本文關(guān)鍵詞：臍帶血單個(gè)核細(xì)胞遷移體外實(shí)驗(yàn)研究　出處：《山西醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 臍血 細(xì)胞遷移 基質(zhì)細(xì)胞衍生因子-1(SDF-1) 亞族受體-4(CXCR4)

【摘要】： 目的: 異基因造血干細(xì)胞(HSC)移植是根治惡性血液病的唯一方法。對(duì)于缺乏HLA匹配供者的病人,尋找可靠的HSC源非常必要。除骨髓外,胎兒組織如胎兒肝臟、臍帶或胎盤血以及成人動(dòng)員的外周血中都含有HSC,但由于不同來(lái)源的HSC具有各自的特殊性,使臨床應(yīng)用受到局限。與成人血相比,臍血有其獨(dú)特的優(yōu)勢(shì):如含有較高比例的HSC;造血干/祖細(xì)胞中端粒較長(zhǎng);體外對(duì)細(xì)胞因子的刺激敏感;有較強(qiáng)的增殖能力;免疫細(xì)胞功能幼稚;容易獲得;對(duì)供者無(wú)損害;病毒抗原污染機(jī)會(huì)少等。目前臍血被認(rèn)為是最有前途的HSC來(lái)源。 但由于一份臍血中所含的造血干/祖細(xì)胞的數(shù)量有限,臍血移植主要局限于小兒和體重輕的成人患者,極大地限制了臍血移植在臨床的廣泛應(yīng)用。體外擴(kuò)增臍血干/祖細(xì)胞以拓寬臨床應(yīng)用范圍一直是研究的熱點(diǎn)。但大量的臨床實(shí)踐表明,造血干細(xì)胞成功移植不僅取決于造血干/祖細(xì)胞的數(shù)量,而且與其歸巢能力密切相關(guān)。 基質(zhì)細(xì)胞衍生因子—1(stromal cell-derived factor,SDF-1)及其受體(CXCR4)作為a趨化因子家族的一個(gè)成員,不但與調(diào)節(jié)機(jī)體免疫功能、維持胚胎發(fā)育、細(xì)胞增殖等有關(guān),而且SDF-1/CXCR4在免疫細(xì)胞趨化、腫瘤轉(zhuǎn)移以及造血細(xì)胞的動(dòng)員和歸巢等方面發(fā)揮著關(guān)鍵作用。為此,我們探討了趨化因子SDF-1以及其受體CXCR4在細(xì)胞遷移中的作用,旨在為臨床成功進(jìn)行造血干細(xì)胞移植提供一定的理論依據(jù)。 方法: 1.采臍血后應(yīng)用Ficoll淋巴細(xì)胞分離液分離、收集單個(gè)核細(xì)胞,洗滌兩遍后備用。 2.調(diào)整細(xì)胞濃度后將細(xì)胞接種于含有SCF、FL、TPO造血生長(zhǎng)因子的24孔培養(yǎng)板,分別在培養(yǎng)第4、7和10天半定量換液,補(bǔ)充生長(zhǎng)因子,觀察細(xì)胞擴(kuò)增情況。 3.將1×10~5個(gè)細(xì)胞懸浮于含0.1ml平衡液的Transwell上腔,其下腔為含有不同濃度SDF-1的0.6ml趨化液,在37℃、5%CO_2、100%濕度條件下趨化4～5小時(shí),同時(shí)另設(shè)細(xì)胞抗原CXCR4阻斷組。計(jì)數(shù)進(jìn)入下腔的細(xì)胞。 4.將1×10~6個(gè)細(xì)胞接種在含生長(zhǎng)因子SCF、FL、TPO的2ml IMDM培養(yǎng)基中培養(yǎng)。分別在培養(yǎng)的第0、4、7、10、14天,收集細(xì)胞,流式細(xì)胞儀檢測(cè)CXCR4表達(dá)情況。同時(shí),在第3步驟中獲得的SDF-1最佳趨化濃度條件下檢測(cè)細(xì)胞遷移率。 5.統(tǒng)計(jì)分析采用SPSS 13.0統(tǒng)計(jì)軟件,所有檢驗(yàn)結(jié)果采用雙側(cè)檢驗(yàn),P值小于或等于0.05認(rèn)為差別有統(tǒng)計(jì)學(xué)意義。計(jì)量數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示,組間比較采用單因素方差分析,兩變量間關(guān)系研究采用直線相關(guān)分析。 結(jié)果: 通過對(duì)6例標(biāo)本的觀察,我們發(fā)現(xiàn) 1.每份臍血的體積及所含有核細(xì)胞在不同個(gè)體中差異較大。6份臍血體積范圍在44～91ml,平均69.17±17.43ml;有核細(xì)胞范圍為(1.14～4.52)×10~8個(gè),平均2.91±1.27×10~8個(gè)。 2.經(jīng)14天重組人造血生長(zhǎng)因子組合SCF、FL、TPO體外培養(yǎng),各階段的MNC均得到有效擴(kuò)增;至14天時(shí),總有核細(xì)胞數(shù)擴(kuò)增了248倍,細(xì)胞數(shù)量顯著增加。 3.臍血MNC遷移實(shí)驗(yàn)發(fā)現(xiàn):隨著SDF-1濃度增加,新鮮臍血MNC遷移率升高,但SDF-1濃度達(dá)到150ng/ml時(shí)遷移率趨于平穩(wěn);當(dāng)CXCR4阻斷型抗體作用后,MNC遷移率與未加SDF-1組無(wú)差異。 