人外周血淋巴细胞体外扩增培养后细胞表型变化及生物学活性的研究
发布时间：2018-07-20 20:26
【摘要】： 目的：建立一种高效、简便的体外扩增培养人外周血淋巴细胞的方法。 方法：取35例恶性肿瘤患者的外周血单个核细胞(PBMCs)，在GMP实验室体系下，应用OKM-100及OKM-200培养基，经细胞刺激因子OKM-24体外诱导扩增，观察效应细胞体外扩增培养周期、扩增前后细胞总数、扩增倍数以及细胞存活率，同时观察细胞回输患者后的不良反应。 结果：细胞在培养的第2天即可观察到效应细胞增殖；细胞扩增培养周期为13．46±1．20d；扩增培养前外周血分离所得的PBMCs细胞数为6．60±2．23×10~6，扩增培养至细胞成熟收获后所得的效应细胞数为2．99±0．65×10~9，扩增倍数为488．06±191．25；台盼蓝染色检测细胞存活率为97．71±1．07％；内毒素及细菌、真菌、支原体、外来病毒检测均为阴性。患者回输效应细胞后无明显的不良反应。 结论：在本研究体系下体外诱导扩增培养肿瘤患者自体外周血致敏淋巴细胞效率较高，操作简便，生物安全性良好。 目的：观察35例恶性肿瘤病人外周血淋巴细胞在体外经扩增培养后细胞表型的变化。 方法：体外大规模诱导和扩增恶性肿瘤病人外周血淋巴细胞及单个核细胞，然后应用流式细胞术测定扩增前后CD~(3+)、CD19~+、CD28~+、CD25~+、CD29~+、CD45RA~+、CD45RO~+、HLA-DR~+、CD3~+CD4~+、CD3~+CD8~+、CD3~-CD16~-CD56~+、CD3~-CD16~-CD56~+、CD3~-CD16~+CD56~+、CD3~+CD16~-CD56~+、CD3~+CD16~-CD56~+、CD3~+CD16~+CD56~+、CD4~+CD25~+、CD4~+CD29~+、CD8~+CD28~+、CD8~+CD28~-、CD4~+CD45RA~+、CD8~+CD45RA~+、CD4~+CD45RO~+、CD8~+CD45RO~+、CD3~+HLA-DR~+、CD3~+HLA-DR~-、CD4~+HLA-DR~+、CD4~+HLA-DR~-、CD4~+HLA-DR~+、CD4~+HLA-DR~-细胞在淋巴细胞中所占百分比的变化。 结果：经体外扩增培养后，CD3~+，CD3~+CD8~+，，CIK(cytokine-induced killercells)细胞及其亚型CD3~+CD16~-CD56~+、CD3~+CD16~+CD56~-、CD3~+CD16~+CD56~+，CD45RO~+细胞及其亚型CD8~+CD45RO~+，CD8~+CD28~-，CD25~+，CD29~+，CD3~+HLA-DR~-，CD8~+HLA-DR~+和CD8~+HLA-DR~-细胞比例较培养前明显增加(P0．01)；而CD19~+，CD3~+CD4~+，NK(natural killer cells)细胞及其亚型CD3~-CD16~-CD56~+、CD3~-CD16~+CD56~-、CD3~-CD16~+CD56~+，CD45RA~+细胞及其亚型CD4~+CD45RA~+、CD8~+CD45RA~+，CD4~+CD45RO~+，CD28~+细胞及其亚型CD8~+CD28~+，CD4~+CD25~+，CD4~+CD29~+，CD4~+HLA-DR~+和CD4~+HLA-DR~-细胞比例则明显的降低(P0．01)；HLA-DR~+细胞和CD3~+HLA-DR~+细胞培养前后细胞比例变化无统计学意义(P值分别为0．137和0．423)。 结论：经体外扩增培养后，所得细胞主要是以CD3~+细胞为主，CD4~+T细胞比例明显下降，CD8~+T细胞比例显著增高，调节性T细胞(Treg)细胞和CD19~+B细胞的比例极低，活化性T细胞的比例与培养前无显著差异。在增多的CD8~+T细胞中，效应性T细胞(T_E)并未明显增加，但记忆性T细胞(T_M)却显著增多。NK细胞比例下降，但CIK细胞的比例却明显增多。从对培养后细胞表型的分析可知，理论上，培养后细胞不仅具有直接的杀伤肿瘤细胞的细胞毒活性，回输患者体内后经抗原刺激后还具有活化为效应细胞的能力。 目的：①研究人外周血淋巴细胞在体外经扩增培养后细胞在不同保存条件下存活率的变化；②初步研究体外扩增培养细胞杀伤肿瘤细胞的活力。 方法：①按照本课题第一部分中的方法体外扩增3例恶性肿瘤患者外周血淋巴细胞，收获细胞后计算效应细胞浓度，然后将效应细胞分别放置于4℃和25℃下保存，于细胞收获后的不同时间点应用碘化丙啶(PI)对两种存放条件下的细胞进行染色并应用流式细胞仪器检测计算细胞存活率；②按照本课题第一部分中的方法体外扩增3例患者外周血淋巴细胞，按照本课题第二部分中的方法检测扩增后细胞中NK细胞、CIK细胞的百分比，然后应用MTT法检测效应细胞的细胞毒活性。 结果：①患者1效应细胞初始存活率为98．8％，细胞浓度为7．4×10~7／mL，在4℃保存条件下其存活率明显降低的时间点为102h，而在25℃保存条件下其存活率明显下降的时间点为48h；患者2效应细胞初始存活率为95．5％，细胞浓度为5．8×10~7／mL，在4℃保存条件下其存活率明显下降的时间点为60h，在25℃保存条件下其存活率明显降低的时间点为24h；患者3效应细胞初始存活率为97．1％，细胞浓度10．4×10~7／mL，在4℃保存条件下其存活率明显降低的时间点为84h，在25℃保存条件下其存活率明显下降的时间点为24h。②患者1效应细胞中NK细胞的百分比为2．4％，CIK细胞的百分比为53．9％，其12：1、25：1和50：1三个效靶比效应细胞杀伤肿瘤细胞的杀伤率分别为64．9％、70．8％和83．4％；患者2效应细胞中NK细胞的百分比为0．6％，CIK细胞的百分比为69．5％，其12：1、25：1和50：1三个效靶比效应细胞杀伤肿瘤细胞的杀伤率分别为71．6％、75．1％和88．6％；患者3效应细胞中NK细胞的百分比为11．6％，CIK细胞的百分比为41．7％，其12：1、25：1和50：1三个效靶比效应细胞杀伤肿瘤细胞的杀伤率分别为62．9％、67．3％和74．8％。 结论：①4℃条件比25℃条件更适合与保存效应细胞，效应细胞初始存活率低者以及细胞浓度较高者其细胞存活率下降的比较明显；②效应细胞具有较强的杀伤肿瘤细胞的细胞毒活性，且该活性与CIK细胞在效应细胞中所占的百分比的呈正相关。
