胎盤來(lái)源多能細(xì)胞的體外分離培養(yǎng)與生物學(xué)特性研究
發(fā)布時(shí)間：2018-08-29 09:23
【摘要】： 目的間充質(zhì)干細(xì)胞(mesenchymal stem cells，MSCs)具有高度增殖、自我更新的能力，廣泛分布于多種人體組織中，參與機(jī)體組織器官的正常功能活動(dòng)和損傷修復(fù)，是再生醫(yī)學(xué)領(lǐng)域研究的熱點(diǎn)。目前已成功地從人骨髓、外周血、肌肉、脂肪、臍血、羊水及胎兒組織中分離并鑒定出MSCs。其中骨髓來(lái)源間充質(zhì)干細(xì)胞(bone marrow mesenchymal stemcells，BMSCs)研究最多，也取得了一些進(jìn)展。但也發(fā)現(xiàn)臨床采集骨髓有一定困難，可能感染并引起并發(fā)癥，而且隨著供者年齡增加其數(shù)量、增殖和分化能力均顯著下降。近幾年來(lái)有學(xué)者發(fā)現(xiàn)胎盤組織中存在MSCs。與骨髓取材相比胎盤在胎兒娩出后即完成使命，成為“廢棄”物，其取材方便，易于分離，對(duì)其研究不會(huì)涉及倫理道德問(wèn)題，因而可能成為組織修復(fù)和基因治療的新細(xì)胞來(lái)源。本課題旨在探索體外分離培養(yǎng)胎盤來(lái)源多能細(xì)胞(placenta-derived mutipotent cells,PDMCs)的方法和條件，以及對(duì)其生物學(xué)和功能上的特性進(jìn)行初步研究。 對(duì)象和方法 (1)胎盤組織30份：在無(wú)菌條件下，采集足月健康順產(chǎn)分娩孕婦的胎盤。 (2)PDMCs的制備：剪切胎盤蛻膜組織1~2mm~3大小的組織塊，用酶消化法從中分離得到細(xì)胞懸液，通過(guò)人淋巴分離液密度梯度離心得到單核細(xì)胞，接種于培養(yǎng)瓶中。 (3)取原代到15代細(xì)胞分別利用MTT染色技術(shù)，免疫組織化學(xué)染色，HE染色，透射電鏡，以及流式細(xì)胞檢測(cè)儀，從不同層面和角度鑒定胎盤來(lái)源多能細(xì)胞的生物學(xué)特征，并與BMSCs進(jìn)行對(duì)比研究，以觀察兩者之間在生物學(xué)特性方面的異同。 結(jié)果 (1)細(xì)胞培養(yǎng)：通過(guò)混合酶消化法可以得到PDMCs，細(xì)胞形態(tài)良好，傳至15代沒有形態(tài)改變。 (2)細(xì)胞形態(tài)：通過(guò)倒置顯微鏡和HE染色觀察可見細(xì)胞呈長(zhǎng)梭形，漩渦狀生長(zhǎng)，細(xì)胞體積較大。 (3)細(xì)胞生長(zhǎng)規(guī)律：原代細(xì)胞接種后的9-14天出現(xiàn)細(xì)胞克隆。傳代之后的第4天為對(duì)數(shù)生長(zhǎng)期，此后到第7天為平穩(wěn)期。14天左右為衰退期。與文獻(xiàn)報(bào)道的來(lái)自于骨髓的間充質(zhì)干細(xì)胞的生長(zhǎng)規(guī)律類似。 (4)細(xì)胞超微結(jié)構(gòu)：通過(guò)透射電鏡觀察PDMCs和BMSCs，發(fā)現(xiàn)兩者有共同的特點(diǎn)，即核膜不規(guī)則，核仁明顯，核內(nèi)以常染色質(zhì)為主，且分布均勻；胞漿內(nèi)，可見豐富的粗面內(nèi)質(zhì)網(wǎng)，線粒體，，核糖體等細(xì)胞器，并且胞膜完整，表面有大量微絨毛。 (5)表面標(biāo)志：通過(guò)免疫組織化學(xué)染色和流式細(xì)胞檢測(cè)，發(fā)現(xiàn)PDMCs表達(dá)CD105，CD166，CD44，CD29，CD9，HLA-ABC，不表達(dá)CD34，CD40L，和HLA-DR。這與BMSCs表面抗原標(biāo)志表達(dá)相類似。PDMCs表達(dá)胚胎干細(xì)胞表面抗原標(biāo)志SSEA-3，SSEA-4，TRA-1-60，TRA-1-81，Oct-4的量高于BMSCs。 結(jié)論(1)用酶消化法可以從足月分娩的胎盤蛻膜組織中分離獲得長(zhǎng)梭形貼壁生長(zhǎng)的細(xì)胞(PDMCs)，這類細(xì)胞具有與BMSCs相似的生物學(xué)特性。 (2)PDMCs較BMSCs更為原始
[Abstract]:Objective Mesenchymal stem cells (mesenchymal stem cells,MSCs) have the ability of high proliferation and self-renewal. They are widely distributed in a variety of human tissues and participate in the normal function and repair of tissues and organs, which is a hot research topic in the field of regenerative medicine. MSCs. has been successfully isolated and identified from human bone marrow, peripheral blood, muscle, fat, umbilical cord blood, amniotic fluid and fetal tissues. Bone marrow derived mesenchymal stem cells (bone marrow mesenchymal stemcells,BMSCs) have been studied most and some progress has been made. But it is also found that it is difficult to collect bone marrow in clinic, which may cause infection and complications, and with the increase of donor age, the ability of proliferation and differentiation are significantly decreased. In recent years, some scholars have found that MSCs. exists in placenta tissue. Compared with bone marrow, placenta completes its mission and becomes an "abandoned" object. It is convenient and easy to separate. The study of placenta is not related to ethical and moral issues, so it may become a new cell source for tissue repair and gene therapy. The aim of this study was to explore the methods and conditions of