本文選題：半夏瀉心湯 + 胰島β細(xì)胞凋亡��； 參考：《中國(guó)中醫(yī)科學(xué)院》2017年碩士論文

【摘要】：2型糖尿病是一種常見(jiàn)的具有遺傳易感性并受環(huán)境等多種因素影響的慢性非傳染性疾病。胰島β細(xì)胞凋亡是2型糖尿病的關(guān)鍵發(fā)病機(jī)制之一,如何減少胰島β細(xì)胞的凋亡成為防治糖尿病及其并發(fā)癥的重要方法之一。許多中醫(yī)學(xué)者認(rèn)為脾虛胃熱、痰瘀互結(jié),胃腸失調(diào)是導(dǎo)致糖尿病發(fā)生發(fā)展的重要病機(jī)。中醫(yī)調(diào)和胃腸法能夠改善胃腸道功能、健脾清熱、祛瘀化痰,可用于糖尿病及其相關(guān)疾病的治療。本研究為證實(shí)半夏瀉心湯(中醫(yī)調(diào)和胃腸法的經(jīng)典代表方)可用于糖尿病的治療,并探索其分子機(jī)制,分別從綜述和實(shí)驗(yàn)研究?jī)刹糠诌M(jìn)行闡釋。第一部分文獻(xiàn)綜述2型糖尿病的發(fā)病率近年來(lái)呈現(xiàn)不斷攀升的趨勢(shì),嚴(yán)重影響居民的生活質(zhì)量和生命安全,并帶來(lái)巨大的經(jīng)濟(jì)負(fù)擔(dān)。本病可由多種原因引起,諸如遺傳因素、環(huán)境因素,目前對(duì)于2型糖尿病病因研究較為關(guān)注的是胰島β細(xì)胞凋亡對(duì)2型糖尿病發(fā)生發(fā)展的關(guān)鍵作用。越來(lái)越多的研究證實(shí)高糖、高脂、高濃度游離脂肪酸以及多種炎癥因子等因素可加重胰島β細(xì)胞氧化應(yīng)激反應(yīng),進(jìn)而導(dǎo)致胰島β細(xì)胞的凋亡,誘發(fā)或加重2型糖尿病病情和病程。隨著對(duì)糖尿病這一病因研究的深入,許多藥物的相關(guān)治療機(jī)制正在研究過(guò)程中,諸如GLP-1類似物(如利拉魯肽、艾塞那肽)和DPP-IW抑制劑(如沙格列汀),許多研究表明這些藥物可以抑制胰島β細(xì)胞的凋亡并促進(jìn)GLP-1活性的發(fā)揮,有利于2型糖尿病的改善。然而這些藥物可能存在未納入醫(yī)保范圍,價(jià)格昂貴,不方便攜帶和保存等缺點(diǎn)。許多中醫(yī)學(xué)者認(rèn)為脾虛胃熱、痰瘀互結(jié),胃腸失調(diào)是導(dǎo)致糖尿病發(fā)生發(fā)展的重要病機(jī)。調(diào)和胃腸法能夠改善胃腸道功能、健脾清熱、祛瘀化痰。而且“胃-腸-胰島軸”的觀點(diǎn)與半夏瀉心湯從調(diào)和胃腸方面治療糖尿病不謀而合。因此,可以用于糖尿病及其相關(guān)疾病的防治。半夏瀉心湯作為調(diào)和胃腸法的經(jīng)典方,現(xiàn)代臨床及實(shí)驗(yàn)研究表明半夏瀉心湯復(fù)方及其單味藥可改善糖、脂代謝,降低血糖,改善胃腸道激素的分泌,提高機(jī)體的抗氧化能力和免疫功能,抑制胰島細(xì)胞的凋亡,用于治療糖尿病及其相關(guān)疾病。但半夏瀉心湯的作用機(jī)制尚不明確,有待驗(yàn)證。第二部分實(shí)驗(yàn)研究目的:體外培養(yǎng)RIN-m5F胰島細(xì)胞,建立RTN-m5F細(xì)胞凋亡模型并加以藥物干預(yù),通過(guò)CCK8、流式細(xì)胞術(shù)以及蛋白印跡的方法,證實(shí)半夏瀉心湯抗RIN-m5F細(xì)胞凋亡的作用及其作用機(jī)制,從分子水平證明半夏瀉心湯治療2型糖尿病的有效性和科學(xué)性。為2型糖尿病的防治提供新思路和深入研究的技術(shù)平臺(tái)。為臨床治療2型糖尿病提供新的思路和方法。方法:33只雄性SD大鼠隨機(jī)分組灌胃,取血收集含藥血清(正常血清、含半夏瀉心湯藥物血清、含利拉魯肽藥物血清),H_2O_2處理RIN-m5F細(xì)胞建立氧化應(yīng)激細(xì)胞凋亡模型,造模成功后探索半夏瀉心湯最佳濃度后隨機(jī)分組,分別為:空白對(duì)照組、模型組、半夏瀉心湯+H_2O_2組、利拉魯肽+H202組。流式細(xì)胞術(shù)檢測(cè)各組細(xì)胞凋亡率、放免法測(cè)定胰島素含量、蛋白印跡(Western blot)法測(cè)定Bcl-2,Bax,Caspase-3,PI3K,AKT,p-AKT的蛋白表達(dá)量。所得數(shù)據(jù)用Microsoft EXCEL 2010進(jìn)行管理,以均數(shù)土標(biāo)準(zhǔn)差(x一±s)表示,運(yùn)用SPSS20.0統(tǒng)計(jì)軟件,各組數(shù)據(jù)進(jìn)行正態(tài)性檢驗(yàn)和方差齊性檢驗(yàn)后,采用單因素方差分析的方法進(jìn)行統(tǒng)計(jì)學(xué)分析,組間比較采用LSD-t檢驗(yàn),P0.05認(rèn)為有統(tǒng)計(jì)學(xué)意義。結(jié)果:通過(guò)上述實(shí)驗(yàn)得出:(1)建立了 RIN-m5F細(xì)胞的凋亡模型。H_2O_2的適宜造模濃度為300μM;(2)確立了半夏瀉心湯含藥血清的最佳濃度。運(yùn)用CCK8檢測(cè)不同濃度的半夏瀉心湯含藥血清對(duì)RIN-m5F細(xì)胞的保護(hù)作用,發(fā)現(xiàn)半夏瀉心湯含藥血清最佳濃度為10%,差異有統(tǒng)計(jì)學(xué)意義(P0.05);(3)證實(shí)了半夏瀉心湯含藥血清能夠抑制RIN-m5F細(xì)胞的凋亡。流式細(xì)胞術(shù)結(jié)果顯示與模型組相比,藥物干預(yù)后,細(xì)胞活力顯著增強(qiáng),細(xì)胞凋亡率顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05);(4)證實(shí)了半夏瀉心湯含藥血清能夠促進(jìn)胰島素的分泌。放免法檢測(cè)胰島素分泌結(jié)果顯示與模型組相比,藥物干預(yù)后,葡萄糖刺激前后的胰島素分泌均顯著增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05);(5)驗(yàn)證了半夏瀉心湯含藥血清能夠降低Bax,Caspase-3的表達(dá),升高Bcl-2、p-AKT的表達(dá)。Westernblot檢測(cè)相關(guān)蛋白表達(dá)結(jié)果顯示與模型組相比,藥物干預(yù)后各組細(xì)胞Bax,Caspase-3的表達(dá)量明顯降低、Bcl-2的表達(dá)量明顯升高,p-AKT的表達(dá)明顯升高,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:半夏瀉心湯可改善RIN-m5F細(xì)胞凋亡,促進(jìn)胰島素的分泌,其可能是通過(guò)激活PI3K/AKT通路,從而調(diào)節(jié)下游因子表達(dá)有關(guān)。
