本文選題：間充質(zhì)干細(xì)胞 + 臍帶��； 參考：《昆明醫(yī)學(xué)院》2008年博士論文

【摘要】： 目的: 肝臟損傷后的再生和修復(fù)始終是肝臟疾病研究中重要的課題,應(yīng)用干細(xì)胞對(duì)終末期肝病進(jìn)行細(xì)胞移植治療或是作為轉(zhuǎn)基因治療的細(xì)胞載體是目前研究的熱點(diǎn)之一。但干細(xì)胞的來源始終是困擾進(jìn)一步應(yīng)用的原因。間充質(zhì)干細(xì)胞是成體干細(xì)胞中的一種,分布廣泛,具有成體干細(xì)胞的一切共有特征,并且目前研究證實(shí)其在體外具有很強(qiáng)的免疫抑制能力,其作用對(duì)象涉及了NK細(xì)胞、T淋巴細(xì)胞、B淋巴細(xì)胞和樹突狀細(xì)胞等幾乎所有的免疫細(xì)胞。是目前首選的轉(zhuǎn)基因移植的干細(xì)胞。最近幾年發(fā)現(xiàn)從臍帶基質(zhì)中也可以培育出間充質(zhì)干細(xì)胞(臍帶間充質(zhì)干細(xì)胞,UC-MSCs),這為今后間充質(zhì)干細(xì)胞廣泛的臨床應(yīng)用提供了一個(gè)極為簡(jiǎn)便充足的來源。但目前關(guān)于臍帶間充質(zhì)干細(xì)胞的特性的研究還較少,至今為止尚未見到UC-MSCs分化為肝細(xì)胞的報(bào)道。因此,本研究以UC-MSCs為載體細(xì)胞,攜帶HGF基因到受損的肝臟模型,觀察其對(duì)肝損傷修復(fù)再生的作用。 材料與方法: 1.人臍帶間充質(zhì)干細(xì)胞(UC-MSC)的分離和培養(yǎng)(Isolation and culture of humanUC-MSC) 臍帶取自經(jīng)父母授權(quán)同意的醫(yī)院產(chǎn)科的足月產(chǎn)兒,均在取材后24小時(shí)內(nèi)進(jìn)行處理。首先,在無菌條件下用PBS沖洗去除樣本血液,采用組織塊培養(yǎng)法和酶消化法提取wharton's jelly的間充質(zhì)干細(xì)胞。試驗(yàn)比較不同濃度的血清,不同的取材方法來取得的干細(xì)胞,并進(jìn)行流式細(xì)胞儀檢測(cè)和染色體核型分析。 2.用慢病毒載體的構(gòu)建和肝細(xì)胞生長(zhǎng)因子(HGF)和綠色熒光蛋白(GFP)基因的轉(zhuǎn)染 含人肝細(xì)胞生長(zhǎng)因子cDNA的構(gòu)建復(fù)合物PUC-SRct/HGF的NotⅠ片段被純化并被插入到pcDNA3.1(invitrogen)中,繼而從pcDNA3.1-HGF構(gòu)建體中切除肝細(xì)胞生長(zhǎng)因子的PMEⅠ片段并將其克隆入復(fù)制缺陷慢病毒載體pWPI載體,即pWPI-HGF。載體序列的方向和完整性通過測(cè)序證實(shí)。重組體慢病毒通過磷酸鈣轉(zhuǎn)染法瞬時(shí)轉(zhuǎn)染入293T細(xì)胞中,pWPI和pAPI-HGF的滴定度通過轉(zhuǎn)導(dǎo)的293T細(xì)胞的GFP表達(dá)量來評(píng)估。傳代至第三代的MSC(間充質(zhì)干細(xì)胞)在含4μg/mL硫酸魚精蛋白(Sigma)的MOI 20內(nèi)進(jìn)行轉(zhuǎn)導(dǎo)。間充質(zhì)干細(xì)胞的轉(zhuǎn)導(dǎo)效率通過在熒光顯微鏡(Nikon,Japan)下檢測(cè)GFP的表達(dá)量來評(píng)估。用轉(zhuǎn)染后的第三代細(xì)胞進(jìn)行移植。 3.動(dòng)物模型的建立及間充質(zhì)干細(xì)胞移植治療 制作SD雄性大鼠CCL4急性肝損傷加部分肝切除模型,進(jìn)行細(xì)胞移植試驗(yàn)。大鼠染毒24小時(shí)后行部分肝切除術(shù),切除范圍占全肝比例約35%,術(shù)前眼底采血以備測(cè)肝功。SHAM組施予除肝葉切除以外的類似手術(shù)操作作為陰性對(duì)照。移植治療后血清酶學(xué)檢測(cè)肝功能以及免疫組化檢測(cè)綠色熒光蛋白陽性細(xì)胞的數(shù)目、形態(tài)和分布。western blot檢測(cè)肝組織中肝細(xì)胞生長(zhǎng)因子的表達(dá)情況。RT-PCR方法檢測(cè)肝中肝細(xì)胞生長(zhǎng)因子及相關(guān)基因表達(dá)。 結(jié)論: 1,臍帶基質(zhì)中能夠較容易的獲得豐富的間充質(zhì)干細(xì)胞,其細(xì)胞表面標(biāo)志物與其他組織來源的間充質(zhì)干細(xì)胞大致相同。該種間充質(zhì)干細(xì)胞能為今后臨床間充質(zhì)干細(xì)胞的應(yīng)用提供一條簡(jiǎn)便充足的來源; 2,慢病毒作為載體能夠簡(jiǎn)便有效的轉(zhuǎn)染肝細(xì)胞生長(zhǎng)因子基因到臍帶間充質(zhì)干細(xì)胞并獲得穩(wěn)定的表達(dá); 3,用部分肝切除術(shù)加腹腔單次注射CCL4的方法能夠成功的建立大鼠肝損傷再生的復(fù)合型模型。并且該模型建立方法更簡(jiǎn)單,與臨床情況吻合性更好; 4,將轉(zhuǎn)染了肝細(xì)胞生長(zhǎng)因子基因的臍帶間充質(zhì)干細(xì)胞移植到大鼠肝損傷再生復(fù)合型模型中,臍帶間充質(zhì)干細(xì)胞能夠到達(dá)受損肝臟并表達(dá)肝細(xì)胞生長(zhǎng)因子基因參與到受體肝損傷的修復(fù)和再生過程中,對(duì)肝臟的修復(fù)和再生過程產(chǎn)生有益的影響,其機(jī)理尚需要進(jìn)一步的研究。
[Abstract]:Objective:
Regeneration and repair of liver injury has always been an important issue in the study of liver diseases. Cell transplantation for end-stage liver disease by stem cells is one of the hotspots of current research, but the source of stem cells is always the reason for the further application of stem cells. Mesenchymal stem cells are adult cells. A species of stem cells, widely distributed, has all the common characteristics of adult stem cells and has been proved to have strong immunosuppressive ability in vitro. The targets involved in almost all immune cells, such as NK cells, T lymphocytes, B lymphocytes and dendritic cells, are the first preferred transplants. Cell. Mesenchymal stem cells (umbilical cord mesenchymal stem cells, UC-MSCs) have been found in the umbilical cord matrix in recent years. This provides a very simple and sufficient source for the extensive clinical application of mesenchymal stem cells in the future. However, there are few studies on the characteristics of umbilical cord mesenchymal stem cells. So far, UC- has not yet been seen. MSCs is a report of hepatocyte differentiation. Therefore, this study uses UC-MSCs as the carrier cell, carrying the HGF gene into the damaged liver model, and observing its effect on the repair and regeneration of liver injury.
Materials and methods:
Isolation and culture of 1. human umbilical cord mesenchymal stem cells (UC-MSC) (Isolation and culture of humanUC-MSC)
