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