本文選題：celecoxib　切入點(diǎn)：環(huán)氧合酶　出處：《南京醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 研究背景： 經(jīng)皮冠狀動(dòng)脈內(nèi)血管成形術(shù)(percutaneous transluminal coronary angioplasty，PTCA)已被廣泛應(yīng)用于冠心病的治療，但術(shù)后6個(gè)月內(nèi)擴(kuò)張部位再狹窄的發(fā)生率仍高達(dá)33％。關(guān)于再狹窄的機(jī)制主要認(rèn)為和血管中膜平滑肌細(xì)胞增生有關(guān)。環(huán)氧化酶-2(cyclooxygenase-2，COX-2)是誘導(dǎo)型環(huán)氧化酶，其終產(chǎn)物是前列腺素E_2(PGE_2)。COX-2和PGE_2與炎癥時(shí)的炎細(xì)胞反應(yīng)和腫瘤的發(fā)生發(fā)展密切相關(guān)。有學(xué)者認(rèn)為PTCA術(shù)后的血管平滑肌增生是一種炎癥反應(yīng)。celecoxib是第一個(gè)進(jìn)入臨床應(yīng)用的選擇性COX-2抑制劑，已被FDA批準(zhǔn)用于關(guān)節(jié)炎的治療和家族性多發(fā)性腸息肉病的防治，但celecoxib能否抑制血管平滑肌的增生并通過(guò)局部用藥達(dá)到防治PTCA術(shù)后再狹窄的發(fā)生尚不清楚。本課題通過(guò)celecoxib對(duì)血管平滑肌細(xì)胞(vascular smooth muscle cell，VSMC)體外作用的實(shí)驗(yàn)研究，探討了celecoxib抑制培養(yǎng)動(dòng)脈血管平滑肌細(xì)胞增生的作用及其機(jī)制。 研究目的： 1．觀察COX-2在血管平滑肌細(xì)胞中的表達(dá)情況。 2．觀察celecoxib在體外對(duì)血管平滑肌細(xì)胞抑制增殖、誘導(dǎo)凋亡的生物學(xué)效應(yīng)。 3．初步探討celecoxib抑制血管平滑肌細(xì)胞增殖、誘導(dǎo)凋亡可能的分子機(jī)制。 4．制備PLGA-celecoxib緩釋微球。 研究方法： 1．細(xì)胞培養(yǎng)：取大鼠胸主動(dòng)脈做血管平滑肌細(xì)胞原代培養(yǎng)，傳代后按常規(guī)方法培養(yǎng)。 2．Western blot實(shí)驗(yàn)檢測(cè)血管平滑肌培養(yǎng)細(xì)胞中COX-2蛋白表達(dá)水平。 3．倒置顯微鏡觀察celecoxib對(duì)細(xì)胞生長(zhǎng)的影響。 4．細(xì)胞生長(zhǎng)和活性測(cè)定(WST-1)實(shí)驗(yàn)檢測(cè)celecoxib處理后血管平滑肌培養(yǎng)細(xì)胞增殖的變化。 5．流式細(xì)胞儀檢測(cè)藥物處理后血管平滑肌培養(yǎng)細(xì)胞的凋亡指數(shù)。 6．Western blot實(shí)驗(yàn)檢測(cè)celecoxib用藥前后caspase-3的激活片段、Akt(Thr~(308))磷酸化水平的改變。 7．乳劑溶解蒸發(fā)法制備celecoxib-PLGA緩釋微球，倒置顯微鏡下觀察微球形態(tài)。 研究結(jié)果： 1．Western blot實(shí)驗(yàn)結(jié)果顯示，培養(yǎng)的血管平滑肌細(xì)胞中能夠檢測(cè)到COX-2蛋白的表達(dá)。 2．倒置顯微鏡下觀察，celecoxib處理后，隨著時(shí)間和濃度的增加，培養(yǎng)平滑肌細(xì)胞的胞體皺縮，變圓，漂浮細(xì)胞增多。 3．WST-1實(shí)驗(yàn)結(jié)果顯示，以0、6.25、12.5、25、50μM／L celecoxib分別處理細(xì)胞24小時(shí)，相對(duì)應(yīng)的細(xì)胞生長(zhǎng)率分別為100％，81.15％，66.72％，54.93％和11.41％。細(xì)胞生長(zhǎng)抑制作用呈劑量依賴性關(guān)系。 4．流式細(xì)胞儀檢測(cè)結(jié)果顯示，50μM／L Celecoxib作用24小時(shí)時(shí)，培養(yǎng)血管平滑肌細(xì)胞的凋亡率為30.72％。 5．Western blot結(jié)果顯示，，Ce1ecoxib處理24小時(shí)后可以檢測(cè)到caspase-3被激活時(shí)的20kd片段，激活片段的條帶深度與Celecoxib的藥物濃度相關(guān)；Celecoxib處理培養(yǎng)的血管平滑肌細(xì)胞2小時(shí)后Akt(Thr~(308))的磷酸化水平明顯降低。 6．倒置顯微鏡下觀察PLGA-celecoxib微球：微球均勻圓整，不粘連，球體均勻度好，粒徑分布較均勻直徑約為1～5μm，微球形態(tài)可維持至14天以上。 研究結(jié)論： 1．大鼠血管平滑肌培養(yǎng)細(xì)胞中可檢測(cè)到COX-2蛋白表達(dá)。 2．COX-2選擇性抑制劑celecoxib對(duì)血管平滑肌細(xì)胞有明顯的增殖抑制作用，并可誘導(dǎo)細(xì)胞發(fā)生凋亡，該效應(yīng)呈劑量依賴性。 3．Celecoxib誘導(dǎo)細(xì)胞發(fā)生凋亡的機(jī)制可能是通過(guò)抑制Akt(Thr~(308))的磷酸化，進(jìn)而激活caspase-3來(lái)實(shí)現(xiàn)的。 4．Celecoxib-PLGA微球可維持形態(tài)結(jié)構(gòu)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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 姚玉宇,冷靜,彭韜,尹航,黃峻;動(dòng)脈內(nèi)膜損傷后平滑肌細(xì)胞增殖與凋亡及相關(guān)基因表達(dá)[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;2002年01期

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