本文選題：胎肝間充質(zhì)干細(xì)胞 + 誘導(dǎo)分化　； 參考：《重慶醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 研究背景: 冠狀動(dòng)脈粥樣硬化性心臟病,特別是急性心肌梗死,是嚴(yán)重危害人類(lèi)健康的常見(jiàn)病,多發(fā)病。目前雖然能在發(fā)病4-6小時(shí)內(nèi)通過(guò)及時(shí)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(shí)缺血心肌已經(jīng)壞死。此時(shí)再灌注治療只能防止梗死面積的擴(kuò)大,而無(wú)法挽救已經(jīng)壞死的心肌。如何更有效地治療冠心病,成為醫(yī)學(xué)研究的焦點(diǎn)。而新近的研究結(jié)果,為患者帶來(lái)了希望。 傳統(tǒng)觀點(diǎn)認(rèn)為,心肌細(xì)胞出生后即向終末分化,不能夠復(fù)制,并且成年心肌組織中沒(méi)有儲(chǔ)存的心肌祖細(xì)胞,因此心肌受損后心肌細(xì)胞不能再生,被疤痕組織代替,最終導(dǎo)致心臟的收縮功能受損[1 ]。 然而目前很多研究結(jié)果[2]給我們帶來(lái)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(xì)胞是單核細(xì)胞,,25%是雙核細(xì)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(jìn)心肌細(xì)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(xì)胞發(fā)生分裂增生[3]。但分裂的細(xì)胞數(shù)量很少,修復(fù)心肌組織的能力有限。因此,細(xì)胞移植,這一旨在增加心肌細(xì)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(xì)胞、骨骼肌干細(xì)胞、骨髓間充質(zhì)干細(xì)胞等各類(lèi)干細(xì)胞的心肌移植,并屢有移植細(xì)胞在體內(nèi)存活分化,修復(fù)梗死心肌,改善心臟功能的研究報(bào)道。然而要將上述研究最終用于臨床,卻面臨諸多問(wèn)題。 胚胎干細(xì)胞是來(lái)源于哺乳動(dòng)物早期胚胎的內(nèi)細(xì)胞團(tuán)或桑椹胚的二倍體細(xì)胞,從植入子宮內(nèi)膜前的囊胚中提取,在體外分離培養(yǎng)而建立的細(xì)胞系。其顯著特征是可以在體外長(zhǎng)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(xì)胞的穩(wěn)定發(fā)育潛能,有“萬(wàn)能細(xì)胞”之稱(chēng)[4]。近年有些國(guó)家已解除胚胎干細(xì)胞研究的禁令,為其掃除了社會(huì)和倫理方面的障礙。但獲取受限和移植排斥反應(yīng)是限制其發(fā)展的瓶頸。 骨骼肌干細(xì)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(diǎn)。但其數(shù)量極少,且與年齡增長(zhǎng)負(fù)相關(guān),分離純化的技術(shù)難度大。而最大的缺陷在于骨骼肌干細(xì)胞并不能與受體心肌細(xì)胞形成閏盤(pán)連接,不能整合到受體心肌細(xì)胞中形成生理性功能合胞體,導(dǎo)致骨骼肌干細(xì)胞移植后往往發(fā)生心室節(jié)律紊亂,使其應(yīng)用價(jià)值受到不少學(xué)者質(zhì)疑。 骨髓間充質(zhì)干細(xì)胞具有取材方便、體外分離純化相對(duì)簡(jiǎn)單、易擴(kuò)增等特點(diǎn);同時(shí)由于它們存在獨(dú)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(zhì)干細(xì)胞是用于心肌移植的優(yōu)勢(shì)細(xì)胞之一,被廣泛關(guān)注[6,7]。 胎肝干細(xì)胞(Embryonic hepatic stem cells,EHSCs)是近年逐漸被關(guān)注的一類(lèi)間充質(zhì)干細(xì)胞細(xì)胞。有學(xué)者[8]認(rèn)為胚胎肝臟發(fā)育早,胎肝干細(xì)胞可能具有比骨髓干細(xì)胞更強(qiáng)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(lèi)心肌細(xì)胞方向分化做出了初步探討[9,10]。