本文選題：microRNA + 肝細(xì)胞�。� 參考：《第四軍醫(yī)大學(xué)》2013年博士論文

【摘要】：【背景】 終末期肝病在我國(guó)的發(fā)病率呈逐年增高趨勢(shì)，年死亡率達(dá)30-50%。原位肝移植是目前治療終末期肝病的唯一有效手段。但是供肝缺乏為原位肝移植治療的瓶頸。此外原位肝移植治療費(fèi)用昂貴、術(shù)后需長(zhǎng)期使用免疫抑制劑、圍手術(shù)期風(fēng)險(xiǎn)大等問(wèn)題，極大限制了原位肝移植的廣泛應(yīng)用。 成體干細(xì)胞在體內(nèi)外均可以突破胚層的限制，分化為具有功能的肝細(xì)胞。因此，成體干細(xì)胞移植技術(shù)成為一種新興的終末期肝病治療技術(shù)。但是其向肝細(xì)胞分化的機(jī)制仍不清楚。傳統(tǒng)的誘導(dǎo)手段復(fù)雜、誘導(dǎo)周期長(zhǎng)、分化效率低，限制了干細(xì)胞治療技術(shù)在臨床的廣泛推廣與應(yīng)用。 microRNA是一類短的內(nèi)源性非編碼RNA，通過(guò)與靶基因mRNA的3’非編碼區(qū)的結(jié)合位點(diǎn)結(jié)合，參與基因轉(zhuǎn)錄后調(diào)控。以往研究認(rèn)為，microRNA調(diào)控基因表達(dá)并未起到開關(guān)的作用，而是發(fā)揮可變電阻器的作用。近年來(lái)研究發(fā)現(xiàn)microRNA的時(shí)空特異性分布規(guī)律使得microRNA在機(jī)體發(fā)育和細(xì)胞分化過(guò)程中發(fā)揮了重要的調(diào)控作用。利用特定的microRNA組合可以介導(dǎo)細(xì)胞從一種終末分化狀態(tài)轉(zhuǎn)化為另一種終末分化狀態(tài)。盡管已經(jīng)有文獻(xiàn)陸續(xù)報(bào)道了microRNA在肝臟發(fā)育過(guò)程的重要性，但其在干細(xì)胞肝向分化過(guò)程中的作用尚不清楚。 【目的】 本研究的目的是篩選成體干細(xì)胞肝向分化過(guò)程特異性的microRNA表達(dá)譜，并探討關(guān)鍵microRNA分子在肝向分化中的作用，為干細(xì)胞肝向分化機(jī)制的闡明提供理論支持。研究?jī)?nèi)容主要包括三部分：①建立成體干細(xì)胞向肝細(xì)胞分化的體外模型；②篩選并鑒定成體干細(xì)胞肝向分化過(guò)程特異的microRNA表達(dá)譜；③闡明關(guān)鍵microRNA分子在肝向分化過(guò)程中的作用。 【方法】 1．通過(guò)免疫磁珠方法純化外周血造血干細(xì)胞，通過(guò)貼壁方法分選臍帶間充質(zhì)干細(xì)胞（mesenchymal stem cell, MSC）。應(yīng)用電子顯微鏡觀察細(xì)胞形態(tài)、流式細(xì)胞技術(shù)檢測(cè)細(xì)胞免疫表型，誘導(dǎo)成骨、成脂分化潛能方面檢測(cè)分離的干細(xì)胞純度及特性。在成功分離得到干細(xì)胞后，給予肝向分化誘導(dǎo)培養(yǎng)液，利用qRT-PCR方法和細(xì)胞免疫熒光方法分別從RNA、蛋白質(zhì)水平檢測(cè)MSC經(jīng)肝向誘導(dǎo)后肝細(xì)胞特異性基因的表達(dá)變化，利用PAS染色方法、尿素氮檢測(cè)試劑盒以及LDL攝取實(shí)驗(yàn)檢測(cè)肝細(xì)胞特異性功能的水平，從而驗(yàn)證是否成功建立干細(xì)胞肝向分化的體外模型。 2．選取干細(xì)胞肝向分化模型7個(gè)不同時(shí)間點(diǎn)的細(xì)胞，提取RNA，進(jìn)行microRNA芯片檢測(cè)。依據(jù)差異倍數(shù)、表達(dá)量、以及變化規(guī)律篩選microRNA分子進(jìn)行qRT-PCR檢測(cè)以驗(yàn)證芯片結(jié)果。將qRT-PCR與芯片結(jié)果一致的差異表達(dá)的microRNA譜與其在成骨分化過(guò)程中的表達(dá)變化及其在肝癌細(xì)胞和正常肝細(xì)胞系的表達(dá)變化做比較，以明確該microRNA表達(dá)譜的肝向分化特異性。 3.分別構(gòu)建包含miR-1246、miR-1290、miR-148a、mR-30a、miR-424、miR-542-5p干擾序列的慢病毒顆粒，感染入MSC后，給予肝向分化誘導(dǎo)培養(yǎng)液，觀察分別抑制microRNA活性后對(duì)肝向分化過(guò)程的影響。另外分別合成以上microRNA的模擬物，并以肝臟富集的miR-122為對(duì)照，單一轉(zhuǎn)染或共同轉(zhuǎn)染后，利用qRT-PCR、細(xì)胞免疫熒光、PAS染色、尿素氮檢測(cè)以及LDL攝取實(shí)驗(yàn)等方法檢測(cè)外源性增加microRNA表達(dá)后能否啟動(dòng)MSC的肝向分化并將其轉(zhuǎn)化為成熟的有功能的肝細(xì)胞。