【作者】 魏斌；

【导师】 高英茂；

【作者基本信息】 山东大学 ， 人体解剖与组织胚胎学， 2008， 博士

【摘要】 血细胞的发生指的是由造血组织中的干细胞(hematopoietic stem cell,HSC)经增殖、分化直至成熟而形成各种血细胞的过程。所谓造血干细胞,是指存在于造血组织内的一类能分化发育成各种血细胞的原始细胞。现在一般认为,在造血组织中存在一类能生成各种血细胞的最原始的细胞,称之为全能造血干细胞(totipotential hematopoietic stem cell,THSC)。THSC在一定的造血微环境和某些因素的诱导下可增殖分化为多能淋巴干细胞(pludpotential lymphoid stem cell)和多能髓性造血干细胞(pluripotential myeloid stem cell,PMSC或CFU-S)。前者可进一步分化、发育成功能性的淋巴细胞,后者可发育成粒细胞巨噬细胞系、红细胞系、巨核细胞系等造血祖细胞(hematopoietic progenitor cell,HPC),并进一步发育为白细胞、红细胞和血小板等。因而,从造血细胞发生的角度可把造血细胞简要分为三大部分,即造血干细胞、造血祖细胞和可辨认的前体细胞(recognized precursor)。造血的过程就是造血干细胞、祖细胞增殖分化形成血细胞的动态平衡过程,但不包括成熟细胞在体内的储存、释放以及分配的过程。造血活动以造血干细胞及造血祖细胞的活动为主。在胚胎造血系统的发育过程中,首先由中胚层的间充质组织分化为血液成血管细胞(hemangioblast),进而形成HSC和成血管细胞(angioblast)。HSC首先发生的部位见于胚外卵黄囊血岛和胚内主动脉旁脏壁层/主动脉-性腺-中肾区(paraaortic splanchnopleure/aortagonad mesonephros,PAS/AGM)。随着胚体内外血循环的建立,HSC被播散在肝脏和脾脏,最终主要定居在骨髓,维持机体终身造血。胚胎造血发育的研究迄今已有百余年历史,在哺乳动物的胚胎中,卵黄囊,肝脏,胸腺,脾脏和骨髓都已经被看作是具有造血功能的器官。近年来,有些研究认为,胎盘也是一个造血器官,在胚胎期发挥着重要作用。但是目前就胎盘是否具有造血功能仍有激烈争论。本实验应用多种实验方法,检测了胎盘的造血功能,力图进一步明确胎盘是否存在造血功能和胎盘造血的生物学特点。第一部分小鼠胎盘组织中单个核细胞的分离、鉴定和胎盘中固有造血干/祖细胞(HS/PCs)的确认【研究目的】从小鼠胎盘中去除胎盘中所含有的胎儿血和母体血,分离和检测胎盘组织中固有的HS/PCs,探讨小鼠胎盘是否具有造血功能。【研究方法】1、取孕12.5天小鼠,0.2ml水合氯醛腹腔注射麻醉,无菌条件下取出孕子宫,剥离子宫,完整取出胎鼠及其胎盘。在体视镜下观察,通过血流方向辨别脐动脉和脐静脉。将注射器(BD 29 G)针头朝向胎盘方向插入脐动脉,缓慢的将冲洗液肝素生理盐水(0.5mg/ml,290000IU/ml)注入脐动脉。边注射边观察,直至脐静脉中无血细胞回流,脐静脉中的液体变澄清,于是拔出注射器,终止注射。将冲出的胎盘内的胎儿血细胞收集,作为对照。然后将胎盘从母体蜕膜、脐血管及卵黄囊残迹上剥离,彻底冲洗胎盘中的母体血,直至胎盘变成白色。2、将冲洗后的胎盘作石蜡切片,苏木素伊红染色,观察胎盘组织中是否残留血细胞。3、将冲洗后的胎盘充分剪碎,加入0.1%胶原酶Ⅳ在37℃下搅拌消化30min,终止消化,离心。然后再加入0.25%胰酶37℃孵育10min,终止消化,离心。加入1ml新鲜培养基(IMDM),做成细胞悬液,以备通过离心提取单个核细胞。4、将三种密度的Percoll梯度液(1.10g/ml,1.080g/ml,1.055g/ml)用长针头注射器按密度从高到低逐层轻轻放置到10ml离心管中。将细胞悬液轻轻置于最上层,以2000 rpm离心25min。用吸管将上层和中间层分离液之间的细胞带小心取出,再加入1ml新鲜培养液IMDM,制成单个核细胞悬液。5、制作细胞涂片,Giemsa染色。光学显微镜观察是否为单个核细胞。6、运用脾结节形成技术检测从胎盘组织中提取的单个核细胞是否为造血干细胞。【结果】1、冲洗后胎盘胎儿血管内均见不到胎儿血细胞,胎盘中的胎儿血已经被清除干净。2、采用组合酶消化法制备胎盘细胞悬液,Percoll密度梯度离心法分离的细胞,经细胞涂片Giemsa染色光镜观察,确认为单个核细胞,直径在7～10μm,形似小淋巴细胞。3、将分离后得到的单个核细胞经尾静脉输入致死剂量照射的小鼠,观察到受体鼠脾脏上出现大量脾结节。脾脏组织切片苏木素伊红染色光镜观察确认为脾结节形成单位,即造血集落。【结论】采用胎盘冲洗的方法可以将胎盘中的胎儿血细胞和母体血细胞清除干净,Percoll密度梯度离心法可有效分离胎盘中单个核细胞。脾结节实验显示,这些单个核细胞具有形成脾结节的能力,符合HS/PCs的特征,是胎盘中固有的HS/PCs。第二部分运用HS/PCs的表面标志确认胎盘固有HS/PCs的存在及其数量和分布【研究目的】运用免疫荧光技术和流式细胞术检测胎盘组织中单个核细胞的表面标志,以确认胎盘固有HS/PCs的存在及其数量和分布,从而进一步证明胎盘的造血功能。【研究方法】1、运用免疫荧光技术检测胎盘组织中的单个核细胞是否表达HS/PCs特异性抗原CD34、CD117和Sca-1,以便进一步确认胎盘组织中存在固有的HS/PCs。2、运用流式细胞术检测来自胎盘组织中的CD34、CD117和Sca-1阳性细胞的相对数和绝对数,以判断胎盘固有造血细胞的分化状况,并与来自胎盘胎儿血中的单个核细胞的相应检测结果比对。3、胎盘冰冻切片,CD34、CD117和Sca-1免疫组化染色,以观察胎盘固有HS/PCs在胎盘组织中的分布状况。【结果】1、免疫荧光染色显示胎盘组织中的单个核细胞表达HS/PCs特异性抗原CD34,CD117和Sca-1。CD34~+细胞百分率为20.1±5.3%,CD117~+细胞百分率为28.5±3.4%,Sca-1~+细胞百分率为35±8.6%。证明胎盘中的这些单个核细胞确实为HS/PCs。2、流式细胞术显示,胎盘HS/PCs表达HS/PCs特异性抗原CD34,CD117和Sca-1的三个细胞群所占的比例分别为20.2%,24.6%和26.2%。在来自胎盘胎儿血的单个核细胞中,这三个细胞群所占的比例分别为8.2%,6.3%和6.5%。胎盘的单个核细胞中三种阳性细胞的浓度均高于胎盘胎儿血的单个核细胞。胎盘的单个核细胞中,CD34~+、CD117~+、Sca-1~+阳性细胞的绝对数分别为1.4×10~4,1.8×10~4和1.8×10~4,在胎盘胎儿血单个核细胞中则分别为0.7×10~4,0.5×10~4和0.5×10~4。胎盘的单个核细胞中三种阳性细胞数均高于胎盘胎儿血。胎盘的单个核细胞中CD34~+/Sca-1~+细胞所占的比例为15.3%,要比胎盘胎儿血中的比例高,后者为5.1%。3、在E12.5的鼠胚胎盘中,胎盘迷路内可见CD34~+细胞、CD117~+细胞和Sca-1~+细胞,在绒毛膜板也可见CD117~+细胞,Sca-1表达量显著高于CD34和CD117。【结论】运用HS/PCs表面特异性抗原的免疫组化、免疫荧光染色和流式细胞技术,进一步证明了胎盘具有造血功能,胎盘组织中存在固有的HS/PCs,且具有一定的分布规律。