發(fā)布時間：2018-02-14 12:08

  本文關鍵詞： 骨髓基質干細胞 誘導分化 肝細胞 細胞移植 鼠　出處：《青島大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的：目前認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，制約了肝移植的應用。骨髓基質干細胞(BMSCs)具有易獲得、自我復制能力強、在特定的條件下可向各胚層來源的細胞分化的特點。將BMSCs定向誘導分化成為肝細胞，進行細胞移植，以替代損傷的肝細胞，對肝病治療有非常重要的臨床價值。本實驗探索和篩選誘導BMSCs向肝細胞定向分化為的最佳條件、探索Ca~(2+)在BMSCs向肝細胞定向分化過程中的作用、以及體內移植對肝損傷的治療作用，為臨床上應用BMSCs進行細胞移植治療肝損傷提供實驗依據。 方法：用全骨髓法從大鼠骨髓中分離BMSCs，純化和擴增BMSCs。MTT比色法篩選BMSCs的生長曲線，選擇最佳生長狀態(tài)的BMSCs，培養(yǎng)體系中分別加入肝細胞提取液(G，200μg／ml、500μg/ml)、大鼠和人來源的β-神經生長因子—β-NGF from rat(β-NGF-r，20ng/ml、50ng/ml)、β-NGF from human(β-NGF-h 20ng/ml、50ng/ml)和肝細胞生長因子HGF(50μg/ml)誘導BMSCs向肝細胞分化，通過鏡下觀察分化細胞的形態(tài)、RT-PCR法和免疫細胞化學染色法檢測肝細胞的特異性標志物白蛋白(ALB)和a-抗胰蛋白酶(AAT)、吲哚靛青綠(ICG)攝取實驗鑒定肝樣分化細胞。應用流式細胞技術分別檢測BMSCs和G誘導分化細胞內游離[Ca~(2+)]i。將BrdU標記后的BMSCs異體移植入慢性肝損傷和對照組的小鼠體內，免疫組織化學法檢測BMSCs在肝內的遷移和分化，血清學指標觀察BMSCs移植后對慢性肝損傷的治療作用。 結果：(1)全骨髓分離培養(yǎng)法是純化、培養(yǎng)和擴增BMSCs的簡便有效方法；BMSCs標準生長曲線表明體外培養(yǎng)擴增第3-5代的細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)誘導后，，分化細胞逐漸趨短變圓呈圓形和卵圓形，圓形細胞可以攝取ICG，呈現綠色。免疫細胞化學染色顯示分化細胞呈AAT陽性表達；各誘導組分化細胞的AAT表達顯著性高于對照組(P＜0.05)，其中G、β-NGF組AAT表達強于HGF組(P＜0.05)，G、β-NGF組間無顯著性差異(P＞0.05)，兩種不同來源的β-NGF誘導效果無差異顯著性(P＞0.05)。(3)RT-PCR結果顯示四種誘導劑誘導21d后，分化細胞均表達ALB mRNA。(4)G誘導形成的肝樣分化細胞中[Ca~(2+)i較對照組細胞顯著性升高(P＜0.05)，加入尼莫地平可以阻斷BMSCs向肝細胞的誘導分化，并影響細胞的增殖活性。(5)BMSCs移植后7d、14d、28d損傷組受體肝臟內均可見BrdU標記的移植細胞，移植細胞大部分呈現由血管逐漸向肝實質內遷移的過程，BMSCs移植后20d、40d的慢性肝損傷小鼠血清學指標與對照組有明顯的改善(P＜0.05)。 結論：(1)體外培養(yǎng)BMSCs第3-5代的細胞具有較高的活力。(2)G、β-NGF from rat、β-NGF from human和HGF均可誘導BMSCs向肝細胞分化。(3)Ca~(2+)在BMSCs的增殖和向肝細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：1510672