[Abstract]:Objective : To establish an efficient and simple method for culturing human peripheral blood lymphocytes in vitro .
Methods : Peripheral blood mononuclear cells ( PBMCs ) were isolated from 35 patients with malignant tumor . Under GMP laboratory system , OKM - 100 and OKM - 200 medium were used to induce amplification of OKM - 24 cells .
Results : The proliferation of effector cells was observed on the second day of culture .
The cell amplification culture cycle was 13.46 卤 1.20 days .
The number of PBMCs isolated from peripheral blood before amplification culture was 6.60 卤 2.23 脳 10 ~ 6 , the number of effector cells obtained after amplification was 2.99 卤 0.65 脳 10 ~ 9 , and the amplification factor was 488.06 卤 191.25 ;
The survival rate of trypan blue stain was 97.71 卤 1.07 % .
Both endotoxin and bacteria , fungi , mycoplasma and external virus were all negative . There was no obvious adverse reaction after the patient returned to the effector cells .
Conclusion : In vitro induction of autologous peripheral blood - sensitized lymphocytes in cultured tumor patients is high in vitro , and the operation is simple and the biological safety is good .
Objective : To observe the changes of cell phenotype in peripheral blood lymphocytes of 35 patients with malignant tumor after amplification culture in vitro .
Methods : Peripheral blood lymphocytes and mononuclear cells of patients with malignant tumor were induced and amplified on a large scale in vitro . CD ~ ( 3 + ) , CD29 ~ + , CD28 ~ + , CD25 ~ + , CD29 ~ + , CD3 ~ + CD56 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD3 ~ + CD16 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD3 ~ + CD16 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD4 ~ + CD29 ~ + , CD8 ~ + CD28 ~ + , CD8 ~ + CD28 ~ - , CD4 ~ + ~ + , The percentage of CD4 + , CD8 + , CD4 + , CD3 ~ + HLA - DR ~ + , CD3 ~ + HLA - DR ~ - , CD4 ~ + HLA - DR ~ + , CD4 ~ + HLA - DR ~ - , CD4 ~ + HLA - DR ~ + , CD4 ~ + HLA - DR ~ - cells in lymphocytes was changed .
Results : CD3 ~ + , CD3 ~ + CD8 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD3 ~ + CD16 ~ + CD56 ~ - , CD3 ~ + CD16 ~ + CD56 ~ + , CD8 ~ + CD28 ~ - , CD25 ~ + , CD29 ~ + , CD3 ~ + HLA - DR ~ - , CD8 ~ + HLA - DR ~ + and CD8 ~ + HLA - DR ~ - cells increased significantly ( P0.01 ) .
