本文選題：白細胞 + 全白細胞分離液　； 參考：《中國醫(yī)科大學》2007年碩士論文

【摘要】： 目的 從全血中分離和純化白細胞是許多生物學和醫(yī)學應(yīng)用中的基本方法，它廣泛被應(yīng)用于包括骨髓移植患者巨細胞病毒感染的早期診斷和白血病的免疫分型的診斷等許多方面。眾所周知，白細胞分離是許多生物學實驗的第一步，也是至關(guān)重要的一步，因為后續(xù)的研究成果的可信度很大程度上取決于分離的白細胞活性與純度。因此對白細胞分離方法的研究成為當血液學一個頗為熱點的研究課題。 目前國內(nèi)外報道的分離白細胞的方法很多，包括密度梯度離心法、物理吸附法、細胞電泳法、免疫磁珠法、流式細胞法等，其中有的分離方法所提取的細胞純度和細胞濃度已經(jīng)可以達到很高的標準。但這些方法只適用于單種白細胞或單個白細胞亞群的分離，而本實驗研究的主要目的是為了尋找一種適用于白血病免疫分型芯片的細胞分離方法，它的最終結(jié)果是要將外周血中的全白細胞分離提取出來。 為了分離、提取形態(tài)及功能完整的全白細胞，本實驗在既往細胞分離方法的基礎(chǔ)上作了改進，根據(jù)等密度梯度離心原理，通過改變細胞分離液比重及滲透壓自制了一種全白細胞分離液，經(jīng)過我們反復(fù)對全白細胞分離液條件探索以期達到一次性分離全白細胞的目的。 方法 1、全白細胞分離液的配制 右旋糖苷(或聚蔗糖)和泛影葡胺按不同比例混合配制成1.108g/ml、1.110g/ml、1.112g/ml、1.113g/ml四種比重分離液，并且在不同比重的分離液中分別加入五種不同濃度的氯化鈉來調(diào)節(jié)分離液滲透壓。 2、全白細胞分離液最佳比重及最佳滲透壓探索 取50例正常外周血標本，采用四種比重五種滲透壓全白細胞分離液，比較分離效果，篩選分離液的最佳滲透壓和最佳比重。細胞分離效果測定參數(shù)為自細胞回收率、紅細胞去除率和各種白細胞比例三方面。 3、兩種全白細胞分離液比較 取20例正常外周血標本，，通過我們配制的兩種全白細胞分離液(Dextran-全白細胞分離液和Ficoll-全白細胞分離液)對白細胞分離效果進行比較，篩選最佳全白細胞分離液。細胞分離效果比較參數(shù)為細胞濃度，細胞純度，細胞活性。 4、全白細胞分離液適用的芯片上固定抗體濃度確定 將原始濃度為0.2mg/ml的單克隆抗體按比例1：2；1：4；1：8；1：16稀釋，用MicroGridⅡ芯片點樣儀將其點在氨基硅烷—醛基修飾的玻片上，制備單克隆微陣列；然后用全白細胞分離液分離提取白細胞，制備自細胞懸液；經(jīng)過吖叮橙染色后孵育芯片；通過掃描儀掃描及顯微鏡觀察結(jié)果比較篩選芯片上固定抗體濃度。 5、全白細胞分離液對正常血細胞的分離及芯片對細胞分離結(jié)果的檢測 取臨床50例正常外周血標本，首先用全白細胞分離液進行白細胞分離，芯片孵育及細胞染色，然后通過對顯微鏡下各抗體點捕獲細胞形態(tài)觀察和數(shù)量統(tǒng)計，檢測全白細胞分離液分離白細胞效果。6、全白細胞分離液對白血病細胞的分離及其在白血病免疫分型芯片的應(yīng)用 通過全白細胞分離液對10例白血病標本的分離及芯片對白血病細胞捕獲，檢測全白細胞分離液對腫瘤細胞分離效果。 結(jié)果 1、全白細胞分離液中以比重為1.112g/ml、滲透壓為520m0sm/l左右分離液分離細胞效果最好。白細胞回收率及紅細胞去除率最高，分離所獲得的白細胞組分全，各種白細胞的比例正常。 2、Ficoll全白細胞分離液分離提取細胞的效果好于Dextran全白細胞分離液：兩種分離液分離的細胞濃度及細胞活性無明顯差異，但Ficoll全白細胞分離液分離的細胞純度高于Dextran全白細胞分離液。 3、全白細胞分離液適用的芯片上固定最佳抗體稀釋比例為1：4，即固定的抗體濃度為50μg/ml。此點樣條件的確定不僅使芯片捕獲細胞的密度大，捕獲細胞的特異性好，而且保證了芯片對全白細胞分離液檢測結(jié)果的準確性。 4、芯片對全白細胞分離液分離外周血白細胞檢測結(jié)果：取50例正常外周血標本，用全白細胞分離液分離，再用白血病免疫分型細胞芯片捕獲細胞后，統(tǒng)計分析。在芯片上可見CD3、CD4、CD7、CD8、CD19、CD20、CD21、CD22八個抗體點上捕獲的細胞90％以上為淋巴細胞，CD13、CD14、CD15、CD33四個抗體點上捕獲的細胞90％以上為粒細胞，細胞的形態(tài)正常。CD4、CD15、CD19三個抗體點上捕獲的細胞做免疫細胞化學染色，可見CD4、CD15、CD19陽性。 5、利用全白細胞分離液對10例白血病標本的分離，白血病免疫分型芯片成功地捕獲到白血病細胞。急性T淋巴細胞白血病(T-ALL)4例，在CD4、CD7抗體點上捕獲到幼稚的淋巴細胞，急性B淋巴細胞白血病(B-ALL)3例，在CD19、CD22點上可見幼稚的淋巴細胞，急性髓系白血病(AML)3例，在CD13、CD33、CD15抗體點上捕獲到幼稚的粒細胞。通過白血病免疫分型芯片對白血病診斷的結(jié)果與臨床相符合。 結(jié)論 本實驗自制的全白細胞分離液成功地分離全白細胞。所獲得的細胞濃度及細胞純度高，細胞形態(tài)及細胞活性保持良好，細胞組分全，細胞比例正常，不僅解決了白細胞提取過程中紅細胞和粒細胞不易分離的難題，而且也最大限度的減少了紅細胞污染，完全符合白血病免疫分型芯片對細胞標本的要求。為許多生物學和醫(yī)學的應(yīng)用提供了一種有效的細胞分離方法。
[Abstract]:objective
