| Breast cancer is one of the most common malignant tumor infemale. It is important for revealing the mechanism of breast cancer,making early diagnosis, judging prognosis and making survivalanalysis to identify the protein related to the canceration ofmammary gland and find the protein changing in the progression ofdisease. With impulsing of the human genome project (HGP), peoplehave made extensive research about generating and developing oftumor on gene level. The functional action of gene will be exhibitedby protein ultimately, so proteome and proteomics come into being.Two dimensional polyacrylamide gel electrophoresis is a primarilyprotein separated technique in proteome investigation. The quantityof extraction from the protein is being tried to improve by all kindsof ways.Objective:The protein extracted from the tissue of breast cancer canrepresent all the protein of tissue with pathological changes andreflect the case in real intracorporeal situation, but it can be affectedby blood, fat and connective tissue etc. The present research object istissue beside the tumor of breast cancer infitrating ductal carcinoma(the distance to the tumor >3cm, confirmed by pathologic result). Itwill be optimized on extracting and handling to the samples,quassation of cells and tissue, preparing the solution (principal theassociated employment of denaturing agent, surface active agent andreductant), methods of precipitation and staining to acquireSDS-PAGE electropherogram with superior quality and improvereproducibility and stability. The present study will providefoundation for the next research of 2-DE.Methods:15 patients with breast cancer in breast surgery of our hospitalfrom 2005, September to 2006,March were selected with completeclinical data, who had accepted the treatment of operation andconfirmed to be infiltrating ductal carcinoma stage Ⅱ by pathologicresults. The tissue about 0.040g (the distance to the tumor >3cm)beside the tumor was taken to undertake experiment, it had beenoptimized by sodium dodecylsulphate polyacrylamide gelelectrophoresis technique on some committed steps such asextracting and handling to the samples, quassation of cells and tissue,preparing the solution and methods of precipitation and staining.Results:Extracting the protein from tissue will have much moreinfluencing factor compared to from cells. The different results ofSDS-PAGE electropherogram will be produced with the sametwo-way electrophoretic condition and detected method because ofdiversity in sample processing and the amount of samples. Thedestination of improving the method of two-dimensionalelectrophoresis is to elevate the dissolubility of protein. Since theprotein is easy to degrade in common temperature, we shouldshorten the exposing time of samples in common temperature as faras possible and keep hypothermy in treating process. The sampleswere flushed immediately by normal saline after obtained, then takento the laboratory with a ice holder, it is an effective way to removalconfusing of foreign substance to samples and acquire satisfactoryeffect on extracting the protein and SDS-PAGE electropherogram.Due to the specificity of the tissue of the tumor, grinding addaltrasound method was applied in present experiment. The quartzsand was affiliated in the period of grinding, and satisfactory resultshad been obtained. It is also important to choosing suitablepreparative method of samples for acquiring content 2-weletropherogram. Ideal 2-DE resolve should demolish allnon-covalent bonding protein compounds and form everypolypeptide solution. Through using urea-sulfocarbamide mixturewith different concentration compared to only apply urea, we canfind that the better effect of SDS-PAGE electropherogram would beobtained by separately using urea with higher concentration. Thecapability of dissolving sample had been improved. In the process ofutilization, heating should be avoided as far as possible. If thetemperature surpassed 30 ℃, the protein would occuramido-formylation and the isoelectric point of the protein wouldchange. A series of electropherograms with high quality would beobtained by adjusting the concentration of urea and accommodatingthe concentration of surface acting agent CHAPS and reducing agentDTT, Trips at the same time. PEG method was better thanTCA/acetone method in precipitating protein, because it couldprecipitate most protein of the tissue. TCA/acetone method only hadobviously effect on protein with great molecular weight. Sliverstaining, as a sensitive staining method, were preferably compared toCoomassie brilliant blue staining. But in order to reduce experimenterror and make staining linearization and uniform, the manipulationof staining and the degree of coloration were critically, which wereneeded to grasp gradually.Conclusions:1. extracting and handling the samples: The samples should beflushed immediately by normal saline after obtained and hypothermyshould be kept in treating process. Shortening the exposing time ofsamples in common temperature as far as possible could decreasepollution and degradation of the protein.2. quassation of cells and tissue: The method of grinding addultrasound was applied with quartzsand addition.3. solvent fluid: Modified solvent fluid of the protein RS IV(8M urea(4.8048g), 4% CHAPS(0.4g), 100 mM DTT(0.1542g),40 mM Tris(0.0484g), 2% ampholyte(200ul)pH 3-10)had beentaken to improve the solvent efficiency of the protein.4. PEG method in sedimentation had better effect, it couldprecipitate most of the protein.5. Sliver staining method was advised to apply in stainingmethod.Key words: infiltrative duct carcinoma;proteomics;SDS-PAGE;tissue sample making;protein dissolubility;stainingThe new ideas of present study1. It was a successful way to take the tissue beside the tumor ofbreast cancer as object of research and undertake SDS-PAGEelectrophoresis, which would provide foundation for the continuedstudy.2. Better effect of SDS-PAGE electropherogram had beenacquired by using higher concentration urea separately because of itsbetter capability of dissolvent to the protein in tissue.
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