本文選題：骨髓間充質(zhì)干細(xì)胞 + 明膠海綿　； 參考：《河北醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 第一部分:骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)、鑒定及其自身特性的初步研究 目的:通過本實(shí)驗(yàn),對(duì)骨髓間充質(zhì)干細(xì)胞進(jìn)行分離、培養(yǎng)、鑒定并對(duì)其自身特性加以研究,同時(shí)為下一步明膠海綿載體搭載移植實(shí)驗(yàn)提供搭載細(xì)胞。 方法:首先,利用差速貼壁培養(yǎng)法將骨髓間充質(zhì)干細(xì)胞從大鼠的骨髓之中分離、純化出來。之后,利用流式細(xì)胞學(xué)方法對(duì)骨髓間充質(zhì)干細(xì)胞的表面標(biāo)記物加以鑒定。在鑒定其為骨髓間充質(zhì)干細(xì)胞后,以C6細(xì)胞系做為對(duì)照繪制其在不同代數(shù)時(shí)期的生長曲線,對(duì)其致瘤性加以研究。利用免疫熒光學(xué)的方法,研究骨髓間充質(zhì)干細(xì)胞在一定條件下自發(fā)向神經(jīng)細(xì)胞轉(zhuǎn)化的特性。利用Transwell方法,研究骨髓間充質(zhì)干細(xì)胞可向受損神經(jīng)組織定向遷移的特性。最后,研究骨髓間充質(zhì)干細(xì)胞簡便的凍存與復(fù)蘇保存方法。 結(jié)果:從大鼠骨髓腔中沖出的混雜細(xì)胞,在倒置相差顯微鏡下顯示為大量單個(gè)或成團(tuán)均勻分布的小圓形細(xì)胞。4～8小時(shí)之后部分細(xì)胞沉降并鋪至培養(yǎng)皿底部。24小時(shí)后觀察可見鋪于培養(yǎng)皿底部的大部分細(xì)胞變扁并逐漸伸出突起。3天后未貼壁細(xì)胞及其它髓腔內(nèi)物質(zhì)經(jīng)換液被大部分去除。此后貼壁細(xì)胞不斷擴(kuò)增,至10～14天可見大量細(xì)胞匯集成片,形態(tài)不一,成團(tuán)生長。其中可見大扁平形、多角形、星形、梭形等多種細(xì)胞形態(tài)。經(jīng)過傳代細(xì)胞形態(tài)和類型逐漸趨向單一,至第5代細(xì)胞大小一致,形態(tài)呈梭形,已基本得到純化。利用流式細(xì)胞學(xué)方法的檢測,細(xì)胞體系表達(dá)表面標(biāo)志物CD31、CD34、CD45為陰性,表達(dá)表面標(biāo)志物CD71為陽性。對(duì)其不同時(shí)期的生長曲線與C6細(xì)胞系相比較,骨髓間充質(zhì)干細(xì)胞生長曲線有很明顯的壓低。通過倒置相差顯微鏡的觀察和免疫熒光學(xué)的檢測,發(fā)現(xiàn)骨髓間充質(zhì)干細(xì)胞在一定條件下可自發(fā)分化出神經(jīng)樣細(xì)胞并表達(dá)神經(jīng)細(xì)胞標(biāo)志物GFAP、NSE。通過Transwell實(shí)驗(yàn),可以觀察到在相同的時(shí)間內(nèi)損傷組骨髓間充質(zhì)干細(xì)胞穿過率明顯高于對(duì)照組骨髓間充質(zhì)干細(xì)胞的穿過率,經(jīng)統(tǒng)計(jì)P0.05有顯著差異。經(jīng)過簡便方法凍存的骨髓間充質(zhì)干細(xì)胞在-80℃條件下至少可以存活3個(gè)月,在液氮中保存則至少可以存活半年。骨髓間充質(zhì)干細(xì)胞經(jīng)過復(fù)蘇后數(shù)量會(huì)有較大的損失,但只要復(fù)蘇后存活的骨髓間充質(zhì)干細(xì)胞達(dá)到一定的數(shù)量和密度,這些細(xì)胞仍可迅速擴(kuò)增并在較短的時(shí)間內(nèi)獲得大量、穩(wěn)定,可以利用的骨髓間充質(zhì)干細(xì)胞。 結(jié)論:通過最常應(yīng)用的差速貼壁培養(yǎng)法可以很穩(wěn)定地培養(yǎng)出骨髓間充質(zhì)干細(xì)胞,第5代細(xì)胞已達(dá)到純化要求。通過細(xì)胞表面標(biāo)志物的鑒定可以確定其為骨髓間充質(zhì)干細(xì)胞。10代以內(nèi)的骨髓間充質(zhì)干細(xì)胞致瘤性極小或者不存在,可以自發(fā)分化出神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞。另外,其具有獨(dú)特的歸巢性可向受損的神經(jīng)組織遷移。骨髓間充質(zhì)干細(xì)胞通過簡便的凍存與復(fù)蘇方法能夠在較長時(shí)間內(nèi)穩(wěn)定的保存,可為后續(xù)應(yīng)用提供儲(chǔ)備細(xì)胞。 第二部分:骨髓間充質(zhì)干細(xì)胞經(jīng)明膠海綿載體搭載向神經(jīng)組織移植可行性的初步研究 目的:研究骨髓間充質(zhì)干細(xì)胞被明膠海綿載體搭載后其存活率是否受到影響和其自身特性有否改變,并進(jìn)一步研究骨髓間充質(zhì)干細(xì)胞經(jīng)過明膠海綿載體搭載后能否與神經(jīng)細(xì)胞共存。 方法:首先,觀察經(jīng)明膠海綿搭載后骨髓間充質(zhì)干細(xì)胞形態(tài)有否改變并統(tǒng)計(jì)經(jīng)明膠海綿搭載移植后的骨髓間充質(zhì)干細(xì)胞貼壁存活的數(shù)量與未經(jīng)搭載細(xì)胞相比是否有差異。