心肌細胞共培養(yǎng)誘導骨髓基質(zhì)干細胞分化為心肌細胞的研究
發(fā)布時間：2018-11-27 13:15
【摘要】： 目的 骨髓中存在一定數(shù)量的骨髓基質(zhì)干細胞(BMSCs, bone marrow stromal stem cells),這些細胞具有自我復制能力和巨大的分化潛能,在不同的條件下,可分化為骨、軟骨、神經(jīng)、心肌和脂肪等中胚層來源的組織。有研究結(jié)果表明,胞嘧啶類化學物質(zhì)5-氮胞苷可誘導骨髓基質(zhì)干細胞向心肌細胞分化,這使BMSCs成為細胞移植治療缺血性心肌病最有前途的細胞之一。 本實驗旨在通過對大鼠骨髓基質(zhì)干細胞進行體外分離、培養(yǎng)、動態(tài)觀察BMSCs的生長特點;觀察在不經(jīng)過任何誘導,將擴增的BMSCs與心肌細胞共同培養(yǎng),觀察BMSCs分化為心肌細胞的能力。并且通過GFP質(zhì)粒轉(zhuǎn)染骨髓基質(zhì)干細胞,觀察GFP在標記BMSCs中的表達能力,以期進一步了解骨髓基質(zhì)干細胞的生物學特性,為其應用于臨床提供更可靠的理論依據(jù)。 實驗方法 1.全骨髓貼壁培養(yǎng)法體外分離,培養(yǎng)大鼠骨髓基質(zhì)干細胞,倒置相差顯微鏡下逐日觀察細胞形態(tài)及生長狀。 2.差速貼壁法體外分離,培養(yǎng)心肌細胞,倒置相差顯微鏡下逐日觀察細胞形態(tài)及生長狀況。 3.將提取的GFP質(zhì)粒轉(zhuǎn)染培養(yǎng)好的第3代骨髓基質(zhì)干細胞,Olympus熒光顯微鏡下觀察細胞的轉(zhuǎn)染效率。 4.將轉(zhuǎn)染后的骨髓基質(zhì)干細胞離心收集后按1∶3的比例加入到培養(yǎng)好的心肌細胞中共培養(yǎng),Olympus熒光顯微鏡逐日觀察細胞變化。 實驗結(jié)果 1.大鼠骨髓基質(zhì)干細胞的形態(tài)學觀察:骨髓基質(zhì)干細胞接種于培養(yǎng)瓶中后24h用倒置顯微鏡觀察大部分細胞已貼壁,貼壁生長的細胞以分散、克隆集落的方式增殖,細胞大多呈梭形,類似成纖維細胞,偶有寬大平坦的多角形細胞。7-10d后克隆集落逐漸增多,隨著集落生長的不斷擴大,細胞融合。傳代培養(yǎng)后細胞不再以集落方式生長,而呈分布均勻的紡錘形細胞生長。 2、心肌細胞的形態(tài)學觀察:心肌細胞一般在24 h后會自動從組織塊周圍遷出,剛開始從組織塊中遷出時,心肌細胞呈現(xiàn)梭形,伴隨著遷出細胞的增多,組織塊周圍的心肌細胞呈現(xiàn)出不規(guī)則的圓形、三角形。細胞間緊密連接,整體形如鋪路石樣,隨著細胞生長密度的不斷增加,細胞間伸出的偽足會交織成網(wǎng)狀。有時一些細胞緊密結(jié)合形成細胞簇,呈現(xiàn)出放射狀或同心圓狀,經(jīng)過2～3 d培養(yǎng),這些細胞簇會自發(fā)地搏動。 3、GFP轉(zhuǎn)染骨髓基質(zhì)干細胞的結(jié)果:轉(zhuǎn)染GFP后,細胞仍貼壁生長,呈梭形或多角形,仍分裂增殖,但增殖速度有所降低。熒光顯微鏡下, 24 h可見有綠色熒光表達,但數(shù)量較少,強度較低,48 h可見細胞發(fā)出強烈的熒光。 4、共培養(yǎng)后BMSCs形態(tài)學與生長行為變化:在共培養(yǎng)后,BMSCs增殖速度緩慢,并且容易變成多角形或扁平,隨著共培養(yǎng)時間的延長,有相當數(shù)量的BMSCs發(fā)生細胞崩解等死亡現(xiàn)象。24h后發(fā)現(xiàn)有很多貼壁的未跳動的尖梭形細胞靠近搏動的心肌細胞;48h后,這些尖梭形細胞呈集落樣生長,并且在熒光顯微鏡下觀察到個別被標記的BMSCs呈肌管樣結(jié)構(gòu),這些BMSCs通�？拷珓拥奈礃擞浀男募〖毎�;在第4天,部分與心肌細胞接觸的被標記的BMSCs開始呈微弱心肌樣搏動。同時,也有些BMSCs開始死亡,至64h已有相當一部分BMSCs發(fā)生死亡。 結(jié)論 1.可以從大鼠骨髓中分離出骨髓基質(zhì)干細胞,經(jīng)體外培養(yǎng)可大量擴增。 2.GFP轉(zhuǎn)染BMSC后可以長期高效表達,而且對宿主細胞毒性很小,是一個非常理想的組織工程種子細胞的示蹤工具。 3.大鼠骨髓基質(zhì)干細胞在不用誘導劑的情況下,與心肌細胞共培養(yǎng)后可以分化為心肌細胞。
[Abstract]:Purpose The bone marrow has a certain number of bone marrow stromal stem cells (BMSCs, bone marrow stem cells), which have self-replicating ability and great differentiation potential, and can be differentiated into the source of the mesoderm such as bone, cartilage, nerve, myocardium and fat under different conditions. The results of the study show that the 5-N-cell-like chemical can induce the differentiation of bone marrow stromal stem cells into the cardiac muscle cells, which makes the BMSCs become the most promising cells for the treatment of ischemic cardiomyopathy by cell transplantation. The purpose of this study was to observe the growth characteristics of BMSCs by in vitro separation, culture and dynamic observation of bone marrow stromal stem cells in rats. The ability of the cells to be enhanced and the expression of GFP in the labeled BMSCs was observed by the transfection of the bone marrow stromal cells with the GFP plasmid, with a view to further understanding the biological characteristics of the bone marrow stromal stem cells and to provide more reliable clinical application the theory of According to the method, the whole bone marrow adherent culture method is isolated and cultured in vitro to culture the bone marrow stromal stem cells of the rat, and the inverted phase difference microscope day-to-day observation of cell morphology and growth. 2. In vitro separation of the differential adhesion method, culture of the cardiac muscle cells and the inversion of the phase-difference microscope The morphology and growth of the cells were observed on a day-to-day basis. 