本文選題：非病毒載體 + 重編程��； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：自2006年Yamanaka首次利用“四因子”組合(p SOX2、p OCT4、p C-MYC、p KLF4)成功誘導(dǎo)生成i PSCs后,細(xì)胞重編程技術(shù)始終是研究熱點(diǎn)之一。由于i PSCs來源于患者自身體細(xì)胞,并具有自我更新和多向分化能力,因此,將i PSCs作為一種替代胚胎干細(xì)胞(ESCs)的細(xì)胞應(yīng)用于臨床中,能夠有效避免很多倫理及法律問題,且i PSCs源于患者,能夠?qū)崿F(xiàn)個(gè)體化治療。原位肝移植是目前唯一能有效治療肝癌的方法。然而,由于器官短缺,近年來因?yàn)楦伟┧劳龅娜藬?shù)不斷增加;此外,世界范圍內(nèi)的肝癌發(fā)病率也在不斷上升。肝細(xì)胞移植的臨床試驗(yàn)帶來振奮人心的成果。但作為替代肝臟移植的肝細(xì)胞移植仍然需要解決肝細(xì)胞來源的問題。目前,很多研究都致力于尋找一種能夠產(chǎn)生穩(wěn)定的功能性肝細(xì)胞的可行性方法。其中,可能會(huì)成為肝細(xì)胞不竭來源的細(xì)胞就是人源誘導(dǎo)多能干細(xì)胞(hi PSCs)。hi PSCs具有自我更新和多向分化能力,與人源胚胎干細(xì)胞(h ESCs)相比,hi PSCs涉及到的倫理問題較少,且完全不受來源限制。因此,hi PSCs來源肝細(xì)胞(hi PSC-HEPs)在藥物篩選、細(xì)胞治療以及體外疾病模型等方面具有重要的意義。本研究課題是以磷酸鈣納米粒作為基因載體,通過攜帶兩種不同因子組合將人臍帶間充質(zhì)干細(xì)胞(h UMSCs)重編程為hi PSCs,繼而進(jìn)行肝細(xì)胞定向誘導(dǎo)分化,并篩選優(yōu)化出最佳誘導(dǎo)分化培養(yǎng)基,采用最佳誘導(dǎo)方案對(duì)其進(jìn)行培養(yǎng),使其分化為肝細(xì)胞,為肝細(xì)胞移植提供理論基礎(chǔ),從而更好地解決肝細(xì)胞短缺問題。第一章重編程法制備人源肝臟細(xì)胞及其應(yīng)用研究進(jìn)展通過查閱大量資料,對(duì)人源肝細(xì)胞重編程方法進(jìn)行了綜述,總結(jié)并分析人源肝細(xì)胞重編程的影響因素;闡述了人源肝細(xì)胞重編程技術(shù)的研究現(xiàn)狀及其在再生醫(yī)學(xué)、藥物篩選及體外疾病模型等方面的應(yīng)用,并指出人源肝細(xì)胞應(yīng)用于臨床實(shí)踐的局限性。為本論文實(shí)驗(yàn)工作的順利開展和進(jìn)行提供了理論依據(jù)。第二章非病毒納米基因傳遞系統(tǒng)的hi PSCs重編程研究采用磷酸鈣納米粒作為基因載體,對(duì)比兩種不同的因子組合,即p SOCK(p SOX2、p OCT4、p C-MYC、p KLF4)與p OS+mi R(p OCT4、p SOX2、mi R302b-367),制備得到磷酸鈣納米粒C-p SOCK與C-p OS+mi R,通過瓊脂糖凝膠電泳、透射電鏡觀察、毒性評(píng)價(jià)、粒徑以及Zeta電位測(cè)定等一系列檢測(cè)進(jìn)行表征;以人臍帶間充質(zhì)干細(xì)胞(h UMSCs)為源細(xì)胞,進(jìn)行重編程研究。結(jié)果顯示:兩種因子組合所制備得到的納米粒均呈現(xiàn)形態(tài)規(guī)整、分散均勻、表面帶正電荷的球形或者橢球型;細(xì)胞毒性實(shí)驗(yàn)表明兩種不同因子組合制備得到的磷酸鈣納米粒無毒或低毒;通過堿性磷酸酶染色,統(tǒng)計(jì)陽性克隆數(shù),從而考察兩種納米粒的重編程效率;免疫熒光、Western blot均說明納米粒C-p OS+mi R誘導(dǎo)生成的hi PSCs能夠表達(dá)多能性標(biāo)記蛋白,且能進(jìn)行體內(nèi)分化(內(nèi)、中、外)。第三章hi PSCs的肝細(xì)胞定向誘導(dǎo)分化研究設(shè)置4種不同肝細(xì)胞誘導(dǎo)分化培養(yǎng)基(MediumⅠ、Ⅱ、Ⅲ、Ⅳ),對(duì)重編程生成的hi PSCs進(jìn)行肝細(xì)胞定向誘導(dǎo)分化,采用酶聯(lián)免疫檢測(cè)法(ELISA)分別在誘導(dǎo)分化第0,3,7,11,15,19,23,27,32天檢測(cè)AFP和ALB表達(dá)情況,從而篩選出最佳誘導(dǎo)方案;光學(xué)顯微鏡下觀察細(xì)胞形態(tài)變化;用免疫熒光法、Western blot檢測(cè)肝臟特征性蛋白(AFP、ALB、CK8、CK18、SOX17)的表達(dá)。結(jié)果顯示:誘導(dǎo)培養(yǎng)基MediumⅡ、誘導(dǎo)方案“三步法”的肝細(xì)胞誘導(dǎo)效果較好;顯微鏡下觀察細(xì)胞形態(tài)發(fā)生明顯變化,從“克隆團(tuán)”形態(tài)逐漸變化為“鋪路石”樣形態(tài),誘導(dǎo)分化結(jié)束后,細(xì)胞形態(tài)基本一致,為多角多邊形或者類圓形,且細(xì)胞核較明顯,部分細(xì)胞表現(xiàn)出典型肝細(xì)胞形態(tài):出現(xiàn)雙核甚至多核現(xiàn)象,且誘導(dǎo)分化的肝細(xì)胞能夠表達(dá)肝臟肝臟特征性蛋白(AFP、ALB、CK8、CK18、SOX17)。第四章二維培養(yǎng)體系中肝細(xì)胞藥物代謝酶表達(dá)及細(xì)胞活性研究在二維培養(yǎng)體系下,選擇Ⅰ相代謝酶細(xì)胞色素P450(CYP450)、Ⅱ相代謝酶葡萄糖醛酸轉(zhuǎn)移酶(UGT)與谷胱甘肽硫轉(zhuǎn)移酶(GST),用Western blot測(cè)定誘導(dǎo)后細(xì)胞蛋白表達(dá),以人胚胎干細(xì)胞來源肝細(xì)胞(h ESCs-HEPs)為陽性對(duì)照;考察在誘導(dǎo)分化不同時(shí)間藥物代謝酶的動(dòng)態(tài)變化,同時(shí)比較hi PSCs誘導(dǎo)形成的肝細(xì)胞與胚胎干細(xì)胞來源肝細(xì)胞(h ESCs-HEPs)藥物代謝酶的表達(dá)差異;不同時(shí)間取樣,檢測(cè)谷草轉(zhuǎn)氨酶(AST)、谷丙轉(zhuǎn)氨酶(ALT)和乳酸脫氫酶(LDH)的質(zhì)量濃度;檢測(cè)白蛋白分泌、尿素合成及葡萄糖消耗含量,分別采用溴甲酚綠法、二乙酰-肟法及葡萄糖氧化酶-過氧化物酶法。結(jié)果顯示:hi PSCs誘導(dǎo)形成的肝細(xì)胞(hi PSCs-HEPs)能夠較好表達(dá)CYP450、UGT與GST,且與h ESCs-HEPs表達(dá)無明顯差異;而誘導(dǎo)前hi PSCs很少表達(dá)甚至不表達(dá)CYP450、UGT與GST;hi PSCs的肝細(xì)胞誘導(dǎo)分化過程中ALT、AST、LDH變化趨勢(shì)與h ESCs基本一致;hi PSCs-HEPs與h ESCs-HEPs測(cè)定的白蛋白分泌水平、尿素合成濃度以及葡萄糖消耗量基本一致。