然后,以未經(jīng)搭載第5代骨髓間充質(zhì)干細(xì)胞為對(duì)照繪制經(jīng)過搭載骨髓間充質(zhì)干細(xì)胞的生長曲線,并對(duì)其致瘤性加以研究。之后誘導(dǎo)其向神經(jīng)細(xì)胞方向分化,觀察經(jīng)過搭載的骨髓間充質(zhì)干細(xì)胞與未經(jīng)搭載的細(xì)胞在誘導(dǎo)之后形態(tài)的不同,并統(tǒng)計(jì)二者所分化出的神經(jīng)元比例是否有差異。最后,建立體外神經(jīng)細(xì)胞培養(yǎng)體系,并通過倒置相差顯微鏡研究觀察經(jīng)明膠海綿搭載后的骨髓間充質(zhì)干細(xì)胞能否與神經(jīng)細(xì)胞共同培養(yǎng)。 結(jié)果:倒置相差顯微鏡觀察,明膠海綿組與對(duì)照組相比較細(xì)胞的形態(tài)未有明顯變化,多呈長梭形,扁平老化的現(xiàn)象也很少見。無論是在明膠海綿中部或是邊緣部均有骨髓間充質(zhì)干細(xì)胞貼壁,而且在明膠海綿中部貼壁的細(xì)胞數(shù)量更多一些。通過對(duì)明膠海綿組和對(duì)照組未貼壁細(xì)胞的計(jì)數(shù)比較P0.05,無統(tǒng)計(jì)學(xué)差異。經(jīng)搭載后的第5代骨髓間充質(zhì)干細(xì)胞的生長曲線與未經(jīng)搭載第5代細(xì)胞的生長曲線相比較沒有觀察到明顯的變化。骨髓間充質(zhì)干細(xì)胞經(jīng)誘導(dǎo)6～8小時(shí)后可以觀察到細(xì)胞胞體開始收縮并伸出突起,部分突起相互連接形成網(wǎng)絡(luò)狀結(jié)構(gòu)。經(jīng)免疫熒光學(xué)檢測細(xì)胞可表達(dá)神經(jīng)膠質(zhì)細(xì)胞標(biāo)記物GFAP和神經(jīng)元細(xì)胞標(biāo)記物NSE。通過流式細(xì)胞學(xué)方法對(duì)明膠海綿組與對(duì)照組表達(dá)NSE陽性率做比較,經(jīng)統(tǒng)計(jì)P0.05,無顯著差異。新生大鼠神經(jīng)細(xì)胞種植后12～24小時(shí)后大部分細(xì)胞可貼壁并伸出突起。隨著培養(yǎng)時(shí)間的延長神經(jīng)元胞突主干和分枝明顯延長形成稠密的網(wǎng)絡(luò),神經(jīng)膠質(zhì)細(xì)胞數(shù)量則不斷增長。原代神經(jīng)細(xì)胞可在體外維持培養(yǎng)14～30天以上。經(jīng)過搭載的骨髓間充質(zhì)干細(xì)胞移植入神經(jīng)細(xì)胞培養(yǎng)體系以后,倒置相差顯微鏡下觀察可見二者能夠共同生長、繁殖。 結(jié)論:骨髓間充質(zhì)干細(xì)胞經(jīng)過明膠海綿搭載后,細(xì)胞的貼壁、存活未受到明顯影響。其低致瘤性的增殖特性以及可分化為神經(jīng)細(xì)胞的特性也未有明顯改變。經(jīng)明膠海綿搭載移植后骨髓間充質(zhì)干細(xì)胞可以與神經(jīng)細(xì)胞共存。
[Abstract]:Part one: isolation, culture, identification and characterization of bone marrow mesenchymal stem cells
Objective: through this experiment, the bone marrow mesenchymal stem cells were isolated, cultured, identified and studied on their own characteristics. At the same time, the cells were provided for the next step of Gelfoam carrier carrying transplantation.
Methods: first, the bone marrow mesenchymal stem cells were isolated and purified from the bone marrow of rats by differential adherence culture. Then, the surface markers of bone marrow mesenchymal stem cells were identified by flow cytometry. After identifying the bone marrow mesenchymal stem cells, the C6 cell line was used as the control to draw their different algebra. The growth curve of the period was used to study the tumorigenicity of the bone marrow mesenchymal stem cells. The characteristics of the spontaneous migration of bone marrow mesenchymal stem cells to the nerve cells under certain conditions were studied. The characteristics of the directional migration of bone marrow mesenchymal stem cells to the damaged nerve tissue were studied by Transwell method. Finally, the bone marrow mesenchymal stem cells were studied. A simple method of cryopreservation and resuscitation.