最后將誘導(dǎo)的肝細(xì)胞移植入肝損傷小鼠體內(nèi)，通過(guò)檢測(cè)小鼠肝功能的變化、肝組織形態(tài)學(xué)的變化以及人源性肝細(xì)胞和干細(xì)胞在肝臟的定植最終明確microRNA誘導(dǎo)的肝細(xì)胞能否在小鼠體內(nèi)發(fā)揮肝細(xì)胞的功能。 【結(jié)果】 1.成功建立了成體干細(xì)胞肝向分化體外細(xì)胞模型 從外周血分離CD34+細(xì)胞后經(jīng)流式細(xì)胞儀分析，，99%以上的細(xì)胞為CD34+細(xì)胞，細(xì)胞呈圓形不貼壁生長(zhǎng)。經(jīng)四種不同培養(yǎng)體系培養(yǎng)與擴(kuò)增后，發(fā)現(xiàn)CD34+細(xì)胞在SFM34+IL3+SCF+GM-SCF培養(yǎng)液中擴(kuò)增效率最高，可以擴(kuò)增6倍。但是，擴(kuò)增后的細(xì)胞失去了CD34的免疫表型，即SFM34+IL3+SCF+GM-SCF培養(yǎng)液雖然可以高效的擴(kuò)增CD34細(xì)胞，但是，擴(kuò)增的CD34細(xì)胞失去了造血干細(xì)胞的特征。 從臍帶間質(zhì)分離的MSC具有典型的長(zhǎng)索狀形態(tài)，呈旋渦狀生長(zhǎng)。流式細(xì)胞分析結(jié)果顯示，分離的細(xì)胞88.7%可以表達(dá)MSC標(biāo)志分子CD105，不表達(dá)內(nèi)皮細(xì)胞標(biāo)志分子CD31和造血干細(xì)胞標(biāo)志分子CD34。分離的細(xì)胞經(jīng)成骨誘導(dǎo)培養(yǎng)液誘導(dǎo)14天后，具有鈣鹽沉積。即我們成功地從臍帶間質(zhì)分離到MSC。 qRT-PCR結(jié)果顯示肝向誘導(dǎo)1周后，肝細(xì)胞特異性基因HNF4α、ALB和CK18的mRNA表達(dá)水平上調(diào)，于26天時(shí)達(dá)到高峰。免疫細(xì)胞熒光染色結(jié)果顯示MSC經(jīng)肝向誘導(dǎo)14天后可以檢測(cè)到肝細(xì)胞特異性基因AFP、ALB、CK18的蛋白質(zhì)的表達(dá)。PAS染色結(jié)果顯示，MSC經(jīng)肝向誘導(dǎo)14天后可以檢測(cè)到紫紅色的糖原顆粒。LDL攝取實(shí)驗(yàn)結(jié)果顯示，MSC經(jīng)肝向誘導(dǎo)14天時(shí)，大多數(shù)細(xì)胞可以攝取DIL標(biāo)記的LDL。尿素氮檢測(cè)結(jié)果顯示，MSC肝向誘導(dǎo)6天后其合成尿素的能力逐漸提高，于22天時(shí)達(dá)到高峰。即我們成功地建立了臍帶間質(zhì)來(lái)源MSC肝向分化的體外細(xì)胞模型。 2．肝向分化特異性microRNA表達(dá)譜的篩選與鑒定 本研究中共檢測(cè)了1205個(gè)人類已知的和144個(gè)人類病毒相關(guān)microRNA分子在肝向分化7個(gè)不同時(shí)間點(diǎn)的表達(dá)變化情況。共61個(gè)microRNA分子在肝向分化前后變化倍數(shù)大于2倍，且在7個(gè)時(shí)間點(diǎn)中呈規(guī)律性表達(dá)。在此基礎(chǔ)上，依據(jù)差異倍數(shù)和表達(dá)含量共篩選了7個(gè)差異倍數(shù)大于4倍的上調(diào)的microRNA、6個(gè)表達(dá)含量最高的上調(diào)的microRNA以及6個(gè)差異倍數(shù)大于4倍的下調(diào)的microRNA、10個(gè)表達(dá)含量最高的下調(diào)的microRNA進(jìn)行了qRT-PCR驗(yàn)證。qRT-PCR結(jié)果顯示，上調(diào)microRNA中miR-542-5p、miR-148a、miR-1290、miR-424、miR-30a和miR-1246的表達(dá)模式與芯片結(jié)果一致。下調(diào)microRNA中，除miR-3646外，其他下調(diào)microRNA均呈下調(diào)表達(dá)。但除miR-146a外，其他microRNA的表達(dá)模式與芯片結(jié)果吻合度較差。 qRT-PCR和芯片結(jié)果一致的6個(gè)上調(diào)的microRNA與8個(gè)下調(diào)的microRNA在成骨分化過(guò)程中的表達(dá)結(jié)果顯示，部分在肝向分化過(guò)程中上調(diào)的microRNA在成骨分化過(guò)程中卻呈下降趨勢(shì)，部分在肝向分化過(guò)程中下調(diào)的microRNA在成骨分化過(guò)程中卻呈上升趨勢(shì)。