半夏蛋白的提取分离、含量测定及对Hela细胞的作用研究

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半夏蛋白的提取分离、含量测定及对Hela细胞的 作

用研究

作者姓名

冯瑞娟

学科(专业）生物化学与分子生物学 指导老师 所在学院 提交日期

檢涛副教授 生命科学学院 年

中国杭州

I *

I

浙江理工大学硕士学位论文

ZHEJIANG SCI-TECH UNIVERSITY MASTER DISSERTATION

Extraction, Isolation, Assaying of Pinellin and Its Effect on Hela

Cell

RuiJuan Feng

Directed by Dr. Tao Xu, vice professor

College of Life Sciences, Zhejiang Sci-Tech University

Mar., 2012 HANGZHOU, CHINA

分^^号： ______________ UDCf

密级:

半夏蛋白的提取分离、含量测定及对Hda细胞的

作用研究

浙江理工大学硕士学位论文

论文答辩日期

答辩委员会主席 jcj 4-川 论文评阅人；

答辩委员会成员

後傳

课题来源

国家自然科学基金

(30772712)

搞要

半夏(?/?^///a/mia,a(Thunb.)Breit.)为天南星科半夏属植物，是我国传统的中药材，具 有燥湿化疲，降逆止呕，消痛散结的功效。主要活性成分有留醇、生物碱、活性多糖、氧 基酸、有机酸和凝集素等。其中，半夏凝集素是半夏总蛋白的主要成分之一*近年来的研 究发现，半夏蛋白具有多种生物活性，是半夏抗虫、抗肿瘤及抗生育的主要有效成分，成 为半夏研究的热点。本文的研究内容是半夏总蛋白及半夏凝集素，主要包括半夏总蛋白的 动态变化及半夏凝集素对Hela细胞的生长的影响。

本研究采用4种常用的总蛋白质提取方法(丙翻沉淀法、95%(NH4)2S04盐杴法、TCA- 丙嗣法和Tris-饱和臉法)，同等条件下分别提取半夏总蛋白质，对其进行十二炼基硫酸纳

- 聚丙炼酸胺凝胶(SDS-PAGE)电泳，电泳结果显示：四种方法提取的总蛋白电泳行为无

明鼠 差导。采用Bradford蛋白含量测定试剂盒测定总蛋白含量，四种方法提取的总蛋白

浙江理工大学硕士学位论文

含量结 果为：TCA-丙銅法(1.082 mg/ml)>95% (NH4)2S04 盐杴法(0.678 mg/ml)>Tris-饱和粉法 (0.493 佳。

利用TCA-丙爾法分别提取半夏不同生长时期(出芽期、全苗期、珠芽期、佛焰范期及 倒苗期)采集到的样品总蛋白质,SDS-PAGE电泳检测及Bradford蛋白含量测定试剂盒测定 半夏不同生长时期总蛋白质含量。其中，不同时期采集到的样品分为新鲜和千燥处理两组。 SDS-PAGE电泳结果比较分杴发现：新鲜组样品中，不同生长时期的总蛋白成分有差异；

新鲜组样品多千燥处理组样品10条蛋白带，且干燥处理组样品中出现了新鲜组样品中没 有的蛋白条带(如表观分子量为26 kDa和35 kDa的蛋白),而表观分子量为12 kDa和24 kDa 的蛋白成分在两个处理及各个生长时期中占的比例均较大。含量测定结果为：新鲜半夏不 同生长时期中，块莲中总蛋白含量变化表现为倒苗期>佛焰苟；期>出芽期>珠芽期>全苗 期；不同生长时期干燥处理半夏中，块基中总蛋白含量变化表现为倒苗期>珠芽期>出芽 期>佛焰箱期>全苗期。含量测定结果显示倒苗期总蛋白含量最高，全苗期总蛋白含量最 低。结果表明，半夏不同生长时期总蛋白成分发生了一定变化，同一蛋白成分在不同的生 长时期含量有差异，新鲜和干燥处理样品中蛋白含量和种类也有一定差异。

同时，本实验室根据半夏凝集素的甘露糖专一结合的性质，设计制备了甘露糖

丙飼法(0.369 mg/ml),比较分杴发现TCA-丙雨法提取半夏总蛋白效果最

-Sepharose4B凝胶，从半夏总蛋白中成功分离純化出一种半夏凝集素，SDS-PAGE凝胶电

泳检测，表观分子量为12kDa,纯度达到电泳纯，研究表明该凝集素是一种糖蛋白，能凝 集小鼠红细胞。

用不同浓度(0.004 mg/mL、0.02 mg/mL. 0.1 mg/mL, 0.5mg/mL 和 I mg/mL)半夏凝集 素处理Hela细胞，用MTT法测定细胞活力，结果表明低浓度(0.004 mg/mL、0.02 mg/mL 及