第三部分胎盘单个核细胞的体外培养和造血调控基因表达的检测确认胎盘的造血功能【研究目的】通过对胎盘组织中单个核细胞的体外培养、造血集落形成和造血调控基因表达的观察和检测,进一步证明胎盘的造血功能。【研究方法】1、将胎盘单个核细胞接种在甲基纤维素半固体培养体系中进行培养。培养14d后观察是否形成造血集落,并确定集落的属性,检测高增殖潜能集落形成状况。取自胎盘胎儿血液的单个核细胞作上述同样检测,作为阳性对比。2、RT-PCR检测胎盘组织中单个核细胞Scl、Runx1、Tel/Etv6、GATA-2的表达状况。【结果】1、胎盘单个核细胞培养14天后,可见BFU-E、CFU-GM、CFU-GEMM的集落形成。胎盘胎儿血单个核细胞培养14天后,也可见BFU-E、CFU-GM、CFU-GEMM的集落生成,但前者的BFU-E、CFU-GM和CFU-GEMM集落数均高于后者,前者BFU-E的形态特点为多中心,高度血红蛋白化;后者BFU-E则为弱血红蛋白化。前者CFU-GM集落较致密,大小为中度或较大,有时集落巨大,好像是几个集落融合而成;后者CFU-GM集落则较小。两者的CFU-GEMM集落都很大,都可以用作为HPP-CFCs的来源。2、来自胎盘组织和来自胎盘胎儿血细胞的HPP-CFCs培养60天后,均形成直径超过0.5mm的集落。10~5个胎盘单个核细胞再植后可获得35个CFU-GEMM集落,胎盘胎儿血单个核细胞再植后也可以获得CFU-GEMM集落,但数量仅为胎盘细胞的2/3,而且胎盘胎儿血单个核细胞形成的集落要比胎盘单个核细胞形成的集落小。3、造血调控相关基因Scl、Runxl、Tel/Etv6、GATA-2在胎盘组织的单个核细胞上均有表达。【结论】胎盘组织中的单个核细胞在体外培养中可形成多种造血祖细胞克隆,并可检测到HPP-CFCs(体外培养的最原始的多潜能造血前体细胞之一)的活性,说明胎盘中的单个核细胞是HS/PCs,并有多向分化潜能。造血调控相关基因Scl、Runxl、Tel/Etv6、GATA-2在胎盘组织的单个核细胞上均有表达这进一步证明了小鼠胎盘具有造血功能。更多还原

【Abstract】 BackgroundHematopoiesis is a process that hematopoietic stem cells(HSCs) in the hematopoietic tissues expanse,differentiate,mature and give rise to all kinds of blood cells.The so-called HSCs are a type of primitive ceils which exist in the hematopoietic tissues, develop and differentiate into a variety of blood cells.They are also called totipotentiai hematopoietic stem cells(THSC).Under the induction of certain hematopoietic microenviroment and some factors,THSC can proliferate and differentiate into pluripotential lymphoid stem cells and pluripotential myeloid stem cell(PMDC).The former can differentiate and development into functional lymphocytes,while the latter can develop into CFU-GM,BFU-E,and CFU-GEMM, which will further give rise to white blood cells,red blood cells and platelets. Therefore,on the aspect of hematopoiesis,hematopoietic ceils can be typically classified into three types:hematopoietic stem cells,hematopoietic progenitor cells (HPC) and recognized precursors.The process of hematopoiesis is a dynamic balance of HSCs and HPCs expansion,differentiation and blood cells formation.However,the storage,release and distribution of mature blood cells in the body are not included in this process.HSCs and HPCs play a major role in the hematopoiesis.During the development of embryonic hematopoietic system,the mesenchymal tissues of mesoderm first differentiate into hemangioblast,and then HSCs and angioblast.The first HSC is found in the extraembryonic blood island of yolk sack and paraaortic splanchnopleure/aortagonad mesonephros(PAS/AGM).With the establishment of intra and extra embryonic circulation,HSCs were seeded into liver and spleen,and lastly into bone marrow to maintain the hematopoiesis during the lift time.The study on the development of embryonic hematopoiesis has been carried on for over a century.In mammal embryos,yolk sac,liver,thymus have been thought to be hematopoietic organs.Recently,several studies indicated that placenta is also a hematopoietic organ,which plays an important role on the embryonic stage.However, at present,it is still controversial about the hematopoiesis of palcenta.In our studies, various empirical methods were used to detect the hematopoietic ability