發(fā)布時(shí)間：2018-05-25 15:27

  本文選題：外周血單個(gè)核細(xì)胞 + 肝細(xì)胞��； 參考：《大連醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 干細(xì)胞是一類具有無限或永生的自我更新能力的細(xì)胞,能產(chǎn)生至少一種類型的、高度分化的子代細(xì)胞。在個(gè)體發(fā)育的不同階段及成體不同組織中均存在著干細(xì)胞,來源于各種組織的成體干細(xì)胞在適當(dāng)?shù)奈h(huán)境中能分化為其他類型的細(xì)胞,即成體干細(xì)胞具有可塑性。 人外周血單個(gè)核細(xì)胞包括單核細(xì)胞、干細(xì)胞和淋巴細(xì)胞,通過密度梯度離心法,收集外周血單個(gè)核細(xì)胞,利用單核細(xì)胞可粘著于塑料培養(yǎng)器皿表面的特性,可去除未貼壁細(xì)胞,進(jìn)一步純化單核細(xì)胞。巨噬細(xì)胞集落刺激因子(macrophage colony stimulating factor,M-CSF)可促進(jìn)單核細(xì)胞增殖分化,白細(xì)胞介素-3(interleukin-3,IL-3)可促進(jìn)單核細(xì)胞分化及多能干細(xì)胞自我更新。這兩種因子的聯(lián)合應(yīng)用對(duì)單核細(xì)胞的可塑性具有協(xié)同作用,可使外周血單核細(xì)胞產(chǎn)生部分未分化,具有重新分化能力并對(duì)刺激它們終末分化的因子反應(yīng)下調(diào),使這些單核細(xì)胞來源的細(xì)胞被誘導(dǎo)分化為肝樣細(xì)胞成為可能。 細(xì)胞的分化和再生受細(xì)胞因子或生長(zhǎng)因子的控制,在大多數(shù)的研究中,肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor,HGF)、表皮生長(zhǎng)因子(epidermal growth factor, EGF)、轉(zhuǎn)化生長(zhǎng)因子(transforming growth factor,TGF)、成纖維細(xì)胞生長(zhǎng)因子(fibroblast growth factor,FGF)應(yīng)用最多。曾有報(bào)道,在研究多潛能人祖細(xì)胞(hMAPCs)轉(zhuǎn)分化實(shí)驗(yàn)時(shí),分析比較了多種細(xì)胞因子的作用,如FGF、白血病抑制因子(leukaemia inhibitory factor,LIF)、HGF、制瘤素M(oncostatin M,OSM)、EGF等,得出在HGF和FGF-4存在的情況下分化為肝樣細(xì)胞的培養(yǎng)條件較好。 目前對(duì)急性或晚期肝病引起的肝功能衰竭,最佳治療方法是肝移植,原位肝移植由于供體器官緊缺,其應(yīng)用受到限制。采用細(xì)胞移植技術(shù)來修復(fù)、治療終末期肝病患者,幫助患者度過危險(xiǎn)期等待肝移植或者直接修復(fù)衰竭肝臟,是一個(gè)理想的方法,因此尋找合適的肝細(xì)胞來源備受關(guān)注。外周血有收集方便、供體不需要麻醉、無骨髓穿刺痛苦、易于患者接受等優(yōu)點(diǎn),因此用外周血干細(xì)胞移植的方法來獲得轉(zhuǎn)分化后的肝樣細(xì)胞,其臨床應(yīng)用前景非常突出。 目的:探索人外周血單個(gè)核細(xì)胞在HGF及FGF-4誘導(dǎo)下分化為肝樣細(xì)胞的可行性。 方法: 1.采集正常健康志愿獻(xiàn)血者的外周血,密度梯度離心法分離外周血單個(gè)核細(xì)胞。 2.用RPMI1640培養(yǎng)液對(duì)外周血單個(gè)核細(xì)胞培養(yǎng)2小時(shí)后,去除未貼壁細(xì)胞。 3.在含10%胎牛血清、140uM ?-巰基乙醇、5ng/ml M-CSF、0.4ng/ml IL-3的RPMI1640培養(yǎng)液中對(duì)貼壁細(xì)胞培養(yǎng)6天。 4.6天后,用含10%胎牛血清、20ng/ml HGF、10ng/ml FGF-4的RPMI1640培養(yǎng)液繼續(xù)誘導(dǎo)培養(yǎng)20天,即對(duì)外周血單個(gè)核細(xì)胞總共培養(yǎng)約26天。 5.觀察細(xì)胞培養(yǎng)過程中的形態(tài)變化,加入HGF、FGF-4誘導(dǎo)前及誘導(dǎo)后的第6、10、14、20天采用免疫熒光法檢測(cè)肝細(xì)胞標(biāo)志物甲胎蛋白(α-fetoprotein,AFP)、白蛋白(albumin,ALB)的表達(dá)情況。 結(jié)果: 1.剛分離的單個(gè)核細(xì)胞形態(tài)上呈較為均一的圓形,臺(tái)盼蘭染色法測(cè)定活細(xì)胞率 95 %,培養(yǎng)2小時(shí)后去除未貼壁細(xì)胞,貼壁細(xì)胞形態(tài)呈毛毛糙糙的圓形。 2.在M-CSF、IL-3存在的條件下,對(duì)貼壁細(xì)胞培養(yǎng)6天后,可見細(xì)胞呈集落樣生長(zhǎng),細(xì)胞趨向融合。未用M-CSF、IL-3培養(yǎng)或單用M-CSF培養(yǎng)的對(duì)照組細(xì)胞呈單個(gè)細(xì)胞生長(zhǎng),隨培養(yǎng)時(shí)間延長(zhǎng),貼壁伸展,培養(yǎng)2周后大部分呈纖維樣或梭形,少部分呈圓形。 3.加入HGF、FGF-4培養(yǎng)后4天左右,可見部分細(xì)胞體積變大,向類圓形細(xì)胞轉(zhuǎn)變,隨培養(yǎng)時(shí)間延長(zhǎng),類圓形細(xì)胞比例增加。培養(yǎng)20天后,細(xì)胞數(shù)量較未加入HGF、FGF-4培養(yǎng)前減少。未加入HGF、FGF-4培養(yǎng)的對(duì)照組細(xì)胞,隨培養(yǎng)時(shí)間的延長(zhǎng),部分細(xì)胞貼壁伸展,培養(yǎng)20天后大部分呈梭形或纖維樣,少部分呈圓形。 4.實(shí)驗(yàn)組加入HGF、FGF-4培養(yǎng)后第6、10、14、20天免疫熒光法檢測(cè)AFP均為陽(yáng)性表達(dá)。實(shí)驗(yàn)組未加入HGF、FGF-4培養(yǎng)前及對(duì)照組于實(shí)驗(yàn)組相應(yīng)檢測(cè)日期免疫熒光法檢測(cè)AFP均為陰性表達(dá)。 5.實(shí)驗(yàn)組加入HGF、FGF-4培養(yǎng)后第10、14、20天免疫熒光法檢測(cè)ALB均為陽(yáng)性表達(dá)。實(shí)驗(yàn)組未加入HGF、FGF-4培養(yǎng)前及對(duì)照組于實(shí)驗(yàn)組相應(yīng)檢測(cè)日期免疫熒光法檢測(cè)ALB均為陰性表達(dá)。 結(jié)論:外周血單個(gè)核細(xì)胞在一定誘導(dǎo)條件下具有向肝樣細(xì)胞分化的潛能,有可能成為干細(xì)胞移植治療肝病的細(xì)胞來源。
[Abstract]:Stem cells are a class of cells with unlimited or permanent self - renewal ability to produce at least one type of differentiated , highly differentiated progeny . Stem cells are present in different stages of ontogeny and in different tissues of the adult body , and adult stem cells derived from various tissues can be differentiated into other types of cells in an appropriate microenvironment , i.e . adult stem cells have plasticity .


