臺灣博碩士論文加值系統

誘導性多能幹細胞 (induced pluripotent stem cells, iPSCs) 是自成體細胞誘導逆分化程序所獲得。從病患自體細胞所得來的多能幹細胞，在移植時減少了異體免疫排斥的顧慮，提升移植安全性。但是誘導性多能幹細胞的發展，存在兩個主要障礙；其一是自成體細胞重新編程 (reprogramming) 為多能性幹細胞的效率不佳，雖然已有許多研究團隊指出了一些提高效率的方法，其效率仍然未達能廣泛應用的程度；第二是多能性幹細胞的致腫瘤特性，若將多能性幹細胞移入動物體內，有相當的機率會發育成腫瘤，使得多能性幹細胞治療法的安全性增添了許多疑慮。
一般常用的誘導方法，是透過病毒感染來表現四個重設因子 (reprogramming factor)：Oct4、Sox2、Klf4、c-Myc；然而透過病毒感染表現的方法可能導致內源性 (endogenous) 基因的異常表現，也可能造成致腫瘤的發生。目前已有研究證實利用蛋白質轉導方式，可以將四個重設因子之重組蛋白送入細胞中，以誘導其重新編程生成誘導性多能幹細胞，避免染色體修飾可能造成的病變。但是蛋白質純化的繁複步驟及質量控管，卻成為蛋白質轉導方式的另一項障礙。
在本研究中，同樣以蛋白質轉導方法，作為平台開發的基礎，但選用毛囊乳突細胞 (Dermal papilla cells, DPCs) 作為細胞材料。DPCs已被發現可利用外來表現單一個重設因子 (Oct4)，使其生成誘導性多能幹細胞。實驗結果證實，利用蛋白質轉導的方式，可藉由單一個重設因子 (Oct4) 之重組蛋白，誘導小鼠毛囊乳突細胞進行重新編程，獲得誘導性多能幹細胞；保留蛋白質方法的優點，省去反覆的蛋白質純化步驟。
改善誘導效率部分，過去的研究中已提到自我吞噬作用會參與在細胞的重塑行為(rearrangement)，且也有報導指出細胞自噬的活化劑可以提升iPSCs的生成效率，改善效率不佳的問題。本研究以蛋白質誘導DPCs重新編程的平台，透過抑制或促進細胞自噬作用，觀察細胞自噬作用對於誘導性多能幹細胞生成的影響。另外，由本實驗室從中草藥何首烏中所純化分離出的化合物EH-201，已在過去的研究中被證實能提升細胞中的細胞自噬作用，在本研究中也將其作為誘導細胞重新編程的調控之一，在EH-201的協助之下，可望提高誘導性多能幹細胞的生成效率。
在本實驗室過去研究中，指出Cirhin參與在正常胚胎發育過程，且具抑制腫瘤的發生的能力；顯示Cirhin也是一個胚胎時期的標誌基因，可能參與在重新編程的程序，並抑制多能幹細胞的致腫瘤性。本實驗則在誘導細胞重新編程的過程中，弱化或者額外表現Cirhin，探討所獲得之誘導性多能幹細胞在致腫瘤特性的差異。
實驗結果顯示，我已建構出能以大腸桿菌來表現重設因子重組蛋白 (recombinant reprogramming protein) 的質體；並以RosettaTM(DE3)pLys大腸桿菌菌株成功表現目標蛋白；透過管柱層析，也成功分離出足夠濃度的Oct4重組蛋白可供實驗使用。誘導重新編程部分則證實，毛囊乳突細胞確實能在單一個Oct4重組蛋白的作用下，生成誘導性多能幹細胞，並且表現內源性胚胎幹細胞特有基因 (embryonic specific genes)。並且也觀察到EH-201確實能夠增進重新編程與誘導多能性幹細胞的生成效率。
透過此一平台，便能進一步的檢測細胞自噬的活化劑或抑制劑，對誘導細胞重新編程及誘導性多能幹細胞生成效率的影響；並且探討Cirhin基因表現，對參與細胞重新編程的角色，和所生成之誘導性多能幹細胞致腫瘤性的調控情形。

