【摘要】：近年來,干細(xì)胞由于其自我更新及多向分化潛能的雙重特性,在再生醫(yī)學(xué)、細(xì)胞替代治療及藥物篩選等領(lǐng)域顯示出了廣闊的應(yīng)用前景。并且隨著干細(xì)胞以及組織工程相關(guān)基礎(chǔ)及臨床研究的深入,已經(jīng)在一部分疾病中顯示出了良好的治療效果。但當(dāng)代醫(yī)學(xué)中,仍然存在著許多以目前醫(yī)療水平所不能徹底治愈的疾病,其中有一部分有關(guān)細(xì)胞、組織甚至器官的壞死如糖尿病、免疫功能障礙、阿爾茲海默病及帕金森等。干細(xì)胞的出現(xiàn)及逐漸深入研究,也為這些疾病的治愈帶來的嶄新的希望。目前干細(xì)胞的主要來源及潛在來源包括胚泡,胎兒組織,臍帶血及成人組織。在諸多種類干細(xì)胞的臨床試驗(yàn)及應(yīng)用中,造血干細(xì)胞移植的理論基礎(chǔ)和技術(shù)方法相對(duì)較成熟。造血干細(xì)胞移植目前對(duì)于大多數(shù)惡性血液系統(tǒng)腫瘤及一些免疫缺陷病是很有潛力的一種根治性治療方法,在臨床上已有廣泛的應(yīng)用。造血干細(xì)胞移植主要包括自體移植和異體移植兩種方式。在造血干細(xì)胞的來源方面,過去比較成熟理想的來源為骨髓及外周血,但其來自于成體因而干性較弱。研究者們一直在堅(jiān)持不懈的尋找更為理想并容易獲得的細(xì)胞來源。20世紀(jì)80年代起,臍血因其豐富的造血干細(xì)胞含量吸引了研究者們的注意。臍血來源干細(xì)胞在臨床上的應(yīng)用迅速發(fā)展,在造血重建及免疫系統(tǒng)重建方面有較理想的效果,擴(kuò)展了造血干細(xì)胞的來源。臍血干細(xì)胞來源豐富且免疫原性較低,但單份臍血含的細(xì)胞數(shù)量較少限制了其臨床應(yīng)用范疇。因此,找到更加廣泛,更加適合的細(xì)胞來源成為造血干細(xì)胞移植臨床應(yīng)用的突破口。胎盤為圓盤形,嵌于子宮壁中,是孕期支持胎兒和母體之間進(jìn)行各種物質(zhì)交換并兼具內(nèi)分泌等功能的器官,由底蛻膜、葉狀絨毛膜和羊膜三部分組成。通常,胎盤屬于分娩過程中所產(chǎn)生的廢棄物,然而近年來由于許多研究發(fā)現(xiàn)多種干、祖細(xì)胞存在于胎盤中,即胎盤本身便是一個(gè)含量豐富的干、祖細(xì)胞庫,因而受到越來越多的關(guān)注和探索。大量細(xì)胞組群的存在顯示出其在未來再生醫(yī)學(xué)臨床應(yīng)用中的巨大潛能。研究顯示胎盤中含有許多不同種類的干細(xì)胞,近些年來,諸多研究的主要方向及重點(diǎn)針對(duì)于其所含間充質(zhì)干細(xì)胞方面,對(duì)于造血干細(xì)胞相關(guān)的發(fā)掘研究較少�；谝延械膶�(shí)驗(yàn)研究及相關(guān)結(jié)果,我們可以推斷,對(duì)胎盤來源的細(xì)胞加以更為深入和細(xì)致的研究,充分了解其生物學(xué)特性,對(duì)于選擇和促進(jìn)其盡早應(yīng)用于臨床有著至關(guān)重要的作用,可以解決臨床多種相關(guān)疾病干細(xì)胞來源短缺或不合的問題。并且胎盤來源的細(xì)胞較體細(xì)胞而言,更為原始干性更強(qiáng),可能獲得更優(yōu)的臨床療效。本研究的主要目的即為檢測(cè)胎盤中單個(gè)核細(xì)胞總數(shù)以及CD34+細(xì)胞數(shù)量并與單份臍血中細(xì)胞數(shù)量做比較,觀察胎盤及臍血來源細(xì)胞的集落形成情況,分別檢測(cè)胎盤來源細(xì)胞、母血來源細(xì)胞和臍血來源細(xì)胞的HLA型別,應(yīng)用STR-PCR技術(shù)檢測(cè)和探索胎盤來源細(xì)胞與母血、臍血細(xì)胞的嵌合程度,為今后應(yīng)用于臨床提供相關(guān)理論基礎(chǔ)。本研究主要分以下兩部分內(nèi)容:1.胎盤和臍血中細(xì)胞總數(shù)及其中CD34+細(xì)胞所占比例研究。胎盤及臍血來源細(xì)胞在體外接種培養(yǎng)的集落形成情況。在產(chǎn)婦及其家屬知情同意并簽署知情同意書的前提下,在研究獲得解放軍307醫(yī)院倫理委員會(huì)審批的情況下,我們收集了五位健康足月剖宮產(chǎn)產(chǎn)婦的臍血及其對(duì)應(yīng)胎盤。分娩過程中將臍血抽出置入一次性塑料血袋暫時(shí)保存,取回后離心并取下層細(xì)胞計(jì)數(shù)。胎盤用生理鹽水反復(fù)沖洗以去除其表面殘存血液及雜質(zhì),用注射器推注并收集胎盤脈管中的血液,應(yīng)用含有10g/L AMD3100的生理鹽水灌注胎盤脈管系統(tǒng),夾閉臍動(dòng)脈并孵育30分鐘,收集灌注液,胎盤脈管中血液及灌注液離心并取下層細(xì)胞計(jì)數(shù)。胎盤剩余組織部分用剪切加酶消化法、研磨法處理,由100目鋼網(wǎng)過濾,反復(fù)沖洗離心后計(jì)細(xì)胞數(shù)。將胎盤脈管血液、灌注液及組織來源的細(xì)胞計(jì)總數(shù)。用流式細(xì)胞儀檢測(cè)臍血來源細(xì)胞及胎盤來源細(xì)胞中CD34+細(xì)胞的數(shù)量及所占比例。以5×104將臍血來源及胎盤來源細(xì)胞分別添加進(jìn)1.1m L培養(yǎng)基中并接種于培養(yǎng)皿,于含5%CO2的培養(yǎng)箱中以37℃培養(yǎng)并定期觀察。實(shí)驗(yàn)結(jié)果顯示,胎盤中所獲取的細(xì)胞總數(shù)、CD34+細(xì)胞數(shù)及其所占比例均明顯高于從臍血所獲取的細(xì)胞數(shù)。胎盤和臍血的集落培養(yǎng)皿均在接種后第六天可以觀察到集落的出現(xiàn),第十四天可以觀察到集落形成良好。但與臍血細(xì)胞相同的接種密度下,胎盤來源細(xì)胞所形成的集落數(shù)量較少。胎盤中所含細(xì)胞數(shù)量上明顯的優(yōu)勢(shì)提示我們,胎盤可能是未來臨床干細(xì)胞移植治療各類疾病很有潛力的來源。集落形成實(shí)驗(yàn)部分顯示胎盤來源細(xì)胞具有多種集落形成的能力,而集落形成能力是造血干祖細(xì)胞分化以及增殖潛能的一項(xiàng)重要評(píng)估內(nèi)容。2.胎盤來源細(xì)胞的HLA檢測(cè)及其與母血、臍血來源細(xì)胞嵌合的檢測(cè)。于產(chǎn)婦進(jìn)入產(chǎn)房前自貴要靜脈抽取外周血5m L,離心后取下層細(xì)胞。分別檢測(cè)胎盤來源細(xì)胞、母血細(xì)胞及臍血細(xì)胞的HLA-A、HLA-B、HLA-C、HLA-DRB1、HLA-DQB1位點(diǎn)。HLA檢測(cè)結(jié)果顯示本研究五例樣本中有兩例胎盤細(xì)胞HLA位點(diǎn)檢測(cè)提示其含有母體成分,其余三例結(jié)果顯示胎盤來源細(xì)胞與臍血細(xì)胞位點(diǎn)完全相合。STR-PCR方法檢測(cè)嵌合率的結(jié)果顯示五例胎盤樣本中均有母體成分嵌合,而HLA檢測(cè)提示有母體成分的兩例母體成分嵌合率尤其高。因此我們推斷,HLA檢測(cè)結(jié)果不同可能與其檢測(cè)靈敏度及胎盤組織根據(jù)解剖結(jié)構(gòu)肉眼分離有偏差相關(guān),但與STR檢測(cè)結(jié)果綜合考慮提示,就目前的分離方法,胎盤中確實(shí)混有母體來源的成分,而其是否對(duì)臨床移植的效果產(chǎn)生影響,還需要進(jìn)一步的研究。本研究的創(chuàng)新性在于系統(tǒng)并完整的將臍血、胎盤和母血來源細(xì)胞以同樣的檢測(cè)方法檢測(cè),并得出可以對(duì)比并有一定意義的檢測(cè)結(jié)果。同時(shí),將HLA檢測(cè)和STR-PCR檢測(cè)方法共同應(yīng)用于檢測(cè)胎盤細(xì)胞來源及其與母體成分和胎兒成分嵌合的探索研究中,更加精準(zhǔn)的檢測(cè)出微小的嵌合比率,為潛在的臨床應(yīng)用可行性及方法的選擇及奠定了基礎(chǔ)。
