本文關(guān)鍵詞：人羊膜上皮細(xì)胞橫向分化為肝細(xì)胞樣細(xì)胞及脾內(nèi)移植的初步研究　出處：《中南大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人羊膜上皮細(xì)胞 干細(xì)胞 標(biāo)記 人羊膜上皮細(xì)胞 肝細(xì)胞樣細(xì)胞 誘導(dǎo)分化 人羊膜上皮細(xì)胞 移植 肝功能損傷 肝細(xì)胞功能 表達(dá)

【摘要】： 在胚胎發(fā)育過(guò)程中,肝臟的整個(gè)發(fā)育過(guò)程是一個(gè)涉及多基因、多環(huán)節(jié)、多途徑的網(wǎng)絡(luò)調(diào)控過(guò)程。1965年Farber的研究揭開(kāi)了對(duì)于肝細(xì)胞研究的新篇章,他首次提出在肝內(nèi)存在小上皮細(xì)胞,即卵圓細(xì)胞。他的發(fā)現(xiàn)推動(dòng)了肝再生及肝細(xì)胞肝癌發(fā)病機(jī)制的深入研究。目前認(rèn)為肝干細(xì)胞(hepaticstem cell,HSC)來(lái)源于前腸內(nèi)胚層,在胚胎發(fā)育過(guò)程中以未成熟的肝細(xì)胞的形式存在,其形態(tài)與膽管上皮細(xì)胞相似,其生化特征類似于胚胎干細(xì)胞。20世紀(jì)末,大量研究表明,成體干細(xì)胞可以跨越胚層限制分化為肝細(xì)胞。美國(guó)科學(xué)家近年宣布,他們?cè)谘蛩邪l(fā)現(xiàn)了和胚胎干細(xì)胞一樣有效的干細(xì)胞,并在實(shí)驗(yàn)室中使這種干細(xì)胞分化成肌肉、骨頭、脂肪、血管、神經(jīng)和肝臟細(xì)胞。和胚胎干細(xì)胞相比,這種干細(xì)胞更容易分化成各種組織,且不會(huì)形成一種名為畸胎瘤的良性腫瘤。來(lái)源于人廢棄胎盤的羊膜上皮細(xì)胞(human amnioticepithelial cells,hAECs)是這樣一類極具研究潛力的細(xì)胞。目前國(guó)內(nèi)外這方面的研究尚處于起步階段。 本課題對(duì)人羊膜上皮細(xì)胞完成了體外檢測(cè)和鑒定,對(duì)其向肝細(xì)胞樣細(xì)胞體外分化的條件進(jìn)行了摸索和優(yōu)化,在成功誘導(dǎo)生成肝細(xì)胞樣細(xì)胞的基礎(chǔ)上,將誘導(dǎo)獲得的肝細(xì)胞樣細(xì)胞移植入裸小鼠體內(nèi),檢測(cè)了誘導(dǎo)獲得的肝細(xì)胞樣細(xì)胞是否具有生物學(xué)功能。本研究分為3個(gè)部 目的:建立人羊膜上皮細(xì)胞的體外分離方法及培養(yǎng)體系,檢測(cè)人羊膜上皮細(xì)胞的生物學(xué)特性,檢測(cè)人羊膜上皮細(xì)胞的AAV-EGFP感染效率。 方法:使用胰蛋白酶從人羊膜上消化人羊膜上皮細(xì)胞進(jìn)行培養(yǎng),細(xì)胞免疫熒光檢測(cè)胚胎干細(xì)胞表面SSEA-1、SSEA-3、SSEA-4和TRA-1-60、TRA-1-81標(biāo)記;RT-PCR檢測(cè)全能性相關(guān)基因TERF、THY1、LEFTYA、Rex-1、Sox-2、Oct-4和nanog等的表達(dá);腺相關(guān)病毒感染檢測(cè)人羊膜上皮細(xì)胞的AAV-EGFP感染效率。 結(jié)果:人羊膜上皮細(xì)胞表達(dá)SSEA3、SSEA4、TRA-1-60、TRA-1-81等胚胎干細(xì)胞的特異性標(biāo)記;表達(dá)TERF1、THY、EFTYA、Rex-1、SOX2、Oct-4和Nanog等全能性相關(guān)基因;人羊膜上皮細(xì)胞的AAV-GFP感染效率在病毒濃度為10~(-3)pfu/ml時(shí)可達(dá)58.2%±1.3%。 結(jié)論:建立了hAECs的細(xì)胞分離方法及適合hAECs生長(zhǎng)和增殖的體外培養(yǎng)體系;hAECs具干細(xì)胞特征;體外培養(yǎng)的hAECs細(xì)胞可以被AAV-GFP感染,并表達(dá)GFP,且表達(dá)效率達(dá)50%以上。 目的:研究Dex,HGF,IGF等細(xì)胞因子聯(lián)合使用的誘導(dǎo)體系對(duì)hAECs向肝細(xì)胞樣(hepatocyte-like)細(xì)胞誘導(dǎo)分化的影響。 方法:采用Dex,HGF,IGF等細(xì)胞因子聯(lián)合使用方法誘導(dǎo)培養(yǎng)hAECs向肝細(xì)胞樣細(xì)胞分化,誘導(dǎo)周期為兩周。誘導(dǎo)過(guò)程中采用RT-PCR鑒定細(xì)胞ALB、CYP1A1、CYP1A2、IGFR、c-met等肝細(xì)胞相關(guān)關(guān)鍵功能基因的表達(dá)和HNF3、HNF4和C/EBPa三種轉(zhuǎn)錄因子的表達(dá)。流式細(xì)胞術(shù)分析集落細(xì)胞表面標(biāo)記ALB、AFP和CK18的時(shí)程變化;為了優(yōu)化誘導(dǎo)體系,檢測(cè)了Dex、HGF、IGF等細(xì)胞因子的劑量依賴性;比較不同代數(shù)的hAECs在誘導(dǎo)向肝細(xì)胞樣細(xì)胞分化的ALB基因表達(dá)情況。 結(jié)果:在誘導(dǎo)兩周后的hAECs中可以檢測(cè)到ALB、CYP1A1、CYP1A2、IGFR、c-met等肝細(xì)胞相關(guān)關(guān)鍵功能基因的表達(dá),且這些功能基因的表達(dá)呈現(xiàn)逐漸遞增的趨勢(shì)。提示hAECs可能已經(jīng)被成功誘導(dǎo)為肝細(xì)胞樣細(xì)胞。