本文關(guān)鍵詞： 人骨髓間充質(zhì)干細(xì)胞 生物學(xué)特性 細(xì)胞培養(yǎng) 細(xì)胞表面標(biāo)志物 骨髓間充質(zhì)干細(xì)胞 肝細(xì)胞生長因子 表皮細(xì)胞生長因子 細(xì)胞分化 肝樣細(xì)胞　出處：《南方醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 肝臟疾病在我國屬臨床常見病、多發(fā)病,嚴(yán)重地危害人類的健康。大量肝臟疾病患者由于缺乏理想的、有效的治療手段而死亡。肝細(xì)胞移植是治療終末期肝功能衰竭的有效方法之一,卻面臨細(xì)胞來源缺乏、體外難以大量增殖、傳代后無法保持其原有特性、免疫排斥等眾多因素的限制,因此尋找新的肝細(xì)胞來源尤為迫切。而人骨髓間充質(zhì)干細(xì)胞(human bone marrow mesenchymal stem cells, HMSCs)是一類具有自我增殖和分化潛能的多能干細(xì)胞,因其具有可塑性、取材方便、低免疫原性以及易于外源基因的轉(zhuǎn)染和表達(dá)等優(yōu)點(diǎn)而被廣泛用于肝臟疾病的研究。近年研究表明,在適宜的微環(huán)境下,HMSCs具有向肝樣細(xì)胞分化的潛能,使其在肝細(xì)胞治療的臨床應(yīng)用中越來越受到人們的重視,也為臨床上應(yīng)用HMSCs移植治療肝損傷和急慢性肝功能衰竭展示了一個可喜的前景。當(dāng)前,HMSCs移植被認(rèn)為有可能替代原位肝移植(orthotopic liver transplantation, OLT),為肝衰竭的有效治療帶來新的希望。因此,本實(shí)驗(yàn)主要研究HMSCs在體外定向誘導(dǎo)分化為肝樣細(xì)胞的能力,實(shí)驗(yàn)內(nèi)容大致分為兩部分：第一部分主要通過研究HMSCs的生物學(xué)特性,建立分離培養(yǎng)和擴(kuò)增細(xì)胞的方法,為以后的研究提供足夠的細(xì)胞來源；第二部分主要研究利用肝細(xì)胞生長因子(hepatocyte growth factor, HGF)、表皮細(xì)胞生長因子(epidermal growth factor, EGF)以及HGF+EGF聯(lián)合誘導(dǎo)HMSCs向肝細(xì)胞方向分化,并采用免疫細(xì)胞化學(xué)染色、逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcription-polymerase chain reaction, RT-PCR)及酶聯(lián)免疫吸附法(ELISA)等技術(shù)鑒定細(xì)胞分化能力,試圖為自體骨髓間充質(zhì)干細(xì)胞移植治療終末期肝病的臨床應(yīng)用提供理論依據(jù)。 第一部分人骨髓間充質(zhì)干細(xì)胞的生物學(xué)特性 目的建立HMSCs體外分離培養(yǎng)和擴(kuò)增的方法,研究HMSCs的生物學(xué)特性,為利用HMSCs作為種子細(xì)胞治療失代償性肝硬化等終末期肝病提供實(shí)驗(yàn)基礎(chǔ)。 方法采用密度梯度離心聯(lián)合貼壁篩選法分離培養(yǎng)HMSCs,測定其接種貼壁率；在倒置顯微鏡下連續(xù)觀察細(xì)胞的形態(tài)變化；并應(yīng)用流式細(xì)胞儀測定細(xì)胞表面標(biāo)記物鑒定所培養(yǎng)的細(xì)胞。 結(jié)果HMSCs體外培養(yǎng)生長狀況良好,具有活躍的增殖能力,接種12h后細(xì)胞貼壁率達(dá)95%以上,細(xì)胞外觀呈紡錘形、長梭形和多角形等,第4 d細(xì)胞數(shù)量開始增多,以克隆集落方式增殖,細(xì)胞形態(tài)呈長梭形,細(xì)胞排列成漩渦狀,細(xì)胞間界限不清,10-14d鋪滿瓶底；培養(yǎng)的HMSCs陽性表達(dá)β1-整合素CD29(99.4%)和基質(zhì)受體CD44(97.2%),陰性表達(dá)造血細(xì)胞標(biāo)志CD34(4.7%),CD45(5.6%)。 結(jié)論HMSCs是區(qū)別于人骨髓中造血干細(xì)胞的另一類細(xì)胞,通過密度梯度離心聯(lián)合貼壁篩選法可在體外培養(yǎng)出高純度的HMSCs,獲得的HMSCs在體外生長穩(wěn)定,且增殖較快,可作為組織工程的種子細(xì)胞。 第二部分體外誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞向肝樣細(xì)胞分化 目的探討HGF和EGF體外誘導(dǎo)HMSCs向肝樣細(xì)胞分化的可行性,為生物人工肝及肝細(xì)胞移植尋找理想的種子細(xì)胞。 方法取食管癌手術(shù)患者肋骨,采用密度梯度離心聯(lián)合貼壁篩選法獲取HMSCs。取第3代HMSCs,分為4組,肝細(xì)胞生長因子組(A組)加入20μg/L HGF,表皮細(xì)胞生長因子組(B組)加入20μg/L EGF,聯(lián)合組(C組)同時加入20μg/LHGF和20μg/L EGF,空白對照組(D組)不加任何生長因子。倒置顯微鏡下觀察細(xì)胞形態(tài)的變化,在誘導(dǎo)7、14d RT-PCR檢測甲胎蛋白(alpha fetoprotein,AFP)和白蛋白(albumin, ALB) mRNA的表達(dá),誘導(dǎo)7、14、21、28d免疫細(xì)胞化學(xué)染色檢測AFP,細(xì)胞角蛋白18 (cytokeratin 18, CK18)及誘導(dǎo)7、14、21dELISA檢測培養(yǎng)上清液中ALB水平。 結(jié)果HGF組、EGF組、聯(lián)合組HMSCs誘導(dǎo)后向肝樣細(xì)胞轉(zhuǎn)化,細(xì)胞形態(tài)由長梭形變?yōu)轭悎A形或多角形,誘導(dǎo)第7,14d AFP、ALB mRNA均呈陽性表達(dá)；空白對照組未見多角形細(xì)胞,AFP、ALB mRNA均呈陰性表達(dá)；免疫細(xì)胞化學(xué)染色各誘導(dǎo)組均可檢測出AFP和CK18表達(dá),并以時間依賴方式產(chǎn)生ALB,且細(xì)胞上清液ALB的含量在A、B、C三組間的差異有顯著性意義(F=9160.446,P=0.000),而空白對照組則沒有檢測到上述指標(biāo)。 