本文選題：胎盤 + 造血干細(xì)胞��； 參考：《鄭州大學(xué)》2007年碩士論文

【摘要】： 研究目的近年來，造血干細(xì)胞移植成為治療惡性血液病、再生障礙性貧血、自身免疫性疾病，某些遺傳性疾病及實(shí)體瘤的一種重要手段。據(jù)有關(guān)資料顯示，僅我國就有400多萬各類疾病患者等待造血干細(xì)胞移植，而造血干細(xì)胞的來源卻很有限。骨髓、外周血和臍血是目前造血干細(xì)胞的主要來源，骨髓和外周血造血干細(xì)胞移植要求HLA配型完全一致，這即使是同胞間也只有25％的機(jī)率，在非血緣關(guān)系的人群中僅有1/400—1/10000的可能性，遠(yuǎn)遠(yuǎn)不能滿足臨床治療的需要；臍血雖免疫原性較弱，移植后移植物抗宿主病(GVHD)的發(fā)生率低，但單份臍血中所含的造血干細(xì)胞數(shù)量有限，難以使一個(gè)成人獲得造血干細(xì)胞重建，需要擴(kuò)增后才能用于成人。所以尋找新的造血干細(xì)胞來源是個(gè)極待解決的問題。已有研究證實(shí)：胎盤中含有較多的造血干細(xì)胞，胎盤組織有支持造血干細(xì)胞生存的微環(huán)境。這表明通常丟棄的胎盤也有望成為HSC移植的資源。但胎盤組織中造血干細(xì)胞的含量有限，故胎盤造血干細(xì)胞進(jìn)行體外擴(kuò)增成為胎盤造血干細(xì)胞用于移植的一個(gè)前提，國內(nèi)外已有相關(guān)報(bào)道。移植治療后GVHD是影響移植治療效果的重要原因之一，本研究比較了擴(kuò)增后的胎盤及臍血單個(gè)核細(xì)胞和動(dòng)員后的外周血的免疫活性，以了解胎盤作為干細(xì)胞源用于移植時(shí)GVHD的發(fā)生情況，為胎盤組織源造血干細(xì)胞用于移植提供理論依據(jù)。 對象與方法 (一)對象 1．胎盤組織和與其相對應(yīng)的臍血8份：在無菌條件下，分別采集鄭州大學(xué)第一附屬醫(yī)院足月健康順產(chǎn)分娩孕婦的胎盤及與其相對應(yīng)的臍血。 2．外周血8份：在無菌條件下采集鄭州大學(xué)第一附屬醫(yī)院和河南省腫瘤醫(yī) 院血液科干細(xì)胞移植健康供者的經(jīng)G-CSF動(dòng)員4-5后的外周血。 (二)方法 1．單個(gè)核細(xì)胞(MNC)制備：剪切胎盤組織呈1—2mm~3大小的組織塊，用酶消化的方法經(jīng)320目的細(xì)胞篩過濾制備胎盤組織源單個(gè)細(xì)胞懸液；用淋巴細(xì)胞分離液分別分離胎盤組織源、臍血源的MNC。 2．胎盤和臍血MNC的體外培養(yǎng)：將分離的胎盤MNC、臍血MNC用DMEM培養(yǎng)液懸浮，加入刺激因子，置37℃、體積濃度為5％CO_2、飽和濕度的培養(yǎng)箱中培養(yǎng)7天。 3．用淋巴細(xì)胞分離液將培養(yǎng)后的胎盤和臍血細(xì)胞再次分離MNC，同時(shí)分離動(dòng)員后的外周血的MNC，，并為后續(xù)實(shí)驗(yàn)調(diào)好細(xì)胞濃度。 4．采用CCK—8檢測混合淋巴細(xì)胞反應(yīng)中胎盤、臍血和外周血三種來源細(xì)胞的增殖情況；采用流式細(xì)胞儀技術(shù)檢測胎盤、臍血和外周血三種來源細(xì)胞中CD_3、CD_(19)、CD_(16)、CD_(56)、HLA-DR的表達(dá)。 結(jié)果 1．單向混合淋巴細(xì)胞反應(yīng)混合淋巴細(xì)胞反應(yīng)中，擴(kuò)增后的胎盤MNC組的細(xì)胞增殖顯著低于其他兩組(P0.001)，擴(kuò)增后的臍血MNC組的細(xì)胞增殖顯著高于擴(kuò)增后的胎盤MNC，但卻顯著低于動(dòng)員后的外周血(P0.001)。 2．表型分析擴(kuò)增后的胎盤MNC中T淋巴細(xì)胞(CD_3~+細(xì)胞)、B淋巴細(xì)胞(CD_(19)~+細(xì)胞)比例少于擴(kuò)增后的臍血MNC和動(dòng)員后的外周血，與擴(kuò)增后臍血MNC和動(dòng)員后的外周血均有顯著差異。擴(kuò)增后的胎盤MNC中NK細(xì)胞(CD_(16)~+CD_(56)~+細(xì)胞)和HLA-DR+細(xì)胞比例最高，與擴(kuò)增后臍血MNC和動(dòng)員后的外周血有顯著差異；臍血中NK細(xì)胞的含量最少。 結(jié)論 1．單向混合淋巴細(xì)胞培養(yǎng)中，以擴(kuò)增后的胎盤MNC組的細(xì)胞增殖最低，表明擴(kuò)增后的胎盤MNC免疫活性最低。 2．?dāng)U增后的胎盤MNC中CD_3~+細(xì)胞，CD_(19)~+細(xì)胞的比例最低，表明擴(kuò)增后的胎盤MNC免疫活性最低。 3．?dāng)U增后的胎盤MNC中成熟型NK細(xì)胞比例最高，可能有助于增加移植物抗白血病作用，但并不增加GVHD的發(fā)生率和嚴(yán)重程度。 4．?dāng)U增后的胎盤MNC可用于移植，若其用于移植，GVHD的發(fā)生率及嚴(yán)重 4．?dāng)U增后的胎盤MNC可用于移植，若其用于移植，GVHD的發(fā)生率及嚴(yán)重程度較擴(kuò)增后的臍血MNC和動(dòng)員后的外周血用于移植時(shí)低。
[Abstract]:In recent years, hematopoietic stem cell transplantation has become an important means for the treatment of malignant hematological diseases, aplastic anemia, autoimmune diseases, some hereditary diseases and solid tumors. According to the relevant data, only about 4000000 of the patients in our country are waiting for the transplantation of blood stem cells, while the source of hematopoietic stem cells is very good. Bone marrow, peripheral blood and umbilical blood are the main sources of hematopoietic stem cells at present. Bone marrow and peripheral blood stem cell transplantation require the same HLA matching, which is only 25% of the probability between the siblings, and the only possibility of 1/400 1/10000 in the unrelated population is far from meeting the needs of clinical treatment; umbilical blood is exempt. Pestilence is weak and the incidence of graft versus host disease (GVHD) after transplantation is low, but the number of hematopoietic stem cells in single cord blood is limited. It is difficult for one adult to reestablish hematopoietic stem cells and need to be amplified to be used in adults. Therefore, finding new hematopoietic stem cells is a problem to be solved. There are more hematopoietic stem cells in the disc and the placental tissue has a microenvironment to support the survival of the hematopoietic stem cells. This indicates that the usually discarded placenta is also expected to be a resource for HSC transplantation. However, the content of hematopoietic stem cells in placental tissue is limited, so the placental hematopoietic stem cells are amplified into a pre transplant of placental hematopoietic stem cells for transplantation. It has been reported at home and abroad. GVHD is one of the important factors affecting the effect of transplantation treatment after transplantation. This study compares the immune activity of the expanded placenta and cord blood mononuclear cells and mobilized peripheral blood in order to understand the occurrence of GVHD in the placenta as a stem cell source for transplantation and to stem the hematopoietic stem of placenta tissue. The use of cell for transplantation provides a theoretical basis.
