鸦胆子生物肽的分离纯化及其对人乳腺癌细胞MCF-7作用机制研究

发布时间：2018-03-17 20:47

本文选题：鸦胆子　切入点：抗肿瘤　出处：《北京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：恶性肿瘤严重威胁人类健康,然而,大部分抗肿瘤药物存在着选择性差、毒副作用大等缺点。因此,寻找高效低毒、天然来源的抗肿瘤成分迫在眉睫。鸦胆子为苦木科植物鸦胆子[Brucea javanica(Linnaeus)Merrill]的干燥成熟果实,主产于我国广东、广西、海南和福建等地。该药为清热解毒类中药,味苦、性寒,临床上常用来治疗肺癌、前列腺癌和胃肠道癌等。近年来,已从鸦胆子中分离获得多种抗肿瘤有效成分,然而关于鸦胆子蛋白质及多肽抗肿瘤的研究却鲜少报道。乳腺癌是造成女性肿瘤患者死亡的首要因素,而中医药对于乳腺癌的治疗有着明显的特色与优势,清热解毒便是重要治则之一。本论文以鸦胆子蛋白质为研究对象,通过酶解法获得生物肽,然后采用超滤、色谱等方法进行离纯化,最终获得显著抑制人乳腺癌MCF-7细胞株增殖的多肽组分F9-9。本文的主要研究成果如下:鸦胆子主要成分及蛋白质组成研究。采用旋光法、索氏提取法、凯氏定氮法、烘干法、马弗炉法分别进行鸦胆子淀粉、粗脂肪、蛋白质、水分和灰分含量的测定,含量依次为 49.26 ±0.02%,16.16 ±0.13%,17.47 ±0.12%,6.74 ±0.05%和5.61 ±0.10%;采用顺序抽提法提取鸦胆子清蛋白、球蛋白、醇溶蛋白和谷蛋白四类蛋白组分,并用凯氏定氮法进行定量,各蛋白组分占总蛋白含量的百分比依次为 15.01%、8.11%、2.47%和 44.92%。鸦胆子抗肿瘤肽的制备工艺研究。以含量相对较高的谷蛋白、清蛋白和球蛋白为材料,采用胃蛋白酶进行酶解,将酶解产物超滤(MWCO = 3kDa)后获得滤过液,MTT结果显示这三类蛋白的小分子产物(≤3 kDa)在浓度为50 μg·mL-1时,对MCF-7细胞的增殖抑制率分别是71.12%、75.26%和17.04%,由于球蛋白组分中蛋白质含量高达79.28%,因此确定球蛋白为获得抗肿瘤肽的最优蛋白质来源。然后,采用正交实验法确定球蛋白的最佳提取工艺参数为:NaCl浓度5%,提取时间1.5 h,固液比1:12。最后,在底物浓度[S]为1%,酶与底物浓度比[E]/[S]为1:10,水解时间为48h的条件下,采用多种蛋白酶水解球蛋白,最终确定胃蛋白酶为水解球蛋白的最优酶。抗肿瘤肽的分离纯化。依次采用超滤截留、SephadexG-10凝胶色谱和C18反相高效液相色谱对鸦胆子球蛋白的胃蛋白酶酶解产物进行分离纯化。酶解产物经G4漏斗过滤后,进行超滤(MWCO = 3kDa),获得小分子产物,并命名为鸦胆子球蛋白酶解产物(BJGH)。研究发现BJGH对MCF-7细胞株具有较强的增殖抑制活性,经BCA定量后,作用于MCF-7细胞72h,IC50值为2.0050.04μg·mL-1。BJGH 经 SeperdexG10 分离后,获得 9 个组分(F1～F9),作用 MCF-7细胞72 h后,IC50值分别为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,组分F6、F7、F8、F9与BJGH相比,对MCF-7细胞株的增殖抑制活性显著增强(P0.05)。进而,采用半制备反相高效液相色谱对活性最高的组分F9进行分离,共获得9个亚组分(F9-1～F9-9),其中,F9-7,F9-8和F9-9显示出较强的细胞增殖抑制活性,在样品浓度为0.25μg·mL-1,作用MCF-7细胞72h后,它们对MCF-7细胞的增殖抑制率分别为50.20%、46.91%和62.74%;进一步测得三组分的IC50值分别为 0.25 ± 0.01μg·mL-1,0.32 ± 0.01μg·ml-1 和 0.124 ± 0.004μg·mL-1。组分F9-9抗肿瘤作用机制研究。采用流式细胞术考察组分F9-9对MCF-7细胞株细胞凋亡及细胞周期的影响,设置F9-9浓度为0.03125μg·mL-1、0.0625μg·mL-1和0.125μg·mL-1。Annexin V-FITC/PI双染法结果表明,组分F9-9中浓度及高浓度作用于MCF-7细胞48h后,细胞凋亡数显著高于阴性对照组(P0.05),细胞早期凋亡总数由对照组的4.47%分别上升为13.30%和25.25%,晚期凋亡总数由对照组的13.40%上升为21.90%和25.40%;而低浓度组未造成明显的凋亡现象(P0.05)。PI单染法的结果表明,F9-9低浓度作用于MCF-7细胞48 h后,与阴性对照组(67.34%)相比,加药组可引起83.79%细胞发生明显的G0/G1期阻滞(P0.05)。因此,F9-9低浓度时可能通过阻断细胞周期(G0/G1期)而抑制细胞增殖,在中、高浓度时,则通过诱导细胞凋亡而抑制细胞增殖。应用qRT-PCR技术检测F9-9对抑癌基因P53和PTEN,以及肿瘤转移抑制基因NM23H-1 mRNA表达的影响。设置F9-9浓度为0.125μg·mL-1,0.25 μg·mL-1和0.5 μg·L-1,与MCF-7细胞作用24 h后,结果表明,与阴性对照组相比,加药组细胞P53,PTEN和NM23H-1mRNA的表达量均明显增加(P0.05)。其中,F9-9低、中、高三个剂量组细胞P53mRNA表达量分别为对照组的1.80、2.02和1.84倍;PTEN mRNA表达量分别为对照组的5.14、3.72和4.76倍;NM23H-1 mRNA表达量分别为对照组的3.54、2.78和2.00倍。因此,F9-9可能通过上调P53,PTEN和NM23H-1 mRNA表达而抑制MCF-7细胞的增殖。本研究从鸦胆子球蛋白中获得了可显著抑制乳腺癌细胞增殖的多肽组分F9-9,为该药的临床引用提供了一定的理论指导。
[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.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285

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