本文選題：云南褐煤 + 黃腐酸鈉�。� 參考：《昆明理工大學(xué)》2014年碩士論文

【摘要】：黃腐酸是一類分子結(jié)構(gòu)復(fù)雜的有機(jī)弱酸混合物,其來(lái)源不同,腐化過(guò)程不同,提取工藝不同等均有可能造成其化學(xué)和藥理學(xué)活性上的差異,這極大的限制了其在藥用上的研究進(jìn)展,因此很有必要在對(duì)黃腐酸的來(lái)源、提純、標(biāo)準(zhǔn)化和治療機(jī)制上下功夫。傳統(tǒng)用藥研究認(rèn)為黃腐酸具有抗?jié)儭⒏纳莆⒀h(huán)、止血活血等功能,其機(jī)制可能與促血管新生有關(guān),褐煤黃腐酸對(duì)血管新生的影響實(shí)際如何還未見(jiàn)報(bào)道,此外黃腐酸對(duì)癌細(xì)胞增殖影響的報(bào)道具有一定的爭(zhēng)議性,有研究認(rèn)為黃腐酸能促進(jìn)癌細(xì)胞的增殖和轉(zhuǎn)移,也有研究認(rèn)為黃腐酸能抑癌抗氧化,這是否與其來(lái)源及血管新生相關(guān)也有待探討。 以此為依據(jù)本文對(duì)云南資源豐富的年青褐煤黃腐酸在同等條件下按照地域的不同采取了多樣本研究,探討了各地黃腐酸鈉對(duì)血管新生的影響,以及黃腐酸鈉的體外抗癌活性,經(jīng)研究比較得出各地黃腐酸鈉藥理活性的特性,為云南各地褐煤資源能依據(jù)實(shí)際情況采取針對(duì)性的開(kāi)發(fā)利用提供一定的基礎(chǔ)數(shù)據(jù)。 本論文的研究以云南峨山、尋甸、昭通三地的年青褐煤為原料,采用H202氧化降解法制備,001×7型陽(yáng)離子交換樹(shù)脂純化,得到終產(chǎn)物精制黃腐酸。利用體外人臍靜脈內(nèi)皮細(xì)胞(Human umbilical vein endothelial cells, HUVEC) MTT、劃痕、血管內(nèi)皮細(xì)胞生長(zhǎng)因子(Vascular edothelial growth factor, VEGF)表達(dá)測(cè)定實(shí)驗(yàn),在體雞胚絨毛尿囊膜(Chick chorioallantoic membrane, CAM)模型血管生成實(shí)驗(yàn),測(cè)定各地黃腐酸鈉對(duì)血管新生的影響及可能機(jī)制。此外本文還采用了MTT法測(cè)定各地黃腐酸鈉對(duì)人非小細(xì)胞肺癌細(xì)胞株A549和人食管癌細(xì)胞株KYSE510的影響。主要研究結(jié)果如下： 1.峨山和尋甸的黃腐酸鈉鹽在HUVEC模型上的趨勢(shì)具有一定的相似性,在低濃度時(shí)對(duì)HUVEC的增殖和遷移具有促進(jìn)作用,同時(shí)能上調(diào)VEGF的表達(dá),隨著濃度的增高HUVEC的增殖和遷移受到抑制,分泌的VEGF濃度也降低。但兩者同時(shí)也存在一定的差異性,峨山黃腐酸鈉在50μg·ml-1時(shí)出現(xiàn)的促進(jìn)效應(yīng)最強(qiáng),尋甸在25μg·ml-1使出現(xiàn)的促進(jìn)效應(yīng)最強(qiáng),同時(shí)無(wú)論是促進(jìn)還是抑制尋甸黃腐酸鈉的作用強(qiáng)于峨山黃腐酸鈉。昭通地區(qū)的褐煤黃腐酸鈉在低濃度時(shí)對(duì)HUVEC沒(méi)有明顯的作用,在高濃度時(shí)有抑制效應(yīng),該作用與峨山地區(qū)的褐煤黃腐酸鈉相當(dāng)。 2.CAM實(shí)驗(yàn)統(tǒng)計(jì)結(jié)果顯示云南峨山地區(qū)的褐煤黃腐酸鈉在200μg·ml-1、400μg·ml-1時(shí)對(duì)CAM血管新生具有一定的促進(jìn)作用,當(dāng)濃度達(dá)到1200μg·ml-1時(shí)能夠顯著抑制CAM血管新生。尋甸褐煤黃腐酸鈉在200μg·ml-1時(shí)能明顯促進(jìn)CAM血管新生,當(dāng)濃度達(dá)到800μg·ml;-1時(shí)能顯著抑制CAM血管新生。昭通地區(qū)的褐煤黃腐酸鈉鹽不具備促進(jìn)CAM血管新生的作用,當(dāng)濃度達(dá)到800μg·ml-1時(shí)可抑制CAM血管新生。三者比較無(wú)論促進(jìn)作用還是抑制作用以尋甸黃腐酸鈉的效應(yīng)最強(qiáng)。 3.