本文選題：胎肝間充質干細胞 + 誘導分化　； 參考：《重慶醫(yī)科大學》2009年碩士論文

【摘要】： 研究背景: 冠狀動脈粥樣硬化性心臟病,特別是急性心肌梗死,是嚴重危害人類健康的常見病,多發(fā)病。目前雖然能在發(fā)病4-6小時內通過及時溶栓或介入治療以挽救瀕臨死亡的心肌組織,但由于絕大部分患者就診時缺血心肌已經壞死。此時再灌注治療只能防止梗死面積的擴大,而無法挽救已經壞死的心肌。如何更有效地治療冠心病,成為醫(yī)學研究的焦點。而新近的研究結果,為患者帶來了希望。 傳統(tǒng)觀點認為,心肌細胞出生后即向終末分化,不能夠復制,并且成年心肌組織中沒有儲存的心肌祖細胞,因此心肌受損后心肌細胞不能再生,被疤痕組織代替,最終導致心臟的收縮功能受損[1 ]。 然而目前很多研究結果[2]給我們帶來觀念上的更新:其一,心臟不是終末分化器官,心肌中有75%的細胞是單核細胞,,25%是雙核細胞,這一比例不受疾病、年齡、性別、心肌肥大及心肌缺血等影響。其二,急性心肌損傷促進心肌細胞代償性增殖。 雖然有研究顯示:急性心肌梗死后也有少量心肌細胞發(fā)生分裂增生[3]。但分裂的細胞數(shù)量很少,修復心肌組織的能力有限。因此,細胞移植,這一旨在增加心肌細胞數(shù)目的方法可能是有效治療心肌損傷的最佳選擇。 目前研究多集中在胚胎干細胞、骨骼肌干細胞、骨髓間充質干細胞等各類干細胞的心肌移植,并屢有移植細胞在體內存活分化,修復梗死心肌,改善心臟功能的研究報道。然而要將上述研究最終用于臨床,卻面臨諸多問題。 胚胎干細胞是來源于哺乳動物早期胚胎的內細胞團或桑椹胚的二倍體細胞,從植入子宮內膜前的囊胚中提取,在體外分離培養(yǎng)而建立的細胞系。其顯著特征是可以在體外長期保持不分化狀態(tài)下的增殖能力,并仍具備分化為三胚層細胞的穩(wěn)定發(fā)育潛能,有“萬能細胞”之稱[4]。近年有些國家已解除胚胎干細胞研究的禁令,為其掃除了社會和倫理方面的障礙。但獲取受限和移植排斥反應是限制其發(fā)展的瓶頸。 骨骼肌干細胞具有能耐受缺血環(huán)境;移植后很快分化成具有收縮性的骨骼肌等特點。但其數(shù)量極少,且與年齡增長負相關,分離純化的技術難度大。而最大的缺陷在于骨骼肌干細胞并不能與受體心肌細胞形成閏盤連接,不能整合到受體心肌細胞中形成生理性功能合胞體,導致骨骼肌干細胞移植后往往發(fā)生心室節(jié)律紊亂,使其應用價值受到不少學者質疑。 骨髓間充質干細胞具有取材方便、體外分離純化相對簡單、易擴增等特點;同時由于它們存在獨特的免疫耐受性(其原因可能是缺乏Ⅱ型HLA和免疫共刺激因子B7[5]),能夠在異種異體環(huán)境中存活,而不被受體免疫系統(tǒng)所排斥。所以骨髓間充質干細胞是用于心肌移植的優(yōu)勢細胞之一,被廣泛關注[6,7]。 胎肝干細胞(Embryonic hepatic stem cells,EHSCs)是近年逐漸被關注的一類間充質干細胞細胞。有學者[8]認為胚胎肝臟發(fā)育早,胎肝干細胞可能具有比骨髓干細胞更強的增殖分化能力和更低的免疫原性和免疫活性。有研究者已就其向類心肌細胞方向分化做出了初步探討[9,10]。胎肝干細胞有望用于心肌移植的優(yōu)勢細胞之一。 研究目的: 1.優(yōu)化小鼠EHSCs的分離、純化、擴增的方法;2.比較不同胎齡的EHSCs生物學性狀,遴選出獲得小鼠EHSCs的合適時期;3.通過體外誘導分化實驗了解EHSCs是否具有分化為類心肌細胞的潛能;4.比較不同的誘導條件下,EHSCs向類心肌細胞方向的分化情況。 方法與結論: 第一部分:探討小鼠EHSCs分離、純化、擴增方法,比較不同胎齡的小鼠EHSCs的生物學性狀。采用膠原酶加EDTA消化法和差速貼壁法分離不同胎齡的小鼠EHSCs,用含15%優(yōu)等胎牛血清的L-DMEM培養(yǎng)液培養(yǎng),通過反復傳代對EHSCs進行純化和擴增培養(yǎng)。結果提示:13.5d胎齡組EHSCs形態(tài)均一,生長狀態(tài)良好,干細胞特性明顯,是相對原始的干細胞。隨胎齡增加,16.5d胎齡組和19.5d胎齡組EHSCs形態(tài)差異漸大,生長狀態(tài)漸次,逐漸向具有肝細胞和膽管細胞標志的雙顯型干細胞方向過渡。 結論:1. 13.5d胎齡的EHSCs,是相對原始的干細胞,可能具有更廣泛的分化潛能。2.通過上述方法,可獲得數(shù)量可觀,性質穩(wěn)定的EHSCs。 第二部分:探討不同的誘導條件下,EHSCs向心肌細胞方向的分化情況。取3-4代細胞以1.5×10~4 /cm~2密度接種于培養(yǎng)板上。當細胞接近80%-90%融合時,用不同濃度的誘導劑誘導細胞24h,然后置于37OC,5%CO_2, 20%O_2 ,飽和濕度的孵箱中培養(yǎng)。倒置顯微鏡下觀察細胞形態(tài)變化,發(fā)現(xiàn)在下述條件下細胞發(fā)生了向心肌細胞方向的分化:誘導劑為5-aza 5μmol/L+DMSO 0.8%,孵育時間為24h,培養(yǎng)液是含有15%FCS、1%非必需氨基酸、1%左旋谷氨酸的L-DMEM培養(yǎng)基。培養(yǎng)條件是37OC,5%CO_2,20%O_2,飽和濕度的孵箱。結果提示:誘導3周后,分化細胞呈小圓形,具有相互聚集形成球形細胞團結構的趨勢;誘導后第4周,細胞免疫組化染色提示轉化細胞表達心肌特異性肌鈣蛋白T ( troponin T, Tn T)和α-肌動蛋白(α-actin)。 結論:1.小鼠EHSCs具有向心肌細胞方向分化的潛能;2.不同種屬來源的細胞發(fā)生分化的時間和過程并不完全相同,轉化為成熟心肌細胞所需時間也不同。 總結: 1.利用改良的差速貼壁法,可以從胎肝中分離出具有良好貼壁能力的干細胞群,方法較為簡單易行。 2.胎齡為13.5 d的小鼠胎肝,單位重量的胎肝中含有更多的具有形成集落能力的干細胞。 3.胎齡為13.5 d的小鼠胎肝干細胞處于相對原始的未分化階段。 4.小鼠EHSCs在5-aza和DMSO聯(lián)合誘導的情況下在體外可向類心肌細胞方向分化。 5.誘導劑組合5-aza 5μmol/L+ DMSO 0.8%能誘導EHSCs在體外向心肌細胞方向分化,加大誘導劑劑量并不能提高誘導效率。 6.誘導劑組合5-aza 5μmol/L+ DMSO 0.8%誘導EHSCs在體外向心肌細胞方向分化的合理作用時間為24h,延長誘導劑的作用時間,并不能提高誘導效率。
[Abstract]:Research background:
Coronary atherosclerotic heart disease, especially acute myocardial infarction, is a common disease that seriously endangers human health. It is often possible to save the dying myocardium by timely thrombolytic or interventional therapy within 4-6 hours of the disease, but the ischemic myocardium has been necrotic at the time of most of the patients. Treatment can only prevent the expansion of the infarct area and can not save the necrotic myocardium. How to treat coronary heart disease more effectively has become the focus of medical research, and recent results have brought hope to the patients.
The traditional point of view is that cardiac myocytes are differentiated into terminal cells after birth and can not be replicated, and there is no stored cardiac progenitor cells in adult myocardium, so myocardial cells can not regenerate after myocardial damage and are replaced by scar tissue, resulting in impaired cardiac contractile function [1).
