本文選題：誘導(dǎo)多能干細(xì)胞 + 表皮干細(xì)胞��； 參考：《深圳大學(xué)》2017年碩士論文

【摘要】：燒傷是臨床常見外傷性疾病,是因?yàn)闊崃�、化學(xué)試劑、電力等因素造成的組織器官的損害癥狀。嚴(yán)重?zé)齻麜斐善鞴俟δ芩ソ?引發(fā)多種并發(fā)癥,還可能會危害患者生命。皮膚是否能夠再生與修復(fù)的關(guān)鍵在于存在皮膚表層的表皮干細(xì)胞。然而大面積或者深度燒傷往往會將皮膚本身的表皮干細(xì)胞破壞掉,從而導(dǎo)致創(chuàng)面修復(fù)的困難。傳統(tǒng)治療方案包括自體、異體或異種皮膚移植,但手術(shù)結(jié)果并不總?cè)缛艘�。皮膚創(chuàng)面修復(fù)需要組織工程構(gòu)建的皮膚替代物,表皮干細(xì)胞則是皮膚替代物常用的種子細(xì)胞。然而表皮干細(xì)胞的純化率低,不能大量獲取。因此如何在體外大量擴(kuò)增表皮干細(xì)胞則是皮膚組織工程研究的關(guān)鍵點(diǎn)。2006年日本科學(xué)家發(fā)現(xiàn)將c-Myc、Klf4、Sox2和Oct-3/4四個與胚胎干細(xì)胞多樣性相關(guān)的轉(zhuǎn)錄因子導(dǎo)入小鼠胚胎成纖維細(xì)胞(Mouse embryonic fibroblast,MEF),獲得具有類似胚胎干細(xì)胞多樣性的細(xì)胞,并稱之為誘導(dǎo)多能干細(xì)胞(Induced pluripotent stem cells,iPSCs)。由于iPSCs避免了胚胎干細(xì)胞導(dǎo)致的道德倫理與法律問題,而且容易獲得細(xì)胞,因此成為了組織工程的重要細(xì)胞來源。近十年來,iPSCs已被證實(shí)能夠通過運(yùn)用細(xì)胞因子誘導(dǎo)的方法定向分化為表皮干細(xì)胞。在現(xiàn)有iPSCs定向誘導(dǎo)分化為表皮干細(xì)胞的方法中,有使用羊膜誘導(dǎo)的、改變培養(yǎng)環(huán)境的、頻頻更換培養(yǎng)基的等等,這些培養(yǎng)方法的局限性在于誘導(dǎo)分化方法的不可控性以及操作的繁瑣。本論文研究致力于使用添加成分明確的誘導(dǎo)表皮干細(xì)胞分化培養(yǎng)基(Epidermal stem cell differentiation medium,ESCDM),以提高表皮干細(xì)胞分化以及增殖的效率。本論文中,我將使用倒置顯微鏡(Inverted microscope,IM)、免疫細(xì)胞化學(xué)技術(shù)(Immunocytochemistry,ICC)方法對人iPSCs(Human iPSCs,hiPSCs)定向誘導(dǎo)分化為表皮干細(xì)胞的過程進(jìn)行初步的探討和研究。在本實(shí)驗(yàn)當(dāng)中,iPSCs使用mTeSR?1完全培養(yǎng)基培養(yǎng),避免與動物免疫蛋白接觸。iPSCs傳代之后分為實(shí)驗(yàn)組與對照組,實(shí)驗(yàn)組在誘導(dǎo)分化iPSCs采用添加維甲酸(Retinoic acid,RA)和骨形成蛋白4(Bone morphogenetic protein 4,BMP4)的方法,而對照組則采用羊膜提取液(Amnion extract,AETRA)進(jìn)行誘導(dǎo)的方法。兩組每天更換分化培養(yǎng)基,使用表皮干細(xì)胞無血清培養(yǎng)基(Defined keratinocyte serum free medium,DK-SFM)作為分化培養(yǎng)基。最終獲得的細(xì)胞經(jīng)過形態(tài)結(jié)構(gòu)觀察與ICC進(jìn)行鑒定。同時設(shè)立人正常表皮干細(xì)胞(Normal human epidermal keratinocytes,NHEK)作為對照組,設(shè)立為觀察分化得到的表皮干細(xì)胞與正常人表皮干細(xì)胞的差異。結(jié)果發(fā)現(xiàn),誘導(dǎo)分化第6天之后,實(shí)驗(yàn)組的表皮干細(xì)胞的增殖數(shù)目顯著多于對照組,免疫熒光技術(shù)檢測誘導(dǎo)第6、8、10、12天兩組的表皮干細(xì)胞的細(xì)胞角化蛋白CK19以及α6整合素的表達(dá)量,實(shí)驗(yàn)組的CK19以及α6整合素的陽性表達(dá)細(xì)胞均顯著多于對照組的,可以得知實(shí)驗(yàn)組的誘導(dǎo)表皮干細(xì)胞分化的效率高于對照組。同時與正常表皮干細(xì)胞比較發(fā)現(xiàn),實(shí)驗(yàn)組的形態(tài)與特性并無明顯差別,增殖速度與正常表皮干細(xì)胞相同。而AETRA培養(yǎng)的表皮干細(xì)胞形態(tài)不一致、少量細(xì)胞出現(xiàn)分化、CK19以及α6整合素的表達(dá)量也偏低。綜上所述,使用添加RA和BMP4的ESCDM縮短了hiPSCs定向誘導(dǎo)分化為表皮干細(xì)胞需要的時間,提高表皮干細(xì)胞分化的效率以及增殖的速度,同時維持了正常表皮干細(xì)胞的形態(tài)與特征。
[Abstract]:Burn is a common clinical traumatic disease, which is a symptom of tissue damage caused by heat, chemical reagents, electricity and other factors. Severe burns can cause organ failure, cause a variety of complications, and may also harm the patient's life. The key to the regeneration and repair of the skin is the epidermal stem cells in the skin surface. A large area or deep burn often destroys the epidermal stem cells of the skin itself, which leads to the difficulty of repairing the wound. Traditional treatments include autologous, allogenic or heterogeneous skin transplantation, but the results are not always desirable. Skin wound repair requires tissue engineered skin substitutes, and epidermal stem cells are skin. However, the purification rate of epidermal stem cells is low and can not be obtained in large quantities. Therefore, how to amplify epidermal stem cells in vitro is the key point of skin tissue engineering in vitro..2006 Japanese scientists found that four transcriptional factors related to c-Myc, Klf4, Sox2 and Oct-3/4 were introduced into small number of transcription factors related to embryonic stem cell diversity. Mouse embryonic fibroblasts (Mouse embryonic fibroblast, MEF), which have cells that have the diversity of