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Gecko anti- tumor research

Author: HouXinZuo
Tutor: WangChunMei
School: Beijing University of Traditional Chinese Medicine
Course: Chinese medicine pharmacy
Keywords: gecko protein anti-tumor purification pharmacological
CLC: R285.5
Type: Master's thesis
Year: 2008
Downloads: 299
Quote: 3
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Abstract

The thesis consists of three parts: The first is to study the anti-tumor activity of gecko. The second is to purify the gecko anti-tumor proteins. The third is to try to explain the pharmacological mechanism of the anti-tumor proteins.The transplant tumor model of H22 hepatocarcinoma in mice was established and used to investigate the effects of natural and processed gecko on tumor. In vitro, the inhibitory effect of fresh and dried gecko on proliferation of different cancer cells was measured by MTT assay. The gecko fresh and processed all has the suppression growth function to the tumor in vivo and vitro. There were no significant different between fresh gecko and processed one. Beside the moderate and high groups of the fresh gecko, the indexes of the thymus and spleen of the others were obviously more increasing than positive control group. The artillery product might enhance the experimental animal’s immunity. Because the natural product is not easy to obtain and be transported or stored. In addition, the artillery product is widely used in the traditional Chinese medicine clinic. So the gecko’s artillery product was selected for deeply research.The proteins were purified by following methods. Firstly, the gecko water extract was purified by ammonium sulfate precipitant. There are four groups, including 20%、40%、60%、80%. And three of them were measured by MTT assay. The protein of the group 40% is less content and it is a large number of oil containing. The protein of the groups 60% and 80% were choused for next purification. Secondly, the ammonium sulfate precipitant was purified by gel filtration chromatography. It was separated as three peaks by Sephadex G-25 and two peaks by Sephadex G-75. The activity of three peaks separated by Sephadex G-25 was measured by MTT assay. It was show that group1 (G1) had the best activity. The G1 was then isolated by ion-exchange chromatography using two packing CM spharose Fast Flow and DEAE cellulose DE-52.CM-SFF is cation exchange medicinal preparation. DE-52 is the anionic exchange medicinal. The result showed that the G1 was not adsorbed by CM and adsorbed by DE-52.So the major part in the G1 component is the acidic protein and the bioelectric point is smaller than 7.DE-52 was chosen for further G1 separation. After three steps separation the separation effect was not good enough. The purification methods were optimized. The samples were separated as seven groups by ammonium sulfate precipitant. Six of them were measured by MTT assay. It was found that the group 80% and 90% had better activity. The group 70-90% was separated by Sephadex G-25. Then G1 was separated by three kinds of hydrophobic interaction chromatography separately Phenyl FF, Octyl FF, and Butyl FF. The result showed that Phenyl FF was the most suitable material of three. After several steps of separation, the protein was partly purified.To assay the cell apoptosis effects of the gecko protein extracts, cells treated with gecko proteins were observed after stained by giemsa, AO/EB and measured by flow cytometry. The flow cytometry showed that the gecko proteins could induct apoptosis of HeLa cells, but the results need to be confirmed.

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