Induced pluripotent stem cells (iPSCs) were derived from somatic cells that have been induced to reprogram, which offered an appealing solution to the likely immune rejection of ESC-derived cells, thus providing newer and safer avenues for patient-specific cell therapy. However, there are two main hurdles in iPSCs applications. First is the efficiency of reprogramming is extremely low. And although many research groups have shown some strategies to enhance the reprogramming efficiency, the results remain unsatisfied. The other hurdle is the tumorigenicity of iPSCs. The high possibility of tumor formation from transplantation of iPSCs prevent the in usage in stem cell therapy.
Usually, reprogramming of somatic cells were induced by the expression of four reprogramming factors (Oct4、Sox2、Klf4、c-Myc) introduced by viral infection. But the virus genome might insert into chromosomes, thus altering the endogenous gene expression, to increase tumorigenicity. Previous studies have proved that recombinant reprogramming proteins can be transferred into cells through protein transduction, and induced their reprogramming to become iPSCs. Although promising, the recombinant proteins used in these approaches are usually difficult to be reproducibly purified in sufficient amount, making them difficult to be used routinely in the clinic.
In this study, I also used protein transduction as the base for platform development, but chose dermal papilla cells (DPCs) as the model. Because DPCs have been reported to be reprogrammed by single reprogramming factor gene- Oct4. The result has showed that DPCs can be reprogrammed by transduction of Oct4 protein.

Previous reports have mentioned that autophagy would participate in cell rearrangement, and also, autophagy inducer can enhance the iPSCs production.. My study was based on the platform of protein induce DPCs reprogramming, through inhibit or enhance autophagy, to verify the effect of autophagy in induce iPSC. Moreover, the compound purified by our lab from Polygonum multiflorum- EH-201, which previously confirmed the ability to increase autophagy, was also treated in the reprogramming process, and may enhance the iPSCs production.
In addition, previous studies of our lab have reported that CIRH1A gene regulate embryonic development, and it also has the ability to suppress the tumor formation; shows that Cirhin is one of the embryonic specific gene, and may inhibit tumorigenicity while reprogramming of iPSCs. In this study, Cirhin was knockdown or overexpressed in DPCs, thus to investigate the tumorigenicity of observed iPSCs.
The result of my study shows that I have successfully construct the plasmid, which can express recombinant reprogramming protein by E. coli strain RosettaTM(DE3)pLys. Through Ni-column chromatography, I also successfully purified enough amount of recombinant Oct4 protein for research. The experimental approach shows DPCs indeed can be reprogrammed into iPSCs, which express endogenous embryonic specific genes. EH-201 also shows it’s efficacy of enhancing reprogramming process.
With this platform, we can additionally evaluate the effect of enhancer and inhibitor of autophagy in reprogramming; and to investigate the role of Cirhin in reprogramming process, and it’s regulation of tumorigenecity of iPSCs.

目錄 I
圖表目錄 II
縮寫檢索表 IV
中文摘要 1
Abstract 3
緒論 5
試藥及器材 12
實驗方法 17
實驗結果 28
實驗討論 31
參考文獻 34
圖表 38
附圖 50

圖次
Figure 1. The sequence of designed fragment for modifying of protein expression vector: bFGF/pET3b. 38
Figure 2. Construction of the expression vector for recombinant proteins 39
Figure 3. Maps of the expression vector for various reprogramming factors 40
Figure 4. Induction of the expression of various recombinant proteins in BL21(DE3)-pLys 41
Figure 5. Induction proteins expression in Rosetta™(DE3)pLysS 42
Figure 6. Induction of recombinant Sox2 protein expression in Rosetta™(DE3)pLysS 43
Figure 7. Prurification of the recombinant Oct4 protein by Ni-column 44
Figure 8. Protocol for transducing the recombinant Oct4 proteins into DPCs and detection of the cellular Oct4 proteins 45
Figure 9. Protein induced reprogramming process co-treat with growth factors 46
Figure 10. Protein induced reprogramming process co-treat with EH-201 47
Figure 11. Protein induced reprogramming process with Cirhin knockdown or Cirhin over-expression. 48


表次
Table 1. Primers used for PCR amplification of the genes encoding various recombinant reprogramming factors. 49


附圖
Supplementary Figure 1. Gene expression level analysis of Cirhin in mouse tissues and cell lines by online analysis database BioGPS. 50
Supplementary Figure 2. Gene expression level analysis of p53 in mouse tissues and cell lines by online analysis database BioGPS. 51

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