[Abstract]:In recent years, stem cells have shown broad application prospects in regenerative medicine, cell replacement therapy and drug screening due to their dual characteristics of self-renewal and multi-directional differentiation potential. But in modern medicine, there are still many diseases which can not be cured completely by the current medical level. Some of them are related to the necrosis of cells, tissues and even organs, such as diabetes, immune dysfunction, Alzheimer's disease and Parkinson's disease. The emergence and gradual in-depth study of stem cells also bring about the cure of these diseases. The main sources and potential sources of stem cells include blastocysts, fetal tissues, umbilical cord blood and adult tissues. In clinical trials and applications of many types of stem cells, the theoretical basis and technical methods of hematopoietic stem cell transplantation are relatively mature. Hematopoietic stem cell transplantation is currently used in most malignant hematological malignancies. Hematopoietic stem cell transplantation mainly includes autologous transplantation and allogeneic transplantation. In the past, the ideal source of hematopoietic stem cells was bone marrow and peripheral blood, but it came from adult and therefore dry. Since the 1980s, umbilical cord blood has attracted the attention of researchers because of its abundant hematopoietic stem cell content. The clinical application of umbilical cord blood-derived stem cells has developed rapidly, and they are ideal for hematopoietic reconstruction and immune system reconstruction. Umbilical cord blood stem cells have abundant sources and low immunogenicity, but the small number of cells contained in a single umbilical cord blood limits the scope of its clinical application. Therefore, to find a wider and more suitable cell source becomes a breakthrough in the clinical application of hematopoietic stem cell transplantation. The placenta is usually a waste produced during childbirth. However, in recent years, many studies have found that many kinds of stem, progenitor cells exist in the placenta, that is, the placenta. As a rich stem-progenitor cell bank, it has attracted more and more attention and exploration. The existence of a large number of cell groups shows its great potential in the future clinical application of regenerative medicine. Based on the existing experimental studies and related results, we can infer that more in-depth and detailed study of placental-derived cells and a full understanding of their biological characteristics are crucial to the selection and promotion of their early clinical application. The main purpose of this study is to detect the total number of mononuclear cells in the placenta and the number of CD34 + cells in the placenta and to compare them with a single umbilical cord. The number of cells in blood was compared, the colony formation of cells from placenta and umbilical cord blood was observed, the HLA types of placenta-derived cells, maternal blood-derived cells and umbilical cord blood-derived cells were detected respectively, and