寿胎丸对自然流产患者滋养层细胞凋亡蛋白表达的影响

发布时间：2018-06-09 20:37

本文选题：寿胎丸 + 滋养层细胞　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景及目的自然流产(Spontaneous abortion,SA)是妊娠常见并发症,按自然流产发展的不同阶段,可分为先兆流产、难免流产、不全流产、完全流产。对自然流产患者及时有效的治疗,可进一步影响妊娠结局。故自然流产预防及治疗所展开的研究具有一定的社会意义。清代医家张锡纯在《医学衷中参西录》创立的寿胎丸方现广泛用于自然流产的防治。前期实验研究发现寿胎丸含药血清呈剂量依赖性地增强自然流产患者滋养层细胞的增殖活性、侵袭迁移力,减少细胞凋亡。基于此,本课题探讨寿胎丸含药血清对自然流产患者滋养层细胞凋亡及凋亡蛋白Bcl-2、Bax、Fas、FasL、Survivin表达的影响,以期进一步探讨寿胎丸防治自然流产作用机制及其作用靶点。研究方法1、将36只SPF级雌性SD大鼠随机分为3组,分别予寿胎丸水煎液(6.25g/kg)、地屈孕酮水溶液(2.08 mg/kg)、等量生理盐水灌胃,制备寿胎丸含药血清、地屈孕酮含药血清及空白血清。2、体外分离培养正常妊娠人工流产者及难免流产患者滋养层细胞(Trophoblastic cells,TB),采用免疫细胞化学法检测贴壁细胞中细胞角蛋白(Cytokeratin-7,CK-7)和波形蛋白(Vimentin)表达,证实为滋养层细胞后进行后续实验。3、不同浓度(5%、10%、20%)寿胎丸含药血清干预自然流产患者滋养层细胞,空白血清及地屈孕酮含药血清分别作为空白对照及阳性对照。4、采用流式细胞术(Flow cytometry,FCM)检测各组滋养层细胞凋亡情况。5、采用蛋白质免疫印迹法(Western blot)检测各组凋亡相关蛋白Bcl-2、Bax、Fas、FasL、Survivin表达的差异并分析其相关性。6、统计学方法:采用SPSS20.0统计学软件进行分析,计量资料以(x±s)表示。根据实验设计的不同采用不同统计方法,以P0.05为差异有统计学意义。研究结果1、与正常对照组比较,自然流产患者滋养层细胞早期凋亡、总凋亡比例明显升高(P0.05)。2、与正常对照组相比,自然流产患者滋养层细胞Bcl-2、Survivin蛋白表达量减少,Fas、FasL、Bax蛋白表达量增多(P0.01)。3、与空白血清对照组相比,寿胎丸含药血清、地屈孕酮含药血清能降低自然流产患者滋养层细胞早期凋亡、总凋亡比例(p0.01)。4、与空白血清对照组相比,寿胎丸含药血清、地屈孕酮含药血清能上调Bcl-2、Survivin蛋白水平,下调Fas、FasL、Bax蛋白水平(P0.01)。5、寿胎丸含药血清对自然流产患者滋养层细胞凋亡及凋亡蛋白的影响呈剂量依赖性(p0.05)。6、Bcl-2与Survivin存在密切的正相关关系,Bax、Fas、FasL与Survivin存在密切的负相关关系(P0.01)。结论寿胎丸含药血清呈剂量依赖性减少自然流产患者滋养层细胞凋亡,上调Bcl-2、Survivin蛋白,下调Fas、FasL、Bax蛋白,从内源性途径、外源性途径抑制滋养层细胞凋亡是寿胎丸防治自然流产可能作用机制之一。
[Abstract]:Background and objective spontaneous abortion is a common complication of pregnancy. It can be divided into threatened abortion, incomplete abortion and complete abortion according to different stages of the development of spontaneous abortion. Timely and effective treatment of spontaneous abortion patients can further affect the outcome of pregnancy. Therefore, the study of prevention and treatment of spontaneous abortion has certain social significance. Zhang Xichun, a doctor in Qing Dynasty, founded Shou-Tye Pill prescription in Shen Xilu, widely used in the prevention and treatment of spontaneous abortion. In previous studies, it was found that the serum of Shoufeiwan enhanced the proliferation activity, invasion and migration of trophoblastic cells in a dose-dependent manner, and reduced the apoptosis of trophoblast cells in a dose-dependent manner. Based on this, the purpose of this study was to investigate the effect of Shou-Feiwan on the apoptosis of trophoblastic cells and the expression of apoptosis protein Bcl-2OBax-Fas-FasL+ survivin in the trophoblast of spontaneous abortion patients, in order to further explore the mechanism and targets of Shou-Feiwan in the prevention and treatment of spontaneous abortion. Methods 1. Thirty-six SPF female Sprague-Dawley rats were randomly divided into 3 groups. They were given Shou-Tye Pill decoction (6.25g / kg / kg), Diproprogesterone solution (2.08 mg / kg), and the same amount of normal saline intragastric perfusion to prepare the serum of Shou-Tye Pill. Trophoblastic cells were isolated from trophoblastic cells of normal pregnancy induced abortion and inevitable abortion in vitro. Cytokeratin cytokeratin-7 (CK-7) and vimentin (Vimentin) in adherent cells were detected by immunocytochemistry. It was confirmed that for the trophoblastic cells, the following experiments were carried out. 3. Different concentrations of the trophoblastic cells were used to intervene in the trophoblast cells of the spontaneous abortion patients with the serum of Shou-Feiwan. Blank serum and prodroxyprogesterone serum were used as blank control and positive control respectively. Flow cytometry (FCM) was used to detect the apoptosis of trophoblastic cells in each group. Western blot was used to detect apoptosis correlation in each group. The difference of expression of FasLN survivin in FasL of Bcl-2P and FasL was analyzed and its correlation was analyzed. 6. Statistical method: SPSS 20.0 statistical software was used to analyze the expression of survivin, and the expression of survivin was analyzed by SPSS 20.0 software. The metrological data are expressed as x 卤s. According to the different design of the experiment using different statistical methods, with P0.05 as the difference has statistical significance. Results 1.Compared with the normal control group, the early apoptosis of trophoblastic cells in patients with spontaneous abortion was significantly higher than that in the normal control group, and the ratio of total apoptosis was significantly higher than that in the normal control group. The expression of Bcl-2P survivin protein in trophoblast cells of patients with spontaneous abortion was decreased. The expression of Fas-FasL- Bax protein was increased. Compared with the control group, the serum of Shou-Feiwan and Divergesterone could reduce the early apoptosis of trophoblast cells in the patients with spontaneous abortion, compared with the control group, Shoufeiwan and Diproprogesterone could reduce the early apoptosis of trophoblastic cells. Compared with the control group, the serum of Shou-Li Wan and Diproprogone could upregulate the level of Bcl-2P survivin protein. The level of FasL / Bax protein was down-regulated (P 0.01). 5. The effect of Shouti pills on the apoptosis and apoptotic protein of trophoblast cells in spontaneous abortion patients was found to be in a dose-dependent manner. There was a close positive correlation between FasL and survivin. There was a close negative correlation between FasL and survivin (P 0.01). Conclusion the serum of Shou-Feiwan can decrease the apoptosis of trophoblastic cells in a dose-dependent manner, up-regulate the expression of Bcl-2 survivin protein and down-regulate Fas-FasL- Bax protein in spontaneous abortion patients. Inhibition of trophoblastic cell apoptosis by exogenous pathway is one of the possible mechanisms of Shou-Feiwan in the prevention and treatment of spontaneous abortion.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.21

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