肝細胞生長因子(hepatocyte growth factor, HGF)是一種可以促進細胞生長、分化和遷移的物質，同時因為可以使聚集的細胞離散便發現與早期研究的離散因子(scatter factor)是同一種物質，其會由肝細胞或是部分表皮細胞分泌產生。肝細胞生長因子分為兩個部份: 一個是a鏈，包含有N端區域和四個kringle區域；另一段是b鏈，為一個類似絲胺酸蛋白酶的區域，並且以雙硫鍵和a鏈相聯結。由人類細胞萃取出經過糖基化的b鏈大小是34 kDa。之前研究發現肝細胞生長因子的b鏈(HGFb)單獨時一樣具有和c-Met結合的能力，只是親合力比a鏈單獨時低1000倍。本實驗即是要找出能快速且大量產生的肝細胞生長因子b鏈的方法:將肝細胞生長因子b鏈基因經由聚合酶鏈鎖反應擴增後，轉入pET-22b(+)載體中進行表達，實驗證實有大量的肝細胞生長因子b鏈表達在細胞膜間質區域和培養基上清液中。在肝細胞生長因子b鏈進行細胞增生和遷移測試後，實驗數據顯示重組的肝細胞生長因子b鏈在不同濃度狀況下可以達到不同抑制細胞增生和遷移的程度。實驗證實與在培養基中的肝細胞生長因子b鏈、pET-22b(+)空載體產生的蛋白質和以往論文中提到的1000倍濃度比較起來，10 nM肝細胞生長因子b鏈即可明顯達到抑制細胞增生和遷移的功能。

Hepatocyte growth factor (HGF), a factor which stimulates cell growth, morphogenesis and migration, was found in hepatocytes and other epithelial cells. HGF was found to induce cell scattering, so that it was previously named as “scatter factor” (SF), which turned out to be the same protein. HGF has two chains, a-chain, which contains N-terminal domain and four kringle domains and b-chain, a serine protease-like domain linked with a-chain by disulfide bond. The size of b-chain is 34 kDa after glycosylation in human cells. The function of HGFb was not unknown until recently that HGFb was shown 1000-fold lower affinity to c-Met than HGFa. Thus this project is to create biotechnological approaches for quick and large scale production of HGFb. The DNA fragment amplified by PCR was transferred to pET-22b(+) for expression, and showed that large amount of recombinant HGFb was produced both in periplasmic space and culture supernatant. Assays for cancer proliferation and migration with the HGFb showed that the recombinant protein inhibited cancer cells in a dose-independent manner. Our experiment showed clearly that 10 nM of the E. coli-produced HGFb inhibited proliferation and migration of cancer cells in comparison with the medium and pET controls, 1000-fold higher affinity to c-Met than reported.

Abbreviation
Abstract
Chinese Abstract
Chapter 1 Introduction ……………………………………………..1

1.1 Background …………………………………………...1
1.2 The aim of the experiment…………………………….6

Chapter 2 Materials and methods ..………………………………...7

2.1 Materials.. ……………….……………………………….7
2.2 Approaches in molecular biology………………………10
2.2.1 Plasmid purification…………………………........10
2.2.2 Restriction of enzyme…………………………….11
2.2.3 Agarose gel electrophoresis………….……...……11
2.2.4 Band-preparation…………....……………………12
2.2.5 Polymerase Chain Reaction….…………………...12
2.2.6 T-A cloning…………………………………….…13
2.2.7 Transformation…………………………………...13
2.2.8 Checking the plasmid of pGEM-T and HGFb
2.2.9 Ligation of pET-22b(+) and HGFb
2.2.10 Checking the plasmid of pET-22b(+) and HGFb….15
2.2.11 Purifying midi plasmid…………..……………..…16
2.3 Approaches in recombinant protein detection………….17
2.3.1 Extracting HGFb
2.3.2 SDS-PAGE…………………...…………………18
2.3.3 Western blotting………………………………...19
2.3.4 Purifying HGFb……………………………...…20
2.4 Approaches in cell biology…………………………….20
2.4.1 Cell proliferation………………………………..20
2.4.2 Cell migration…………………………………..21

Chapter 3 Results…………………………………………………22

3.1 PCR and PCR cloning….………………………………22
3.2 Expression of HGFb……………………………….........22
3.3 Cell proliferation.……………………………….………23
3.4 Cell migration….……….……………………………….25

Chapter 4 Discussion………………………………………………27

4.1 Expression of HGFb in periplasmic space……………….27
4.2 Expression of HGFb in supernatants…………………….28
4.3 The failure of HGFb purification……………………..,…28
4.4 Inhibitory effect of HGFb on cancer cell proliferation…..30
4.5 Inhibitory effect of HGFb on cancer migration...……..…30
4.6 Future work……………………………..……………...…30

References ..……………………………………….………………...32
Figures…..………………………………………….………………..40
Figure 1…………………………………………………………...40
Figure 2……………………………………………………………40
Figure 3……………………………………………………………41
Figure 4……………………………………………………………42
Figure 5……………………………………………………………43
Figure 6……………………………………………………………44
Figure 7……………………………………………………………44
Figure 8……………………………………………………………45
Figure 9……………………………………………………………45
Figure 10………………………………………………………….46
Figure 11………………………………………………………….47
Figure 12………………………………………………………….48
Figure 13………………………………………………………….49
Figure 14………………………………………………………….50
Figure 15A………………………………………………………..51
Figure 15B………………………………………………………..52
Figure 16………………………………………………………….53
Figure 17………………………………………………………….54
Figure 18………………………………………………………….55
Figure 19A………………………………………………………..56
Figure 19B………………………………………………………..57
Figure 20………………………………………………………….58
Figure 21…………………………………………………………..59

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