the degree of chimerism between placenta-derived cells and maternal blood and umbilical cord blood cells was detected by STR-PCR, which provided relevant theory for clinical application in the future. This study is divided into the following two parts: 1. The total number of cells in placenta and umbilical cord blood and the proportion of CD34 + cells in them. With the approval of the committee, we collected the umbilical cord blood and its placenta from five healthy full-term cesarean mothers. During delivery, the umbilical cord blood was extracted and placed in a disposable plastic blood bag for temporary preservation, centrifuged and counted the cells in the lower layer. Push and collect the blood in the placental vein, perfuse the placental vein system with saline containing 10g/L AMD3100, clamp the umbilical artery and incubate for 30 minutes, collect the perfusion fluid, centrifuge the blood and perfusion fluid in the placental vein and count the cells in the lower layer. The number and proportion of CD34 + cells in cord blood and placenta derived cells were measured by flow cytometry. The cord blood and placenta derived cells were added into 1.1mL medium by 5 *104 and inoculated into 1.1mL medium respectively. The results showed that the total number of cells, the number of CD34 + cells and their proportion in placenta were significantly higher than those in umbilical cord blood. Colony culture dishes of placenta and umbilical cord blood were observed on day 6 and day 14 after inoculation. Good colony formation can be observed. However, at the same inoculation density as umbilical cord blood cells, the number of colonies formed by placental-derived cells is relatively small. Placenta-derived cells have a variety of colony-forming abilities, and colony-forming ability is an important assessment of the differentiation and proliferation potential of hematopoietic stem-progenitor cells. 2. HLA detection of placenta-derived cells and detection of their chimerism with maternal and umbilical cord blood-derived cells. The HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci of placenta-derived cells, maternal blood cells and umbilical cord blood cells were detected respectively. The results of HLA detection showed that two of the five samples in this study contained maternal components, and the other three showed that placenta-derived cells and umbilical cord blood cell loci were identical. The results of TR-PCR showed that all five placenta samples had maternal component chimerism, and HLA detection suggested that the maternal component chimerism rate was especially high in two cases with maternal component. The results suggest that the current isolation methods do mix maternal-derived components in the placenta, and further research is needed to determine whether the maternal-derived components have an impact on clinical transplantation outcomes. At the same time, HLA and STR-PCR were used to detect the origin of placental cells and their chimerism with maternal and fetal components, and the small chimerism ratio was detected more accurately, which laid a foundation for the potential clinical application feasibility and method selection. Foundation.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R457.7

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