【摘要】：在胚胎的發(fā)育過程中,第一個造血干細胞(hematopoietic stem cells, HSC)產生于胚內的主動脈-性腺-中腎區(qū)(aorta-gonads-mesonephros region,AGM區(qū)),稍后出現(xiàn)在胎盤和卵黃囊,而后遷移到胎肝。出生后骨髓代替胎肝成為造血活動的主要場所。這些不同的造血微環(huán)境為HSC的產生、成熟、擴增和分化提供合適的調控信號。由于體外的培養(yǎng)條件不足以模擬體內復雜的微環(huán)境,HSC在體外容易失去“干性”。組織培養(yǎng)(tissue culture)的方法可保留組織內部三維的、更接近生理狀態(tài)的微環(huán)境,在體外可顯著促進HSC的成熟和增殖。在小鼠體節(jié)期胚胎,HSC在不同的時間和位點發(fā)生。在發(fā)育早期(胚胎期10.5-11.5天),HSC數(shù)目極少(約1-3個/胚胎),且無特異性的表面標志,這為探討HSC的發(fā)育調控機制增加了難度。因此,為了分離和純化HSC、明確HSC的具體定位、揭示其發(fā)育規(guī)律,發(fā)現(xiàn)和鑒定新的造血和/或HSC的特異性標記顯得尤為重要。 本研究的第一部分,我們通過建立特殊的組織培養(yǎng)方法,探討小鼠胚胎AGM區(qū)的造血發(fā)育規(guī)律。選取小鼠E10.5-E11胚胎的AGM區(qū)作為實驗對象,建立氣液交界的組織培養(yǎng)體系,孵育2天后作如下三類分析:應用集落形成實驗在體外觀察髓系祖細胞的數(shù)量,應用脾結節(jié)形成(colony-forming unit of spleen, CFU-S)實驗在體內檢測髓系祖細胞的變化,利用移植(長期重建)實驗考察HSC的發(fā)育。結果表明:組織培養(yǎng)后,平均每個E10.5 AGM區(qū)的細胞可形成104±18個髓系集落;每個E10.5 AGM區(qū)的細胞在致死劑量照射的成年小鼠脾臟中可形成10.8±3.5個CFU-S。更重要的是,經組織培養(yǎng)的E10.5-E11.5 AGM區(qū)能高比例(85.7%)、高嵌合(51.12%±21.17%)、長期(4個月)重建致死劑量照射的成年小鼠的造血系統(tǒng),在受體的外周血、骨髓、脾臟、胸腺中均能檢測到供體來源的淋系或髓系細胞的嵌合。上述數(shù)據(jù)與經典文獻結果相似,證實:體外組織培養(yǎng)確能促進小鼠AGM區(qū)HSC的成熟和擴增,是研究胚胎造血發(fā)育規(guī)律的有效方法。 本研究的第二部分,借助于建立的組織培養(yǎng)和移植體系,我們集中研究CD43在不同發(fā)育時間和位點的HSC的表達特點。CD43,亦稱leukosialin或sialophorin,與CD34、Endomucin等分子都屬于內皮唾液黏蛋白質家族成員,具有完整的細胞膜表面黏蛋白。我們的研究立題主要基于如下幾點:既往研究表明CD43在人和小鼠的骨髓HSC高表達;人胚胎干細胞和誘導多能干細胞來源的造血祖細胞表達CD43;其表達于人胚胎背主動脈腹側造血簇(富集造血干祖細胞的特殊細胞群)。此外,CD43與小鼠胚胎發(fā)育過程中重要的HSC表面標志CD34在結構上相似,兩者標志意義的異同亦是我們感興趣的問題。 為明確CD43在AGM區(qū)HSC中的表達,我們將E10.5、E11.5、E12.5 AGM區(qū)消化成單細胞懸液,利用免疫磁珠分選,獲得CD43+細胞和CD43-細胞,分別按照1-2個胚胎量移植入60Co致死劑量(9 Gy)照射的成年受體小鼠。移植后4個月對外周血進行檢測,計算(GFP+CD45+)/CD45+的比值得到嵌合率,≥10%則視為重建。結果發(fā)現(xiàn)E10.5、E11.5、E12.5 AGM區(qū)CD43+細胞的重建比例分別為2/15、6/12、4/4。相反,所有的CD43-細胞移植受體均未發(fā)現(xiàn)重建(n=26)。受體的外周血、骨髓、脾臟、胸腺中含有供體來源的相當比例的髓系、B淋巴細胞、T淋巴細胞,表明AGM區(qū)CD43+細胞具有多系分化潛能。同時,通過二次移植實驗,證實AGM區(qū)CD43+細胞具有自我更新能力。既往研究表明AGM區(qū)的HSC存在于c-Kit+CD34+細胞中。進一步的流式分析發(fā)現(xiàn)CD43在E11.5AGM的該群細胞中的表達比例為16.2%-22.3%,提示其具有潛在的富集意義。上述數(shù)據(jù)表明:小鼠胚胎AGM區(qū)的HSC均分布在CD43+亞群。 既往研究發(fā)現(xiàn):E11.5 AGM區(qū)經體外組織培養(yǎng)后,HSC的免疫表型發(fā)生變化,從CD34+轉變成CD34-,與成體骨髓HSC相同。為明確CD43是否有類似特點,我們將AGM區(qū)(E10.5-E11.5)進行2天的組織培養(yǎng),磁珠分選獲得CD43+細胞和CD43-細胞,而后移植入致死劑量照射的受體小鼠。結果發(fā)現(xiàn):E10.5、E11.5 AGM區(qū)CD43+細胞的重建比例分別為4/4和6/6,而CD43-細胞均不能重建(n=7)。重建受體的外周血、骨髓、脾臟、胸腺中含有供體來源的相當比例的髓系或淋系細胞,表明經組織培養(yǎng)的AGM區(qū)CD43+細胞仍然具有多系分化潛能。上述數(shù)據(jù)表明:組織培養(yǎng)能促進AGM區(qū)CD43+HSC的擴增;與CD34不同,在HSC的體外成熟和增殖過程中,CD43持續(xù)表達。 近年來,多項研究表明:與AGM區(qū)和卵黃囊相似,胎盤也是HSC發(fā)育的另一重要位點。為進一步明確CD43在胎盤和卵黃囊來源的HSC中的表達,我們分別將E11.5胎盤和卵黃囊的CD43+細胞和CD43-細胞移植入致死劑量照射的受體小鼠。結果發(fā)現(xiàn)胎盤CD43+細胞重建比例為7/7,卵黃囊為5/8,而CD43-細胞均重建失敗(n=13)。此外,E11.5胎盤和卵黃囊的CD43+細胞亦具有多系分化潛能。既往研究表明胎盤和卵黃囊中的HSC存在于c-Kit+CD34+細胞中,我們的流式分析發(fā)現(xiàn)E11.5胎盤和卵黃囊的該群雙陽性細胞中分別有21.2%-33.7%和14.9%-24.0%表達CD43,提示可通過CD43進一步富集上述組織中的HSC。以上數(shù)據(jù)表明胎盤和卵黃囊中的HSC均表達CD43。 胚胎造血時期,AGM區(qū)、胎盤、卵黃囊來源的HSC可通過血液循環(huán)定居于胎肝,后者成為最重要的HSC擴增位點。