發(fā)布時(shí)間：2018-02-14 12:08

  本文關(guān)鍵詞： 骨髓基質(zhì)干細(xì)胞 誘導(dǎo)分化 肝細(xì)胞 細(xì)胞移植 鼠　出處：《青島大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的：目前認(rèn)為治療肝功能衰竭的有效方法是肝器官移植，但由于供肝缺乏和免疫排斥反應(yīng)，制約了肝移植的應(yīng)用。骨髓基質(zhì)干細(xì)胞(BMSCs)具有易獲得、自我復(fù)制能力強(qiáng)、在特定的條件下可向各胚層來(lái)源的細(xì)胞分化的特點(diǎn)。將BMSCs定向誘導(dǎo)分化成為肝細(xì)胞，進(jìn)行細(xì)胞移植，以替代損傷的肝細(xì)胞，對(duì)肝病治療有非常重要的臨床價(jià)值。本實(shí)驗(yàn)探索和篩選誘導(dǎo)BMSCs向肝細(xì)胞定向分化為的最佳條件、探索Ca~(2+)在BMSCs向肝細(xì)胞定向分化過程中的作用、以及體內(nèi)移植對(duì)肝損傷的治療作用，為臨床上應(yīng)用BMSCs進(jìn)行細(xì)胞移植治療肝損傷提供實(shí)驗(yàn)依據(jù)。 方法：用全骨髓法從大鼠骨髓中分離BMSCs，純化和擴(kuò)增BMSCs。MTT比色法篩選BMSCs的生長(zhǎng)曲線，選擇最佳生長(zhǎng)狀態(tài)的BMSCs，培養(yǎng)體系中分別加入肝細(xì)胞提取液(G，200μg／ml、500μg/ml)、大鼠和人來(lái)源的β-神經(jīng)生長(zhǎng)因子—β-NGF from rat(β-NGF-r，20ng/ml、50ng/ml)、β-NGF from human(β-NGF-h 20ng/ml、50ng/ml)和肝細(xì)胞生長(zhǎng)因子HGF(50μg/ml)誘導(dǎo)BMSCs向肝細(xì)胞分化，通過鏡下觀察分化細(xì)胞的形態(tài)、RT-PCR法和免疫細(xì)胞化學(xué)染色法檢測(cè)肝細(xì)胞的特異性標(biāo)志物白蛋白(ALB)和a-抗胰蛋白酶(AAT)、吲哚靛青綠(ICG)攝取實(shí)驗(yàn)鑒定肝樣分化細(xì)胞。應(yīng)用流式細(xì)胞技術(shù)分別檢測(cè)BMSCs和G誘導(dǎo)分化細(xì)胞內(nèi)游離[Ca~(2+)]i。將BrdU標(biāo)記后的BMSCs異體移植入慢性肝損傷和對(duì)照組的小鼠體內(nèi)，免疫組織化學(xué)法檢測(cè)BMSCs在肝內(nèi)的遷移和分化，血清學(xué)指標(biāo)觀察BMSCs移植后對(duì)慢性肝損傷的治療作用。 結(jié)果：(1)全骨髓分離培養(yǎng)法是純化、培養(yǎng)和擴(kuò)增BMSCs的簡(jiǎn)便有效方法；BMSCs標(biāo)準(zhǔn)生長(zhǎng)曲線表明體外培養(yǎng)擴(kuò)增第3-5代的細(xì)胞具有較高的增殖活力。(2)添加G(200μg/ml，500μg/ml)、β-NGF from rat(20ng/ml，50ng/ml)、β-NGF from human(20ng/ml，50ng/ml)和HGF(50μg/ml)誘導(dǎo)后，，分化細(xì)胞逐漸趨短變圓呈圓形和卵圓形，圓形細(xì)胞可以攝取ICG，呈現(xiàn)綠色。免疫細(xì)胞化學(xué)染色顯示分化細(xì)胞呈AAT陽(yáng)性表達(dá)；各誘導(dǎo)組分化細(xì)胞的AAT表達(dá)顯著性高于對(duì)照組(P＜0.05)，其中G、β-NGF組AAT表達(dá)強(qiáng)于HGF組(P＜0.05)，G、β-NGF組間無(wú)顯著性差異(P＞0.05)，兩種不同來(lái)源的β-NGF誘導(dǎo)效果無(wú)差異顯著性(P＞0.05)。(3)RT-PCR結(jié)果顯示四種誘導(dǎo)劑誘導(dǎo)21d后，分化細(xì)胞均表達(dá)ALB mRNA。(4)G誘導(dǎo)形成的肝樣分化細(xì)胞中[Ca~(2+)i較對(duì)照組細(xì)胞顯著性升高(P＜0.05)，加入尼莫地平可以阻斷BMSCs向肝細(xì)胞的誘導(dǎo)分化，并影響細(xì)胞的增殖活性。(5)BMSCs移植后7d、14d、28d損傷組受體肝臟內(nèi)均可見BrdU標(biāo)記的移植細(xì)胞，移植細(xì)胞大部分呈現(xiàn)由血管逐漸向肝實(shí)質(zhì)內(nèi)遷移的過程，BMSCs移植后20d、40d的慢性肝損傷小鼠血清學(xué)指標(biāo)與對(duì)照組有明顯的改善(P＜0.05)。 結(jié)論：(1)體外培養(yǎng)BMSCs第3-5代的細(xì)胞具有較高的活力。(2)G、β-NGF from rat、β-NGF from human和HGF均可誘導(dǎo)BMSCs向肝細(xì)胞分化。(3)Ca~(2+)在BMSCs的增殖和向肝細(xì)胞定向分化過程中發(fā)揮重要作用。(4)靜脈移植的BMSCs可遷移至異種動(dòng)物損傷的肝臟，可以改善受損肝臟的功能。