However, a lot of current research results [2] bring us a conceptual update: first, the heart is not a terminal differentiation organ, 75% of the cells in the myocardium are mononuclear cells, and 25% are binuclear cells. This proportion is not affected by disease, age, sex, myocardial hypertrophy and myocardial ischemia.
Although studies have shown that a small number of cardiomyocytes have split proliferative [3]. after acute myocardial infarction, the number of divided cells is small and the ability to repair myocardial tissue is limited. Therefore, cell transplantation, a method aimed at increasing the number of cardiomyocytes, may be the best choice for effective treatment of myocardial injury.
At present, many studies have focused on the transplantation of stem cells, such as embryonic stem cells, skeletal muscle stem cells, bone marrow mesenchymal stem cells and other kinds of stem cells, and the research reports on the survival and differentiation of the transplanted cells in the body, repair the infarcted myocardium and improve the heart function. However, the above research should be finally used in clinical practice, but many problems are faced.
The embryonic stem cell is a diploid cell derived from the inner cell mass or morula of the early mammalian embryo, extracted from the blastocyst before the implantation of the endometrium and isolated and cultured in vitro, which is characterized by the ability to maintain the proliferation in an undifferentiated state for a long time in vitro, and still have a differentiation of three germ cells. In recent years, some countries have lifted the prohibition of embryonic stem cell research and removed the social and ethical barriers in [4].. However, access to restriction and graft rejection is the bottleneck to restrict its development.
Skeletal muscle stem cells have the ability to tolerate ischemic environment and quickly differentiate into contractile skeletal muscles. But the number of skeletal muscle cells is very small and is negatively related to age growth. The technical difficulty of separating and purifying is very difficult. The biggest defect is that skeletal muscle stem cells do not form intercalated disc connections with receptor cardiac myocytes and can not be integrated into the receptor. The physiological function syncytial body is formed in the cardiac myocytes, which leads to the ventricular rhythm disorder after the transplantation of skeletal muscle stem cells, which has been questioned by many scholars.
Bone marrow mesenchymal stem cells (MSCs) have the advantages of convenient extraction, relatively simple separation and purification in vitro, and their unique immune tolerance (which may be due to the lack of type II HLA and immuno stimulating factor B7[5]) and can survive in xenoallogenic environment without rejection by the receptor immune system. Mesenchymal stem cells are one of the dominant cells for myocardial transplantation. They are widely concerned about [6,7]..
Embryonic hepatic stem cells (EHSCs) is a kind of mesenchymal stem cell cells which have been paid more attention in recent years. Some scholars believe that embryonic liver is early developed, and fetal liver stem cells may have stronger proliferation and differentiation ability and lower immunogenicity and immune activity than bone marrow stem cells. Cell differentiation has made a preliminary study. [9,10]. fetal liver stem cells are expected to be one of the dominant cells in myocardial transplantation.
The purpose of the study is:
1. to optimize the isolation, purification and amplification of EHSCs in mice; 2. to compare the EHSCs biological characters of different gestational ages and to select the appropriate time to obtain EHSCs in mice; 3. through the induction of differentiation in vitro to understand the potential of EHSCs to differentiate into the myocyte like cells; 4. compare the differentiation of EHSCs into the direction of the myocardial like cells under different induction conditions. Situation.