isolation and culture of placental pluripotent cells (placenta-derived mutipotent cells,PDMCs) in vitro and to study their biological and functional characteristics. Objects and methods (1) 30 samples of placental tissue: under aseptic conditions, The placenta of pregnant women with full term healthy delivery was collected. (2) the preparation of PDMCs: the tissue blocks of 1~2mm~3 in decidual tissue of placenta were cut off, and the cell suspension was isolated by enzyme digestion. Mononuclear cells were obtained by density gradient centrifugation of human lymphoid isolate and inoculated in culture flask. (3) Primary to 15th passage cells were stained by MTT staining, immunohistochemical staining with HE staining, transmission electron microscopy (TEM). And flow cytometry was used to identify the biological characteristics of placental pluripotent cells from different levels and angles, and to compare with BMSCs to observe the similarities and differences of biological characteristics between them. Results (1) Cell culture: PDMCs, cells could be obtained by mixed enzyme digestion. No morphological changes were observed at the 15th generation. (2) the morphology of cells was observed by inverted microscope and HE staining. (3) Law of cell growth: cell clone appeared 9-14 days after primary cell inoculation. The logarithmic growth period was on the 4th day after passage and the decline period was about 14 days from the 7th day. The growth pattern of mesenchymal stem cells from bone marrow was similar to that reported in the literature. (4) Ultrastructure of cells: PDMCs and BMSCs, showed the same characteristics by transmission electron microscope, that is, irregular nuclear membrane. In the cytoplasm, there are abundant organelles such as rough endoplasmic reticulum, mitochondria, ribosome and so on, and the cell membrane is intact. A large number of microvilli were found on the surface. (5) Surface markers: by immunohistochemical staining and flow cytometry, it was found that PDMCs expressed CD105,CD166,CD44,CD29,CD9,HLA-ABC, and did not express CD34,CD40L, and HLA-DR.. This is similar to the expression of BMSCs surface antigen marker. PDMCs express more embryonic stem cell surface antigen marker SSEA-3,SSEA-4,TRA-1-60,TRA-1-81,Oct-4 than BMSCs.. Conclusion (1) long fusiform adherent cell (PDMCs), can be obtained from placental decidua tissue of term delivery by enzyme digestion. (2) PDMCs is more primitive than BMSCs.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329.2
本文編號(hào)：2210781
[Abstract]:Objective Mesenchymal stem cells (mesenchymal stem cells,MSCs) have the ability of high proliferation and self-renewal. They are widely distributed in a variety of human tissues and participate in the normal function and repair of tissues and organs, which is a hot research topic in the field of regenerative medicine. MSCs. has been successfully isolated and identified from human bone marrow, peripheral blood, muscle, fat, umbilical cord blood, amniotic fluid and fetal tissues. Bone marrow derived mesenchymal stem cells (bone marrow mesenchymal stemcells,BMSCs) have been studied most and some progress has been made. But it is also found that it