细胞的胞体皱缩，变圆，漂浮细胞增多。 3．WST-1实验结果显示，以0、6.25、12.5、25、50μM／L celecoxib分别处理细胞24小时，相对应的细胞生长率分别为100％，81.15％，66.72％，54.93％和11.41％。细胞生长抑制作用呈剂量依赖性关系。 4．流式细胞仪检测结果显示，50μM／L Celecoxib作用24小时时，培养血管平滑肌细胞的凋亡率为30.72％。 5．Western blot结果显示，，Ce1ecoxib处理24小时后可以检测到caspase-3被激活时的20kd片段，激活片段的条带深度与Celecoxib的药物浓度相关；Celecoxib处理培养的血管平滑肌细胞2小时后Akt(Thr~(308))的磷酸化水平明显降低。 6．倒置显微镜下观察PLGA-celecoxib微球：微球均匀圆整，不粘连，球体均匀度好，粒径分布较均匀直径约为1～5μm，微球形态可维持至14天以上。研究结论： 1．大鼠血管平滑肌培养细胞中可检测到COX-2蛋白表达。 2．COX-2选择性抑制剂celecoxib对血管平滑肌细胞有明显的增殖抑制作用，并可诱导细胞发生凋亡，该效应呈剂量依赖性。 3．Celecoxib诱导细胞发生凋亡的机制可能是通过抑制Akt(Thr~(308))的磷酸化，进而激活caspase-3来实现的。 4．Celecoxib-PLGA微球可维持形态结构至14天以上。
[Abstract]:Research background:
Percutaneous transluminal coronary angioplasty (percutaneous transluminal coronary angioplasty, PTCA) has been widely used in the treatment of coronary heart disease, but 6 months after the expansion site restenosis rate is as high as 33%. on the mechanism of restenosis is considered mainly in vascular smooth muscle cell proliferation and cyclooxygenase (cyclooxygenase-2 -2., COX-2) is an inducible isoform of cyclooxygenase, the end product of prostaglandin E_2 (PGE_2) is closely related to the occurrence and development of inflammatory cell reaction and tumor.COX-2 and PGE_2 and the inflammation. Some scholars believe that the PTCA postoperative vascular smooth muscle hyperplasia is an inflammatory reaction of.Celecoxib is the first to enter the clinical application of selective COX-2 inhibitor, has been approved by FDA for the treatment of arthritis and of multiple familial polyposis of the control, but the celecoxib can inhibit vascular smooth muscle proliferation and the local use of the drug was The prevention and treatment of restenosis after PTCA is not yet clear. In this study, we studied the effect of celecoxib on the proliferation of cultured vascular smooth muscle cells (celecoxib) through vascular in vitro, and discussed the mechanism of celecoxib inhibiting the proliferation of cultured vascular smooth muscle cells (vascular).
The purpose of the study is:
1. the expression of COX-2 in vascular smooth muscle cells was observed.
2. the biological effects of celecoxib on inhibiting proliferation and inducing apoptosis in vascular smooth muscle cells were observed in vitro.
3. the possible molecular mechanism of celecoxib to inhibit the proliferation and induce apoptosis of vascular smooth muscle cells was preliminarily investigated.
4. PLGA-celecoxib sustained-release microspheres were prepared.
Research methods:
1. cell culture: the rat thoracic aorta was taken as the primary culture of vascular smooth muscle cells and cultured in accordance with the conventional method.
2.Western blot test was used to detect the expression of COX-2 protein in cultured vascular smooth muscle cells.
3. inverted microscope was used to observe the effect of celecoxib on cell growth.
4. cell growth and activity assay (WST-1) test was used to detect the proliferation of cultured vascular smooth muscle cells after celecoxib treatment.
5. flow cytometry was used to detect the apoptosis index of cultured vascular smooth muscle cells after treatment.
6.Western blot test detected the activation of Caspase-3 in celecoxib before and after celecoxib, and the changes in the phosphorylation level of Akt (Thr~ (308)).
Celecoxib-PLGA microspheres were prepared by 7. emulsion dissolution and evaporation method, and the morphology of microspheres was observed under inverted microscope.
The results of the study:
The results of 1.Western blot experiment showed that the expression of COX-2 protein could be detected in cultured vascular smooth muscle cells.
Under 2. inverted microscope, after celecoxib treatment, with the increase of time and concentration, the cells of cultured smooth muscle cells shrink, round and floating cells increase.
3.WST-1 experiment results showed that cell growth rate was 100%, 81.15%, 66.72%, 54.93% and 11.41%. in 24 hours treated with 0,6.25,12.5,25,50 / M / L celecoxib, respectively.
The results of 4. flow cytometry showed that the apoptosis rate of cultured vascular smooth muscle cells was 30.72%. when the action of 50 M / L Celecoxib was 24 hours.
5.Western blot results showed that Ce1ecoxib treatment after 24 hours can be detected by caspase-3 activated 20kD fragment, a drug concentration with depth and the activation of Celecoxib fragments; Celecoxib treatment of cultured vascular smooth muscle cells after 2 hours of Akt (Thr~ (308)) the phosphorylation level was significantly decreased.
6. the PLGA-celecoxib microspheres were observed under inverted microscope. The microspheres were round and round without adhesion, the spheroid was homogeneous, the particle size distribution was uniform, the diameter was about 1~5 m, and the morphology of microspheres could be maintained for more than 14 days.
The conclusions are as follows:
The expression of COX-2 protein could be detected in the cultured cells of vascular smooth muscle of 1. rats.
2.COX-2 Selective Inhibitor Celecoxib can inhibit proliferation of vascular smooth muscle cells and induce cell apoptosis, which is dose dependent.
The mechanism of 3.Celecoxib inducing cell apoptosis may be achieved by inhibiting the phosphorylation of Akt (Thr~ (308)) and activating caspase-3.
4.Celecoxib-PLGA microspheres can maintain the morphological structure to more than 14 days.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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