同樣經(jīng)肝向誘導(dǎo)后部分表達(dá)上調(diào)（下調(diào)）的microRNA在L02和HepG2中的表達(dá)水平低于（高于）其在MSC中的表達(dá)。說(shuō)明在肝向分化過(guò)程中差異表達(dá)的microRNA表達(dá)譜不同于其在成骨分化過(guò)程中的變化也并非肝癌細(xì)胞或肝細(xì)胞所特異性的，而是肝向分化過(guò)程所特異性的。 3.關(guān)鍵microRNA分子在MSC肝向分化中的作用 未感染病毒的MSC以及感染miR-nc-lev的MSC在肝向誘導(dǎo)6天后白蛋白的表達(dá)水平上調(diào)，而感染了miR-1246-RNAi-lev、miR-1290-RNAi-lev、miR-148a-RNAi-lev、miR-30a-RNAi-lev、miR-424-RNAi-lev、miR-542-5p-RNAi-lev的MSC經(jīng)過(guò)肝向誘導(dǎo)后，其白蛋白的水平并未上調(diào)。肝向誘導(dǎo)12天后，miR-nc-lev組無(wú)論是否成功感染慢病毒均可以攝取LDL。而miR-1246-RNAi-lev、 miR-1290-RNAi-lev、miR-148a-RNAi-lev、miR-30a-RNAi-lev、miR-424-RNAi-lev、miR-542-5p-RNAi-lev組，只有在未成功感染慢病毒的細(xì)胞中出現(xiàn)了LDL的攝取。此外，這些慢病毒顆�？梢砸种艸epG2細(xì)胞肝細(xì)胞特異性白蛋白和G6P的表達(dá)。 將miR-122、miR-1246、miR-1290、miR-148a、miR-30a、 miR-424和miR-542-5p模擬物分別轉(zhuǎn)染入MSC后，可以提高相應(yīng)microRNA的水平，但并不能促使MSC高表達(dá)白蛋白。7種microRNA模擬物共同轉(zhuǎn)染入MSC后，不僅可以同時(shí)增加上述7種microRNA的含量，而且可以促使MSC高表達(dá)肝臟特異性早、中、晚期標(biāo)志分子，不表達(dá)肝祖細(xì)胞、胰膽上皮標(biāo)志分子。使MSC的細(xì)胞形態(tài)從間質(zhì)樣向上皮樣轉(zhuǎn)化，并具有肝細(xì)胞特異性的功能。移植入肝損傷小鼠體內(nèi)后，不僅可以發(fā)揮肝細(xì)胞的功能即改善小鼠肝功能，還可以發(fā)揮部分MSC的功能，修復(fù)小鼠肝組織。 【結(jié)論】 我們成功掌握了分離純化外周血造血干細(xì)胞和臍帶MSC技術(shù)；在成功建立MSC肝向分化體外細(xì)胞模型基礎(chǔ)上，通過(guò)芯片和qRT-PCR技術(shù)篩選并鑒定了一組MSC肝向分化過(guò)程所特異性的microRNA表達(dá)譜。在6個(gè)上調(diào)的microRNA中，抑制任意一個(gè)的活性均可以抑制MSC向肝細(xì)胞分化，而單純過(guò)表達(dá)某一種microRNA并不能啟動(dòng)MSC向肝細(xì)胞分化。同時(shí)過(guò)表達(dá)時(shí)，可以促使MSC轉(zhuǎn)化為在體內(nèi)外均具有功能的肝細(xì)胞。本研究首次系統(tǒng)篩選了肝向分化特異性的microRNA譜并詳細(xì)探討了其在肝向分化中的作用。首次證實(shí)7-microRNA組合可以使MSC直接轉(zhuǎn)化為有功能的肝細(xì)胞。為肝向分化機(jī)制理解提供了一定的理論支持。
[Abstract]:[background]
The incidence of end-stage liver disease in China is increasing year by year. The annual mortality rate is 30-50%. in situ liver transplantation is the only effective means to treat end-stage liver disease. However, the lack of donor liver is the bottleneck of orthotopic liver transplantation. In addition, the orthotopic liver transplantation is expensive, the long-term use of immunosuppressive agents should be used after operation, and the perioperative risk is great. These problems greatly restrict the wide application of orthotopic liver transplantation.