of mouse placenta and try to identify whether the placenta possesses the hematopoietic activity and what the characteristics of placental hematopoiesis are.PARTⅠISOLATION AND IDENTIFICATION OF MONONUCLEAR CELLS IN MOUSE PLACENTAObjectiveClear all the fetal and maternal blood out of the mouse placenta,isolate and purify the proper hematopoietic stem/progenitor cells(HS/PCs) in the placenta,and explore if the mouse placenta possesses the ability of hematopoiesis.Methods1.Develop the technique of placenta flushing to clear the fetal blood contained in the placenta.Pregnant females of E12.5 were killed and the uterine horns were removed. The embryos(including the fetus and the placenta) were harvested.Under a stereomicroscope,the umbilical artery and vein were identified by the direction of blood flow.A fine needle(BD 29 G) was inserted into the umbilical artery with the tip pointing to the placenta.Heparin saline(concentration 0.5 mg/ml,290000 IU/ml) was used as flushing fluid and injected into the umbilical artery slowly(0.1ml/min) to flush the embryonic vessel system of the placenta.The flushed embryonic blood was collected as control.Then the placentas were separated from the maternal decidua, umbilical vessels and remnants of the yolk sac.Wash the placenta repetitively,until the palcenta turned pale,to remove the maternal blood.2.The paraffin sections of the flushed placenta were prepared.After HE stain,the sections were observed whether there is residued blood cells in the placental tissue.3.The placentas were mechanically dissected free and drawn through a 16 G needle and incubated with 0.1%collagenase in 10%fetal calf serum(FCS) / phosphate-buffered saline(PBS) for 30 minutes at 37℃and trypsin for 10 minutes at 37℃.Terminate the digestion and centrifugate.Then the cells were resuspensensed with 1 ml IMDM for the subsequent isolation of the mononuclear cells by centrifugation.4.Then the Percoll fluids of different densities(1.1.g/ml,1.080g/ml,1.055g/ml) were added into the centrifuge tube one by one from higher density to less,and then the cell suspensions were gently placed to the utmost upper layer.After centrifugalization of 25 minutes with 2000 rpm,the cells residing between the top layer and the middle layer were harbored and resuspensed with 1 ml IMDM to prepare the mononuclear cells suspension.5.Make cell film preparations and stain with Giemsa.Under microscope,observe if the obtained cells were mononuclear.6.Use CFU-S assay to identify if the extracted mononuclear cells from the placenta were hematopoietic stem cells.Results1.We flushed each placenta with 0.2 ml flushing fluid.After flushing,the fetal circulation blood was all cleared out of the placenta.2.After the combined enzymatic digestion and density gradient centrifugation with Percoll fluids,the cell film preparations showed that the obtained isolated cells were mononuclear and small lymphocyte-like,with a diameter of 7～10μm.3.The isolated mononuclear cell suspensions were injected via the tail vein after the recipient mice were irradiated lethally.CFU-Ss were detected in the spleens of recipient mice.After the