3. The extracted GFP plasmid was transfected into a cultured third generation of bone marrow stromal stem cells, Olymp. the transfection efficiency of the cells is observed under the us fluorescence microscope. Olym The changes of the cells were observed on a day-to-day basis by the pus fluorescence microscope. The results of the experiment were as follows: 1. The morphological observation of the bone marrow stromal stem cells in the rat: The bone marrow stromal stem cells were inoculated into the culture flask and then the cells were observed with an inverted microscope. The grown cells proliferate in a dispersed and colony-like manner, with the majority of the cells in the form of a shuttle, similar to a fibroblast, even with a large, flat polygonal cell. 7-10d After the growth of the colonies, the colonies grew, and the cells were fused with the growth of the colonies. the cells were no longer grown in a colony-like manner, and the distribution of the spindle-shaped cells was uniform. The morphological observation of the cardiac muscle cells: the cardiac muscle cells usually move out of the tissue mass after 24 h, and when the cells move out of the tissue mass, the cardiac muscle cells exhibit a shuttle-shape, with a companion. with moving out The cell is increased, and the heart muscle surrounding the tissue mass presents an irregular circular, triangular shape. The cells are closely connected, and the whole body is like a stone-laying stone. and as the growth density of the cells is increasing, the pseudo-feet that extend between the cells are woven into a net. Sometimes, some cells are tightly combined to form a cell cluster, radial or concentric circles, after 2-3 days of culture, these cell clusters spontaneously beat. 3. Results of GFP transfection of bone marrow stromal cells: after transfection of GFP The cell is still adherent to grow, is in the form of a shuttle or a polygon, but the proliferation is still split, but the proliferation rate is reduced. Under the fluorescence microscope, 24 h can be seen The expression of green fluorescence was small, the intensity was low, and the cells of 48 h showed strong fluorescence. 4. The morphology and growth behavior of BMSCs after co-culture: after co-culture, The proliferation of BMSCs was slow, and it was easy to be polygonal or flat. With the extension of co-culture time, there was a significant number of BMSCs in cell disintegration and the like. The colony-like growth was observed and the individual labeled BMSCs were observed with a myotube-like structure under a fluorescence microscope, which were usually close to the unlabeled cardiomyocytes of the beat. At the fourth day, the labeled BMSCs, which were partially in contact with the cardiomyocytes, began to have a weak heart-like beat. in that case of the present invention, Some BMSCs started to die, and a significant portion of BMSCs died in 64h. Conclusion: 1. The bone marrow stromal cells can be isolated from the bone marrow of the rat. C can be expressed for a long period of time, and the toxicity of the host cells is very small, which is an ideal tissue engineering seed cell.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329
本文編號：2360912
[Abstract]:Purpose The bone marrow has a certain number of bone marrow stromal stem cells (BMSCs, bone marrow stem cells), which have self-replicating ability and great differentiation potential, and can be differentiated into the source of the mesoderm such as bone, cartilage, nerve, myocardium and fat under different conditions. The results of the study show that the 5-N-cell-like chemical can induce the differentiation of bone marrow stromal stem cells into the cardiac muscle cells, which makes the BMSCs become the most promising cells for the treatment of ischemic cardiomyopathy by cell transplantation. The purpose of this study was to observe the growth characteristics of BMSCs by in vitro separation, culture and dynamic observation of bone marrow stromal stem cells in rats. The ability of the cells to be enhanced and the expression of GFP in the labeled BMSCs was observed by the transfection of the bone marrow stromal cells with the GFP plasmid, with a view to further understanding the biological characteristics of the bone marrow stromal stem cells and to provide more reliable clinical application the theory of According to the method, the whole bone marrow adherent culture method is isolated and cultured in vitro to culture the bone marrow stromal stem cells of the rat, and the inverted phase difference microscope day-to-day observation of cell morphology and growth. 