肝細胞生長因子(Hepatocyte growth factor, HGF)經由其專一性受體MET調控了包括細胞的增生、移動、侵入和形態發生等生理過程。最近，我們實驗室的研究結果發現HGF缺失體(NK1、NK2、NK3及NK4)可以成為HGF的拮抗劑(Antagonist)去抑制乳癌細胞(MDA-MB-435S，MDA)的增生、移動及侵入，而將HGF缺失體的基因轉染到MDA細胞中也會有相似的結果產生。為了探討其中的分子機制，我們利用二維電泳技術對MDA細胞、空載體轉染的MDA細胞突變株(MDA-GFP)以及四種HGF缺失體轉染的MDA細胞突變株(MDA-NK1、MDA-NK2、MDA-NK3及MDA-NK4)進行蛋白質體的分析，完成了15組蛋白質體的比對結果。以MDA細胞作為對照組的五組蛋白質差異分析中發現共有56個蛋白質的表現產生差異。MDA和MDA-GFP細胞之間的比對發現共有17個表現差異的蛋白質，其中11個是負調控蛋白；6個是正調控蛋白。MDA和MDA-NK1細胞之間的比對顯示有18個表現差異蛋白，其中15個是負調控蛋白；3個是正調控蛋白。MDA和MDA-NK2細胞之間的比對發現14個負調控蛋白；8個正調控蛋白。相同的比對方法還發現MDA-NK3細胞中出現了11個負調控蛋白和5個正調控蛋白，而MDA-NK4細胞中則產生了15個負調控蛋白和3個正調控蛋白。以MDA-GFP細胞作為對照組的四組蛋白質差異分析(MDA-GFP和MDA-NK1，MDA-GFP和MDA-NK2，MDA-GFP和MDA-NK3，以及MDA-GFP和MDA-NK4)中顯示共有43個表現差異的蛋白。為了了解不同HGF缺失體轉染MDA細胞突變株之間的蛋白質表現差異，我們也完成了三組蛋白質差異分析(MDA-NK1和MDA-NK2，MDA-NK1和MDA-NK3，MDA-NK1和MDA-NK4)，結果顯示共有37個表現差異的蛋白。以MDA-NK2細胞作為對照組與MDA-NK3及MDA-NK4細胞比對時發現有34個蛋白質的表現產生顯著差異。最後經由MDA-NK3和MDA-NK4細胞之間的比對發現共有19個顯著差異的蛋白。我們的結果顯示這些在MDA-MB-435S細胞和HGF缺失體轉染的MDA細胞突變株中表現差異的蛋白與HGF缺失體抑制乳癌細胞的增生和移動有關。

Hepatocyte growth factor (HGF) and its specific receptor MET play a role in many physiological functions including proliferation, migration and morphogenesis. Recently, research results in our laboratory showed that recombinant HGF variants (NK1, NK2, NK3 and NK4) became antagonists to HGF/MET pathway by suppressing proliferation, migration and invasion in human breast cancer cells (MDA-MB-435S, MDA). Similar results were achieved when HGF variants genes were introduced in MDA cells. To understand the molecular mechanism of breast cancer cells metastasis suppressed by HGF variants, MDA and five transfectants, including MDA-GFP, MDA-NK1, MDA-NK2, MDA-NK3 and MDA-NK4 cells were used for proteomic analysis using two-dimensional electrophoresis (2-DE). Differential analysis revealed that a total of 56 polypeptides were differentially expressed through five sets of comparison using wild-type MDA cells as a control. A total of 17 polypeptides were shown differential expression between MDA and MDA-GFP cells, with 11 down-regulated and 6 up-regulated. Eighteen polypeptides were differentially expressed between MDA and MDA-NK1 cells, with 15 down-regulated and 3 up-regulated. There were 22 differentially expressed polypeptides found between MDA and MDA-NK2 cells, in which 14 were down-regulated and 8 were up-regulated. Sixteen polypeptides were shown differentially expressed between MDA and MDA-NK3 cells, with 11 down-regulated and 5 up-regulated. A total of 18 polypeptides were shown differential expression between MDA and MDA-NK4 cells, with 15 down-regulated and 3 up-regulated. Proteomic analysis showed that a total of 43 polypeptides were differentially expressed through four sets of comparison (MDA-GFP and MDA-NK1, MDA-GFP and MDA-NK2, MDA-GFP and MDA-NK3, and MDA-GFP and MDA-NK4). To understand the differential expression among different HGF variants-transfected MDA cells, three sets of cross analysis were also carried out (MDA-NK1 and MDA-NK2, MDA-NK1 and MDA-NK3, and MDA-NK1 and MDA-NK4) and the results showed that a total of 37 differentially expressed polypeptides were found in the three sets of comparison. Similarly, when MDA-NK2 cells were used as a control to compare with MDA-NK3 and MDA-NK4 cells, 34 significantly differential expressed polypeptides were found. The last set of comparison between MDA-NK3 and MDA-NK4 cells, 19 polypeptides were found significantly differential expression. Therefore, our current results revealed that the differentially expressed polypeptides in MDA-MB-435S cells and HGF variants-transfected MDA cells could be related to the inhibition of proliferation and migration of human breast cancer cells by HGF variants.