sections of the spleen were stained with HE,the CFU-Ss were confirmed under microscope.ConclusionPlacenta flushing is effective to clear the fetal and maternal blood in the placenta. Continuous enzymatic digestion with collagenase and trypsin and Percoll density gradient centrifugation method were effective to isolate the mononuclear cells of the placenta.CFU-S assay demonstrated that these mononuclear cells harbored the ability to form colonies in spleen,which was the characteristics of HS/PCs,and were the proper HS/PCs of mouse placenta.PARTⅡUTILIZATION OF HS/PCS SURFACE MARKERS TO DETERMINE EXISTENCE,FREQUENCY AND DISTRIBUTION OF HS/PCs IN MOUSE PLACENTAObjectiveExamine the surface markers of the mononuclear cells of placenta with immunofluorescent technique and flow cytometry to determine the existence, frequency and distribution of the proper HS/PCs of mouse palcenta and provide more evidences of placental hematopoiesis.Methods1.The expressions of HS/PCs-specific antigens---CD34,CD117 and Sea-1---were examined in the mononuclear cells of mouse placenta by immunofluorescence to determine the existence of proper HS/PCs in mouse placenta.2.FACS was applied to examine the absolute and relative numbers of CD34,CD117 and Sea-1 positive cells to judge the differentiation condition of proper HS/PCs of mouse placenta.Compare the results between the proper HS/PCs of mouse placenta and the isolated mononuclear cells from the fetal blood in the placenta.3.Placentas of E12.5 were sectioned and immumohistochemically stained with antibodies of CD34,CD117 and Sca-1 to display the distribution of placental proper HS/PCs in the mouse placenta.Results1.The immunohistochemistry showed that the HS/PCs-specific antigens---CD34, CD117 and Sca-1---were expressed by the placental mononuclear cells.The percentages of CD34,CD117 and Sea-1 positive cells in the total cells were 20.1±5.3%,28.5±3.4%,35±8.6%,respectively.These proved thate the placental mononuclear cells were definite HS/PCs.2.FACS showed that the placental HS/PCs expressed HS/PCs-specific antigens,CD34, CD117 and Sca-1.The percentages of the three cell populations in the total purified placental cells were 20.2%,24.6%,and 26.2%respectively.For the mononuclear cells of placental blood cells,the percentages of the three populations were 8.2%,6.3%, and 6.5%respectively.The concentration of positive cells in placental cells was higher than in placental blood.The absolute numbers of positive cells of CD34, CD117 and Sca-1 were 1.4×10~4,1.8×10~4,and 1.8×10~4 respectively in placental mononuclear cells,and were 0.7×10~4,0.5×10~4,and 0.5×10~4 respectively in the mononuclear cells of placental blood.The number of positive cells in the placental mononuclear cells was higher than in the mononuclear cell of placental blood.For the cell subpopulation of CD34~+/Sca-1~+,the percentage in placental mononuclear cells (15.3%) was also higher than in the mononuclear cells of placental blood(5.1%).3.In the E12.5 mouse placenta,CD34~+ cells,CD117~+ cells and Sca-1~+ cells can be found in the palcenta labyrinth.CD117~+ cells can also be observed in the chorionic