[Abstract]:Objective: the effective method for the treatment of liver failure is liver transplantation, but because of the lack of donor liver and immune rejection, which restricts the application of liver transplantation. Bone marrow stromal stem cells (BMSCs) are easy to obtain, self replication ability, characteristics under certain conditions to the source of the germ cell differentiation the BMSCs directional induced differentiation into liver cells, cell transplantation, to replace the damaged liver cells, has very important clinical value for the treatment of liver diseases. The optimal experimental conditions to explore and screening to induce BMSCs to differentiate into liver cells, to explore the Ca~ (2+) differentiation into hepatocyte function in the process of BMSCs and, in vivo therapeutic effect on liver injury and provide experimental basis for clinical application of BMSCs damage on cell transplantation in the treatment of liver.
Methods: BMSCs were isolated from rat bone marrow by whole bone marrow method, growth curve of purification and amplification of BMSCs.MTT assay BMSCs, select the best growth status of BMSCs culture system were added to the hepatocyte extracts (G, 200 g / ml, 500 g/ml), rat and human derived beta nerve growth factor beta -NGF from rat (beta -NGF-r, 20ng/ml, 50ng/ml), -NGF from human (beta beta -NGF-h, 20ng/ml, 50ng/ml) and hepatocyte growth factor HGF (50 g/ml) to induce BMSCs differentiation into liver cells, observe the differentiation of cell morphology by microscope, RT-PCR assay and immune cell chemical staining method for the detection of liver cell specific markers albumin (ALB) and a- antitrypsin (AAT), indocyanine green (ICG) uptake experiments to identify liver like cells differentiation. BMSCs and G were used to detect the differentiation of intracellular [Ca~ by flow cytometry (2+)]i. BrdU labeled BMSCs after allogeneic implantation In vivo, the migration and differentiation of BMSCs in liver were detected by immunohistochemical method in mice with chronic liver injury and control group. Serological indexes were used to observe the therapeutic effect of BMSCs after transplantation on chronic liver injury.
緇撴灉錛

本文編號(hào)：1510672