is difficult to collect bone marrow in clinic, which may cause infection and complications, and with the increase of donor age, the ability of proliferation and differentiation are significantly decreased. In recent years, some scholars have found that MSCs. exists in placenta tissue. Compared with bone marrow, placenta completes its mission and becomes an "abandoned" object. It is convenient and easy to separate. The study of placenta is not related to ethical and moral issues, so it may become a new cell source for tissue repair and gene therapy. The aim of this study was to explore the methods and conditions of isolation and culture of placental pluripotent cells (placenta-derived mutipotent cells,PDMCs) in vitro and to study their biological and functional characteristics. Objects and methods (1) 30 samples of placental tissue: under aseptic conditions, The placenta of pregnant women with full term healthy delivery was collected. (2) the preparation of PDMCs: the tissue blocks of 1~2mm~3 in decidual tissue of placenta were cut off, and the cell suspension was isolated by enzyme digestion. Mononuclear cells were obtained by density gradient centrifugation of human lymphoid isolate and inoculated in culture flask. (3) Primary to 15th passage cells were stained by MTT staining, immunohistochemical staining with HE staining, transmission electron microscopy (TEM). And flow cytometry was used to identify the biological characteristics of placental pluripotent cells from different levels and angles, and to compare with BMSCs to observe the similarities and differences of biological characteristics between them. Results (1) Cell culture: PDMCs, cells could be obtained by mixed enzyme digestion. No morphological changes were observed at the 15th generation. (2) the morphology of cells was observed by inverted microscope and HE staining. (3) Law of cell growth: cell clone appeared 9-14 days after primary cell inoculation. The logarithmic growth period was on the 4th day after passage and the decline period was about 14 days from the 7th day. The growth pattern of mesenchymal stem cells from bone marrow was similar to that reported in the literature. (4) Ultrastructure of cells: PDMCs and BMSCs, showed the same characteristics by transmission electron microscope, that is, irregular nuclear membrane. In the cytoplasm, there are abundant organelles such as rough endoplasmic reticulum, mitochondria, ribosome and so on, and the cell membrane is intact. A large number of microvilli were found on the surface. (5) Surface markers: by immunohistochemical staining and flow cytometry, it was found that PDMCs expressed CD105,CD166,CD44,CD29,CD9,HLA-ABC, and did not express CD34,CD40L, and HLA-DR.. This is similar to the expression of BMSCs surface antigen marker. PDMCs express more embryonic stem cell surface antigen marker SSEA-3,SSEA-4,TRA-1-60,TRA-1-81,Oct-4 than BMSCs.. Conclusion (1) long fusiform adherent cell (PDMCs), can be obtained from placental decidua tissue of term delivery by enzyme digestion. (2) PDMCs is more primitive than BMSCs.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329.2
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