人非小細(xì)胞肺癌細(xì)胞A549MTT結(jié)果顯示,峨山、尋甸的褐煤黃腐酸鈉對(duì)A549的增殖具有一定的抑制作用,且隨著濃度的增大時(shí)間的延長(zhǎng)藥物效應(yīng)越強(qiáng),兩者比較峨山黃腐酸鈉的活性強(qiáng)于尋甸,昭通地區(qū)的黃腐酸鈉對(duì)A549幾乎無(wú)效應(yīng),當(dāng)濃度增大到800μg.ml-1時(shí)間延長(zhǎng)至42h或72h時(shí)才呈現(xiàn)一定的抑制作用。人食管癌細(xì)胞KYSE510MTT實(shí)驗(yàn)結(jié)果顯示,尋甸褐煤黃腐酸鈉在低濃度50μg·ml-1、100μg·ml-1時(shí)反而能促進(jìn)細(xì)胞的增殖,隨著濃度的增大到400μg.m1-1時(shí)才出現(xiàn)抑制效應(yīng),峨山、昭通兩地的褐煤黃腐酸鈉能抑制細(xì)胞的增長(zhǎng),隨著濃度的增大時(shí)間的延長(zhǎng)藥物效應(yīng)越強(qiáng)。 上述研究結(jié)果表明,三地的褐煤黃腐酸鈉對(duì)血管新生的影響不盡相同,對(duì)實(shí)驗(yàn)所選取的兩株癌細(xì)胞的作用也呈現(xiàn)出一定的差異性,且黃腐酸鈉促進(jìn)血管新生與促進(jìn)癌細(xì)胞增殖之間并沒(méi)有必然的聯(lián)系,本研究進(jìn)一步的說(shuō)明了黃腐酸鈉的藥理活性研究應(yīng)該以固定的產(chǎn)地及來(lái)源為依據(jù),并表明了黃腐酸鈉的作用機(jī)理是一個(gè)多級(jí)分、多靶點(diǎn)、多因素作用的過(guò)程。對(duì)于云南褐煤資源的藥用開(kāi)發(fā)應(yīng)根據(jù)實(shí)際情況采取針對(duì)性的開(kāi)采利用。
[Abstract]:Fulvic acid is a kind of complex organic weak acid mixture with different molecular structures. Its origin is different, the process of decay is different, and the different extraction processes may cause the difference in chemical and pharmacological activity, which greatly limits its research progress in medicine. Therefore, it is necessary for the source, purification, standardization and treatment machine of fulvic acid. On the basis of traditional medicine, it is believed that fulvic acid has the function of resisting ulcers, improving microcirculation, stopping blood and activating blood and so on. Its mechanism may be related to the promotion of angiogenesis, and the effect of brown coal yellow fulvic acid on angiogenesis has not yet been reported. In addition, the reports of the effect of fulvic acid on cancer cell proliferation are controversial. Fulvic acid can promote the proliferation and metastasis of cancer cells. Some studies also suggest that fulvic acid can inhibit cancer and anti-oxidation. Whether this is related to its source and angiogenesis is also to be explored.