2. In vitro separation of the differential adhesion method, culture of the cardiac muscle cells and the inversion of the phase-difference microscope The morphology and growth of the cells were observed on a day-to-day basis. 3. The extracted GFP plasmid was transfected into a cultured third generation of bone marrow stromal stem cells, Olymp. the transfection efficiency of the cells is observed under the us fluorescence microscope. Olym The changes of the cells were observed on a day-to-day basis by the pus fluorescence microscope. The results of the experiment were as follows: 1. The morphological observation of the bone marrow stromal stem cells in the rat: The bone marrow stromal stem cells were inoculated into the culture flask and then the cells were observed with an inverted microscope. The grown cells proliferate in a dispersed and colony-like manner, with the majority of the cells in the form of a shuttle, similar to a fibroblast, even with a large, flat polygonal cell. 7-10d After the growth of the colonies, the colonies grew, and the cells were fused with the growth of the colonies. the cells were no longer grown in a colony-like manner, and the distribution of the spindle-shaped cells was uniform. The morphological observation of the cardiac muscle cells: the cardiac muscle cells usually move out of the tissue mass after 24 h, and when the cells move out of the tissue mass, the cardiac muscle cells exhibit a shuttle-shape, with a companion. with moving out The cell is increased, and the heart muscle surrounding the tissue mass presents an irregular circular, triangular shape. The cells are closely connected, and the whole body is like a stone-laying stone. and as the growth density of the cells is increasing, the pseudo-feet that extend between the cells are woven into a net. Sometimes, some cells are tightly combined to form a cell cluster, radial or concentric circles, after 2-3 days of culture, these cell clusters spontaneously beat. 3. Results of GFP transfection of bone marrow stromal cells: after transfection of GFP The cell is still adherent to grow, is in the form of a shuttle or a polygon, but the proliferation is still split, but the proliferation rate is reduced. Under the fluorescence microscope, 24 h can be seen The expression of green fluorescence was small, the intensity was low, and the cells of 48 h showed strong fluorescence. 4. The morphology and growth behavior of BMSCs after co-culture: after co-culture, The proliferation of BMSCs was slow, and it was easy to be polygonal or flat. With the extension of co-culture time, there was a significant number of BMSCs in cell disintegration and the like. The colony-like growth was observed and the individual labeled BMSCs were observed with a myotube-like structure under a fluorescence microscope, which were usually close to the unlabeled cardiomyocytes of the beat. At the fourth day, the labeled BMSCs, which were partially in contact with the cardiomyocytes, began to have a weak heart-like beat. in that case of the present invention, Some BMSCs started to die, and a significant portion of BMSCs died in 64h. Conclusion: 1. The bone marrow stromal cells can be isolated from the bone marrow of the rat. C can be expressed for a long period of time, and the toxicity of the host cells is very small, which is an ideal tissue engineering seed cell.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329
【引證文獻】
中國碩士學位論文全文數(shù)據(jù)庫 前1條
1 羅建紅;人臍血間充質(zhì)干細胞移植治療DCM大鼠的實驗研究[D];湖南師范大學;2012年
，本文編號：2360912
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