本文選題：骨髓間充質干細胞 + 肝細胞�。� 參考：《中國協(xié)和醫(yī)科大學》2010年碩士論文

【摘要】： 研究背景和目的： 經(jīng)過20多年的發(fā)展,肝細胞移植作為肝移植的替代療法,已經(jīng)成為一種治療急、慢性肝功能衰竭和肝臟相關遺傳代謝疾病的有效方法。但是目前肝細胞移植仍面臨許多問題,如肝細胞的來源短缺、移植細胞數(shù)量有限、肝細胞移植后的增殖、受損肝細胞的替代程度及免疫排斥等諸多問題。2002有人報道,BMSC可以在體外誘導成肝細胞。而BMSC以自身獨特優(yōu)點有望使細胞移植在肝病治療方面取得實質性的突破,臨床應用前景突出。 本實驗室已經(jīng)掌握各種方法誘導BMSC向肝細胞分化,并取得了成功。為了臨床應用方便,我們探討了更簡單、可控、成分明確的培養(yǎng)方法。我們用人血清白蛋白代替了FBS,用無血清的培養(yǎng)方法來提供肝細胞移植臨床應用更好的細胞來源。 肝臟生理功能的實現(xiàn)有賴于肝細胞極化的形成和維持。但是,目前我們對肝細胞極化的認識非常有限。肝細胞極化研究在很大程度上受限于缺乏理想的體外肝細胞極化模型。因此我們應用了骨髓間充質干細胞來源的肝細胞,對體外肝細胞的極化進行了研究。以便用骨髓間充質干細胞來源的肝細胞建立良好的體外肝細胞極化模型。 熊去氧膽酸(UDCA)具有利膽、抗肝細胞凋亡和拮抗疏水性膽汁酸的細胞毒性等多種作用,在臨床上用于治療肝膽系統(tǒng)多種疾病。但目前對UDCA細胞內(nèi)作用機制認識有限,UDCA對肝細胞極化的調節(jié)作用尚缺少研究。本研究選擇了SR-B1、MRP2和NTCP三個極化分子進行檢測,來研究UDCA在mRNA水平對肝細胞極化分子表達的調節(jié)作用的作用。 實驗方法： 1.提取成人骨髓間充質干細胞在無血清培養(yǎng)下向肝細胞誘導分化。通過與有血清誘導分化組和非誘導分化組比較,使用光學顯微鏡觀察細胞分化0天、7天、14天、21天的細胞形態(tài)；使用白蛋白生化檢測試劑盒檢測肝細胞特異性標記物ALB的蛋白表達；使用免疫熒光染色,熒光顯微鏡下觀察白蛋白在肝細胞內(nèi)的合成、分布情況,觀察無血清培養(yǎng)誘導的肝細胞是否具有與有血清誘導分化組細胞相似的成熟肝細胞的特征。 2.使用實時定量PCR方法來檢測分化7天、14天、21天和最后7天加用50μmol/L UDCA(21+U組)誘導的肝細胞的MRP2、NTCP、SR-B1三個極化分子的表達；使用免疫熒光染色方法在共聚焦顯微鏡下觀察MRP2、SR-B1在細胞上的定位,以此觀察骨髓間充質干細胞相肝細胞誘導分化過程中MRP2、NTCP、SR-B1的mRNA表達和MRP2、SR-B1的定位情況。 研究結果： 1.成人骨髓間充質干細胞在無血清培養(yǎng)下誘導分化21天后具有成熟肝細胞形態(tài)(無血清誘導分化組,A組),與非誘導分化組(C組)差別明顯,而與有血清的誘導分化組(B組)相近。分別檢測三組細胞的白蛋白表達,在分化7,14,21天的A組和B組白蛋白逐漸升高,14天后維持在較高水平,三個時間點A組和B組白蛋白表達量明顯高于C組(p0.05)。A組和B組細胞在誘導分化14天后,白蛋白免疫熒光染色發(fā)現(xiàn),細胞內(nèi)有大量點狀、顆粒狀或者均勻分布的綠色熒光,較自身未加一抗的對照及C組比較明顯為陽性。 2.實時定量PCR檢測分化7天、14天、21天和最后7天加用50μmol/L UDCA(21+U組)誘導的肝細胞的MRP2、NTCP、SR-B1極化分子的mRNA表達發(fā)現(xiàn),7天、14天、21天和21+U組四組細胞MRP2、NTCP、SR-B1的mRNA表達隨時間增加而升高,14天、21天組明顯高于7天組(p0.05),21+U組明顯高于21天組(p0.05)。免疫熒光染色顯示細胞誘導14天后MRP2、SR-B1兩種極化蛋白分別定位于肝細胞膜的不同部位,呈現(xiàn)明顯的極化特征,而對照組細胞未觀察到特異性分布。 研究結論： 1.無血清條件下培養(yǎng)骨髓間充質干細胞在特定誘導因子下可以分化為有一定功能的肝細胞,與誘導分化培養(yǎng)基誘導的肝細胞類似。從而為肝細胞臨床移植準備了的良好的細胞來源。 2.成人骨髓間充質十細胞具有向極化肝細胞分化的潛能。骨髓問充質十細胞分化的肝細胞可以作為研究肝細胞極化形成和調節(jié)機制的良好模型。UDCA可以促進肝細胞極化分子的表達。
[Abstract]:Research background and purpose:
After more than 20 years of development, hepatocyte transplantation, as a replacement therapy for liver transplantation, has become an effective method for the treatment of acute, chronic liver failure and liver related genetic metabolic diseases. However, hepatocyte transplantation still faces many problems, such as the shortage of liver cells, the limited number of transplanted cells, and the proliferation of liver cells after transplantation. .2002 has been reported that BMSC can induce hepatocytes in vitro, and BMSC is expected to make a substantial breakthrough in the treatment of liver diseases with its own unique advantages, and the prospect of clinical application is prominent.