On the basis of this, a multi sample study was taken to study the effect of sodium fulvate on angiogenesis and the anticancer activity of sodium fulvate in Yunnan under the same conditions under the same conditions, and the antitumor activity of sodium fulvate in vitro was discussed. The characteristics of the pharmacological activity of sodium fulvate in various parts of Yunnan were compared. Lignite resources can provide some basic data according to the actual situation.
The study in this paper was made of young lignite in three areas of Yunnan, Xundian and Zhaotong, prepared by H202 oxidation degradation, purified by 001 x 7 type cation exchange resin, and obtained refined fulvic acid from the final product. Using human umbilical vein endothelial cells (Human umbilical vein endothelial cells, HUVEC) MTT, scratches and vascular endothelial cell growth The Vascular edothelial growth factor (VEGF) expression test was used to determine the effect and possible mechanism of sodium fulvate on angiogenesis in the chick chorioallantoic membrane (Chick chorioallantoic membrane, CAM) model. In addition, the MTT method was used to determine the effect of sodium fulvate on human non-small cell lung cancer. The effects of cell line A549 and human esophageal cancer cell line KYSE510 were as follows:
1. the trend of the sodium fulvic acid in the 1. mount and Xundian has a certain similarity on the trend of the HUVEC model. At the low concentration, it can promote the proliferation and migration of HUVEC, and increase the expression of VEGF. With the increase of concentration, the proliferation and migration of HUVEC are inhibited, and the secretion of VEGF is also reduced. The promotion effect of Eshan sodium fulvate at 50 g. Ml-1 is the strongest. The promotion effect of Xundian in 25 mu g. Ml-1 is the strongest. At the same time, the effect of promoting or inhibiting sodium fulvate of Xundian is stronger than the sodium fulvic acid sodium. The lignite sodium fulvic acid in Zhaotong area has no obvious effect on HUVEC at low concentration, and at high concentration. The inhibition effect is similar to that of lignite in the mountain area of E Shan.
The 2.CAM experimental results show that the lignite sodium fulvate in the Yunnan area has a certain promotion effect on CAM angiogenesis at 200 g. Ml-1400 mu g. Ml-1, when the concentration reaches 1200 g. Ml-1, it can significantly inhibit the neovascularization of CAM. In the case of 200 mu g ml-1, Xundian lignite sodium fulvic can obviously promote angiogenesis, when the concentration reaches 8. 00 mu g. Ml; -1 can significantly inhibit the angiogenesis of CAM. The sodium salt of brown fulvic acid in Zhaotong area does not have the effect on promoting the angiogenesis of CAM. When the concentration reaches 800 g. Ml-1, it can inhibit the angiogenesis of CAM. The three, no matter the promotion or inhibition, the effect of sodium fulvate in Xundian is the strongest.
The results of 3. non small cell lung cancer cells A549MTT showed that the sodium fulvate of Xundian and the sodium fulvate of Xundian had a certain inhibitory effect on the proliferation of A549, and the stronger the effect of the drug effect with the time of increasing the concentration, the activity of the sodium fulvate was stronger than that in Xundian. The sodium fulvic acid in Zhaotong region had almost no effect on A549, when the concentration increased. When the time of 800 mu g.ml-1 was extended to 42h or 72h, the results of KYSE510MTT experiment in human esophageal cancer cells showed that the sodium fulvic acid of Xundian lignite could promote cell proliferation at a low concentration of 50 mu g. Ml-1100 mu g. The inhibitory effect appeared with the increase of concentration to 400 u. Lignite sodium fulvate inhibited cell growth, and the longer the concentration increased, the stronger the drug effect.
The results showed that the effect of sodium fulvate on the angiogenesis of three areas was different, and the effect on the two cancer cells selected by the experiment was also different. And the sodium fulvate promoted the angiogenesis and the proliferation of cancer cells. This study further explained the sodium fulvate. The study of pharmacological activity should be based on the fixed origin and source, and shows that the mechanism of the action of sodium fulvate is a multi-stage, multi target and multi factor process. The exploitation of Yunnan lignite resources should be exploited according to the actual situation.

【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96;TQ463

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