Methods and conclusions:
The first part: To explore the isolation, purification and amplification of EHSCs in mice, to compare the biological characters of EHSCs in mice of different gestational ages. Using collagenase, EDTA digestion and differential adherence to separate EHSCs in mice of different gestational ages, the L-DMEM culture medium containing 15% superior fetal bovine serum was cultured, and EHSCs was purified and amplified by repeated passages. The results showed that the EHSCs morphology of 13.5d gestational age group was uniform, the growth state was good, the stem cell characteristics were obvious, it was relatively primitive stem cells. As the gestational age increased, the difference of EHSCs morphology between the 16.5d gestational age group and the 19.5d fetal age group gradually increased, and the growth state gradually shifted to the direction of the double developing stem cells with the markers of hepatocytes and bile duct cells.
Conclusion: the EHSCs of 1. 13.5d gestational age is relatively primitive stem cells and may have a wider range of differentiation potential.2. through the above method, which can obtain a considerable quantity and stable EHSCs..
The second part: To investigate the differentiation of EHSCs to myocardial cells under different induction conditions. 3-4 generation cells were inoculated on the culture plate at 1.5 x 10~4 /cm~2 density. When the cells were close to 80%-90% fusion, the cells were induced with different concentrations of inducers to induce cell 24h, and then placed in the incubator of 37OC, 5%CO_2, 20%O_2, saturated humidity. Under the microscope, it was found that the cells were differentiated into cardiomyocyte direction under the following conditions: the inducer was 5-aza 5 mu mol/L+DMSO 0.8%, the incubation time was 24h, and the culture medium was the L-DMEM medium containing 15%FCS, 1% non essential amino acids and 1% L-glutamic acid. The culture condition was the incubator of 37OC, 5%CO_2,20%O_2, saturated humidity. The results suggest that after 3 weeks of induction, the differentiated cells are small round and have a tendency to form spherical cell clusters with each other. After fourth weeks of induction, cell immuno histochemical staining suggests that the transformed cells express cardiac specific troponin T (troponin T, Tn T) and alpha actin (alpha -actin).
Conclusion: 1. EHSCs in mice has the potential of centripetal muscle cell differentiation. 2. the time and process of differentiation of cells from different species are not exactly the same, and the time needed to transform into mature cardiomyocytes is also different.
Summary:
1. by using the modified differential adherence method, stem cells with good adherence ability can be isolated from fetal liver.
2. fetal liver with 13.5 gestational age of 13.5 D contains more stem cells with colony forming ability.
The fetal liver stem cells of 3. gestational age of 13.5 D were in relatively primitive undifferentiated stage.
4. mouse EHSCs can differentiate into cardiomyocytes in vitro under the combined induction of 5-aza and DMSO.
5. inducer combination 5-aza 5 mu mol/L+ DMSO 0.8% can induce EHSCs to differentiate into cardiomyocytes in vitro. Increasing the dose of inducer can not improve the induction efficiency.
6. inducer combination 5-aza 5 mol/L+ DMSO 0.8% induced the rational time of EHSCs to differentiate into cardiomyocytes in vitro, which is 24h, prolonging the action time of inducer, and not improving the induction efficiency.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

