本文選題：鴉膽子　切入點：抗腫瘤　出處：《北京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：惡性腫瘤嚴(yán)重威脅人類健康,然而,大部分抗腫瘤藥物存在著選擇性差、毒副作用大等缺點。因此,尋找高效低毒、天然來源的抗腫瘤成分迫在眉睫。鴉膽子為苦木科植物鴉膽子[Brucea javanica(Linnaeus)Merrill]的干燥成熟果實,主產(chǎn)于我國廣東、廣西、海南和福建等地。該藥為清熱解毒類中藥,味苦、性寒,臨床上常用來治療肺癌、前列腺癌和胃腸道癌等。近年來,已從鴉膽子中分離獲得多種抗腫瘤有效成分,然而關(guān)于鴉膽子蛋白質(zhì)及多肽抗腫瘤的研究卻鮮少報道。乳腺癌是造成女性腫瘤患者死亡的首要因素,而中醫(yī)藥對于乳腺癌的治療有著明顯的特色與優(yōu)勢,清熱解毒便是重要治則之一。本論文以鴉膽子蛋白質(zhì)為研究對象,通過酶解法獲得生物肽,然后采用超濾、色譜等方法進(jìn)行離純化,最終獲得顯著抑制人乳腺癌MCF-7細(xì)胞株增殖的多肽組分F9-9。本文的主要研究成果如下:鴉膽子主要成分及蛋白質(zhì)組成研究。采用旋光法、索氏提取法、凱氏定氮法、烘干法、馬弗爐法分別進(jìn)行鴉膽子淀粉、粗脂肪、蛋白質(zhì)、水分和灰分含量的測定,含量依次為 49.26 ±0.02%,16.16 ±0.13%,17.47 ±0.12%,6.74 ±0.05%和5.61 ±0.10%;采用順序抽提法提取鴉膽子清蛋白、球蛋白、醇溶蛋白和谷蛋白四類蛋白組分,并用凱氏定氮法進(jìn)行定量,各蛋白組分占總蛋白含量的百分比依次為 15.01%、8.11%、2.47%和 44.92%。鴉膽子抗腫瘤肽的制備工藝研究。以含量相對較高的谷蛋白、清蛋白和球蛋白為材料,采用胃蛋白酶進(jìn)行酶解,將酶解產(chǎn)物超濾(MWCO = 3kDa)后獲得濾過液,MTT結(jié)果顯示這三類蛋白的小分子產(chǎn)物(≤3 kDa)在濃度為50 μg·mL-1時,對MCF-7細(xì)胞的增殖抑制率分別是71.12%、75.26%和17.04%,由于球蛋白組分中蛋白質(zhì)含量高達(dá)79.28%,因此確定球蛋白為獲得抗腫瘤肽的最優(yōu)蛋白質(zhì)來源。然后,采用正交實驗法確定球蛋白的最佳提取工藝參數(shù)為:NaCl濃度5%,提取時間1.5 h,固液比1:12。最后,在底物濃度[S]為1%,酶與底物濃度比[E]/[S]為1:10,水解時間為48h的條件下,采用多種蛋白酶水解球蛋白,最終確定胃蛋白酶為水解球蛋白的最優(yōu)酶�？鼓[瘤肽的分離純化。依次采用超濾截留、SephadexG-10凝膠色譜和C18反相高效液相色譜對鴉膽子球蛋白的胃蛋白酶酶解產(chǎn)物進(jìn)行分離純化。酶解產(chǎn)物經(jīng)G4漏斗過濾后,進(jìn)行超濾(MWCO = 3kDa),獲得小分子產(chǎn)物,并命名為鴉膽子球蛋白酶解產(chǎn)物(BJGH)。研究發(fā)現(xiàn)BJGH對MCF-7細(xì)胞株具有較強(qiáng)的增殖抑制活性,經(jīng)BCA定量后,作用于MCF-7細(xì)胞72h,IC50值為2.0050.04μg·mL-1。BJGH 經(jīng) SeperdexG10 分離后,獲得 9 個組分(F1～F9),作用 MCF-7細(xì)胞72 h后,IC50值分別為33.68 ± 0.03μg·mL-1,69.87 ± 0.04μg.mL-1,32.60 ± 0.04μg·mL-1,2.03 ± 0.04 μg·mL-1,2.44 ± 0.02μg·mL-1,0.47 ± 0.02μg·mL-1,0.42 ± 0.03μg·mL-1,0.30±0.02μg·mL-1和0.25±0.02μg·mL-1,其中,組分F6、F7、F8、F9與BJGH相比,對MCF-7細(xì)胞株的增殖抑制活性顯著增強(qiáng)(P0.05)。進(jìn)而,采用半制備反相高效液相色譜對活性最高的組分F9進(jìn)行分離,共獲得9個亞組分(F9-1～F9-9),其中,F9-7,F9-8和F9-9顯示出較強(qiáng)的細(xì)胞增殖抑制活性,在樣品濃度為0.25μg·mL-1,作用MCF-7細(xì)胞72h后,它們對MCF-7細(xì)胞的增殖抑制率分別為50.20%、46.91%和62.74%;進(jìn)一步測得三組分的IC50值分別為 0.25 ± 0.01μg·mL-1,0.32 ± 0.01μg·ml-1 和 0.124 ± 0.004μg·mL-1。組分F9-9抗腫瘤作用機(jī)制研究。采用流式細(xì)胞術(shù)考察組分F9-9對MCF-7細(xì)胞株細(xì)胞凋亡及細(xì)胞周期的影響,設(shè)置F9-9濃度為0.03125μg·mL-1、0.0625μg·mL-1和0.125μg·mL-1。Annexin V-FITC/PI雙染法結(jié)果表明,組分F9-9中濃度及高濃度作用于MCF-7細(xì)胞48h后,細(xì)胞凋亡數(shù)顯著高于陰性對照組(P0.05),細(xì)胞早期凋亡總數(shù)由對照組的4.47%分別上升為13.30%和25.25%,晚期凋亡總數(shù)由對照組的13.40%上升為21.90%和25.40%;而低濃度組未造成明顯的凋亡現(xiàn)象(P0.05)。PI單染法的結(jié)果表明,F9-9低濃度作用于MCF-7細(xì)胞48 h后,與陰性對照組(67.34%)相比,加藥組可引起83.79%細(xì)胞發(fā)生明顯的G0/G1期阻滯(P0.05)。因此,F9-9低濃度時可能通過阻斷細(xì)胞周期(G0/G1期)而抑制細(xì)胞增殖,在中、高濃度時,則通過誘導(dǎo)細(xì)胞凋亡而抑制細(xì)胞增殖。應(yīng)用qRT-PCR技術(shù)檢測F9-9對抑癌基因P53和PTEN,以及腫瘤轉(zhuǎn)移抑制基因NM23H-1 mRNA表達(dá)的影響。設(shè)置F9-9濃度為0.125μg·mL-1,0.25 μg·mL-1和0.5 μg·L-1,與MCF-7細(xì)胞作用24 h后,結(jié)果表明,與陰性對照組相比,加藥組細(xì)胞P53,PTEN和NM23H-1mRNA的表達(dá)量均明顯增加(P0.05)。其中,F9-9低、中、高三個劑量組細(xì)胞P53mRNA表達(dá)量分別為對照組的1.80、2.02和1.84倍;PTEN mRNA表達(dá)量分別為對照組的5.14、3.72和4.76倍;NM23H-1 mRNA表達(dá)量分別為對照組的3.54、2.78和2.00倍。因此,F9-9可能通過上調(diào)P53,PTEN和NM23H-1 mRNA表達(dá)而抑制MCF-7細(xì)胞的增殖。本研究從鴉膽子球蛋白中獲得了可顯著抑制乳腺癌細(xì)胞增殖的多肽組分F9-9,為該藥的臨床引用提供了一定的理論指導(dǎo)。
