臺灣博碩士論文加值系統

臨床上對於大腸直腸癌的治療，除了手術之外，搭配化療藥物，是最常見的方式。然而，復發的情形，時有所聞，這被認為與癌症類幹細胞有關。在這些化療藥物中，抗癌妥(Irinotecan)是其中的用藥之一，是一種作用於DNA topoisomerase I[1, 2]之專一性抑制劑的抗腫瘤藥物。在大多數的身體組織內，它會經carboxylesterase代謝成SN-38。SN-38作用於純化之DNA topoisomerase I比Irinotecan更具活性。然而，SN-38的毒性相當強，容易任意毒殺正常的細胞，且SN-38並非水溶性的藥物，不適用直接用在人體身上。因此，在這篇論文中，選擇具水溶性的CPT-11作為化療藥物。除了具有毒殺腫瘤細胞的功能外，如果我們以針對癌症類幹細胞作為目標，則可以有效的遏止復發的可能性，進而達到治癒的效果。
為了達到針對腫瘤幹細胞治療，我選擇以CD133抗原作為目標。在許多研究中指出，癌症幹細胞的CD133表現量特別的明顯。為了模擬癌症幹細胞的表面抗原表現，我選用大腸直腸癌HCT-116細胞株（CD133　over-expression）作為模型。
蛋白質載體是一種新型的材料，在我的研究中，選擇脫鐵蛋白（apoferritin）它是由24個亞基組成的球狀蛋白。脫鐵蛋白會在中性環境下自我形成中空奈米載體，具外徑12nm以及內徑８nm的奈米性質，使得其擁有更好的循環半衰期。在酸性(pH值 = 2)的環境下脫鐵蛋白會崩解，形成24個亞基，這個過程是可逆的反應，將pH值調回7.4時，脫鐵蛋白回到原本的球體型態。藉著這樣的方式，可將藥物包在其中。除此之外，我在蛋白質上，用麩胺酸以及天門冬胺酸接上CD133的專一附著之胜肽鏈，做為針對具CD133表面抗原的細胞株，以達到針對治療的效果。

It is common that we combine chemotherapy with surgery for colorectal cancer clinically. However, we can find out several recurrence cases in some time, and it is thought to be related with cancer-stem like cells. In these drugs we use on chemotherapy, irinotecan is one of the used-drug for colorectal cancer. It is the drug that plays on DNA topoisomerase I specific prohibited anti-cancer drug. In most our organs, irinotecan metabolizes to SN-38 through carboxylesterase. SN-38 is more powerful on inhibiting DNA topoisomerase I than irinotecan. It is because of its toxicity on normal cells and of its hydrophobic characteristic that we can’t use SN-38 in human body without other assisted materials. In this study, I choose hydrophilic drug, irinotecan, as chemotherapeutic drug. If I can target on cancer stem-like cells and kill them, I can minimize the recurrence rate and make the treatment more efficiency.
In order to achieve the aim of targeting cancer stem-like cells and having some treatment on them, I choose CD133 marker as the target site. It is pointed out that CD133 marker presents obviously on cancer stem-like cells in many researches. For imitating the surface antibody on cancer stem-like cells, I choose colorectal cancer cells, HCT-116 (CD133 over-expression), as my model.
Protein cage is a novel material. I choose apoferritin in my study. It is a sphere shape parotein composed of 24 subunits. Apoferritin can form a hollow nanoparticle in neutral environment, which is 12 nm in the exterior and 8 nm in the interior of protein. It is because of the nano-character that apoferritin has better circulation period in body. Apoferritin will discompose to 24 subunits in pH 2, and it will re-compose to sphere shape after turning pH back to neutral environment. Through this process, I can encapsulate drug into apoferritin. Besides, I graft CD133 specific-binding peptide to glutamic acid and asparatic acid on the surface of apoferritin so that it can target on CD133 over-expression cell line and get the effect of targeting treatment.