誘導(dǎo)前的hAECs表達(dá)HNF1(一種肝細(xì)胞轉(zhuǎn)錄因子),不表達(dá)HNF3、HNF4和C/EBPa;而誘導(dǎo)后的hAECs不僅可以表達(dá)HNF1,還可以表達(dá)HNF3,HNF4和C/EBPa,并且HNF1的表達(dá)在誘導(dǎo)前后的hAECs中也有所不同,誘導(dǎo)后的hAECs中HNF1的表達(dá)水平明顯高于沒(méi)有經(jīng)過(guò)誘導(dǎo)的hAECs。細(xì)胞表面標(biāo)記檢測(cè)發(fā)現(xiàn):誘導(dǎo)6天時(shí),hAECs主要表達(dá)AFP+,約為15.1±2.1%;隨著誘導(dǎo)時(shí)間的延長(zhǎng),10天時(shí)hAECs表達(dá)AFP+/ALB+,約為6.5±1.4%;而誘導(dǎo)14天時(shí),hAECs基本上只表達(dá)ALB+,為13.9±2.3%。誘導(dǎo)10天時(shí)的hAECs開(kāi)始表達(dá)ALB+/CKl8+;約為2.5±1.4%;誘導(dǎo)14天時(shí),細(xì)胞依然表達(dá)ALB+/CK18+,且這種雙陽(yáng)性的細(xì)胞量有明顯增加,為18.9±3.1%;同時(shí)CK18+細(xì)胞數(shù)并沒(méi)有明顯減少,誘導(dǎo)10天時(shí),CK18+細(xì)胞數(shù)為16.1±1.2%;誘導(dǎo)14天時(shí),CK18+細(xì)胞數(shù)為21.3±4.6%。提示,誘導(dǎo)過(guò)程中的hAECs隨著誘導(dǎo)時(shí)間的延長(zhǎng)有一個(gè)逐漸成熟的過(guò)程。劑量依賴性實(shí)驗(yàn)結(jié)果顯示,IGF、HGF均存在劑量依賴性,而Dex的劑量依賴性實(shí)驗(yàn)結(jié)果不具有統(tǒng)計(jì)學(xué)意義,認(rèn)為不存在劑量依賴性。對(duì)不同代數(shù)的hAECs在誘導(dǎo)向肝細(xì)胞樣細(xì)胞分化的ALB基因表達(dá)情況進(jìn)行比較,結(jié)果顯示,傳代三代的hAECs誘導(dǎo)后均能表達(dá)ALB基因,且在表達(dá)強(qiáng)度上沒(méi)有明顯區(qū)別 結(jié)論:hAECs確實(shí)可以在體外向肝細(xì)胞樣細(xì)胞進(jìn)行誘導(dǎo)分化,并能被成功誘導(dǎo)分化為有功能的肝細(xì)胞樣細(xì)胞;hAECs向肝細(xì)胞樣細(xì)胞的誘導(dǎo)過(guò)程中,對(duì)于HGF和IGF具有劑量依賴性,而對(duì)于Dex的這種劑量依賴性并不明顯;傳代三代內(nèi)的hAECs誘導(dǎo)后,在肝細(xì)胞樣細(xì)胞特異性功能基因ALB的表達(dá)上沒(méi)有明顯區(qū)別。 目的:觀察人羊膜上皮細(xì)胞(hAECs)在肝臟受損裸鼠脾內(nèi)移植后是否具有肝細(xì)胞樣功能的表達(dá)并探討其治療肝功能損傷的可行性。 方法: 40只裸小鼠,隨機(jī)分為三組,A組:20只,行2/3肝葉切除后,自脾下極移植5×10~6綠色熒光蛋白標(biāo)記的hAECs;B組:10只,不行肝葉切除,自脾下極移植5×10~6綠色熒光蛋白標(biāo)記的hAECs;C組:10只,行2/3肝葉切除后,自脾下極注射生理鹽水0.2 mL。熒光顯微鏡hAECs體內(nèi)示蹤并觀察其是否表達(dá)肝細(xì)胞特異性蛋白;術(shù)后2周A、B組各10只裸小鼠采血定量檢測(cè)裸鼠血中人白蛋白及肝功能,術(shù)后4周A組另10只及C組10只裸小鼠做以上相同檢測(cè)。 結(jié)果: A組鏡下肝臟和脾臟組織中均發(fā)現(xiàn)綠色熒光信號(hào)的hAECs,大部分表達(dá)人白蛋白;B、C組鏡下均未見(jiàn)綠色熒光細(xì)胞。A組血中可檢測(cè)到人白蛋白且術(shù)后4周明顯高于術(shù)后2(3.9±1.34vs0.37±0.14,P0.01);術(shù)后4周A組肝功能(ALB、ALT、AST)明顯優(yōu)于C組(p0.05),A組肝功能術(shù)后4周亦優(yōu)于術(shù)后2周(p＜0.05)。 結(jié)論:肝損傷可以有效誘導(dǎo)hAECs向肝遷移增殖,大部分hAECs在肝細(xì)胞損傷的微環(huán)境下有肝細(xì)胞樣功能表達(dá),hAECs移植可以在一定程度上修復(fù)受損肝功能。
[Abstract]:In the process of embryonic development, the development of the whole process of the liver is a multi gene, multi link, network control process.1965 Farber multi way opened a new chapter for stem cell research, he first proposed the existence of small epithelial cells in hepatic oval cells. Namely, promote the research of his discovery the pathogenesis of liver regeneration and hepatic cell carcinoma. It is believed that liver stem cells (hepaticstem cell, HSC) from the foregut endoderm, in immature