[Abstract]:Malignant tumor of a serious threat to human health. However, most anticancer drugs has poor selectivity, toxic side effects and other disadvantages. Therefore, looking for high efficiency and low toxicity, a natural source of antitumor component is imminent. Java Brucea Javanica [Brucea javanica (Linnaeus) Merrill] is the dried ripe fruit produced in China, Guangdong, Guangxi. In Hainan and Fujian. The drug is Qingrejiedu Chinese medicine, bitter, cold, clinically used in the treatment of lung cancer, prostate cancer and gastrointestinal cancer. In recent years, has obtained many anticancer active ingredients from Brucea javanica, but the study on the anti tumor protein and polypeptide of Brucea Javanica is rarely reported. Breast cancer is the first cause of death among female patients, while Chinese medicine has distinctive features and advantages for the treatment of breast cancer, detoxification is one of the important treatment. In this paper, Brucea Javanica protein as the research object, to obtain biological peptides by enzymatic hydrolysis, then by ultrafiltration, chromatography and other methods of isolation and purification, the main research results obtained in peptide group significantly inhibit the proliferation of human breast cancer cell line MCF-7 F9-9. points of this paper are as follows: the main composition of Brucea Javanica and protein. Using optical method, Soxhlet extraction method, Kjeldahl method, drying method, Brucea starch, respectively by muffle furnace of crude fat, protein, determination of moisture and ash content. The content was 49.26 + 0.02%, 16.16 + 0.13%, 17.47 + 0.12%, 6.74 + 0.05% and 5.61 + 0.10%; sequential extraction of Brucea javanica albumin and globulin. Gliadin and glutenin four protein fractions, and quantified using the Kjeldahl method, the protein percentage of total protein content were 15.01%, 8.11%, 2.47% and 44.92%. anti-tumor peptide of Brucea javanica Study on preparation process. With relatively higher content of protein, albumin and globulin as materials, using pepsin hydrolysis, the enzymatic hydrolysate of ultrafiltration (MWCO = 3kDa) obtained after filtration, MTT results showed that the product of this three kinds of small molecule protein (less than 3 kDa) at a concentration of 50 g - mL-1 when, on MCF-7 cell proliferation inhibition rates were 71.12%, 75.26% and 17.04%, as the ball protein components in the protein content reaches 79.28%, therefore the determination of globulin is the best source of protein for anti tumor peptide. Then, using the orthogonal experimental method to determine the optimum extraction parameters of protein: NaCl concentration 5%, extraction time 1.5 h, solid-liquid ratio 1:12. finally, the substrate concentration is 1% [S], the ratio of enzyme and substrate [E]/[S] for 1:10, hydrolysis time was 48h, the hydrolysis ball kinds of protease protein, and ultimately determine the optimal pepsin hydrolysates of anti globulin. Separation and purification of peptide. Followed by ultrafiltration, pepsin of Brucea Javanica globulin SephadexG-10 gel chromatography and C18 reversed-phase HPLC hydrolysis products were isolated and purified. Enzymatic hydrolysis products by G4 funnel after filtration, ultrafiltration (MWCO = 3kDa), small molecular products, and named the ball hydrolysates of Brucea javanica (BJGH). The study found that BJGH has a strong inhibitory effect on the proliferation of MCF-7 cells were