CONTENTS
口試委員審定書............................................................................................................ i
致謝............................................................................................................................... ii
中文摘要.......................................................................................................................iii
ABSTRACT................................................................................................................. iv
CONTENTS................................................................................................................. vi
LIST OF SCHEMES....................................................................................................ix
LIST OF TABLES........................................................................................................ix
LIST OF FIGURES......................................................................................................ix
Chapter 1 Introduction...................................................................................................1
1.1Clinical treatment for colorectal cancer.....................................................................1
1.2 Apoferritin and its characteristic..............................................................................1
1.3 Cancer stem cell.......................................................................................................2
1.4 CD133 marker..........................................................................................................2
1.5 CPT-11(irinotecan)...................................................................................................3
1.6 Aim...........................................................................................................................3
Chapter 2 Materials and Methods..................................................................................4
2.0 Experiment procedure..............................................................................................4
2.1 Materials...................................................................................................................4
2.2 Conjugate FITC-CD133 peptide onto apoferritin....................................................4
2.3 Encapsulation of CPT-11 into APO-CD133.............................................................5
2.4 Characterization of APO and APO(CPT-11)-CD133...............................................6
2.5 Stability of APO(CPT-11)-CD133...........................................................................6
2.6 Drug releasing profile of APO(CPT-11)-CD133......................................................6
2.7 Cell culture...............................................................................................................7
2.8 Expression of CD133 marker in different cell lines.................................................7
2.9 Binding affinity of APO(CPT-11)-CD133 onto cells...............................................8
2.10 Cellular uptake.......................................................................................................8
2.11 In vitro cytotoxicity................................................................................................9
2.12 Colony formation...................................................................................................9
2.13 Pharmacokinetics.................................................................................................10
2.14 In vivo, growth inhibition studies of tumors........................................................10
2.15 Biodistribution......................................................................................................11
2.16 H&E staining........................................................................................................11
Chapter 3 Results.........................................................................................................12
3.0 Icons of each carrier types and their symbols……………………………………12
3.1 Conjugate FITC-CD133_peptide onto apoferritin.................................................12
3.2 Encapsulation of CPT-11 into APO-CD133...........................................................12
3.3 Characterization of APO and APO(CPT-11)-CD133.............................................13
3.4 Stability of APO(CPT-11)-CD133..........................................................................13
3.5 Drug releasing profile of APO(CPT-11)-CD133....................................................14
3.6 Expression of CD133 marker in different cell lines...............................................14
3.7 Binding affinity of APO(CPT-11)-CD133 onto cells.............................................14
3.8 Cellular uptake.......................................................................................................15
3.9 In vitro cytotoxicity................................................................................................15
3.10 Colony formation.................................................................................................16
3.11 Pharmacokinetics..................................................................................................16
3.12 In vivo, growth inhibition studies of tumors........................................................16
3.13 Biodistribution......................................................................................................17
3.14 H&E staining........................................................................................................18
Chapter 4 Conclusions and Discussions.......................................................................19
4.1 Conclusions............................................................................................................19
4.2Discussions..............................................................................................................19
REFERENCE...............................................................................................................21
























LIST OF SCHEMES
Scheme 1. Icons of each carrier types and their symbols……………………………23
Scheme2. The procedure of composing and forming APO(CPT-11)-CD133 ............24
LIST OF TABLES
Table 1. CD133 specific-binding peptide conjugated apoferritin................................25
Table 2. The rate of CPT-11 encapsulated in apoferritin..............................................25
Table 3. Size and zeta potential of apoferritin and APO(CPT-11)-CD133 measured by
DLS................................................................................................................26
Table 4. Drug released from APO(CPT-11)-CD133 by the time passed......................26
LIST OF FIGURES
Figure 1. Images measured by transmission electron micrograph (a)Apoferritin
(b)APO(CPT-11)-CD133 and the size distribution images measured by
(c)Apoferritin (d) APO(CPT-11)-CD133.......................................................27
Figure 2. Stability of APO(CPT-11)-CD133 (a) PDI of APO(CPT-11)-CD133 (b)size
of APO(CPT-11)-CD133................................................................................28
Figure 3. Drug releasing profile of APO(CPT-11)-CD133 at different conditions......29
Figure 4. CD133 performance of different cell lines analyzed by flow cytometer
(a)HT-29 (b)SW620 (c)HCT-116(d)HCT-116 (K.O. CD133)........................30
Figure 5. Expression of CD133 on different cell lines by western blot.......................30
Figure 6. Binding affinity of APO(CPT-11)-CD133 on different cell lines analyzed by
flow cytometer (a)HT-29 (b)SW620 (c)HCT-116 (d)HCT-116(K.O.
CD133). .........................................................................................................31
Figure 7. Cellular uptake of different drug types to different cell lines measured by
detecting the florescence of CPT-11(a)combination (b)HCT-116 (c)HT-29
(d)SW620(e)HCT-116(K.O. CD133).............................................................32
Figure 8. IC50 of different drug types on different cell lines analyzed by MTT assay
(a)SW620 (b)HT-29 (c)HCT-116 (d)HCT-116(K.O. CD133) (e)sorted out to be a chart...............................................................................................................33
Figure 9. Colony formation after treated with (a)control (b)CPT-11, (c)APO(CPT-11)
and (d)APO(CPT-11)-CD133. (e)after normalizing control to 1000 and
compare to other groups...................................................................................34
Figure 10. Pharmacokinetics measured by collection blood from nude mice after
intravenous injection of CPT-11 or APO(CPT-11)-CD133 into different
mice................................................................................................................35
Figure11. In vivo tumor growth inhibition after tumors growth up to 100mm3 and
intravenous treatment of no treatment(control), APO-CD133, CPT-11,
APO(CPT-11), APO(CPT-11)-CD133 in different groups (a)tumor size
(b)weight changes............................................................................................36
Figure 12.biodistribution after intravenous injection of APO(CPT-11)-CD133 or
CPT-11 into nude mice, and sacrifice them at 24 and 48 hour respectively...37
Figure 13. H&E staining after sacrificing one mouse for each group. (a)Heart (b)
Liver (c)Spleen (d)Lung (e)Kidney. The order of each subfigure follows : Control, APO-CD133, CPT-11, APO(CPT-11), APO(CPT-11)-CD133 from left to right and top to bottom…….………………………………………………38

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