人胎盤源間充質干細胞對臍血單個核細胞體外生長的支持作用及體內移植促進造血重建的實驗研究
發(fā)布時間：2018-09-06 07:20
【摘要】： 間充質干細胞(mesenchymal stem cells,MSCs)是目前倍受關注的一類具有多向分化潛能的成體干細胞。在不同的誘導條件下,MSCs可分化為成骨細胞、軟骨細胞、脂肪細胞等。MSCs的主要來源為成人骨髓,但成人骨髓源MSCs的細胞數量及增殖分化潛能會隨著年齡的增大而下降,且病毒感染率較高,此外供者MSCs的采集需行骨髓穿刺術,因而MSCs的臨床應用存在很大的局限。近年的研究表明,從胎盤中可分離出MSCs,它們和骨髓MSCs一樣,在合適的培養(yǎng)條件下具有多向分化能力。 MSCs作為骨髓造血微環(huán)境中的重要組成成分,它通過分泌多種造血生長因子以及細胞與細胞間的直接接觸等方式作用于造血干細胞(hematopoietic stem cells,HSCs),因而在造血調控中發(fā)揮重要的作用。目前,MSCs已被用來體外支持HSCs擴增,體內聯合HSCs移植促進造血重建。本研究在成功分離培養(yǎng)人胎盤源間充質干細胞(placenta-derived mesenchymal stem cells,PMSCs)的基礎上,探討人PMSCs在體內和體外支持造血的功能效應。 本文分為兩部分。第一部分闡述了人PMSCs的分離培養(yǎng)和鑒定,并探討人PMSCs對臍血單個核細胞(MNCs)體外生長的支持作用。將人胎盤組織經膠原酶消化、貼壁和傳代培養(yǎng)獲得人PMSCs,運用流式細胞儀檢測其表面標志,分別聯合應用β-甘油磷酸鈉、維生素C、地塞米松體系和3-異丁基-1-甲基黃嘌呤、胰島素、吲哚美辛、地塞米松體系將其誘導分化為成骨細胞和脂肪細胞,繼而采用堿性磷酸酶檢測和vonkossa染色對其進行成骨分化鑒定,采用油紅染色對其進行脂肪分化鑒定。將用密度梯度離心法分離的臍血MNCs和傳代后的PMSCs進行共培養(yǎng),通過細胞計數和流式細胞儀分別檢測造血細胞的生長情況。結果顯示:1.從人胎盤組織中成功分離和培養(yǎng)PMSCs,貼壁細胞呈成纖維細胞狀,經流式細胞儀檢測其表型為CD29~+、CD44~+、CD105~+、CD106~+、CD166~+、CD34~-、CD45~-、HLA-DR~-;2.PMSCs經成骨誘導3周后,堿性磷酸酶染色呈強陽性,von kossa染色可見明顯鈣結節(jié)。PMSCs經成脂誘導2周后,油紅染色呈陽性,有明顯的脂滴出現;3.人PMSCs+臍血MNCs共培養(yǎng)組的細胞總數和CD45~+細胞數在各培養(yǎng)時間點均明顯高于對照組,在培養(yǎng)第7天,CD45~+、CD14~+及CD19~+細胞數共培養(yǎng)組也明顯高于對照組。結果表明,人PMSCs可以作為滋養(yǎng)層細胞有效支持臍血MNCs的體外生長。 第二部分利用同種異基因小鼠骨髓移植模型,比較經骨髓腔內注射(intra-bonemarrow injection,IBMI)和外周靜脈輸注(intravenous injection,Ⅳ)兩種途徑移植小鼠骨髓單個核細胞(BMNCs)后受體早期的造血重建情況,建立了小鼠IBMI技術平臺,并利用IBMI技術初步探討了人PMSCs聯合臍血MNCs共移植入SCID鼠體內后的早期造血重建效應。以BALB/c小鼠為供體,通過IBMI和Ⅳ兩種途徑將其BMNCs移植入經致死量輻照預處理的C57BL/6受鼠體內。60只受鼠隨機分為3組:骨髓腔內注射高劑量組1(IBM1組)、骨髓腔內注射低劑量組2(IBM2組)、尾靜脈注射組(Ⅳ組),每組20只。在骨髓移植后1、3、6、9 d分別計數各組受鼠脛骨骨髓腔內細胞總數,并用流式細胞儀檢測供體植入水平(供體來源細胞總數、供體來源髓系細胞數)。以人PMSCs和臍血MNCs為供體,通過IBMI方法或結合Ⅳ方法,將兩種細胞聯合或單獨移植入經亞致死量輻照預處理的SCID受鼠體內。9只受鼠隨機分為3組:聯合移植A組(PMSCs+臍血MNCs,IBMI)、單獨移植B組(臍血MNCs,IBMI)、聯合移植C組(PMSCs經IBMI,臍血MNCs經Ⅳ),每組3只。在移植后14 d取各組SCID小鼠注射側和對側脛骨腔內骨髓細胞,并用流式細胞儀檢測分析人CD34~+、CD45~+細胞的植入水平。結果顯示:1.IBM1組和IBM2組注射側于移植后6 d脛骨骨髓腔內細胞總數、供體來源細胞總數、供體來源髓系細胞總數均明顯高于Ⅳ組;2.SCID小鼠移植后14 d,B組注射側及對側脛骨骨髓腔內的人CD34~+、CD45~+細胞的百分比均明顯低于A組小鼠的注射側和對側。結果表明,IBMI較Ⅳ更能促進同種異基因骨髓移植后的早期造血功能重建;人PMSCs可以增加臍血MNCs的植入,促進造血重建。 綜上所述,本實驗從人胎盤組織中成功分離培養(yǎng)獲得PMSCs,人PMSCs在體外對臍血MNCs的生長有明顯的支持作用。通過IBMI技術將人PMSCs與臍血MNCs共移植于SCID小鼠體內,人PMSCs可有效促進臍血MNCs的早期造血重建。這為進一步研究人PMSCs體內和體外支持造血的相關分子機理奠定了實驗基礎。