為了明確胎肝中的HSC是否表達CD43,我們將E12.5胎肝的CD43+細胞和CD43-細胞移植入致死劑量照射的受體小鼠。結果發(fā)現(xiàn)只有胎肝CD43+細胞能重建成體造血(重建比例為5/5),且具有造血多系分化潛能。之前研究表明CD45在E11.5以后開始在HSC上表達。為進一步確定CD43在不同發(fā)育階段胎肝HSC中的表達,我們利用CD43和CD45抗體標記E13.5和E16.5的胎肝細胞,經流式分選獲得CD45+CD43high、CD45+CD43mid和CD45+CD43-三群細胞。將其移植后發(fā)現(xiàn)僅CD45+CD43high細胞能重建成體造血(E13.5和E16.5的重建比例分別為3/3和6/7),該現(xiàn)象與成體骨髓HSC集中于CD43high亞群相似。與之吻合的是,流式分析發(fā)現(xiàn):富集胎肝HSC的亞群,即Lin-Sca-1+c-Kit+細胞中,CD43的陽性比例95%。 總結:既往認為CD43是一個類似于CD45的造血標志,而本研究將其表達與早期的造血發(fā)生緊密的關聯(lián)起來。作為一個新的胚胎造血發(fā)育時期的特異性標記,CD43的獨特性表現(xiàn)為:持續(xù)表達于HSC發(fā)育的各個重要階段(出現(xiàn)、成熟、擴增)、各個造血組織(AGM區(qū)、胎盤、卵黃囊、胎肝)。因此,CD43的標志作用為今后研究基因敲除小鼠的胚胎造血活動提供了一個靈敏的標識,其生理功能亦待進一步深入研究。
[Abstract]:In the development of embryo, the first hematopoietic stem cell (hematopoietic stem cells, HSC) is produced in the aorta sex gland and the middle kidney region (aorta-gonads-mesonephros region, AGM region) in the embryo, and later appears in the placenta and the yolk sac and then migrates to the fetal liver. The hematopoietic microenvironment is the production of HSC, mature, amplified and differentiated to provide appropriate regulatory signals. Because the culture conditions in vitro are not sufficient to simulate the complex microenvironment in the body, HSC is easy to lose the "dry" in vitro. The method of tissue culture (tissue culture) can retain the three-dimensional inner part of the tissue, which is closer to the physiological state, and in vitro It can significantly promote the maturation and proliferation of HSC. In mice, HSC occurs at different time and loci. In early development (10.5-11.5 days of embryo stage), the number of HSC is very few (about 1-3 / embryos), and there is no specific surface marker. This makes it difficult to explore the development and regulation mechanism of HSC. Therefore, to separate and purify HSC and clarify HSC It is particularly important to find out and identify new hematopoietic and / or HSC specific markers.
In the first part of this study, we explored the regulation of hematopoiesis in the AGM region of mouse embryos by establishing a special tissue culture method. The AGM area of E10.5-E11 embryos of mice was selected as the experimental object, and the tissue culture system of the boundary of gas and liquid was established. After 2 days of incubation, the following three types of analysis were made: using colony formation experiment to observe the medullary lineage in vitro The number of colony-forming unit of spleen (CFU-S) was used to detect the changes of myeloid progenitor cells in the body. The development of HSC was investigated by transplantation (long term reconstruction). The results showed that after tissue culture, the cells of each E10.5 AGM area could form 104 + 18 medullary colony, and the cells in each E10.5 AGM region were lethal. In the spleen of adult mice irradiated with dose, 10.8 + 3.5 CFU-S. are more important, and the