4.重組人造血生長(zhǎng)因子組合SCF、FL、TPO體外培養(yǎng)MNC時(shí),培養(yǎng)的早期,趨化因子SDF-1受體CXCR4的表達(dá)升高,同時(shí)臍血MNC遷移率也升高。但隨著培養(yǎng)時(shí)間的延長(zhǎng),CXCR4表達(dá)量漸漸降低,而且臍血MNC遷移率隨之降低。 結(jié)論: 1.經(jīng)有核細(xì)胞計(jì)數(shù)證明,在含血清、無(wú)基質(zhì)細(xì)胞支持的條件下,采用重組人造血生長(zhǎng)因子SCF、FL、TPO聯(lián)合擴(kuò)增系統(tǒng),可以有效地對(duì)新鮮臍血MNC進(jìn)行體外擴(kuò)增。 2.通過Transwell Plate可以有效地模擬細(xì)胞穿越內(nèi)皮現(xiàn)象。隨著趨化因子SDF-1濃度的增高,MNC遷移率遞增,但當(dāng)SDF-1濃度達(dá)一定高時(shí),細(xì)胞趨化率達(dá)穩(wěn)定,繼續(xù)增高SDF-1濃度,細(xì)胞遷移率變化不顯著。 3.MNC遷移率與趨化因子受體CXCR4表達(dá)量間存在相關(guān)性,應(yīng)用CXCR4阻斷抗體后MNC趨化率與未加SDF-1無(wú)顯著差異。
[Abstract]:Objective:
Allogeneic hematopoietic stem cell transplantation (HSC) is the only way to cure malignant hematological diseases. For the lack of HLA matched donors for patients, it is necessary to find a reliable source of HSC. In addition to the bone marrow, fetus liver, adult umbilical cord or placental blood and mobilized peripheral blood are contained in HSC, but because of different sources of HSC have their own specialty, the clinical application is limited. Compared with the adult blood, umbilical cord blood has its unique advantages such as: with a higher proportion of HSC; hematopoietic stem / progenitor cells in longer telomeres; stimulation of cytokine in vitro sensitivity; have a strong proliferation ability; immature immune function; easy to get; no damage to the donor; less pollution. At present the opportunity of virus antigen of umbilical cord blood is considered as the most promising source of HSC.
But because of a contained in cord blood hematopoietic stem / progenitor cell number is limited, umbilical cord blood transplantation mainly confined to children and adults with light weight, which greatly limits the clinical application of umbilical cord blood transplantation. The in vitro expansion of umbilical cord blood stem / progenitor cells to broaden the scope of clinical application has been the focus of research. But in clinical practice a large number of hematopoietic stem cell transplantation show that success depends not only on the hematopoietic stem / progenitor cell number, and the ability of homing.