[Abstract]:At present, mesenchymal stem cells (MSCs) are a kind of adult stem cells with multipotent differentiation potential. Under different induction conditions, MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and so on. The main source of MSCs is adult bone marrow, but the number and proliferation and differentiation potential of MSCs derived from adult bone marrow. In addition, the collection of MSCs from donors requires bone marrow aspiration, so the clinical application of MSCs is limited. Recent studies have shown that MSCs can be isolated from placenta, and they, like bone marrow MSCs, can differentiate into multiple cells under suitable culture conditions.
As an important component of bone marrow hematopoietic microenvironment, MSCs act on hematopoietic stem cells (HSCs) by secreting a variety of hematopoietic growth factors and direct contact between cells. MSCs play an important role in hematopoietic regulation. At present, MSCs have been used to support HSCs amplification in vitro and in vivo. Combined HSCs transplantation promotes hematopoietic reconstitution. In this study, human placenta-derived mesenchymal stem cells (PMSCs) were successfully isolated and cultured to investigate the functional effects of human PMSCs on supporting hematopoiesis in vivo and in vitro.
The first part describes the isolation, culture and identification of human PMSCs, and the supporting effect of human PMSCs on the growth of umbilical cord blood mononuclear cells (MNCs) in vitro. Sodium, vitamin C, dexamethasone system and 3-isobutyl-1-methylxanthine, insulin, indomethacin, dexamethasone system were induced to differentiate into osteoblasts and adipocytes, then alkaline phosphatase detection and vonKossa staining were used to identify the osteogenic differentiation, and oil red staining was used to identify the adipogenic differentiation. The results showed that: 1. PMSCs were successfully isolated and cultured from human placenta tissues. The adherent cells were fibroblast-like, and their phenotypes were CD29~+, CD44~+, CD1 detected by flow cytometry. 05~+, CD106~+, CD166~+, CD34~-, CD45~-, HLA-DR~-; 2. Alkaline phosphatase staining and von Kossa staining were strongly positive in PMSCs after 3 weeks of osteogenesis induction. Oil red staining was positive in PMSCs after 2 weeks of lipogenesis induction, and obvious lipid droplets appeared. 3. The total number of cells and CD45~+ cells in human PMSCs + umbilical cord blood MNCs co-culture group were strongly positive in all cultures. The number of CD45~+, CD14~+ and CD19~+ cells co-cultured on the 7th day was also significantly higher than that of the control group. The results showed that human PMSCs could be used as trophoblast cells to support the growth of umbilical cord blood MNCs in vitro.