Results: the hybrid cells washed out from the marrow cavity of the rat were displayed on the inverted phase contrast microscope for a large number of small round cells with uniform distribution of a large number of single or mass groups.4 ~ 8 hours after the cell settlement and spread to the bottom of the culture dish for.24 hours, and observed that the large part of the cell spread at the bottom of the culture dish became flat and gradually extended out for.3 days. The adherent cells and other intramedullary substances were removed most of the fluid. After that, the adherent cells were continuously amplified, and a large number of cell lines were visible to 10~14 days, with different morphologies, such as large flattened, polygonal, star and spindle shape. Through the form and type of the passages, the cells gradually tended to be single, to the fifth generation. The cell size is the same, the shape is spindle shaped, and it has been basically purified. By flow cytometry, the expression surface markers CD31, CD34, CD45 are negative, and the expression surface marker CD71 is positive. The growth curve of bone marrow mesenchymal stem cells has obvious pressure compared with the C6 cell line at different times. Low. Through the observation of inverted phase contrast microscope and the detection of immunofluorescence, it was found that bone marrow mesenchymal stem cells can spontaneously differentiate into the neuro cell and express the nerve cell marker GFAP under certain conditions. Through the Transwell experiment, NSE. can observe that the rate of bone marrow mesenchymal stem cells in the injury group is obviously higher in the same time. The cross rate of bone marrow mesenchymal stem cells in the control group was significantly different by statistical P0.05. The bone marrow mesenchymal stem cells stored in the simple method can survive at least 3 months at -80 C, and can survive for at least half a year in liquid nitrogen. The survival of the bone marrow mesenchymal stem cells in the post Soviet Union reaches a certain amount and density, and these cells can still rapidly expand and obtain a large, stable, and available bone marrow mesenchymal stem cells in a relatively short period of time.
