臺灣博碩士論文加值系統

肝癌衍生生長因子( hepatoma-derived growth factor ; HDGF)，是從人類肝癌細胞株的培養液所純化出，為一種新型的生長因子。在許多類型的癌症HDGF的表現是正向調節。除此之外，HDGF是眾所周知的可刺激各類細胞，包括成纖維細胞，肝癌細胞和血管內皮細胞的增殖和遷移。HDGF是由240個胺基酸所組成，包括從N端的1~100個胺基酸序列，有完整結構稱之PWWP (conserved Pro-Trp-Trp-Pro motif ) domain 。及從胺基酸序列101至240的C端，是高度可變的區域。HDGF的PWWP domain會與細胞膜外的肝素( heparin )產生鍵結，會讓外生性的人類肝癌衍生生長因子更容易進入細胞；而C端可能可以幫助蛋白質由細胞質進入細胞核中，讓此因子在細胞核中發生細胞增生或活化的反應。HDGF的兩端都各有一個入核序列( nuclear localization sequence；NLS)。 迄今尚未確定HDGF細胞表面接受體，81 到100 的胺基酸序列被認為是HDGF與NIH3T3細胞膜接受器結合的位置，其中Lys96扮演重要的接受器結合角色。在本研究中，我們在大腸桿菌中表現並純化重組的HDGF，重組的HDGF蛋白會刺激子宮頸癌HeLa細胞的生長和移行。隨後，利用定點突變進行生產重組蛋白，包括同時將兩個NLSs基因突變和受體結合（ K96A ）突變的HDGF蛋白。刪除NLS1和NLS2會徹底破壞HDGF進入細胞核及促進生長的能力，也影響它促進細胞移行的能力。HDGF的胜肽中替換單一氨基酸（ K96A ）足以削弱其黏合細胞表面和其增殖活性。總之，對HDGF蛋白的生物學功能而言NLSs和K96殘基是很重要。

Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from conditioned medium of hepatoma cell line. HDGF expression is upregulated in many types of cancer. Besides, HDGF it is well known that stimulate the proliferation and migration of various types including fibroblasts, hepatoma cells, and endothelial cells. HDGF is composed of 240 amino acids with a well conserved, N-terminal PWWP (conserved Pro-Trp-Trp-Pro motif) domain (residues 1-100) and a highly variable C-terminal domain (residues 101-240). PWWP domain binds to heparin and heparin sulfate located outside the surface of cell membrane and facilitated internalization of the protein into cells. There are two putative bipartite nuclear localization signals (NLSs) in HDGF. The cell surface receptor of HDGF is not been identified so far. The amino acids 81 to 100 are responsible for NIH3T3 membrane receptor binding domain and Lys96 played a major role in this domain. In the present study, I expressed and purified a functional recombinant HDGF protein from E. coli. Recombinant HDGF protein stimulated the growth and migration of cervical cancer HeLa cells. Subsequently, site-directed mutagenesis was been completed on produce recombinant HDGF protein with mutations in both NLSs and receptor binding (K96A). Respectively, Deletion of NLS1 and NLS2 abolished the nucleus targeting of HDGF and abrogated the growth promoting as well as the chemotaxic capability of recombinant HDGF. Substitution of a single amino acid (K96A) within this peptide was sufficient to diminish it is binding to the cell surface and it is proliferated activity. In summary, both NLSs and K96 residue are important to biological functions of HDGF protein.