第五章三維培養(yǎng)體系中hi PSCs定向肝細(xì)胞誘導(dǎo)分化在三維培養(yǎng)體系下,對(duì)重編程生成的hi PSCs進(jìn)行肝細(xì)胞定向誘導(dǎo)分化。以Ⅰ型膠原和殼聚糖作為支架材料,采用冷凍干燥-熱固化法制備膠原/殼聚糖三維支架,考察不同質(zhì)量比與不同固化時(shí)間對(duì)支架性能的影響,優(yōu)化三維支架制備工藝,通過形態(tài)觀察、吸水率與保水率測(cè)定等對(duì)制備得到的支架進(jìn)行表征;將納米粒C-p OS+mi R與空白膠原/殼聚糖支架相結(jié)合,得到三維載基因納米粒-膠原/殼聚糖支架;人臍帶間充質(zhì)干細(xì)胞(h UMSCs)在三維載基因納米粒-膠原/殼聚糖支架中進(jìn)行重編程后,形成邊界清晰、形態(tài)規(guī)整的克隆團(tuán);對(duì)三維支架上重編程生成的hi PSCs進(jìn)行肝細(xì)胞定向誘導(dǎo)分化,用ELISA法對(duì)比二維與三維培養(yǎng)體系中肝細(xì)胞功能;分別用溴甲酚綠法、二乙酰-肟顯色法、葡萄糖氧化酶-過氧化物酶法比較二維與三維培養(yǎng)體系中肝細(xì)胞代謝活性。結(jié)果顯示:在三維培養(yǎng)體系誘導(dǎo)21天后,細(xì)胞形態(tài)呈現(xiàn)明顯的肝細(xì)胞樣細(xì)胞形態(tài);在三維培養(yǎng)體系下,誘導(dǎo)形成的肝細(xì)胞功能沒有“損傷”,仍處于正常肝功能狀態(tài)。
[Abstract]:After the successful induction of I PSCs by the "four factor" combination (P SOX2, P OCT4, P C-MYC, P KLF4) in 2006, cell reprogramming has always been one of the hotspots of research. Because I originates from the body and has self renewal and multi differentiation energy, it is used as a substitute for embryonic stem cells. The use of cells in the clinic can effectively avoid many ethical and legal problems, and I PSCs is derived from patients and can achieve individualized treatment. Orthotopic liver transplantation is the only effective treatment for liver cancer. However, the number of deaths due to liver cancer has increased in recent years due to the shortage of organs; in addition, the incidence of liver cancer in the world The rate is also rising. The clinical trials of hepatocyte transplantation have brought exciting results. But as a substitute for liver transplantation, liver cell transplantation still needs to solve the problem of hepatocyte origin. Many studies are currently working to find a feasible way to produce stable functional liver cells. The cell inexhaustible source is the ability of human induced pluripotent stem cells (HI PSCs).Hi PSCs to have self-renewal and multidirectional differentiation. Compared with human embryonic stem cells (H ESCs), the ethical problems involved in hi PSCs are less and are completely unrestricted from sources. Therefore, hi PSCs comes from liver cells (HI PSC-HEPs) in drug screening, cell therapy, and body. The model of external disease is of great significance. This research focuses on the use of calcium phosphate nanoparticles as a gene carrier to reprogram human umbilical cord mesenchymal stem cells (H UMSCs) into hi PSCs by carrying two different combinations of factors. The induction program can be cultured to differentiate into liver cells, provide a theoretical basis for hepatocyte transplantation, and thus better solve the problem of liver cell shortage. Chapter 1 reprogramming a human source liver cell and its application research progress through consulting a large number of data, summarizing and analyzing human liver cell heavy cell reprogramming methods, summarizing and analyzing people. The influence factors of reprogramming of source hepatocyte, the current status of human hepatocyte reprogramming and its application in regenerative