Research Of Proteome Differential Expression And Its Significance In Hepatic Metastasis Of Large Intestine Cancer Using Technology Of Two-dimensional Electrophoresis

Large intestine cancer is one of the popular malignant tumors in our country. A large number of statistical data indicates that the rates of incidence and mortality of the disease in our country have been going up in recent years. Hepatic metastasis caused severe and fatal effect on patients that underwent radical resecsion for large intestine primary cancer. Early forecast and diagnosis of liver metastasis is the key to improving survival rate for large intestine cancer patients. Now proteome study methods and techniques, especially two-dimensional electrophoresis (2-DE), provide useful and important support for the research of hepatic metastasis of large intetine cancer. 【Objective】 To detect and analyse proteome differential expression among normal intestine mucosa, primary foci and hepatic metastasis of large intestine cancer with two-dimensional electrophoresis, to identify associated proteins with hepatic metastasis and study molecular biology mechanisms of hepatic metastasis of large intestine cancer, and to select tumor marks for clinical forecast and early diagnosis of hepatic metastasis. 【Methods】 (1) Extract proteome samples from the three kinds of tissue specimens of normal large intestine mucosa, primary foci and liver metastasis of large intestine cancer with different lysis buffer and with different procdures, one-step procedure and sequential extraction procedure. (2) Protein quantification about every extraction sample was performed with Bradford's method. (3) Two-dimensional electrophoresis: a) isoelectric focusing was executed using modern technology, IPG and in-gel rehydration. The total Volt-hour is 35000Vh～40000Vh. b) Equilibration: we used high-powereddetergent, TBP, instead of DTT once popularly used, to improve extrction effect, reduce protein loss, and simplify operation steps. c) SDS-PAGE: Use symmetrical gels with the character of 12.5% and 1.5mm. (4) Staining: with silver and coomassie brilliant blue staining. (5) Image collection and analysis: using software of Melanie 3.0. a) Illustrate the difference of electrophoresis between one-step procedure and sequential extraction procedure; b) Evaluate the repeatability of the electrophoresis. (6) Display and analyze the proteome differential expression among the three kinds of tissue. (7) PMF analysis: Choosing significant protein spots, destaining, in-gel digestion, desalting, and preparing sample for mass spectrum analysis. After that, get PMF map with analysis of MALDI-TOF, and use Profound searching process to search information about the protein spots in the data-base named NCBInr. 【Results】 (1) Comparison of the two protein extraction methods indicated that with sequential extraction procedure we got more proteins than with one-step procedure, and decreased protein loss in extracting process. (2) With normal intestine mucosa tissue protein samples, repeatability of 2-DE was carried out and the results revealed that: a) Protein spots in different electrophoresis images distributed the same; b) The protein spot match rate was 72% and the coefficient of protein matching was 0.912; c) Statistic analysis showed no significant difference between repetitive 2-DE experiments (t=1.97, P=0.56); d) Protein position repeatability in 2-DE was good. (3) Proteome differntial expression and image analysis: Under the same condition and with Melanie 3.0, protein spot number on the images of 2DE taken from primary cancer foci, normal intestine mucosa and hepatic metastasis was 206±22, 390±28, 236±19 respectively. Compared with reference normal mucosa 2-DE, matching protein spots in normal intestine mucosa, primary foci and hepatic metastasis were 313±45, 195±38, 184±24 respectively, and the matching rate was 80.3%, 77.2%, 78.0%. (4) PMF analysis: Differential protein expression among normal intestine mucosa, primary foci and hepatic matastasis was found. 9 difference expression protein spots and their PMF map were analysed by searching NCBInr database and the representing proteins were recognized. A calmodulin named "calmod...