The separation and purification of white blood cells from the whole blood is a basic method in many biological and medical applications. It is widely used in many aspects, including early diagnosis of cytomegalovirus infection in bone marrow transplant patients and diagnosis of immunophenotyping of leukemia. It is well known that leukocyte separation is the first step of many biological experiments. An important step, because the reliability of subsequent research results depends largely on the separation of leukocyte activity and purity. Therefore, the study of leukocyte isolation has become a hot research topic in hematology.
There are many methods to separate white cells at home and abroad, including density gradient centrifugation, physical adsorption, cell electrophoresis, immunomagnetic beads and flow cytometry, among which the purity and cell concentration of some separation methods can reach very high standard. But these methods are only suitable for single white cells or single cells. The main purpose of this study is to find a cell separation method suitable for leukemic immunophenotyping chips. The final result is to isolate and extract all white blood cells from peripheral blood.
In order to isolate and extract complete white cells with complete morphology and function, this experiment was improved on the basis of previous cell separation methods. According to the principle of ISO density gradient centrifugation, a kind of full white cell separation liquid was made by changing the proportion of cell separation fluid and osmotic pressure. After repeated exploration of the conditions of the whole white cell separation solution, we could reach a period of time. The purpose of one-off separation of all leukocytes.
Method
1, the preparation of the total leukocyte separation solution
Dextran (or polysucrose) and meglumine are mixed into four kinds of specific gravity separation liquid, 1.108g/ml, 1.110g/ml, 1.112g/ml, 1.113g/ml, and five different concentrations of sodium chloride are added to the separation liquid of different specific gravity to regulate the osmotic pressure of the separation liquid.
2, the best proportion and optimal osmotic pressure of total leukocyte separation fluid.
50 normal peripheral blood samples were taken, and four specific gravity and five osmotic total leukocyte separation fluid were used to compare the separation effect. The optimum osmotic pressure and optimum specific gravity were screened. The parameters of cell separation effect were AutoCyte recovery rate, red cell removal rate and all kinds of white blood cell ratio in three aspects.