Adult stem cells can break through the restriction of the germ layer and differentiate into functional hepatocytes in vivo and in vivo. Therefore, adult stem cell transplantation has become a new technique for the treatment of end-stage liver disease. However, the mechanism of its differentiation to hepatocytes is still unclear. The traditional induction method is complex, the induction cycle is long, the differentiation efficiency is low, and the dry fine is limited. Cell therapy is widely popularized and applied in clinical practice.
MicroRNA is a short endogenous non coding RNA, which is involved in post transcriptional regulation by binding to the binding site of the 3 'non coding region of the target gene mRNA. Previous studies suggest that microRNA regulation gene expression does not play the role of switching, but plays the role of variable resistors. In recent years, the spatial and temporal distribution of microRNA has been discovered. Rules make microRNA play an important role in the development of body and cell differentiation. The use of specific microRNA combinations can mediate cells from a terminal differentiation to another terminal differentiation state. Although the literature has reported the importance of microRNA in the development of the liver, it is in stem cells. The role of liver differentiation in the process of liver differentiation is not clear.
[Objective]
The purpose of this study is to screen the specific microRNA expression profiles of adult stem cell liver differentiation process, and to explore the role of key microRNA molecules in liver differentiation, and provide theoretical support for the clarifying mechanism of stem cell differentiation. The main contents of this study include three parts: (1) establishing an in vitro model for differentiation of adult stem cells into hepatocytes. (2) to screen and identify the specific microRNA expression profiles of adult stem cells in the process of liver differentiation; (3) clarify the role of key microRNA molecules in the process of liver differentiation.