[Abstract]:Type 2 diabetes is a common chronic non communicable disease with genetic susceptibility and environmental factors. The apoptosis of islet beta cells is one of the key pathogenesis of type 2 diabetes. How to reduce the apoptosis of islet beta cells is one of the important methods to prevent diabetes and its complications. Deficiency of stomach heat, phlegm and stasis, gastrointestinal disorders are important pathogenesis for the development of diabetes. Traditional Chinese medicine harmonization gastrointestinal function can improve gastrointestinal function, invigorate the spleen and heat, remove stasis and phlegm, and can be used in the treatment of diabetes and related diseases. This study confirms that the Banxia Xiexin Decoction (classical representative of Chinese medicine and gastrointestinal method) can be used in diabetes Treatment, and explore its molecular mechanism, from the two parts of the review and experimental research respectively. Part 1 literature review the incidence of type 2 diabetes in recent years has been rising trend, seriously affecting the quality of life and life safety of residents, and bring huge economic burden. This disease can be caused by a variety of reasons, such as genetic factors. Environmental factors are concerned about the key role of islet beta cell apoptosis to the development of type 2 diabetes in the study of the etiology of type 2 diabetes. More and more studies have confirmed that high glucose, high fat, high concentration of free fatty acids and many inflammatory factors can aggravate the oxidative stress reaction of islet beta cells, and then lead to islet beta cells. Apoptosis, inducing or aggravating the disease and course of type 2 diabetes. With the in-depth study of this cause of diabetes, many related mechanisms are being studied, such as GLP-1 analogues (such as lyalalu, iserin) and DPP-IW inhibitors (such as Shah Glenn Dean), and many studies have shown that these drugs can inhibit islet beta cells The apoptosis and the promotion of GLP-1 activity are beneficial to the improvement of type 2 diabetes. However, these drugs may not be included in medical insurance coverage, expensive, inconvenient to carry and preserve. Many Chinese scholars believe that spleen deficiency stomach heat, phlegm and stasis are interconnected, gastrointestinal disorders are important pathogenesis for the development of glycuria. Improving the function of gastrointestinal tract, invigorating the spleen and clearing heat, removing stasis and eliminating phlegm, and the idea of "stomach bowel islet axis" and Banxia Xiexin Decoction in the treatment of diabetes from the harmonization of stomach and intestines. Therefore, it can be used for the prevention and treatment of diabetes and its related diseases. Banxia Xiexin soup is a classic recipe for harmonization of gastrointestinal methods. Modern clinical and experimental studies show that Pinellia Xiexin Decoction compound and its single flavor medicine can improve sugar, lipid metabolism, reduce blood sugar, improve the secretion of gastrointestinal hormones, improve the body's antioxidant capacity and immune function, inhibit the apoptosis of islet cells, and use it for the treatment of diabetes and related diseases. However, the mechanism of Banxia Xiexin soup is not clear and needs to be verified. Second parts of experimental research items are still needed. The RIN-m5F islet cells were cultured in vitro, the RTN-m5F cell apoptosis model was established and drug intervention was established. The effect and mechanism of Banxia Xiexin Decoction