Object and method
(I) objects
1. placental tissue and 8 umbilical cord blood corresponding to it: under aseptic conditions, the placenta and its corresponding umbilical cord blood were collected from the First Affiliated Hospital of Zhengzhou University.
2. peripheral blood 8: under sterile conditions, the First Affiliated Hospital of Zhengzhou University and Henan oncology doctor were collected.
Stem cells transplantation from healthy Department of Hematology donors were mobilized by G-CSF for 4-5 of peripheral blood.
(two) method
1. mononuclear cell (MNC) preparation: the cut placenta tissue is 1 - 2mm~3 size tissue block. The placental tissue source suspension is prepared by the enzyme digestion method through 320 cell sieves, and the placental tissue source and the MNC. of the umbilical blood source are separated by the lymphocyte separation solution.
2. in vitro culture of placenta and umbilical cord blood MNC: the isolated placenta MNC, umbilical cord blood MNC was suspended in DMEM medium, added to the stimulating factor, placed at 37 C, the volume concentration was 5%CO_2, and the saturated humidity incubator was cultured for 7 days.
3. the MNC was separated from the cultured placenta and cord blood cells by lymphocyte separation solution, and the MNC of the mobilized peripheral blood was separated, and the cell concentration was adjusted for the follow-up experiment.
4. CCK - 8 was used to detect the proliferation of three kinds of cells from the placenta, umbilical blood and peripheral blood in mixed lymphocyte reaction. The flow cytometry was used to detect CD_3, CD_ (19), CD_ (16), CD_ (56), and HLA-DR in the placenta, umbilical cord blood and peripheral blood.
Result
In 1. unidirectional mixed lymphocyte reaction mixed lymphocyte reaction, the proliferation of the expanded placental MNC group was significantly lower than that of the other two groups (P0.001). The proliferation of MNC group after amplification was significantly higher than that after the amplification of placental MNC, but it was significantly lower than that of the mobilized peripheral blood (P0.001).
2. phenotypic analysis of T lymphocyte (CD_3~+ cells) and B lymphocyte (CD_ (19) + cells) in the placental MNC after amplification, the proportion of MNC and mobilized peripheral blood after amplification is less than that of amplified umbilical blood and mobilized peripheral blood. There is a significant difference between the MNC of umbilical cord blood and the peripheral blood after the amplification. The ratio of NK cells (CD_ (16) ~+CD_ (56) ~ + cells) and HLA-DR+ cells in the amplified placental MNC The highest cases were significantly different from those of the expanded cord blood MNC and mobilized peripheral blood, and the NK cells in umbilical cord blood were the least.
conclusion
1. in unidirectional mixed lymphocyte culture, the proliferation of the placental MNC group was the lowest, indicating that the MNC activity of the expanded placenta was the lowest.
2. the percentage of CD_ (19) ~ + cells in CD_3~+ cells after MNC amplification was the lowest, indicating that the MNC activity of the expanded placenta was the lowest.
The proportion of mature NK cells in MNC after 3. amplification is the highest, which may help to increase the anti leukemic effect of the graft, but does not increase the incidence and severity of GVHD.
4. the expanded placenta MNC can be used for transplantation. If it is used for transplantation, the incidence and severity of GVHD are serious.
4. expanded placental MNC can be used for transplantation. If it is used for transplantation, the incidence of GVHD and the severity of MNC in umbilical blood after amplification and the mobilization of peripheral blood are low for transplantation.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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