[method]
1. the peripheral blood stem cells (mesenchymal stem cell, MSC) were purified by the immunomagnetic bead method. The cell morphology was observed by the electron microscope, the cell immunophenotype was detected by flow cytometry, and the purity and characteristics of the separated stem cells were detected by the induction of osteogenesis and the potential of adipogenic differentiation. After the stem cells were successfully isolated, the hepatocyte differentiation induction culture was given. The expression of specific genes in the hepatocyte was detected by qRT-PCR and cell immunofluorescence methods from the RNA and protein levels, respectively. The specificity of the liver cell specific gene was detected by the PAS staining method, the urea nitrogen detection kit and the LDL uptake test for the detection of the liver cell specificity of MSC. The level of function, thereby verifying whether successful establishment of stem cell hepatocyte differentiation model in vitro.
2. the cells of 7 different time points of stem cell liver differentiation model were selected, RNA was extracted and microRNA chip was detected. According to the difference multiplier, the expression and the change rule, the microRNA molecules were screened by qRT-PCR to verify the results of the chip. The microRNA spectrum of differential expression of qRT-PCR and the result of the chip was consistent with the process of osteogenesis differentiation. The expression changes and their expression changes in hepatoma cells and normal hepatocyte lines were compared to clarify the liver differentiation specificity of the microRNA expression profile.
3. the lentivirus particles including miR-1246, miR-1290, miR-148a, mR-30a, miR-424, miR-542-5p interference sequences were constructed respectively. After infection into MSC, the liver differentiation induction culture was given, and the effects of inhibiting the activity of microRNA on the liver differentiation process were observed respectively. After single transfection or co transfection, qRT-PCR, cell immunofluorescence, PAS staining, urea nitrogen detection and LDL uptake test were used to detect the liver differentiation of MSC and convert it into mature and functional liver cells after exogenous microRNA expression, and then the induced hepatocytes were transplanted into the liver injury mice. By detecting the changes in the liver function of mice, the changes of liver histomorphology and the colonization of human hepatocytes and stem cells in the liver, the liver cells of microRNA induced liver cells can be played in mice.
[results]
1. successfully established adult stem cell hepatocyte differentiation model in vitro.
After the separation of CD34+ cells from peripheral blood, more than 99% of the cells were CD34+ cells, and the cells were round and non wall growth. After four different cultures and amplification, it was found that the amplification efficiency of CD34+ cells in the SFM34+IL3+SCF+GM-SCF culture solution was the highest, which could be expanded by 6 times. But the expanded cells lost the CD34 immunity. Although the SFM34+IL3+SCF+GM-SCF culture medium can amplify CD34 cells efficiently, the expanded CD34 cells lose the characteristics of hematopoietic stem cells.
The MSC separated from the umbilical cord was a typical long cable shaped form and was vortexed. Flow cytometry showed that the isolated cell 88.7% could express the MSC marker molecule CD105, and the cells that did not express the endothelial cell marker molecule CD31 and the hematopoietic stem cell marker CD34. were induced for 14 days after the induction of the osteogenic induction culture, with calcium salts. Deposition. That is, we succeeded in separating MSC. from umbilical cord stroma.
QRT-PCR results showed that after 1 weeks of liver induction, the mRNA expression level of hepatocyte specific gene HNF4 a, ALB and CK18 increased and reached the peak at 26 days. The results of immunofluorescence staining showed that the hepatocyte specific gene AFP, ALB, CK18 protein expression of MSC showed that MSC via the liver to the liver after 14 days of liver induction. 14 days after induction, the.LDL uptake test of purple red glycogen granules showed that when MSC was induced 14 days after liver induction, most cells could take the LDL. urea nitrogen test of DIL markers. The ability of MSC liver to synthesize urea gradually increased after 6 days of induction, and reached the peak at 22 days. That is, we successfully established the umbilical cord between the umbilical cord. A cell model of MSC liver differentiation in vitro.