In the second part, we compared the early hematopoietic reconstitution of bone marrow mononuclear cells (BMNCs) recipients by intra-bone marrow injection (IBMI) and peripheral intravenous injection (IV) in allogeneic bone marrow transplantation mice, and established a mouse IBMI platform. The early hematopoietic reconstitution effect of human PMSCs combined with umbilical cord blood MNCs co-transplanted into SCID mice was studied by I technique. BMNCs were transplanted into C57BL/6 recipients by IBMI and IV in BALB/c mice. Intramedullary injection of low dose group 2 (IBM2 group), tail vein injection group (IV group), each group of 20. After bone marrow transplantation 1, 3, 6, 9 days were counted the total number of tibial marrow cells in each group, and flow cytometry was used to detect the level of donor implantation (total number of donor-derived cells, number of donor-derived myeloid cells). Human PMSCs and umbilical cord blood MNCs were used as donors, through Nine SCID recipients were randomly divided into three groups: group A (PMSCs + umbilical cord blood MNCs, IBMI), group B (umbilical cord blood MNCs, IBMI), group C (PMSCs via IBMI, umbilical cord blood MNCs via IV), and three in each group. The results showed that: 1. The total number of cells in the tibial bone marrow cavity, the total number of donor-derived cells and the total number of donor-derived myeloid cells in the IBM 1 and IBM 2 groups were significantly higher than those in the IV group 6 days after transplantation. The percentage of CD34~+ and CD45~+ cells in the injected and contralateral tibial bone marrow cavities of D mice was significantly lower than that of A mice 14 days after transplantation.
To sum up, PMSCs were successfully isolated from human placenta and cultured in vitro. Human PMSCs can significantly support the growth of umbilical cord blood MNCs. Human PMSCs and umbilical cord blood MNCs were co-transplanted into SCID mice by IBMI technology. Human PMSCs can effectively promote the early hematopoietic reconstruction of umbilical cord blood MNCs. This is a further study of human PMSCs in vivo and in vitro. In vitro support for hematopoietic related molecular mechanisms laid the experimental foundation.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329
本文編號：2225651
[Abstract]:At present, mesenchymal stem cells (MSCs) are a kind of adult stem cells with multipotent differentiation potential. Under different induction conditions, MSCs can differentiate into osteoblasts, chondrocytes, adipocytes and so on. The main source of MSCs is adult bone marrow, but the number and proliferation and differentiation potential of MSCs derived from adult bone marrow. In addition, the collection of MSCs from donors requires bone marrow aspiration, so the clinical application of MSCs is limited. Recent studies have shown that MSCs can be isolated from placenta, and they, like bone marrow MSCs, can differentiate into multiple cells under suitable culture conditions.
As an important component of bone marrow hematopoietic microenvironment, MSCs act on hematopoietic stem cells (HSCs) by secreting a variety of hematopoietic growth factors and direct contact between cells. MSCs play an important role in hematopoietic regulation. At present, MSCs have been used to support HSCs amplification in vitro and in vivo. Combined HSCs transplantation promotes hematopoietic reconstitution. In this study, human placenta-derived mesenchymal stem cells (PMSCs) were successfully isolated and cultured to investigate the functional effects of human PMSCs on supporting hematopoiesis in vivo and in vitro.