quantified by BCA, in MCF-7 cells 72h, IC50 value of 2.0050.04 g mL-1.BJGH by SeperdexG10 after the separation of 9 components (F1 ~ F9), MCF-7 cells after 72 h IC50, respectively 33.68 + 0.03 g mL-1,69.87 + 0.04 g.mL-1,32.60 + 0.04 g mL-1,2.03 + 0.04 g mL-1,2.44 + 0.02 g mL-1,0.47 + 0.02 g mL-1,0.42 + 0.03 g mL-1,0.30 + 0.02 g mL-1 and 0.25 + 0.02 g mL-1, the group F6, F7, F8, F9 and BJGH compared to MCF-7 cell proliferation inhibition activity was significantly enhanced (P0.05). Then, using semi preparative HPLC separation of the most active component F9, obtained a total of 9 subfractions (F9-1 ~ F9-9), among them, F9-7. F9-8 and F9-9 showed strong inhibitory activity on cell proliferation, the concentration of sample was 0.25 g, mL-1, MCF-7 cells after 72h on MCF-7 cell proliferation inhibition rates were 50.20%, 46.91% and 62.74%; further measured the three components of the IC50 values were 0.25 + 0.01 g mL-1,0.32 + 0.01 g ml-1 and 0.124 + 0.004 u g mL-1. component F9-9 study on the anti tumor mechanism. Flow cytometry was used to investigate effects of fraction F9-9 on MCF-7 cell apoptosis and cell cycle, setting the concentration of F9-9 was 0.03125 ~ g ~ mL-1,0.0625 ~ g ~ mL-1 and 0.125 ~ g mL-1.Annexin V-FITC/PI double staining. Results table The concentration of the components and high concentration of F9-9 in MCF-7 cells after 48h, apoptosis is significantly higher than the negative control group (P0.05), the total number of apoptosis were increased by 4.47% in the control group were 13.30% and 25.25%, the total number of late apoptosis increased from 13.40% in the control group were 21.90% and 25.40%; and the low concentration group due to distinct apoptosis (P0.05).PI single staining results showed that low concentration of F9-9 in MCF-7 cells after 48 h, and the negative control group (67.34%) compared to 83.79% cell dosing group can cause significant G0/G1 phase arrest (P0.05). Because of this, probably by blocking the cell cycle of F9-9 cells at low concentration (G0/G1) and inhibition of cell proliferation, in high concentrations, induces apoptosis and inhibit cell proliferation. Detection of F9-9 qRT-PCR application on tumor suppressor gene P53 and PTEN, and the effects of the tumor metastasis suppressor gene NM23H-1 mRNA expression. F9-9 The concentration of 0.125 G - mL-1,0.25 G - mL-1 and 0.5 g, L-1, and MCF-7 cells after 24 h results show that compared with the negative control group, the cells treated with P53, the expression of PTEN and NM23H-1mRNA were significantly increased (P0.05). Among them, F9-9 low, high dose groups the expression of P53mRNA were 1.80,2.02 and 1.84 times higher than the control group; the expression of mRNA PTEN were 5.14,3.72 and 4.76 times higher than that of the control group; the expression of mRNA in NM23H-1 group were 3.54,2.78 and 2 times of control. Therefore, F9-9 may regulate P53, PTEN and NM23H-1 mRNA expression and inhibit the proliferation of MCF-7 cells in this study. The peptide group can significantly inhibit the proliferation of breast cancer cells from F9-9 Bruceolic globulin, to provide a theoretical guidance for clinical reference for the medicine.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284;R285