medicine, drug screening and in vitro disease model were introduced, and the limitations of the application of human hepatocytes in clinical practice were pointed out. The theoretical basis for the successful development and development of this paper was provided. Second The hi PSCs reprogramming of non viral nanoscale gene transfer system uses calcium phosphate nanoparticles as the gene carrier, and compares two different combinations of factors, namely, P SOCK (P SOX2, P OCT4, P C-MYC, P KLF4) and the agarose gel electrophoresis. A series of tests, such as transmission electron microscopy, toxicity evaluation, particle size and Zeta potential measurement, were characterized by a series of tests. Human umbilical cord mesenchymal stem cells (H UMSCs) were used as the source cells for reprogramming. The results showed that the nanoparticles prepared by the two factors were uniformly distributed, dispersed evenly, with a spherical or ellipsoid with positive charges on the surface. The cytotoxicity test showed that the calcium phosphate nanoparticles prepared by two different combinations were non-toxic or low toxic; the reprogramming efficiency of the two nanoparticles was investigated by the alkaline phosphatase staining and the number of positive clones, and the immunofluorescence and Western blot showed that the hi PSCs induced by the nanoparticles C-p OS+mi R could express the multienergy standard. Third hi PSCs induced differentiation of hepatocytes and differentiation medium (Medium I, II, III, IV) were set up in 4 different hepatocytes (Medium, II, III, IV). The induced differentiation of liver cells was induced by reprogrammed hi PSCs, and the differentiation of 0,3,7,11 was induced by enzyme immunoassay (ELISA), respectively. The expression of AFP and ALB was detected by 15,19,23,27,32 days, and the optimal induction scheme was screened. The morphological changes of cells were observed under the optical microscope, and the expression of liver characteristic proteins (AFP, ALB, CK8, CK18, SOX17) was detected by immunofluorescence and Western blot. The results showed that the inducible medium Medium II was induced by the induction program "three steps" of hepatocyte induction. The morphological changes were observed under the microscope, and the morphology of the cells changed from "cloned group" to "pave" form. After the induction of differentiation, the cell morphology was basically the same, it was polygonal polygon or round, and the nucleus was more obvious. Some cells showed typical liver cell morphology: Double nucleus and even multinuclear appearance appeared. AFP, ALB, CK8, CK18, SOX17 in liver liver cells can be expressed in the differentiated hepatocytes. Fourth in two dimensional culture system, the expression of drug metabolizing enzymes and cell activity of hepatocytes in the two dimensional culture system, the selection of cytochrome P450 (CYP450), phase II metabolic enzyme glucuronotransferase (UGT) and Valley in the two-dimensional culture system Cystamine thiotransferase (GST) was used to determine the