0.1 mg/mL)具有促进Hda细胞增殖的作用，且随浓度和时间增加促增殖作用减弱，48h 时

有最大促增殖效率为23.36%;高浓度(0.5 mg/mL、I mg/mL)有抑制Hela细胞增殖的作 用，并且这种作用有时间和剂量依赖性，72 h时有最大抑制率为71.89%。

同时，选取对细胞有促增殖和抑制作用的两个浓度0.004 mg/mL和I mg/mL,作用于 对数期生长的Hda细胞，PBS作为对照，分别培养24 h、485和7211。倒置显微镜下观察 摄影，结果显示：浓度为0.004 mg/mL时，在三个时间段观察的Hela细胞生长状态均较 PBS对照组好，72h时，细胞己铺满瓶底；浓度为I mg/mL,作用细胞24 h时，部分细 胞死亡，

浙江理工大学硕士学位论文

部分细胞有分裂增殖的现象，但细胞形态已明显异于PBS对照组，作用细胞48 h 和72 h时，细胞生长能力全部被抑制，皱缩死亡，悬浮于培养液中。

关键词：半夏总蛋白；动态变化；含量测定；Hela细胞；MTT法

Extraction, Isolation, Assaying of Pinellin and Its Effect on Hela Cell

Abstract

Rhizoma Pinelliae {Pinellia ternata (Thunb.)Breit.) is a plant of the Pinellia Tenore of Araceae. As a

traditional Chinese herbal medicine, Pinellia ternata has many founctions such as drying

dampness to eliminate phlegm, calming the adverse-rising energy to stop vomiting and dissipating palpable and removing stasis. And mainly active components of which are sterols, alkaloids, active polysaccharides, amino acids, organic acids and proteins, but Pinellia ternata agglutinin (PTA) is one of the main components of total protein of Pinellia ternata. Recent studies show that pinellin with multiple biological activites is the main active ingredient of Pinellia ternata in

insect resistance, anti-tumour and anti.fertility, which make it a research focus. In this paper, the center of attention is protein and

PTA including the dynamic changes of total protein of Pinellia ternata and the effect of PTA on Hela cells in vitro.

In this study, four kinds of commonly used total protein extraction methods (acetone precipition method, 95% (NH4)2S04 salting-out method, TCA-acetone precipitation method and Tris-saturated phenol method) were used to

extract total protein of Pinellia ternata in equal conditions.Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE)

electrophoresis was used to detect the total protein extracted by the four methods. The electrophoresis results showed that: the electrophoretic behavior of the total protein extracted by the four methods had no significant difference. Also, Bradford protein assay kit was used to determine the total protein content and the results showed thatrTCA-acetone precipitation method (1,082 mg/mL)>95% (NH4)2S04 salting-out method (0,678 mg/mL) >Tris-saturated phenol method

(0.493 mg/mL) > acetone precipition method (0.369 mg/mL), So TCA-acetone precipitation method is the best one for

total protein extraction of Pinellia ternata.

TCA-acetone precipitation method was used to extract the total protein of Pinellia ternata samples of different growth stages (bud stage, seeding stage, bulbil stage, spathe stage and maturation stage). And the samples collected in different periods were divided into two groups of fresh and dried. SDS-PAGE electrophoresis and Bradford protein assay kit

were used to detect

浙江理工大学硕士学位论文

the total protein in different growth stages. The results of SDS-PAGE electrophoresis of the two groups showed that: the total protein composition was different in different growth periods in fresh group. And the protein bands of samples of fresh group were at least 10 bands more than that of the dried group. But samples of dried group had some protein bands which were not exist in fresh group, such as the proteins with the apparent molecular weight of 26 kDa and 35 kDa. However, the proteins with the apparent molecular weight of 12 kDa and 24 kDa were in large proportion in all the samples of the two groups.

The determination results showed that: total protein content of samples of fresh group changes in a manner like this: maturation stage>spathe stage>bud stage>bulbil stage> seeding stage, while that of dried group as maturation stage > bulbil stage > bud stage >spathe stage〉seeding stage. It can be concluded that total protein content is highest in maturation stage but lowest in seeding stage. The results showed that total protein conponents were different in different

growth periods of Pinellia temata, so does the same protein content. In addition, total protein content and types also had some

differences between the fresh and dried groups.

One kind of mannose-Sepharose 4B affinity gel was designed to isolate PTA from total protein of Pinellia temata.