【相似文獻】

相關期刊論文 前10條

1 ;[J];;年期

2 ;[J];;年期

3 ;[J];;年期

4 ;[J];;年期

5 ;[J];;年期

6 ;[J];;年期

7 ;[J];;年期

8 ;[J];;年期

9 ;[J];;年期

10 ;[J];;年期

相關會議論文 前10條

1 宋今丹;孫寶棟;正紅梅;玉維琴;王蕓慶;;經維甲酸誘導分化的大腸癌細胞的功能改變[A];中國細胞生物學學會第七次會議論文摘要匯編[C];1999年

2 郝長來;王敏;唐克晶;王建祥;;急性髓系白血病M2b型誘導分化和凋亡的體內和體外研究[A];第九屆全國實驗血液學會議論文摘要匯編[C];2003年

3 陳蘇寧;薛永權;岑建農;吳亞芳;潘金蘭;王勇;吳德沛;;人單核細胞系SHI-1的誘導分化和凋亡[A];中華醫(yī)學會第八次全國血液學學術會議論文匯編[C];2004年

4 魯大鵬;劉思思;張浩;;包殼蛋白與As2O3對KB和Tca8113細胞誘導分化的關系[A];第八次全國口腔頜面—頭頸腫瘤會議論文匯編[C];2009年

5 景黎君;賈永林;韓瑞;魯晶晶;李盡義;王繼先;張博愛;彭濤;賈延R，

本文編號：1986361