The full-term birth of the umbilical cord in the obstetrics and Gynecology of the obstetrics and Gynecology which was authorized by their parents by their parents has been processed within 24 hours after taking the material. First, the sample blood was removed by PBS washing under the aseptic condition. The mesenchymal stem cells of wharton's jelly were extracted by tissue mass culture and enzyme digestion. The tests were made to compare the different concentrations of serum and different methods of sampling. Stem cells were obtained and analyzed by flow cytometry and karyotype analysis.
2. construction of lentiviral vector and transfection of hepatocyte growth factor (HGF) and green fluorescent protein (GFP) gene.
The Not I fragment of the complex PUC-SRct/HGF containing human hepatocyte growth factor cDNA was purified and inserted into the pcDNA3.1 (Invitrogen), and then the PME I fragment of the hepatocyte growth factor was removed from the pcDNA3.1-HGF construction and cloned into the replication defective lentivirus vector pWPI vector, that is, the direction and integrity of the pWPI-HGF. vector sequence. It was confirmed by sequencing that the recombinant lentivirus was transiently transfected into 293T cells through calcium phosphate transfection, and the titrations of pWPI and pAPI-HGF were evaluated by the GFP expression of the transduced 293T cells. The generation of MSC (mesenchymal stem cells) to third generations was transduced in MOI 20 of the 4 mu g/mL protamine (Sigma). Mesenchymal stem cells were transduced. The transduction efficiency was assessed by detecting the expression of GFP under Nikon (Japan). The third generation of transfected cells was transplanted.
3. establishment of animal models and transplantation of mesenchymal stem cells
A model of CCL4 acute liver injury and partial hepatectomy in SD male rats was made to carry out a cell transplantation test. The rats were treated with partial hepatectomy after 24 hours of exposure. The resection range accounted for about 35% of the whole liver, and the preoperative fundus blood sampling was used to measure the liver function in group.SHAM. Enzyme detection of liver function and immunohistochemistry detection of the number of green fluorescent protein positive cells, morphology and distribution of.Western blot to detect the expression of hepatocyte growth factor in liver tissue..RT-PCR method was used to detect the expression of hepatocyte growth factor and related genes in liver.
Conclusion:
1, abundant mesenchymal stem cells can be obtained easily in the umbilical cord matrix, and the cell surface markers are roughly the same as those of other tissue derived mesenchymal stem cells. This kind of mesenchymal stem cells can provide a simple and sufficient source for the application of mesenchymal stem cells in the future.
2, lentivirus can be used as a vector to transfect hepatocyte growth factor gene into umbilical cord mesenchymal stem cells effectively and obtain stable expression.
3, the method of partial hepatectomy and single intraperitoneal injection of CCL4 can successfully establish a compound model of rat liver injury regeneration, and the method is more simple and better anastomosed with the clinical situation.
4, the umbilical cord mesenchymal stem cells transfected with the hepatocyte growth factor gene are transplanted into the rat liver regeneration complex model. The umbilical cord mesenchymal stem cells can reach the damaged liver and express the hepatocyte growth factor gene in the repair and regeneration process of the recipient liver injury, which is beneficial to the repair and regeneration of the liver. The mechanism needs further study.

【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 蔣潔;人臍帶沃頓膠間充質(zhì)干細(xì)胞的分離培養(yǎng)及其誘導(dǎo)分化[D];中南大學(xué);2010年

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本文編號(hào)：1795721