The results of SDS-PAGE electrophoresis showed that its apparent molecular weight was 12 kDa.

Further studies showed that the lectin is a glycoprotein, and ^an agglutinate erythrocytes in mice.

In this paper, anti-tumor activity of the lectin was further studied. Hela cells were treated by five different concentrations (0.004 mg/mL, 0.02 mg/mL, 0.1 mg/mL, 0.5 mg/mL and I mg/mL) ofPTA, and MTT method was used to detect cell viability. The results showed that low concentrations lectin (0.004 mg/mL, 0.02 mg/mL and 0.1 mg/mL) can promote Hela cell proliferation, but the promoting proliferation effect weakended with concentration and time increased, the maximum efficiency of promoting proliferation 23.36% was at the time when cells were cultured for 48 hours. But high concentrations lectin (0.5 mg/mL and I mg/mL) inhibited Hela cell proliferation, and the effect increased in a dose and time dependent manner, the maximum inhibition rate 71.89% was at the time when cells were cultured for

72 hours.

At the same time, Hela cells of logarithmic phase growth were treated with the two

IV

concentrations lectin (0.004 mg/mL and I mg/mL) with promoting and inhibition proliferation effect, PBS as the control, and cultured for 24 hours, 48 hours and 72 hours, respectively. Then the cells growth state was observed under inverted microscope photography, the results showed that: cell treated by 0.004 mg/mL lectin was all growth better than the control group at the three periods. While cells treated by I mg/mL lectin were in a poor state. When cultured 24 hours, some cells

浙江理工大学硕士学位论文

were still in division and proliferation state, and some cells dead. But cell morphology was significantly different from the control group. When cultured 48 hours and 72 hours, all the cells were inhibited, and began to shrink to death, suspending in the culture medium.

Key words: Total protein of Pinellia ternata; Dynamic change; Protein determination; Hela cells; MTT method

目录

摘要........................................................................................................................................................................................................................................................................................................................... I Abstract .................................................................................................................................... . ........................................................................................................................................................................... m 目录 .................................................................................................................................... ............................................................ . ................................................................................................................ VI 略语表 .................................................................................................................................................................................................................................................................................................... 第一章文献综述 ................................................................................................................................................................................................................................................................................. I

1.1 ............................................................................................................................................................................................................................................................................................... I 1.1.1半夏的种类及分布 ........................................................................................................................................................................................................................... .1 1.1.2半夏的形态特征 ..................................................................................................................................................................................................................................... 2 1.1.3半夏的遗传多样性研究 ............................................................................................................................................................................................................ 4 1.1.4半夏的人工栽培 ..................................................................................................................................................................................................................................... 6

1- ..................................................................................................................................................................................................................................1.5半夏的

炮制及真伪辨别 ................................................................................................................................................................................................................................................ 7 1.1.6半夏的药理作用 ..................................................................................................................................................................................................................................... 8 1.2植物凝集素研究进展 ........................................................................................................................................................................................................................................ 9

1,2.1植物凝集素的研究 ............................................................................................................................................................................................................................ 9 1.2.2植物凝集素结构与功能 ............................................................................................................................................................................................................ 9 1.2.3植物凝集素的分离纯化 ......................................................................................................................................................................................................... 10 1.2.4植物凝集素基因的克隆 ......................................................................................................................................................................................................... 11 1.3半夏蛋白研究进展 ............................................................................................................................................................................................................................................. 11

1.3.1半夏蛋白的理化性质 ................................................................................................................................................................................................................. 11 1.3.2半夏蛋白的生物学特性 ......................................................................................................................................................................................................... 12 1.3.3半夏蛋白的药理作用 ................................................................................................................................................................................................................. 12 1.3.4半夏总蛋白含量測定 ................................................................................................................................................................................................................. 12 1.3.5半夏蛋白的分离纯化 ................................................................................................................................................................................................................. 13 1.3.6半夏凝集素 .................................................................................................................................................................................................................................................. 13 1.4 小结 ... ............................................................................................................................................................................................................................................................................................ 14 第二章半夏总蛋白的提取及含量测定 ................................................................................................................................................................................................. 15

2.1实验材料、器材与试剂............................................................................................................................................................................................................................. 15

2.1.1 ^ 验材料 ...................................................................................................................... .. ......................................................................................................................... 15 2.1.2实验器材 ........................................................................................................................................................................................................................................................... 15 2.1.3实验试剂及其配制 ......................................................................................................................................................................................................................... 15 2.2实验方法..................................................................................................................................................................................................................................................... ......................... 16