CD3 ~ + , CD3 ~ + CD4 ~ + , CD3 ~ - CD16 ~ - CD56 ~ + , CD3 ~ - CD16 ~ + CD56 ~ - , CD3 ~ - CD16 ~ + CD56 ~ + , CD56 ~ + , CD3 ~ - CD16 ~ + , CD4 ~ + CD25 ~ + , CD4 ~ + CD29 ~ + , CD4 ~ + HLA - DR ~ + and CD4 ~ + HLA - DR ~ - cells decreased significantly ( P0.01 ) .
There was no significant difference between HLA - DR ~ + cells and CD3 ~ + HLA - DR ~ + cells before and after cell culture ( P = 0.137 and 0.423 , respectively ) .
Conclusion : After cultured in vitro , the percentage of CD4 ~ + T cells was decreased , the proportion of CD8 ~ + T cells was significantly increased , the proportion of T _ ( T _ M ) cells was significantly increased , but the proportion of activated T - cells ( T _ M ) increased significantly .
Objective : ( 1 ) To study the changes of survival rate of human peripheral blood lymphocytes in different preservation conditions after amplification culture in vitro ;
( 2 ) Preliminary study on the activity of tumor cells in vitro .
Methods : 鈶
本文编号：2134679
[Abstract]:Objective : To establish an efficient and simple method for culturing human peripheral blood lymphocytes in vitro .
Methods : Peripheral blood mononuclear cells ( PBMCs ) were isolated from 35 patients with malignant tumor . Under GMP laboratory system , OKM - 100 and OKM - 200 medium were used to induce amplification of OKM - 24 cells .
Results : The proliferation of effector cells was observed on the second day of culture .
The cell amplification culture cycle was 13.46 卤 1.20 days .
The number of PBMCs isolated from peripheral blood before amplification culture was 6.60 卤 2.23 脳 10 ~ 6 , the number of effector cells obtained after amplification was 2.99 卤 0.65 脳 10 ~ 9 , and the amplification factor was 488.06 卤 191.25 ;
The survival rate of trypan blue stain was 97.71 卤 1.07 % .
Both endotoxin and bacteria , fungi , mycoplasma and external virus were all negative . There was no obvious adverse reaction after the patient returned to the effector cells .
Conclusion : In vitro induction of autologous peripheral blood - sensitized lymphocytes in cultured tumor patients is high in vitro , and the operation is simple and the biological safety is good .
Objective : To observe the changes of cell phenotype in peripheral blood lymphocytes of 35 patients with malignant tumor after amplification culture in vitro .
Methods : Peripheral blood lymphocytes and mononuclear cells of patients with malignant tumor were induced and amplified on a large scale in vitro . CD ~ ( 3 + ) , CD29 ~ + , CD28 ~ + , CD25 ~ + , CD29 ~ + , CD3 ~ + CD56 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD3 ~ + CD16 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD3 ~ + CD16 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD4 ~ + CD29 ~ + , CD8 ~ + CD28 ~ + , CD8 ~ + CD28 ~ - , CD4 ~ + ~ + , The percentage of CD4 + , CD8 + , CD4 + , CD3 ~ + HLA - DR ~ + , CD3 ~ + HLA - DR ~ - , CD4 ~ + HLA - DR ~ + , CD4 ~ + HLA - DR ~ - , CD4 ~ + HLA - DR ~ + , CD4 ~ + HLA - DR ~ - cells in lymphocytes was changed .
Results : CD3 ~ + , CD3 ~ + CD8 ~ + , CD3 ~ + CD16 ~ - CD56 ~ + , CD3 ~ + CD16 ~ + CD56 ~ - , CD3 ~ + CD16 ~ + CD56 ~ + , CD8 ~ + CD28 ~ - , CD25 ~ + , CD29 ~ + , CD3 ~ + HLA - DR ~ - , CD8 ~ + HLA - DR ~ + and CD8 ~ + HLA - DR ~ - cells increased significantly ( P0.01 ) .
CD3 ~ + , CD3 ~ + CD4 ~ + , CD3 ~ - CD16 ~ - CD56 ~ + , CD3 ~ - CD16 ~ + CD56 ~ - , CD3 ~ - CD16 ~ + CD56 ~ + , CD56 ~ + , CD3 ~ - CD16 ~ + , CD4 ~ + CD25 ~ + , CD4 ~ + CD29 ~ + , CD4 ~ + HLA - DR ~ + and CD4 ~ + HLA - DR ~ - cells decreased significantly ( P0.01 ) .
There was no significant difference between HLA - DR ~ + cells and CD3 ~ + HLA - DR ~ + cells before and after cell culture ( P = 0.137 and 0.423 , respectively ) .
Conclusion : After cultured in vitro , the percentage of CD4 ~ + T cells was decreased , the proportion of CD8 ~ + T cells was significantly increased , the proportion of T _ ( T _ M ) cells was significantly increased , but the proportion of activated T - cells ( T _ M ) increased significantly .
Objective : ( 1 ) To study the changes of survival rate of human peripheral blood lymphocytes in different preservation conditions after amplification culture in vitro ;
( 2 ) Preliminary study on the activity of tumor cells in vitro .
Methods : 鈶
本文编号：2134679
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