liver cells form during embryonic development, the morphology and the bile duct epithelial cells like, its biochemical characteristics similar to embryonic stem cells.20 at the end of the century, a large number of studies show that that adult stem cells can differentiate into liver cells across the ectoderm limits. American scientists announced in recent years, they found the same and embryonic stem cells and effective stem cells in amniotic fluid, and that this in lab The differentiation of stem cells into muscle, bone, fat, blood vessel, nerve and liver cells. Compared with embryonic stem cells, the stem cells differentiate into various organizations more easily, and does not form a benign teratoma. From the abandoned placenta amniotic epithelial cells (human amnioticepithelial cells, hAECs) is such a kind of great research potential of cells. At present domestic and international research in this area is still in its infancy.
This subject has completed the in vitro detection and identification of human amniotic epithelial cells, has carried on the exploration and optimization into hepatocyte like cells in vitro differentiation conditions, based on the generation of hepatocyte like cells induced by success, will be obtained by the hepatocyte like cells transplanted into nude mice, detected the induced hepatocyte like cells whether it has obtained biological function. This research is divided into 3 parts
Objective: to establish an in vitro isolation method and culture system for human amniotic epithelial cells, to detect the biological characteristics of human amniotic epithelial cells, and to detect the AAV-EGFP infection efficiency of human amniotic epithelial cells.
Methods: using trypsin digestion of human amniotic epithelial cells were cultured from human amniotic cells, immunofluorescence of embryonic stem cell surface SSEA-1, SSEA-3, SSEA-4 and TRA-1-60, TRA-1-81; RT-PCR detection of pluripotency related genes TERF, THY1, LEFTYA, Rex-1, Sox-2, Oct-4 and Nanog expression; adeno-associated virus infection detection human amniotic epithelial cells AAV-EGFP infection efficiency.
Results: the expression of SSEA3 in human amniotic epithelial cells, SSEA4, TRA-1-60, TRA-1-81 and other specific markers of embryonic stem cells; the expression of TERF1, THY, EFTYA, Rex-1, SOX2, Oct-4 and Nanog of pluripotency related genes; human amniotic epithelial cells infected by AAV-GFP virus in the efficiency of concentration of 10~ (-3) pfu/ml up to 58.2%. 1.3%.