expression of cell protein after induction of Western blot, and the positive control was taken from human embryonic stem cell derived hepatocytes (H ESCs-HEPs). The dynamic changes of drug metabolizing enzymes at different time of induction of differentiation were investigated, and the drug derived from hi PSCs induced liver cells and embryonic stem cells derived from liver cells (H ESCs-HEPs) were compared. The difference in the expression of metabolic enzymes; sampling at different time, detecting the mass concentration of AST, ALT and LDH, detecting albumin secretion, urea synthesis and glucose consumption, using bromo methyl phenol green method, two acetyl oxime method and glucose oxidase peroxidase method respectively. The results showed: Hi PSCs The induced liver cells (HI PSCs-HEPs) can express CYP450, UGT and GST, and there is no obvious difference from H ESCs-HEPs, but before induction, hi PSCs seldom expresses or even expresses CYP450, UGT and GST. The protein secretion level, the urea synthesis concentration and the glucose consumption are basically the same. In the fifth chapter, in the fifth chapter three dimensional culture system, the induced differentiation of liver cells was induced and differentiated under the three-dimensional culture system. The reprogrammed hi PSCs was directed and differentiated to the liver cells. The type I collagen and chitosan were used as the scaffold material, and the freeze drying heat curing was used. A three-dimensional collagen / chitosan scaffold was prepared by the method. The effects of different mass ratio and curing time on the performance of the scaffold were investigated. The three-dimensional scaffold preparation process was optimized. The prepared scaffolds were characterized by morphological observation, water absorption and water retention ratio. The C-p OS+mi R of the nanoparticles was combined with blank collagen / chitosan scaffold. Gene loaded nanoparticles collagen / chitosan scaffold; human umbilical cord mesenchymal stem cells (H UMSCs) were reprogrammed in three dimensional gene loaded nanoparticles collagen / chitosan scaffold to form a well-defined and orderly cloned cloned group. The hi PSCs generated on the three dimensional stent was directed to induce differentiation of liver cells, and the two dimension was compared with the ELISA method. The liver cell function in the three-dimensional culture system was compared with bromocresol green method, two acetyl oxime colorimetric method and glucose oxidase peroxidase method to compare the metabolic activity of liver cells in two-dimensional and three-dimensional culture systems. The results showed that the morphology of the cells showed obvious liver cell like cells in the three dimensional culture system after the induction of the three-dimensional culture system for 21 days. Under the guidance of the system, the function of induced hepatocytes is not "damaged" and is still in normal liver function.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R329.2

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