on the apoptosis of RIN-m5F cells were confirmed by CCK8, flow cytometry and Western blot. The effectiveness and scientificity of Banxia Xiexin Decoction in the treatment of type 2 diabetes was proved at the molecular level. The prevention and control of type 2 diabetes provides a new idea and a technical platform for deep study. Method: 33 male SD rats were randomly divided into two groups, and collected blood to collect serum containing medicine (normal serum, serum containing Banxia Xiexin Decoction, serum containing relagin drug), and H_2O_2 treatment of RIN-m5F cells After the optimum concentration of the oxidative stress cell apoptosis model, the optimal concentration of Banxia Xiexin Decoction was randomly divided into two groups: blank control group, model group, group +H_2O_2 of Banxia Xiexin Decoction and +H202 group of liararu. Flow cytometry was used to detect the apoptosis rate of each group, radioimmunoassay was used to determine islet content, and Western blot method was used to determine Bcl-2, Bax, Ca. The protein expression of spase-3, PI3K, AKT, p-AKT. The data were managed with Microsoft EXCEL 2010, and the average soil standard deviation (x one + s) was expressed. The SPSS20.0 statistical software was used. The data of each group were tested by the normal test and the variance homogeneity test. The statistical analysis was carried out by the single factor variance analysis method, and the LSD-t test was used among the groups. The results of P0.05 were statistically significant. Results: (1) the optimum model concentration of RIN-m5F cell apoptosis model.H_2O_2 was established. (2) the optimum concentration of Banxia Xiexin Decoction serum was established. The protective effect of serum containing Banxia Xiexin soup with different concentrations on RIN-m5F cells was detected by CCK8, and the Pinellia ternata was found. The best concentration of the serum of Xiexin Decoction was 10%, the difference was statistically significant (P0.05); (3) it was confirmed that the serum of Banxia Xiexin Decoction could inhibit the apoptosis of RIN-m5F cells. The results of flow cytometry showed that compared with the model group, the cell viability was significantly enhanced and the apoptosis rate decreased significantly (P0.05); (4) (4) The results of radioimmunoassay showed that the insulin secretion was significantly increased after the treatment of the model group compared with the model group, and the difference was statistically significant (P0.05). (5) it was proved that the serum of Banxia Xiexin Decoction can reduce the Bax, Caspase-3 table. The expression of.Westernblot detected by the expression of.Westernblot in p-AKT showed that the expression of Bax, Caspase-3 was significantly decreased, the expression of Bcl-2 increased obviously, the expression of p-AKT increased obviously, and the difference of p-AKT was significantly increased, and the difference was statistically significant (P0.05). Conclusion: Banxia Xiexin Decoction can improve RIN-m5F fine. Apoptosis promotes insulin secretion, which may be mediated by activating the PI3K/AKT pathway, thereby regulating the expression of downstream factors.
【學(xué)位授予單位】：中國(guó)中醫(yī)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R259

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