【摘要】：目的:目前肝衰竭的治療方法主要是靠細胞替代和肝移植,但由于供肝缺乏,尚存在免疫排斥反應(yīng),因而制約了肝移植的應(yīng)用。骨髓基質(zhì)干細胞(BMSCs)具有容易獲得,可以自我復(fù)制,高度增殖,在特定的條件下可向各胚層來源的細胞分化的特點,因而將BMSCs細胞移植應(yīng)用于肝病治療有非常重要的臨床價值。本研究摸索適合BMSCs生長的培養(yǎng)體系,探索BMSCs誘導(dǎo)分化為肝細胞的適合條件和機制,為臨床上通過BMSCs細胞移植治療肝臟疾病提供理論依據(jù)。 方法:用全骨髓法從大鼠骨髓中分離出BMSCs,觀察BMSCs的生長特性,MTT法比較不同的培養(yǎng)條件對細胞活力的影響,摸索適合BMSCs生長的培養(yǎng)體系;探索在體外誘導(dǎo)BMSCs向成骨細胞、脂肪細胞分化的條件,證實BMSCs的多向分化潛能;將BMSCs在體外傳代擴增后,培養(yǎng)體系中加入肝細胞提取液(G)和胎肝細胞培養(yǎng)上清液(CM),模擬肝細胞體內(nèi)生長的微環(huán)境,與加入肝細胞生長因子(HGF)、神經(jīng)細胞生長因子(NGF)、維甲酸(RA)的誘導(dǎo)方法作比較,觀察分化細胞的形態(tài)結(jié)構(gòu)變化,并采用免疫細胞化學(xué)法檢測肝細胞的特異性標志α_1-抗胰蛋白酶和白蛋白,ICG染色法鑒定肝細胞的生成;將BrdU標記后的BMSCs進行異體移植,免疫組織化學(xué)法檢測BMSCs在肝內(nèi)的遷移和分化,PAS法檢測所生成肝細胞的功能。 結(jié)果:(1) 使用低糖DMEM加入20%新生牛血清比10%新生牛血清MTT細胞活性增高(P0.01),使用高糖DMEM與低糖DMEM差異無顯著性(P0.05),而用RPMI-1640則MTT活性降低(P0.01)。(2) 本實驗中分離的BMSCs在維生素C50μg/ml、地塞米松10~(-7)mol/L、β-甘油磷酸鈉10mmol/L的誘導(dǎo)條件下可以向成骨細胞分化,在地塞米松10~(-8)mol/L、胰島素10μg/ml的誘導(dǎo)條件下可以向脂肪細胞分化。(3) 免疫細胞化學(xué)法結(jié)果顯示G、CM、NGF、HGF、RA誘導(dǎo)4d、14d、21d后與對照組相比α_1-抗胰蛋白酶和白蛋白表達均呈陽性(P0.01),其中G、CM、NGF組AAT、ALB表達強于HGF、RA組(P0.01)。AAT、ALB陽性的細胞形態(tài)上逐漸趨短變圓。(4) 形態(tài)上呈卵圓形的細胞PAS反應(yīng)胞漿呈強陽性,有分泌儲存糖原的功能。(5) 向肝細胞誘導(dǎo)后,卵圓形細胞能攝取ICG,呈現(xiàn)綠色。誘導(dǎo)3w后ICG陽性率比誘導(dǎo)2w高(P0.01)。(6) BMSCs細胞移植后1周、4周損傷組受體肝臟
[Abstract]:Objective: at present, the treatment of liver failure mainly depends on cell substitution and liver transplantation, but due to the lack of donor liver, there is still immune rejection, which restricts the application of liver transplantation. Bone marrow mesenchymal stem cells (BMSCs) have the characteristics of being readily available, self-replicating, highly proliferating, and differentiating into cells from various embryonic layers under certain conditions. Therefore, the transplantation of BMSCs cells into the treatment of liver disease has very important clinical value. This study explored the culture system suitable for the growth of BMSCs, and explored the suitable conditions and mechanism for the differentiation of BMSCs into hepatocytes, which provided a theoretical basis for the clinical treatment of liver diseases by BMSCs cell transplantation. Methods: BMSCs, was isolated from rat bone marrow by whole bone marrow method to observe the growth characteristics of BMSCs. MTT method was used to compare the effect of different culture conditions on cell viability and to explore a suitable culture system for BMSCs growth. To explore the conditions for inducing BMSCs to differentiate into osteoblasts and adipocytes in vitro and to confirm the potential of BMSCs to differentiate into osteoblasts and adipocytes. After BMSCs was subcultured and amplified in vitro, (G) and (CM), were added to the culture system to simulate the microenvironment of hepatocyte growth in vivo, and the addition of hepatocyte growth factor (HGF), to the culture system. The induction methods of (NGF), retinoic acid (RA) were compared to observe the morphological and structural changes of differentiated cells, and the specific markers 偽 _ 1-antitrypsin and albumin of hepatocytes were detected by immunocytochemistry. The formation of hepatocytes was identified by ICG staining. The BMSCs labeled with BrdU was transplanted, the migration and differentiation of BMSCs in liver were detected by immunohistochemistry, and the function of hepatocytes was detected by PAS. Results: (1) the activity of MTT cells in low glucose DMEM added 20% newborn bovine serum was higher than that in 10% newborn bovine serum (P0.01). There was no significant difference between high glucose DMEM and low glucose DMEM (P0.05). However, the BMSCs isolated by RPMI-1640 reduced MTT activity (P0.01). (2) could differentiate into osteoblasts under the condition of vitamin C 50 渭 g / ml and dexamethasone 10 ~ (-7) mol/L, 尾 -glycerophosphate 10mmol/L. When dexamethasone was induced by 10 ~ (-8) mol/L, insulin (10 渭 g/ml), adipocytes could differentiate into adipocytes. The expression of 偽 _ 1-antitrypsin and albumin were positive after 21 days compared with the control group (P0.01), and the expression of AAT,ALB was stronger in the GG CM-NGF group than in the HGF,RA group (P0.01). AAT,). (4) the PAS reaction cytoplasm of the oval cells was strongly positive, and had the function of secreting and storing glycogen. (5) after induced to hepatocytes, the oval cells could absorb ICG,. Green. The positive rate of ICG after 3 weeks of induction was higher than that of 2 weeks of induction (P0.01). (6) BMSCs cells were transplanted at 1 and 4 weeks after transplantation.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R329

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