Screening and identification of 2. liver specific microRNA expression profiles
In this study, 1205 human and 144 human virus related microRNA molecules were detected at 7 different time points of liver differentiation. A total of 61 microRNA molecules were more than 2 times more than 2 times before and after the liver differentiation, and were regularly expressed in 7 time points. On this basis, the difference multiplier and expression content were based on this. A total of 7 up-regulated microRNA, 6 up-regulated microRNA with the highest expression level, and 6 down-regulation of 4 times more than 4 times, were screened. The 10 down-regulated microRNA with the highest expression content showed that.QRT-PCR results showed that microRNA was up to miR-542-5p, miR-148a, miR-1290, miR-424, miR-3. The expression patterns of 0A and miR-1246 are in accordance with the results of the chip. In the down regulation of microRNA, all the other down-regulated microRNA is down down expression, except for miR-3646, but the expression pattern of other microRNA is not consistent with the chip results except miR-146a.
The expression of 6 up-regulated microRNA and 8 downregulated microRNA in the process of osteogenic differentiation showed that the up regulation of microRNA in the process of liver differentiation showed a downward trend in the process of osteogenesis, and the microRNA in the process of liver differentiation was rising in the process of osteogenesis differentiation. The expression level of microRNA in L02 and HepG2 is lower than (higher) in MSC. It shows that the differential expression of microRNA expressed in the process of hepatic differentiation is different from that in the process of osteogenesis, and not in the liver cells or liver cells, but the liver cells, but the liver. Specific to the process of differentiation.
3. the role of key microRNA molecules in MSC liver differentiation
The MSC of uninfected virus and MSC infected with miR-nc-lev increased in the expression level of albumin for 6 days after liver induction, while miR-1246-RNAi-lev, miR-1290-RNAi-lev, miR-148a-RNAi-lev, miR-30a-RNAi-lev, miR-424-RNAi-lev, miR-542-5p-RNAi-lev MSC were induced by liver direction, and the level of albumin was not up. The liver was induced to 12. Day after, group miR-nc-lev, whether or not successfully infected with lentiviruses, can take LDL. and miR-1246-RNAi-lev, miR-1290-RNAi-lev, miR-148a-RNAi-lev, miR-30a-RNAi-lev, miR-424-RNAi-lev, miR-542-5p-RNAi-lev, and LDL uptake only in cells that have not successfully infected the lentivirus. In addition, these lentivirus particles can inhibit HepG2. The expression of specific albumin and G6P in cell hepatocytes.
MiR-122, miR-1246, miR-1290, miR-148a, miR-30a, miR-424 and miR-542-5p can be transfected into MSC respectively, which can improve the level of the corresponding microRNA, but it can not promote the co transfection of MSC high expression albumin.7 microRNA analogue, which can not only increase the content of these 7 kinds, but also promote high expression. The liver specific early, middle and late marker molecules, which do not express liver progenitor cells and pancreatic bile duct epithelial markers, make the cell morphology of MSC transform from interstitial like to epithelioid and have the function of hepatocyte specific. After transplanted into the liver injury mice, it can not only improve the function of liver cells, but also improve the liver function of mice, and can also play a part of MSC. The function of repairing the liver tissue of mice.
[Conclusion]
We successfully obtained the isolation and purification of peripheral blood hematopoietic stem cells and umbilical cord MSC. On the basis of the successful establishment of MSC hepatocyte differentiation in vitro cell model, the microRNA expression profiles of a group of MSC liver differentiation processes were screened and identified by chip and qRT-PCR technology. In the 6 above adjusted microRNA, the inhibitory activity of any one was inhibited. It can inhibit the differentiation of MSC into hepatocytes, and simply overexpression of a certain kind of microRNA can not initiate MSC to differentiate into liver cells. At the same time, overexpression can induce the transformation of MSC into functional liver cells both in vivo and in vivo. This study first systematically screened the specific microRNA spectrum of liver differentiation and discussed in detail its liver differentiation. For the first time, it is confirmed that 7-microRNA combination can directly transform MSC into functional hepatocytes, providing some theoretical support for understanding the mechanism of liver differentiation.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R575.3

【共引文獻(xiàn)】

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