Conclusion: a cell isolation method for hAECs and an in vitro culture system suitable for hAECs growth and proliferation were established. HAECs has the characteristics of stem cells, and hAECs cells cultured in vitro can be infected by AAV-GFP and express GFP, and the expression efficiency is over 50%.
Objective: To study the effect of the combined use of Dex, HGF, IGF and other cytokines on the induction of differentiation of hAECs to hepatocyte like (hepatocyte-like) cells.
Methods: Dex, HGF, IGF and other cytokines induced by cultured hAECs into hepatocyte like cell differentiation, induction period of two weeks. The identification of RT-PCR ALB cells during induction of CYP1A1, CYP1A2, IGFR, HNF3 and the expression of related key functions such as c-met gene, the expression of HNF4 and C/EBPa three transcription factor. Flow cytometry analysis of colony cell surface markers ALB, AFP and CK18 of the time course of changes; in order to optimize the induction system, detection of Dex, HGF, IGF and other cytokines dose dependent; ALB gene expression comparison of different passages of hAECs cells into hepatocyte like cells in differentiation.
Results: at two weeks after the induction of hAECs was detected in ALB, CYP1A1, CYP1A2, IGFR, c-Met and other related key functional expression of liver cell gene, and the expression of these genes showed a gradual increasing trend. Indicating that hAECs may have been successfully induced into hepatocyte like cells. The expression of HNF1 (hAECs before induction a liver cell transcription factor), the expression of HNF3, HNF4 and C/EBPa; and after the induction of hAECs can not only express HNF1 can also express HNF3, HNF4 and C/EBPa, and the expression of HNF1 in hAECs before and after induction in different levels of HNF1 expression induced by hAECs was significantly higher than that without hAECs. cells surface markers detected by discovery: after 6 days induction, the expression of hAECs AFP+, about 15.1 + 2.1%; with the induction time, the expression of hAECs AFP+/ALB+ in 10 days, about 6.5 + 1.4%; and after 14 days, basically only the expression of ALB+ hAECs, 13.9 + 2.3%. induced 10 days when hAECs began to express ALB+/CKl8+; about 2.5 + 1.4%; after 14 days, cells still express ALB+/CK18+, and the double positive cells were significantly increased, 18.9 + 3.1%; at the same time, the number of CK18+ cells was not significantly reduced, by 10 days, the cell number was CK18+ 16.1 + 1.2%; after 14 days, the number of CK18+ cells was 21.3 + 4.6%., induced by the process of hAECs with the induction time is a mature process. Dose dependent experimental results showed that IGF dose dependently there were HGF, Dex and the dose dependence of experimental results was not statistically meaning that there is no dose dependence. The expression of ALB gene of different generations of the hAECs cells into hepatocyte like cells in differentiation were compared, the results showed that three generations after the induction of hAECs could express ALB gene, and the expression intensity in no obvious area other
Conclusion: hAECs can differentiate into hepatocyte like cells in vitro, and can be induced successfully for functional hepatocyte like cells; hAECs to induction of hepatocyte like cells, in a dose dependent manner for HGF and IGF, and the dose dependence of Dex is not obvious after three; the generation of the hAECs after the induction, there were no significant differences in the expression of hepatocyte like cells specific gene ALB.
Objective: To observe whether human amniotic epithelial cells (hAECs) have hepatocyte like function after liver transplantation in nude mice, and to explore the feasibility of treatment of liver function damage.
Methods: 40 mice were randomly divided into three groups, group A: 20, 2/3 after liver resection, with splenic transplantation 5 x 10~6 green fluorescent protein tagged hAECs; group B: 10, did not receive hepatectomy, with splenic transplantation 5 x 10~6 green fluorescent protein hAECs; group C: 10, 2/3 after liver resection, with splenic injection of saline 0.2 mL. fluorescence microscope hAECs tracing in vivo and to observe the expression of hepatocyte specific protein; after 2 weeks, A, albumin and liver function in B group of 10 mice were detected in blood of nude mice people, after 4 weeks, the other group A 10 and group C 10 nude mice to do more of the same.
Results: the green fluorescence signals of hAECs were found in A group with liver and spleen tissues, most expression of human albumin; B, C group under the microscope showed no green fluorescent cells in group.A blood can be detected in human albumin and after 4 weeks after transplantation was significantly higher than 2 (3.9 + 1.34vs0.37 + 0.14, P0.01); the 4 week A group of postoperative liver function (ALB, ALT, AST) was significantly higher than that of C group (P0.05), after 2 weeks of liver function in A group 4 weeks after surgery was superior to surgery (P < 0.05).
Conclusion: liver injury can effectively induce hAECs to migrate to liver, and most of hAECs has hepatocyte like expression in the microenvironment of liver injury. HAECs transplantation can repair damaged liver function to some extent.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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