Conclusion: bone marrow mesenchymal stem cells can be cultured stably through the most commonly used differential adherent culture method. The fifth generation cells have reached the requirement of purification. Through the identification of cell surface markers, it can be determined that the bone marrow mesenchymal stem cells in the.10 generation of bone marrow mesenchymal stem cells are very small or not, and can be separated spontaneously. Neurons and glial cells, in addition, have a unique homing ability to migrate to damaged nerve tissue. Bone marrow mesenchymal stem cells can be preserved steadily for a long time through a simple cryopreservation and resuscitation method, which can provide reserve cells for subsequent applications.
The second part: preliminary study on the feasibility of bone marrow mesenchymal stem cells transplanted into nerve tissue via gelatin sponge carrier.
Objective: To investigate whether the survival rate of bone marrow mesenchymal stem cells (MSCs) is affected by Gelfoam carrier and their own characteristics, and to further study whether bone marrow mesenchymal stem cells can coexist with nerve cells after carrying Gelfoam carrier.
Methods: first, whether the morphologic changes of bone marrow mesenchymal stem cells (MSCs) were changed after the gelatin sponge was carried and the number of surviving bone marrow mesenchymal stem cells adhered by Gelfoam sponges was compared with that of unloaded cells. Then, the bone marrow mesenchymal stem cells without carrying fifth generations of bone marrow were plotted through the carrying bone. The growth curve of medullary mesenchymal stem cells and its tumorigenicity were studied. Then it was induced to differentiate into the nerve cell direction and observed the difference in the form after the induction of the bone marrow mesenchymal stem cells and the unloaded cells after the induction, and the difference between the two differentiated nerve elements. Finally, the extracorporeal nerve was established. Cell culture system was used to study whether the bone marrow mesenchymal stem cells loaded with gelatin sponge could co cultured with nerve cells by inverted phase contrast microscope.
Results: in the inverted phase contrast microscope, there was no obvious change in the morphology of the cells in the Gelfoam group and the control group, and there was a long spindle shape, and the flat aging phenomenon was rare. The bone marrow mesenchymal stem cells were adhered to the middle or edge of the gelatin sponge, and the number of cells attached to the middle of the gelfoam sponge was more. There was no statistical difference between the number of unadhered cells in the gelatin sponge group and the control group P0.05. There was no significant difference in the growth curve of the fifth generation bone marrow mesenchymal stem cells after carrying on the fifth generation cells. The bone marrow mesenchymal stem cells could be observed after 6~8 hours of induction. The cell bodies began to contract and protruded, and some of the protuberances were connected to form a network structure. By immunofluorescent detection, the expression of glial cell marker GFAP and neuron cell marker NSE. was compared with the control group by flow cytometry, and there was no significant difference between the expression of NSE and the control group by statistical P0.05. Most of the neurons could be adhered to the wall and protruded after 12~24 hours after the nerve cells were planted. With the prolongation of the incubation time, the main stem and branches of the neurons were prolonged to form a dense network, and the number of glial cells increased continuously. The primary nerve cells could be cultured outside the body for more than 14~30 days. After transplantation of bone marrow mesenchymal stem cells into nerve cell culture system, the inverted phase contrast microscope showed that the two could grow together and reproduce.
Conclusion: after the bone marrow mesenchymal stem cells are loaded with gelatin sponge, the cell adhesion and survival have not been significantly affected. The characteristics of its low tumorigenicity and differentiation into neural cells have not changed obviously. The bone marrow mesenchymal stem cells can coexist with the nerve cells after the transplantation of gelfoam.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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