Human peripheral blood mononuclear cells include monocytes , stem cells and lymphocytes , and the peripheral blood mononuclear cells are collected by density gradient centrifugation . The monocytes can be adhered to the surface of the plastic culture vessel to remove non - adherent cells and further purify the monocytes . The macrophage colony stimulating factor ( M - CSF ) can promote the differentiation of monocytes and further purify the monocytes . The macrophage colony stimulating factor ( M - CSF ) can promote the differentiation of monocytes , and can be regulated by the factors that stimulate their terminal differentiation , so that the cells derived from these monocytes are induced to differentiate into liver - like cells .


The differentiation and regeneration of cells were controlled by cytokines or growth factors . In most studies , hepatocyte growth factor ( HGF ) , epidermal growth factor ( EGF ) , transforming growth factor ( TGF ) and fibroblast growth factor ( FGF ) were most widely used .


The present invention has the advantages of convenient collection , no need of anesthesia , no bone marrow puncture pain , easy patient acceptance and the like , and the clinical application prospect of the liver - like cell after the transformation is very prominent .


Objective : To explore the feasibility of differentiation of human peripheral blood mononuclear cells into hepatocyte - like cells induced by HGF and FGF - 4 .


Method :


1 . Peripheral blood and density gradient centrifugation of normal healthy volunteers were collected to separate mononuclear cells from peripheral blood .


2 . After 2 - hour incubation with RPMI1640 medium on peripheral blood mononuclear cells , no adherent cells were removed .


3 . The adherent cells were cultured for 6 days in RPMI1640 medium containing 10 % fetal bovine serum , 140 uM ? - mercaptoethanol , 5 ng / ml M - CSF , 0.4 ng / ml IL - 3 .


After 4 . 6 days , the culture was continued for 20 days with RPMI1640 medium containing 10 % fetal bovine serum , 20 ng / ml HGF , 10 ng / ml FGF - 4 , i.e . , for a total of about 26 days for peripheral blood mononuclear cells .


5 . Observe the morphological changes in the cell culture process , add HGF , FGF - 4 induction and the 6th , 10th , 14th , and 20th days after induction , and detect the expression of alpha - fetal protein ( AFP ) , albumin ( albumin , ALB ) by immunofluorescence .


Results :


1 . The morphology of individual nuclear cells was uniform round , trypan blue staining method was used to determine the viable cell rate of 95 % , the unadherent cells were removed after 2 hours of culture , and the morphology of adherent cells was rough .


2 . In the presence of M - CSF and IL - 3 , the cells showed colony - like growth and the cells tended to fuse .


3 . After adding HGF and FGF - 4 for 4 days , the volume of some cells became larger and the proportion of round cells increased . After 20 days of culture , the number of cells was decreased compared with that of the control group without HGF and FGF - 4 .


4 . The positive expression of AFP was detected by immunofluorescence assay at 6 , 10 , 14 and 20 days after the growth of HGF and FGF - 4 in the experimental group .


5 . The expression of ALB was detected by immunofluorescence assay at 10 , 14 and 20 days after the growth of HGF and FGF - 4 in the experimental group .


Conclusion : Peripheral blood mononuclear cells have the potential to differentiate into liver - like cells under certain induction conditions .
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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