The first part describes the isolation, culture and identification of human PMSCs, and the supporting effect of human PMSCs on the growth of umbilical cord blood mononuclear cells (MNCs) in vitro. Sodium, vitamin C, dexamethasone system and 3-isobutyl-1-methylxanthine, insulin, indomethacin, dexamethasone system were induced to differentiate into osteoblasts and adipocytes, then alkaline phosphatase detection and vonKossa staining were used to identify the osteogenic differentiation, and oil red staining was used to identify the adipogenic differentiation. The results showed that: 1. PMSCs were successfully isolated and cultured from human placenta tissues. The adherent cells were fibroblast-like, and their phenotypes were CD29~+, CD44~+, CD1 detected by flow cytometry. 05~+, CD106~+, CD166~+, CD34~-, CD45~-, HLA-DR~-; 2. Alkaline phosphatase staining and von Kossa staining were strongly positive in PMSCs after 3 weeks of osteogenesis induction. Oil red staining was positive in PMSCs after 2 weeks of lipogenesis induction, and obvious lipid droplets appeared. 3. The total number of cells and CD45~+ cells in human PMSCs + umbilical cord blood MNCs co-culture group were strongly positive in all cultures. The number of CD45~+, CD14~+ and CD19~+ cells co-cultured on the 7th day was also significantly higher than that of the control group. The results showed that human PMSCs could be used as trophoblast cells to support the growth of umbilical cord blood MNCs in vitro.
In the second part, we compared the early hematopoietic reconstitution of bone marrow mononuclear cells (BMNCs) recipients by intra-bone marrow injection (IBMI) and peripheral intravenous injection (IV) in allogeneic bone marrow transplantation mice, and established a mouse IBMI platform. The early hematopoietic reconstitution effect of human PMSCs combined with umbilical cord blood MNCs co-transplanted into SCID mice was studied by I technique. BMNCs were transplanted into C57BL/6 recipients by IBMI and IV in BALB/c mice. Intramedullary injection of low dose group 2 (IBM2 group), tail vein injection group (IV group), each group of 20. After bone marrow transplantation 1, 3, 6, 9 days were counted the total number of tibial marrow cells in each group, and flow cytometry was used to detect the level of donor implantation (total number of donor-derived cells, number of donor-derived myeloid cells). Human PMSCs and umbilical cord blood MNCs were used as donors, through Nine SCID recipients were randomly divided into three groups: group A (PMSCs + umbilical cord blood MNCs, IBMI), group B (umbilical cord blood MNCs, IBMI), group C (PMSCs via IBMI, umbilical cord blood MNCs via IV), and three in each group. The results showed that: 1. The total number of cells in the tibial bone marrow cavity, the total number of donor-derived cells and the total number of donor-derived myeloid cells in the IBM 1 and IBM 2 groups were significantly higher than those in the IV group 6 days after transplantation. The percentage of CD34~+ and CD45~+ cells in the injected and contralateral tibial bone marrow cavities of D mice was significantly lower than that of A mice 14 days after transplantation.
To sum up, PMSCs were successfully isolated from human placenta and cultured in vitro. Human PMSCs can significantly support the growth of umbilical cord blood MNCs. Human PMSCs and umbilical cord blood MNCs were co-transplanted into SCID mice by IBMI technology. Human PMSCs can effectively promote the early hematopoietic reconstruction of umbilical cord blood MNCs. This is a further study of human PMSCs in vivo and in vitro. In vitro support for hematopoietic related molecular mechanisms laid the experimental foundation.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329
【參考文獻】
相關期刊論文 前3條
1 裴雪濤,王立生,徐黎,吳祖澤;造血生長因子對臍血CD_（34）~＋細胞的體外擴增作用[J];中華血液學雜志;1996年06期
2 劉英,莊廣倫,游澤山,李樹濃,侯景徽,孔慶瑜;人臍血造血干細胞經鼠卵黃囊進行宮內移植的實驗研究[J];中華醫(yī)學雜志;2003年01期
3 羅依,梁彬,余勤;成人骨髓間充質干細胞體外培養(yǎng)擴增及其生物學特性研究[J];浙江醫(yī)學;2003年08期
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