Abstract in Chinese----------------------------------------------------------------------------- 2
Abstract in English------------------------------------------------------------------------------4
1 INTRODUCTION------------------------------------------------------------------------11
1.1 Hepatoma Derived Growth Factor (HDGF) -----------------------------------11
1.2 HDGF and tumorigenesis---------------------------------------------------------12
1.3 HDGF and cell migration---------------------------------------------------------13
1.4 Reactive oxygen species (ROS) and oxidative stress-------------------------14
1.5 ROS and ROS and Angiogenesis------------------------------------------------15
2 SPECIFIC AIMS--------------------------------------------------------------------------17
3 MATERIALS AND METHODS--------------------------------------------------------18
3.1 Preparation of competent cell-----------------------------------------------------18
3.2 Eepression and purification of recombinant human HDGF in E. coli-------18
3.3 Western blot analysis--------------------------------------------------------------20
3.4 Cell culture-------------------------------------------------------------------------21
3.5 DNA transfection by lipofectamine---------------------------------------------21
3.6 Proliferation assay-----------------------------------------------------------------22
3.7 Cell migration assay (Invasion) -------------------------------------------------22
3.8 Gelatin zymography---------------------------------------------------------------23
3.9 Scratch wound healing assay----------------------------------------------------24
3.10 Immunofluorescence analysis----------------------------------------------------25
3.11 Flow cytometry assay-------------------------------------------------------------25
3.12 Detection of intracellular reactive oxygen species (ROS) by flow cytometry assay----------------------------------------------------------------------------------26
3.13 Statistic analysis--------------------------------------------------------------------27
4 Results--------------------------------------------------------------------------------------28
4.1 Generation and characterization of various HDGF mutant proteins--------28
4.2 Generation and characterization of various GFP-fused HDGF mutant constructs----------------------------------------------------------------------------28
4.3 Both NLS deletion and K96A mutation of HDGF protein abrogated the stimulating effect of HDGF on cell growth and migration-------------------29
4.4 Both NLS deletion and K96A mutation of HDGF protein had no effect on MMP-2 and MMP-9 secretion of SK-Hep-1 cells-----------------------------29
4.5 HDGF enhances the superoxide anion and hydrogen peroxide production in HeLa cells whereas NLS deletion and K96A mutation of HDGF protein ablate such effect-------------------------------------------------------------------30
4.6 Subcellular localization of NLS deletion and K96A mutation of HDGF protein after transfection in Hela cells.------------------------------------------30
4.7 Effects of HDGF, HDGF-K96A and HDGF-dNLSs gene delivery on the proliferation and migration. ------------------------------------------------------31
4.8 Effect of HDGF, HDGF-K96A and HDGF-dNLSs on hypoxia-inducible factor 1α (HIF- 1α).---------------------------------------------------------------32
4.9 HDGF gene delivery promotes the cell cycle progression of HeLa cells whereas NLS deletion and K96A mutation led to cycle arrest---------------32
5 DISCUSSION-----------------------------------------------------------------------------33
6 FUTURE PERSPECTIVES-------------------------------------------------------------38
7 EFERENCES------------------------------------------------------------------------------40
8 Figures and Legends----------------------------------------------------------------------48
Table 1. The sequences of primers for site-directed mutagenesis.----------------------48
Table 2. Effect of exogenous HDGF, HDGF-K96A and HDGF-dNLSs on the cell cycle progression of Hela cells. --------------------------------------------------------------49
Table 3. Effect of transfection of HDGF, HDGF-K96A and HDGF-dNLSs on the cell cycle progression of HeLa cells. -------------------------------------------------------------50
Figure1. The schematics of HDGF functional domains. ----------------------------------51
Figure 2. To prepare various HDGF mutant proteins and expression vectors.--52
Figure 3. Expression and purification of recombinant HDGF, HDGF-K96A and HDGF-dNLSs proteins in E.coli. ------------------------------------------------------------53
Figure 4. To generate various HDGF mutants by site-directed mutagenesis.----------54
Figure 5. Expression of HDGF, HDGF-K96A and HDGF-dNLSs in Hela cells after transfection. -------------------------------------------------------------------------------------55
Figure 6. Effect of exogenous HDGF, HDGF-K96A and HDGF-dNLSs on the proliferation of SK-Hep-1 cells. -------------------------------------------------------------56
Figure 7. Effect of exogenous HDGF, HDGF-K96A and HDGF-dNLSs on the migration of SK-Hep-1 cells. -----------------------------------------------------------------57
Figure 8. Effect of exogenous HDGF, HDGF-K96A and HDGF-dNLSs on the wound healing of SK-Hep-1 cells. --------------------------------------------------------------------58
Figure 9. Effect of exogenous HDGF, HDGF-K96A and HDGF-dNLSs on MMP-2 and MMP-9 secretion of SK-Hep-1 cells. --------------------------------------------------59
Figure 10. Effect of exogenous HDGF, HDGF-K96A and HDGF-dNLSs on the production of superoxide anion in HeLa cells. --------------------------------------------60
Figure 11. Effect of HDGF, HDGF-K96A and HDGF-dNLSs proteins on the production of hydrogen peroxide in HeLa cells. ------------------------------------------ 61
Figure 12. Subcellular localization of HDGF, HDGF-K96A and HDGF-dNLSs after transfection in Hela cells. ---------------------------------------------------------------------62
Figure 13. Effects of HDGF, HDGF-K96A and HDGF-dNLSs gene delivery on the proliferation and migration of HeLa cells. -------------------------------------------------63
Figure 14. Effect of HDGF, HDGF-K96A and HDGF-dNLSs gene delivery on the wound healing of HeLa cells. ----------------------------------------------------------------64
Figure 15. Effect of HDGF, HDGF-K96A and HDGF-dNLSs on hypoxia-inducible factor 1α (HIF- 1α) of HeLa cells. ----------------------------------------------------------65

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