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 蔡康鵬;吳靖娜;蔡水淋;潘南;蘇永昌;劉智禹;肖美添;;利用酶法制備菲律賓蛤仔抗腫瘤肽的研究[J];漁業(yè)現(xiàn)代化;2016年05期

2 王竹君;張學(xué)武;;螺旋藻水解肽的制備及其抗腫瘤活性研究[J];現(xiàn)代食品科技;2015年11期

3 譚亞芳;李娟;胡樹枝;江仁望;;鴉膽子苦醇抑制人前列腺癌DU145細(xì)胞生長及作用機(jī)制[J];廣西植物;2015年03期

4 周虹;毛建霏;羅玲;郭靈安;黃世群;葉先林;;微波堿水解法測定農(nóng)產(chǎn)品中色氨酸含量[J];食品與發(fā)酵科技;2014年03期

5 韓笑;李娜;杜培革;;抗腫瘤多肽研究進(jìn)展[J];中國生物工程雜志;2013年06期

6 肖佩;王樹鶴;;芳香化酶抑制劑與雌激素依賴性婦科疾病關(guān)系的研究進(jìn)展[J];中國婦產(chǎn)科臨床雜志;2013年03期

7 梅怡;史永照;馮雯;殷慶章;李方明;;雷公藤甲素對乳腺癌細(xì)胞P53、P73基因甲基化的影響以及對細(xì)胞增殖的抑制作用[J];第二軍醫(yī)大學(xué)學(xué)報;2012年04期

8 王健;葉波平;;超濾技術(shù)在蛋白質(zhì)分離純化中的應(yīng)用[J];藥學(xué)進(jìn)展;2012年03期

9 季宇彬;徐博慧;高世勇;;藻紅蛋白誘導(dǎo)乳腺癌MCF-7細(xì)胞凋亡與細(xì)胞周期的關(guān)系[J];中草藥;2009年S1期

10 馮曉梅;韓玉謙;趙志強(qiáng);管華詩;;牡蠣酶解產(chǎn)物中多肽的分離純化及其結(jié)構(gòu)研究[J];中國海洋藥物;2009年02期

，

本文編號：1626411