plate.The expression of Sca-1 was significantly higher than CD34 and CD117.ConclusionPlacental hematopoiesis is further proved with the application of HS/PCs-specific antigens immunohistochemistry,immunofluorescence and flow cytometry.Placental proper HS/PCs exist and distribute regularly in mous placenta. PARTⅢIN VITRO CULTURE OF PLACENTAL MONONUCLEAR CELLS AND DETECTION OF HEMATOPOIESIS-RELATED GENES EXPRESSION TO CONFIRM PLACENTAL HEMATOPOIESISObjectiveConfirm the placental hematopoiesis through in vitro culture of placental mononuclear cells,the observation ofhematopoietic colony forming,and detection of the expression ofhematopoiesis-related genes.Methods1.10~5 purified single cells were plated per 35 mm dish in MethoCult M3434 methylcellulose media.After 14 days of culture,colonies were observed and identified based on their morphology under an inverted microscope.The HPP-CFCs were evalutate.So do the mononuclear cells of placental blood,as positive control.2.RT-PCR was applied to detect the expression of Scl,Runx1,Tel/Etv6,and GATA-2 in the placental mononuclear cells.Results1.Three different types of progenitors-CFU-GEMMs(colonyforming units, granulocytes,erythrocytes,monocytes,macrophages),BFU-Es(burst-forming units, erythroid) and CFU-GMs(colony-forming units,granulocytes,macrophages)-diagnosed from the colony phenotype,were observed on day 14 of culture.The frequencies of placental BFU-Es,CFU-GMs and CFU-GEMMs were higher than that of placental blood.The characteristics of the morphology of placental BFU-Es was multicentered and highly hemoglobinized,while placental blood BFU-Es displayed only weakly hemoglobinized clusters.In the case of CFU-GMs,placental CFU-GMs were dense,medium to large sized,and often actually so large that they seemed to result from the fusion of clusters,whereas placental blood CFU-GMs were small sized.Both placental CFU-GEMMs and placental blood CFU-GEMMs were huge and so qualified more adequately as HPP-CFCs.Compared with placental blood CFU-GEMMs,placental CFU-GEMM appeared larger,denser,and contained more cells,with a very large core of hemoglobinized cells.2.After culturing of 60 days,the colony of HPP-CFCs from both placenta and placental blood exceeded 0.5mm in diameter.When the colonies were scored at 60 days,35 CFU-GEMMs can be obtained from 10~5 replated placental mononuclear cells.By contrast,HPP-CFCs could also be obtained from the mononuclear cells of placental blood and the frequency was about two-thirds of the placenta.Furthermore,the colonies of placental blood were smaller than the placental colonies.3.The expression of hematopoiesis-related genes---Scl,Runx1,Tel/Etv6,and GATA-2---could be detected on the placental mononuclear cells.ConclusionPlacental mononuclear cells harbored the potential to differentiate to CFU-GMs, BFU-Es and CFU-GEMMs.HPP-CFCs activity can also be detected.These indicated that the placental mononuclear cells were HS/PCs and possessed the potential of multipotent differentiation.The placental hematopoiesis was further proved by the hematopoiesis-related genes expression on the placental mononuclear cells.更多还原