2.2.1总蛋白的提収方法 ......................................................................................................................................................................................................................... 16 2.2.2总蛋白的SDS.PAGE电泳检测..................................................................................................................................................................................... 18 2.2.3总蛋白的含量测定 ......................................................................................................................................................................................................................... 19 2.3结果与分杴 ...................................................................................................................................................................................................................... . ............................................. 19

浙江理工大学硕士学位论文

2.3.1不同方法提取半夏总蛋白的SDS-PAGE电泳结果 ............................................................................. . .................................. 19 2.3.2总蛋白的含量测定 ......................................................................................................................................................................................................................... 20 2.4讨论与小结 ...................................................................................................................................................................................................................................................................... 23 第三章不同时期半夏总蛋白含量变化的动态分杴 ....................................................................................................................................................... 25

3.1实验材料、器材与试剂 ..................................................................................................................................................... . .................................................................. 25

3丄I实验材料 ...................................................................................................................................................................................................................................................... 25 3.1.2实验器材 ........................................................................................................................................................................................................................................................... 25

VII

浙江理工大学硕士学位论文

3.1.3实验试剂及其配制 ......................................................................................................................................................................................................................... 25 3.2錢旅 ............................................................................................................................................................................................................................................................................................. 25

3.2.1材料处理 ......................................................................................................................................................................................................................................................... 25 3.2.2半夏的种植 ................................................................................................................................................................................................................................................ 25 3.2.3半夏不同生长时期样品采集及处理 ............................................................................................................................................................. 26 3.2.4半夏总蛋S的提取 ........................................................................................................................................................................................................................ 26 3.2.5不同生长时期半夏总蛋白的SDS-PAGE电泳检测 ............................................................................................................... 26 3.2.6不同生长时期半夏总蛋白的含量测定 ..................................................................................................................................................... 26 3.3结果与分杴 .................................................................................................................................................................................................................................................................... 26

3.3.1半夏不同生长时期采集的样品 .............................................................................................................................................................................. 26 3.3.2不同生长时期半夏总蛋白的SDS-PAGE电泳检测结果 ............................................................................................... 27 3.3.3不同生长时期半夏总蛋白的含量测定结果 .................................................................................................................................... 29 3.4讨论与小结 .................................................................................................................................................................................................................................................................... 32 第四章半夏蛋白对Hela细胞作用的研究 .............................................................................................................................................................................................. 3

4.1实验材料、器材与试剂 ............................................................................................................................................................................................................................. 3

4.1.1实验材料 ............................................................................................................................................................................................................................................................ 3 4.1.2实验器材 ............................................................................................................................................................................................................................................................ 3 4.1.3实验试剂及其配制 .......................................................................................................................................................................................................................... 3 4.2 錢:?? ............................................................................................................................................................................................................................................................................................ 3

4.2.1半夏凝集素单体的制备 .......................................................................................................................................................................................................... 3 4.2.2 Hela细胞培养 ...................................................................................................................................................................................................................................... 3 4.2.3细胞增殖抑制实验(MTT法) ......................................................................................................................................................................................... 36 4.2.4倒置相差显微镜摄影 ............................................................................................................................................ . ................................................................ 36 4.3结果与分杴 .................................................................................................................................................................................................................................................................... 37

4.3.1半夏凝集素单体SDS-PAGE凝胶检测 ......................................................................................................................................................... 37 4.3.2细胞增殖抑制实验分杴 ....................................................................................................................................................................................................... 38 4.3.3细胞形态分杴 ........................................................................................................................................................................................................................................ 40 4,4讨论与小结 ..................................................................................................................................................... .................................... ...................................................................... 41 总结与展望.............................................................................................................................................................................................................................................................................................. 43

.................................................................................................................................................................................................................................................................. . ................................. 45

致谢 ......................................... .......... . ......................................................................... ............ — ................ .................................................... ........................ . ........................................ ................. 52 附录I:重要溶液配制 ............................................................................................................................................................................................................................................................. 53 附录2:攻读硕士学位期间的研究成果 ................................................................................................................................................................................................. 55

浙江理工大学硕士学位论文

略语表

m APS AFLP ddHzO EDTA FBS HPLC ISSR

中文全称 过硫酸按 扩增片段长度多态性技术 双蒸水 乙二胺四乙酸 胎牛血清 高效液相色谱

简单序列重复区间扩增多态性 技术 睡哩蓝 磷酸盐缓冲液

英文全称 Ammonium persulfate Amplled fragment length polymorphism Double distilled Water Ethylene diameminetetraacetic acid Fetal bovine serum