Stromal cell derived factor - 1 (stromal cell-derived, factor, SDF-1) and its receptor (CXCR4) as a member of the a chemokine factor family, not only with the regulation of immune function, maintain the embryonic development, cell proliferation, chemotaxis and SDF-1/CXCR4 in immune cells, play a key role in tumor metastasis and hematopoietic stem cell mobilization homing and etc. Therefore, we explore the role of chemokine SDF-1 and its receptor CXCR4 in cell migration, designed for clinical successful hematopoietic stem cell transplantation provides a theoretical basis.
Method:
1. after umbilical cord blood, Ficoll lymphocyte separation solution was used to separate the mononuclear cells, and the cells were washed for two times.
2., after adjusting the cell concentration, the cells were seeded on 24 hole culture plates containing SCF, FL and TPO hematopoietic growth factors. They were cultured in 4,7 and 10 days respectively for quantitative exchange, supplementation of growth factors and observation of cell expansion.
3., 1 x 10~5 cells were suspended in the Transwell upper chamber containing 0.1ml equilibrium solution, the lower chamber was 0.6ml chemotactic solution containing different concentrations of SDF-1, and it was chemotactic for 4~5 hours at 37 C and 5%CO_2100% humidity. At the same time, another cell antigen CXCR4 blocking group was set up. Counting the cells entering the inferior cavity.
4. will be 1 * 10~6 cells inoculated in containing the growth factor SCF, FL, TPO 2ml IMDM medium. Cells were collected in cultured 0,4,7,10,14 days, respectively, flow cytometry was used to detect the expression of CXCR4. At the same time, in the third step the SDF-1 optimal chemoattractant concentration under the conditions of detection of cell migration rate.
Using SPSS 13 statistical software 5. statistical analysis, all test results using two-sided test, P value is less than or equal to 0.05 that the difference was statistically significant. The mean and standard deviation for the measurement data ((?) + s) said, compared with single factor analysis of variance, linear correlation analysis was used to study the relationship between the two variables.
Result:
Through the observation of 6 specimens, we found that
1., the volume of umbilical cord blood and the nuclear cells contained in each umbilical cord are quite different among different individuals. The volume range of umbilical cord blood of.6 is 44 to 91ml, with an average of 69.17 + 17.43ml, and the range of nucleated cells is (1.14 to 4.52) x 10~8, with an average of 2.91 + 1.27 * 10~8.
2., after 14 days of recombination, artificial blood growth factor combination SCF, FL and TPO were cultured in vitro, and MNC at each stage was effectively amplified. Until 14 days, the total number of nuclear cells increased 248 times, and the number of cells increased significantly.
3. umbilical cord blood MNC migration experiment showed that with the increase of SDF-1 concentration, fresh cord blood MNC migration rate increased, but the concentration of SDF-1 reached 150ng/ml migrated rate tended to be stable; when the CXCR4 blocking antibody, MNC mobility and without SDF-1 group had no difference.
4. recombinant human hematopoietic growth factor SCF, FL, MNC and TPO were cultured in vitro and cultured early expression of chemokine receptor CXCR4 and SDF-1 elevated cord blood MNC migration rate also increased. But with prolonged incubation time, CXCR4 expression gradually decreased, and the umbilical cord blood MNC mobility decreases.
Conclusion:
1., by nuclear cell count, it is proved that the recombinant human blood growth factor SCF, FL and TPO combined amplification system can effectively expand the MNC of fresh umbilical cord blood in serum and without stromal cell support.
2., Transwell Plate can effectively simulate the phenomenon of cell passing through the endothelium. With the increase of SDF-1 concentration, the mobility of MNC increased, but when the SDF-1 concentration reached a certain level, the chemotaxis rate of cells was stable, and the SDF-1 concentration increased, and the mobilities of cell migration did not change significantly.
There was a correlation between the mobility of 3.MNC and the expression of chemokine receptor CXCR4, and there was no significant difference between the chemotactic rate of MNC and the non SDF-1 after the application of CXCR4 to block the antibody.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329

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