embryonic stem cells, are called inducible pluripotent stem cells (Induced pluripotent stem cells, iPSCs). IPSCs avoids ethical and legal problems caused by embryonic stem cells and is easy to obtain cells, thus becoming an organization. The important cell source of engineering. In the past ten years, iPSCs has been proved to be able to differentiate into epidermal stem cells by using cytokine induced methods. In the existing methods of inducing differentiation into epidermal stem cells by iPSCs, there are amniotic membrane induction, change of culture environment, frequency frequency replacement medium and so on, and so on. The limitation lies in the uncontrollability of the induction of differentiation and the cumbersome operation. This paper is devoted to the use of Epidermal stem cell differentiation medium (ESCDM) to improve the differentiation and proliferation of epidermal stem cells. In this paper, I will use inverted microscope (I). Nverted microscope, IM), immunocytochemical Technology (Immunocytochemistry, ICC) method to induce the differentiation of human iPSCs (Human iPSCs, hiPSCs) into epidermal stem cells. In this experiment, iPSCs uses mTeSR? 1 culture to avoid contact with animal immunoglobulin. For the experimental group and the control group, the experimental group was induced to differentiate iPSCs by adding retinoic acid (Retinoic acid, RA) and bone morphogenetic protein 4 (Bone morphogenetic protein 4, BMP4), while the control group was induced by amniotic membrane extraction (Amnion extract, AETRA). The two groups changed the differentiation medium every day, using epidermal stem cells without blood. The clear culture medium (Defined keratinocyte serum free medium, DK-SFM) was used as a differentiation medium. The final cells were identified by morphological structure and ICC, and normal human epidermal stem cells (Normal human epidermal keratinocytes, NHEK) were established as the control group, which was established to observe the differentiation of epidermal stem cells and normal people. The results showed that after sixth days of differentiation, the number of epidermal stem cells in the experimental group was significantly more than that of the control group. The immunofluorescence technique was used to detect the expression of keratin CK19 and alpha 6 integrin in the epidermal stem cells of the two groups on day 6,8,10,12, the CK19 of the experimental group and the positive table of the integrin of alpha 6. The efficiency of the induced epidermal stem cells in the experimental group was higher than that of the control group. At the same time, compared with the normal epidermal stem cells, it was found that the morphology and characteristics of the experimental group were not significantly different from that of the normal epidermal stem cells. The morphology of the epidermal stem cells in the AETRA culture was not consistent, and a small amount of epidermal stem cells were found in the experimental group. The cells were differentiated, and the expression of CK19 and alpha 6 integrin was also low. To sum up, the use of ESCDM adding RA and BMP4 shortened the time required by hiPSCs to induce differentiation into epidermal stem cells, increased the efficiency of epidermal stem cell differentiation and the speed of proliferation, and maintained the morphology and characteristics of normal epidermal stem cells.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R644;R318.08

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 馬松林;張Y，

本文編號：1847065