High performance liquid chromatography

Inter-simple sequence repeat polymerphisms

MTT PBS

Thiazolyl blue tetrazolium bromide

Phosphate buffered saline

PCR-RFLP 多聚酶链反应-限制性片段长 Polymerase chain reaction restriction

度多态性. fragment length polymorphism Phytohem agglutinin Pinellia temata lectin

PHA PTL RAPD SDS

植物凝集素 半夏凝集素

随机扩增DNA多态性技术 十二综基硫酸纳

Random amplified polymorphic DNA Sodium dodecyl sulfate

SDS-PAGE 十二综基硫酸纳-聚丙炼醜胺 Sodium dodecyl sulfate-polyacrylamide gel

凝胶电泳 electrophoresis

Sequence-related amplified polymorphism N,N,KjN-Ietramethylethylenediamine Thin-layer chromatography

SRAP

TEMED TLC

相关序列扩增多态性技术

N,N,N',N'-四甲基乙二胺 薄层色谱

VIII

浙江理工大学硕士学位论文

第一章文献综述

1.1 半*

半MXPineUici ?em?m(Thunb.)Breit.)为天南星科半夏属多年生草本植物，又名三叶半 夏、半

月莲、三步跳、孚田、水玉、羊服等，以千燥块莲入药，性温、味辛、有毒。半夏 是我国中药宝库中一味重要的药材，也是我国重要的出口产品>，在我国已有2000多年的 用药历史。据统计，在558种中药处方中，半夏使用频率居第22位。半夏有水生和陆生 两种，即水半夏和旱半夏(图1.1),旱半夏的药用价值强于水半夏。由于半夏资源日益减少 或因部分地区药用习惯，各地至少有同科3属11种植物充作半夏使用，如水半夏、掌叶半 夏、狗爪半夏等，不少地区在应用中以水半夏等代用。

[】

a:旱半夏；b:水丰夏 Fig. 1,1 Pinellia Tuber and Rhizoma Typhonii Flagelliforinis

i.u半10?1糊及分布

半夏属植物在分类学上属于天南星科南星亚科，该属含9种植物，中国目前发现的有 7种，分

别为石蝴蛛滴水珠半夏(/^corc/flto)、盾叶半夏(/^/?g/,ato)、三叶半夏

r: 圓1.1早半JDm水半Jt

a: Pinellia Tuber; b: Rhizoma Typhonii FlageIIiformis

I Jf _If ^,1

浙江理工大学硕士学位论文

(Rtemata)^ 掌叶半夏三裂叶半夏及大半夏CP;w(vgft_y//fl),其中 6

种为中国特有。我国半夏资源十分丰富，资料显示除内蒙古、青海、西藏、新疆外，我国 其它省市地区均有分布，主要产于辽宁、陕西、湖北、山东、山西、四川、河南、安徽、 甘肃、江苏、浙江等地区，可能多由历史发展起来的。天南星科南星亚科半夏属在我国主 要种如下所示(表U)。

表U夭南星科半K厲在我国的主要种 Table 1.1 The species of PineIIia Ten. of Araceae in China

种名 种拉丁名 别名 分布情况 石觸妹

Rintegrifolia 一面银、白铃子(四川叙 我国特有，散布于湖北宜昌、四川 永）

的叙永和重庆

一滴珠、蛇珠、石半夏、 我国特有，产安徽、浙江、江西、

RcordcUa

滴水珠半夏

水半夏、制蛇子、岩孙、 福建、湖北、湖南、广东、广西、 心叶半夏、独龙珠等 责州，海拔800 m以下

我国特有，产浙江文成、庆元、乐

无

清、雁荡山及福建杳政等地,在《澳 危植物红皮书》中被列为颜危E级

盾叶半夏

Rpeltata

三步跳、洋舉买、老鸭 除内蒙古、新疆、青海、西藏尚未

Rtemata

三叶半夏

眼、燕子尾、地慈姑、 发现野生的外，全国各地广布，

朝

球半夏、和姑、孚田等 鲜、曰本也有

掌叶半夏、绿孙子、天 我国特有，分布于北京、河北、山

虎掌

Rpedatisecta 南星、狗爪半夏、半夏 西、陕西、山东、江苏、上海、安 子、真半夏、大三步跳、 徽、浙江、福建、河南、湖北、湖 南星等

南、广西、四川、责州及云南北部

1.1.2半夏的形态特征

天南星科半夏属植物属多年生草本植物，具块莲，不同种半夏形态特征稍有差异，如 石蝴蛛叶片全缘、滴水珠叶片长三角形、盾叶丰夏叶片盾状、三叶半夏叶片3全裂、虎

浙江理工大学硕士学位论文

掌 叶片鸟足状分裂等。半夏属不同种的形态特征如表1.2及图1.2所示。

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