The laboratory has mastered various methods to induce BMSC to differentiate into hepatocytes and has achieved success. In order to be convenient for clinical application, we have explored a more simple, controllable and well-defined culture method. We use human serum albumin instead of FBS, and use serum-free culture to provide a better cell source for the clinical application of hepatocyte transplantation.
The realization of liver function depends on the formation and maintenance of hepatocyte polarization. However, our understanding of hepatocyte polarization is very limited. The study of hepatocyte polarization is largely limited to the lack of ideal model of hepatocyte polarization in vitro. Therefore, we have applied the liver cells derived from bone marrow mesenchymal stem cells to the liver in vitro. Cell polarization was studied in order to establish a good in vitro hepatocyte polarization model using bone marrow mesenchymal stem cells derived hepatocytes.
Ursodeoxycholic acid (UDCA) has many advantages, such as gallbladder, anti hepatocyte apoptosis, and antagonism to the cytotoxicity of hydrophobic bile acids. It is used in the treatment of various diseases of hepatobiliary system. However, there is limited understanding of the mechanism of intracellular action of UDCA, and the use of UDCA for the regulation of hepatocyte polarization is still lacking. This study chose SR-B1, MRP2 and NTCP. Three polarized molecules were detected to study the role of UDCA in regulating the expression of polarized molecules in hepatocytes at mRNA level.
Experimental methods:
1. the adult bone marrow mesenchymal stem cells were induced to differentiate into hepatocytes in serum-free culture. Compared with the serum induced and non induced differentiation groups, the cell morphology was observed by optical microscope for 0 days, 7 days, 14 days and 21 days by optical microscope, and the liver cell specific marker ALB was detected by the albumin biochemical test kit. The expression of protein, immunofluorescence staining, and fluorescence microscopy were used to observe the synthesis and distribution of albumin in the liver cells, and to observe whether the liver cells induced by serum-free culture have the characteristics of mature hepatocytes similar to those of the cells with serum induced differentiation.
2. using real-time quantitative PCR method to detect the expression of MRP2, NTCP, SR-B1 three molecules in the hepatocytes of 7 days, 14 days, 21 days and the last 7 days with 50 mu UDCA (21+U group), and observe the location of MRP2 and SR-B1 on the cell by using the immunofluorescent staining method under confocal microscope to observe the bone marrow mesenchymal stem cell phase. MRNA expression of MRP2, NTCP, SR-B1 and localization of MRP2 and SR-B1 during hepatocyte differentiation.
The results of the study:
1. adult bone marrow mesenchymal stem cells had mature liver cell morphology (serum free differentiation group, A group) after 21 days of serum-free induction of differentiation (group A), which was significantly different from that of non induced differentiation group (group C), and similar to that of the induced differentiation group (group B) with serum. The expression of albumin in three groups of cells was detected respectively in A and B groups of 7,14,21 days of differentiation. The protein increased gradually and maintained at a higher level in 14 days. The expression of albumin in group A and B was significantly higher than that of group C (P0.05) and group.A and B group for 14 days. Albumin immunofluorescence staining found that there were a large number of dot like, granular or uniformly distributed green fluorescence in the cells, compared with the control and C group that did not add one to one's own resistance and C group. It was obviously positive.
2. real-time quantitative PCR detection of 7 days, 14 days, 21 days and the last 7 days with 50 mol/L UDCA (21+U group) induced MRP2, NTCP, SR-B1 polarizing molecules mRNA expression, 7 days, 14 days, 21 days and 21+U groups of four cells MRP2, NTCP, SR-B1, increased with time, 14 days, 21 days group obviously higher than 7 days group obviously It was significantly higher than the 21 day group (P0.05). Immunofluorescence staining showed that the cells were induced for 14 days after MRP2, and two kinds of SR-B1 polarin were located in different parts of the liver cell membrane, showing obvious polarization characteristics, while the cells in the control group did not observe the specific distribution.
The conclusions are as follows:
1. serum free bone marrow mesenchymal stem cells can differentiate into a certain function of liver cells under specific inducible factors under the condition of serum free conditions, which are similar to those induced by differentiation medium. Thus, a good cell source for the clinical transplantation of hepatocytes is prepared.
2. adult bone marrow mesenchymal cells (ten cells) have the potential to differentiate into polarized hepatocytes. The bone marrow mesenchymal ten cell differentiated hepatocytes can be used as a good model for the study of the formation and regulation mechanism of hepatocyte polarization, which can promote the expression of polarizable molecules in liver cells.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

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