Comparison of 3, two total white blood cell separation fluid
In 20 normal peripheral blood samples, the white cell separation effect was compared through the two total leukocyte separation liquid (Dextran- total leukocyte separation solution and Ficoll- total white cell separation solution), and the optimum total white cell separation solution was screened. The cell separation effect was compared with the cell concentration, cell purity and cell activity.
4, the concentration of antibody immobilized on the whole white cell separation solution is determined.
The monoclonal antibodies with the original concentration of 0.2mg/ml were proportionately 1:2; 1:4; 1:8; diluted at 1:16. The monoclonal microarray was prepared on the glass slides modified by the amino silane aldehyde group by the MicroGrid II chip. Then the white cell suspension was separated and extracted with the total white cell separation solution to prepare the self cell suspension. After staining with acridine orange, it was stained with acridine orange. The chip was incubated, and the concentration of immobilized antibody on the chip was screened by scanning and microscopic observation.
5, separation of normal blood cells by whole leukocyte separation fluid and detection of cell separation results by chips.
In 50 normal peripheral blood samples, white cell separation was first used for leukocyte separation, microchip incubation and cell staining. Then, by observing and counting the cell morphology of the antibody points under the microscope, the leukocyte effect was detected by the total white cell separation solution.6, and the leukocyte separation solution was separated to the leukemia cells. Its application in leukemic immunophenotyping
Isolation of 10 leukemia samples and capture of leukemic cells by leukocyte isolation fluid were used to detect the effect of whole leukocyte separation fluid on tumor cells.
Result
1, the proportion of the total white cell separation liquid was 1.112g/ml and the osmotic pressure of 520m0sm/l separation liquid was the best. The rate of leucocyte recovery and the rate of red cell removal was the highest, the white cell components obtained by separation were all, and the proportion of all kinds of white blood cells was normal.
2, the effect of Ficoll total leucocyte separation solution was better than that of Dextran total white cell separation solution. There was no significant difference in cell concentration and cell activity separated by two kinds of separation solution, but the purity of cell separated by Ficoll total white cell separation solution was higher than that of Dextran total white cell separation solution.
3, the optimal antibody dilution ratio of the full white cell separation solution is 1:4, that is, the fixed antibody concentration is 50 mu g/ml., which not only makes the chip capture cell density, the specificity of the capture cell is good, but also ensures the accuracy of the detection results of the whole white cell separation solution.
4, the microchip was used to separate the peripheral blood white blood cells from the total leukocyte separation solution. 50 cases of normal peripheral blood samples were collected with full white cell separation solution, and then the leukemia immunophenotyping cell chip was used to capture cells. On the chip, CD3, CD4, CD7, CD8, CD19, CD20, CD21, CD22 were found to capture more than 90% cells on the eight antibody points. More than 90% of the cells captured on the four antibody points of the lymphocyte, CD13, CD14, CD15, and CD33 were granulocytes. The cell morphology was normal.CD4, CD15, and CD19 three antibody points were stained by immunocytochemical staining, and CD4, CD15, CD19 positive.
5, the leukemic cells were isolated from 10 leukemic specimens with full leucocyte separation solution. Leukemia cells were successfully captured by leukemic immunophenotyping chips. 4 cases of acute T lymphocytic leukemia (T-ALL) were captured at CD4, CD7 antibody points and 3 cases of acute B lymphocyte leukaemia (B-ALL). The immature lymphoid lymphatic cells were observed at the CD19 and CD22 points. In 3 cases of acute myeloid leukemia (AML), naive granulocytes were captured at the CD13, CD33, and CD15 antibody points. The results of leukemia diagnosis by the leukemia immunophenotyping chip were in accordance with the clinic.
conclusion
The total white cell separation liquid was successfully separated. The cell concentration and cell purity were high, the cell morphology and cell activity remained well, the cell components were complete, and the proportion of cells was normal. It not only solved the difficult problem of the separation of red cells and granulocytes in the process of leukocyte extraction, but also the maximum decrease. The red cell pollution is fully in line with the requirements of the leukemic immunophenotyping chip for the cell specimens. It provides an effective method of cell separation for many biological and medical applications.
【學位授予單位】：中國醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329

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