hematopoietic system of adult mice with high proportion (85.7%), high chimerism (51.12% + 21.17%) and long-term (4 months) reconstruction of lethal dose in the tissue culture of the adult mice can be detected in the peripheral blood, bone marrow, spleen and thymus of the recipient. The above data are similar to those of classical literature. It is proved that tissue culture in vitro can promote the maturation and amplification of HSC in AGM region of mice, and is an effective method to study the rules of embryo hematopoiesis.
In the second part of this study, with the aid of the established tissue culture and transplantation system, we focus on the HSC expression of CD43 at different developmental time and loci.CD43, also known as leukosialin or sialophorin, and CD34, Endomucin and other molecules are members of the endothelial salivary mucin family, with a complete cell membrane surface mucin. The main points of our research are based on the following points: Previous studies have shown that CD43 is highly expressed in human and mouse bone marrow HSC; human embryonic stem cells and hematopoietic progenitors that induce pluripotent stem cells express CD43; they are expressed in the ventral hemopoietic cluster of the human embryonic dorsal aorta (a special cell group enriched with hematopoietic progenitor cells). In addition, CD43 and mouse embryos are also expressed. The important HSC surface marker CD34 is similar in structure. The similarities and differences between the two markers are also interesting issues.
To clarify the expression of CD43 in the AGM region HSC, we digested E10.5, E11.5 and E12.5 AGM into single cell suspension, and obtained CD43+ cells and CD43- cells by immunomagnetic beads, and transplanted into the adult receptor mice irradiated by 60Co lethal dose (9 Gy) according to 1-2 embryos, respectively. After 4 months of transplantation, the peripheral blood was detected and calculated (GFP+CD45+) The ratio of D45+ was found to be chimerism, and more than 10% was regarded as reconstruction. The results showed that the reconstruction ratio of CD43+ cells in E10.5, E11.5, E12.5 AGM region was 2/15,6/12,4/4., and all CD43- cell transplantation recipients were not found to reconstruct (n=26). The peripheral blood of the receptor, bone marrow, spleen, and chest gland contained a considerable proportion of myeloid, B lymphocytes, T lymphocyte, indicating that CD43+ cells in AGM region have multiple differentiation potential. At the same time, two transplanting experiments confirmed that CD43+ cells in AGM region have self-renewal ability. Previous studies showed that HSC in AGM region existed in c-Kit+CD34+ cells. Further flow analysis showed that the proportion of CD43 in this group of E11.5AGM was 16.2%-22.3%. These data indicate that HSC in AGM region of mouse embryos is distributed in CD43+ subsets.