結(jié)論HGF、EGF以及二者聯(lián)合均具有誘導(dǎo)HMSCs向肝樣細(xì)胞分化的能力,以HGF+EGF誘導(dǎo)HMSCs分化為肝樣細(xì)胞的陽性率最高,兩者具有一定程度的聯(lián)合作用。 通過以上實(shí)驗(yàn)研究,我們掌握了HMSCs的體外分離培養(yǎng)和擴(kuò)增的方法,證明了HMSCs具有干細(xì)胞自我更新和強(qiáng)大復(fù)制能力的生物學(xué)特性,具備種子細(xì)胞的先決條件；而經(jīng)過HGF和EGF體外誘導(dǎo)后的HMSCs不但在形態(tài)上轉(zhuǎn)變?yōu)楦螛蛹?xì)胞,而且還能分泌肝細(xì)胞特性蛋白,如AFP和ALB,因此,我們初步認(rèn)為HMSCs具有向肝細(xì)胞分化的能力,將HMSCs用于細(xì)胞移植治療,具有非常重要的理論研究意義和臨床價值。
[Abstract]:Liver disease in China is a common clinical disease, disease, serious harm to human health. A large number of patients with liver disease due to the lack of ideal, effective treatment and death. Liver cell transplantation is one of effective methods for the treatment of end-stage liver failure, are lack of in vitro cell source, to proliferate, cannot keep its original characteristics after passage, many factors such as immune rejection, so to find a new source of liver cells is particularly urgent. But the human bone marrow mesenchymal stem cells (human bone marrow mesenchymal stem cells, HMSCs) is a kind of self proliferation and differentiation of pluripotent stem cells, because of its high plasticity material convenient, low immunogenicity and easy exogenous gene transfection and expression has been used extensively in the study of liver diseases. Recent studies show that in a suitable micro environment, HMSCs has to hepatocyte like cells Cell differentiation potential, its clinical application in liver cell therapy has been paid more attention, but also for the clinical application of HMSCs transplantation for the treatment of liver injury and chronic liver failure shows a promising prospect. At present, HMSCs transplantation is considered a possible alternative to orthotopic liver transplantation (orthotopic liver transplantation, OLT), bring new hope for the effective treatment of liver failure. Therefore, the experimental research of HMSCs in the differentiation into hepatocyte like cells, the experiment is mainly divided into two parts: the first part mainly through the study on the biological characteristics of HMSCs, a method for separation and cultivation and expansion of cells, provide enough cell sources for the future research; the second part mainly studies the use of hepatocyte growth factor (hepatocyte growth, factor, HGF), epidermal growth factor (epidermal growth, factor, and EGF) HGF+EGF induced HMSCs differentiation to hepatocytes and direction by immunocytochemical staining, reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) and enzyme-linked immunosorbent assay (ELISA) technique to identify cell differentiation ability to autologous bone marrow mesenchymal stem cell transplantation provides a theoretical basis for the clinical application of end-stage liver disease.