胎肝干細(xì)胞有望用于心肌移植的優(yōu)勢(shì)細(xì)胞之一。 研究目的: 1.優(yōu)化小鼠EHSCs的分離、純化、擴(kuò)增的方法;2.比較不同胎齡的EHSCs生物學(xué)性狀,遴選出獲得小鼠EHSCs的合適時(shí)期;3.通過(guò)體外誘導(dǎo)分化實(shí)驗(yàn)了解EHSCs是否具有分化為類(lèi)心肌細(xì)胞的潛能;4.比較不同的誘導(dǎo)條件下,EHSCs向類(lèi)心肌細(xì)胞方向的分化情況。 方法與結(jié)論: 第一部分:探討小鼠EHSCs分離、純化、擴(kuò)增方法,比較不同胎齡的小鼠EHSCs的生物學(xué)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(guò)反復(fù)傳代對(duì)EHSCs進(jìn)行純化和擴(kuò)增培養(yǎng)。結(jié)果提示:13.5d胎齡組EHSCs形態(tài)均一,生長(zhǎng)狀態(tài)良好,干細(xì)胞特性明顯,是相對(duì)原始的干細(xì)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(zhǎng)狀態(tài)漸次,逐漸向具有肝細(xì)胞和膽管細(xì)胞標(biāo)志的雙顯型干細(xì)胞方向過(guò)渡。 結(jié)論:1. 13.5d胎齡的EHSCs,是相對(duì)原始的干細(xì)胞,可能具有更廣泛的分化潛能。2.通過(guò)上述方法,可獲得數(shù)量可觀,性質(zhì)穩(wěn)定的EHSCs。 第二部分:探討不同的誘導(dǎo)條件下,EHSCs向心肌細(xì)胞方向的分化情況。取3-4代細(xì)胞以1.5×10~4 /cm~2密度接種于培養(yǎng)板上。當(dāng)細(xì)胞接近80%-90%融合時(shí),用不同濃度的誘導(dǎo)劑誘導(dǎo)細(xì)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(xì)胞形態(tài)變化,發(fā)現(xiàn)在下述條件下細(xì)胞發(fā)生了向心肌細(xì)胞方向的分化:誘導(dǎo)劑為5-aza 5μmol/L+DMSO 0.8%,孵育時(shí)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(jié)果提示:誘導(dǎo)3周后,分化細(xì)胞呈小圓形,具有相互聚集形成球形細(xì)胞團(tuán)結(jié)構(gòu)的趨勢(shì);誘導(dǎo)后第4周,細(xì)胞免疫組化染色提示轉(zhuǎn)化細(xì)胞表達(dá)心肌特異性肌鈣蛋白T ( troponin T, Tn T)和α-肌動(dòng)蛋白(α-actin)。 結(jié)論:1.小鼠EHSCs具有向心肌細(xì)胞方向分化的潛能;2.不同種屬來(lái)源的細(xì)胞發(fā)生分化的時(shí)間和過(guò)程并不完全相同,轉(zhuǎn)化為成熟心肌細(xì)胞所需時(shí)間也不同。 總結(jié): 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(xì)胞群,方法較為簡(jiǎn)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(xì)胞。 3.胎齡為13.5 d的小鼠胎肝干細(xì)胞處于相對(duì)原始的未分化階段。 4.小鼠EHSCs在5-aza和DMSO聯(lián)合誘導(dǎo)的情況下在體外可向類(lèi)心肌細(xì)胞方向分化。 5.誘導(dǎo)劑組合5-aza 5μmol/L+ DMSO 0.8%能誘導(dǎo)EHSCs在體外向心肌細(xì)胞方向分化,加大誘導(dǎo)劑劑量并不能提高誘導(dǎo)效率。 6.誘導(dǎo)劑組合5-aza 5μmol/L+ DMSO 0.8%誘導(dǎo)EHSCs在體外向心肌細(xì)胞方向分化的合理作用時(shí)間為24h,延長(zhǎng)誘導(dǎo)劑的作用時(shí)間,并不能提高誘導(dǎo)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R329

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