Previous studies have found that the immunophenotype of the E11.5 AGM region changes after the culture in vitro, and the immunophenotype of HSC changes from CD34+ to CD34- and is the same as that of adult bone marrow HSC. In order to clarify whether there is a similar characteristic of CD43, we carry out AGM region (E10.5-E11.5) for 2 days of tissue culture, and obtain CD43+ cells and CD43- cells from magnetic beads, and then transplant to lethal dose illumination. The results showed that the reconstruction ratio of CD43+ cells in E10.5, E11.5 AGM region was 4/4 and 6/6 respectively, while CD43- cells were not rebuilt (n=7). The peripheral blood, bone marrow, spleen, and thymus contained a considerable proportion of myeloid or lymphoid cells in the rebuilt receptor, indicating that the tissue culture of AGM region CD43+ cells still had multiple lines. The above data indicate that tissue culture can promote the amplification of CD43+HSC in AGM region. Unlike CD34, CD43 is continuously expressed during HSC maturation and proliferation in vitro.
In recent years, many studies have shown that the placenta is another important site for the development of HSC, similar to the AGM region and the yolk sac. In order to further clarify the expression of CD43 in the HSC from the source of the placenta and the yolk sac, we transplanted the CD43+ and CD43- cells of the E11.5 placenta and the yolk sac into the receptor mice irradiated at the dead dose, and found the placental CD43. The reconstructive ratio of + cells was 7/7, the yolk sac was 5/8, and the CD43- cells were rebuilt (n=13). In addition, the CD43+ cells of the E11.5 placenta and the yolk sac also had multiple differentiation potential. Previous studies showed that the HSC in the placenta and the yolk sac was found in c-Kit+CD34+ cells. Our flow cytometry found the group of double positive cells in the E11.5 placenta and the yolk sac. 21.2%-33.7% and 14.9%-24.0% expressed CD43, respectively, suggesting that CD43 could be further enriched by CD43 in the above tissue, indicating that HSC in the placenta and yolk sac expressed CD43.
In the embryonic period of hematopoiesis, the AGM region, the placenta, and the yolk sac derived from the HSC can be settled in the fetal liver through the blood circulation, and the latter becomes the most important HSC amplification site. In order to determine whether the HSC in the fetal liver is CD43, we transplant CD43+ cells and CD43- cells from the E12.5 fetal liver to the lethal dose irradiated recipient mice. The result is that only the fetal liver CD43+ is fine. The cell can reconstruct the adult hematopoiesis (reconstructive ratio of 5/5) and have the potential of hematopoietic pluripotent differentiation. Previous studies showed that CD45 began to express on HSC after E11.5. To further determine the expression of CD43 in the HSC of fetal liver at different developmental stages, we use CD43 and CD45 antibodies to mark the fetal liver cells of E13.5 and E16.5 and obtain CD45+CD43hig by flow sorting. H, CD45+CD43mid and CD45+CD43- three cells. After transplantation, it was found that only CD45+CD43high cells could reconstruct adult hematopoiesis (the proportion of E13.5 and E16.5 reconstruction was 3/3 and 6/7). This phenomenon was similar to that of adult marrow HSC concentrated in the CD43high subgroup. In the case of CD43, the positive ratio of 95%.
Summary: CD43 was previously thought to be a hematopoiesis marker similar to CD45, and this study associated its expression closely with early hematopoiesis. As a specific marker for a new period of embryonic hematopoiesis, the uniqueness of CD43 is expressed continuously in all important stages of the development of HSC (emergence, maturation, amplification), and every hematopoiesis Tissue (AGM region, placenta, yolk sac, fetal liver). Therefore, the marker function of CD43 provides a sensitive marker for the future study of embryo hematopoiesis in gene knockout mice, and its physiological function should be further studied.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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