The biological characteristics of human bone marrow mesenchymal stem cells
Objective to establish a method for isolation, culture and amplification of HMSCs, and to study the biological characteristics of HMSCs, so as to provide experimental basis for the use of HMSCs as seed cell in the treatment of decompensated cirrhosis and other end-stage liver diseases.
Methods density gradient centrifugation combined with adherence screening method was used to isolate and culture HMSCs. The rate of inoculation adherence was detected. Morphological changes of cells were observed continuously under inverted microscope, and cell surface markers were used to identify the cultured cells.
Results HMSCs in vitro growth in good condition, with active proliferation, after inoculation with 12h cells adherent rate reached more than 95%, the appearance of cells were spindle, spindle and polygon, the number of fourth D cells began to increase, in order to clone colony proliferation, cell morphology of fusiform cells arranged in a spiral shape and between the cells is not clear, 10-14d covered the bottom of the bottle; the culture of HMSCs positive expression of 1- integrin beta CD29 (99.4%) and CD44 (97.2%), matrix receptor negative hematopoietic cell marker CD34 (4.7%), CD45 (5.6%).
Conclusion HMSCs is another kind of cell which is different from human bone marrow hematopoietic stem cells. HMSCs can be cultured in vitro by density gradient centrifugation and adhesion screening. The HMSCs obtained in vitro is stable and fast growing, and can be used as a seeding cell for tissue engineering.
Second part induced differentiation of human bone marrow mesenchymal stem cells into hepatocyte like cells in vitro
Objective to explore the feasibility of inducing HMSCs differentiation into hepatocyte like cells by HGF and EGF in vitro, and to search for ideal seed cells for the transplantation of bioartificial liver and hepatocytes.
Methods rib in patients with esophageal cancer surgery by density gradient centrifugation and adherence screening method of HMSCs. and the third generation of HMSCs, divided into 4 groups, hepatocyte growth factor group (group A) with 20 g/L HGF, epidermal growth factor group (group B) with 20 g/L EGF group (C group at the same time) with 20 g/LHGF and 20 g/L EGF, blank control group (D group) without any growth factors. Morphological changes were observed under the inverted microscope, in 7,14d induced by RT-PCR detection of alpha fetoprotein (alpha fetoprotein, AFP) and albumin (albumin, ALB) mRNA expression induced by 7,14,21,28d immunocytochemistry staining for AFP, cytokeratin 18 (cytokeratin 18, CK18) and 7,14,21dELISA induced by detection of ALB level in the culture medium.
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