Chapter 1: Introduction
1.1 Human hepatocyte growth factor (HGF)……………………...P1
1.1.1 Structure of HGF…………………………………………………P1
1.1.2 HGF/MET signaling…………………………………………...P2
1.1.3 HGF/MET and cancer………………………………………….P4
1.2 The aim…………………………………………………………P8
Chapter 2: Materials and methods
2.1 The formulae of medium and buffers…………………………..P10
2.2 Cell lines and cell cultures………………………………………P12
2.2.1 Subculture…………………………………………………….P12
2.2.2 Defreezing………………………………………………………P13
2.2.3 Preservation……………………………………………………..P13
2.3 Sample preparation for two-dimensional electrophoresis (2-DE)……………………………………………………………...P13
2.3.1 Extraction of cellular proteins from MDA cells and five transfectants…………………………………………………… P13
2.3.2 Proteins precipitation and quantification……………………….P14
2.4 2-DE………………………………………………………………P15
2.4.1 The first dimension: isoelctric focusing (IEF)………………….P15
2.4.2 Equilibration……………………………………………………P15
2.4.3 The second dimension: SDS-polyacrylamide gel electrophoresis (PAGE)………………………………………………………….P16
2.5 Gel image analysis……………………………………………….P17
Chapter 3: Results
3.1 Differentially expressed polypeptides in comparison of wild-type MDA cells with MDA-GFP, MDA-NK1, MDA-NK2, MDA-NK3 and MDA-NK4 cells………………………………………………P18
3.2 Differentially expressed polypeptides in comparison of MDA-GFP cells with HGF variants-transfected MDA cells………………...........................................................................P20
3.3 Differentially expressed polypeptides in comparison of MDA-NK1 cells with MDA-NK2, MDA-NK3 and MDA-NK4 cells…...............................................................................................P21
3.4 Differentially expressed polypeptides in comparison of MDA-NK2 cells with MDA-NK3 and MDA-NK4 cells………………….......................................................................P22
3.5 Comparison of differentially expressed polypeptides between MDA-NK3 and MDA-NK4 cells....................................................P23
Chapter 4: Discussion
4.1 The 2-DE-based proteomic analysis on breast cancer metastasis………………………………………………………P25
4.2 Differential proteomic analysis of HGF variants-transfected MDA cells………………………………………………………P29
4.2.1 The debate of the origin of MDA-MB-435 cells……………….P29
4.2.2 Correlation coefficients among six cell line…………………….P30
4.2.3 Proteome differential expression through MDA cells and five transfectants…………………………………………………….P31

4.2.4 Proteome differential expression through MDA-GFP cells and HGF variants-transfected MDA cells……………………….......P32
4.2.5 Proteome differential expression in different HGF variants-transfected MDA cells…………………………………P33
4.3 Conclusion..................................................................................P35
References…………………………………………………...............P36
Figures……………………………………………………………….P50
Tables………………………………………………………………...P91
Appendixes……………………………………………………….....P99

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