人外周血淋巴細胞體外擴增培養(yǎng)后細胞表型變化及生物學(xué)活性的研究
發(fā)布時間：2018-07-20 20:26
【摘要】： 目的：建立一種高效、簡便的體外擴增培養(yǎng)人外周血淋巴細胞的方法。 方法：取35例惡性腫瘤患者的外周血單個核細胞(PBMCs)，在GMP實驗室體系下，應(yīng)用OKM-100及OKM-200培養(yǎng)基，經(jīng)細胞刺激因子OKM-24體外誘導(dǎo)擴增，觀察效應(yīng)細胞體外擴增培養(yǎng)周期、擴增前后細胞總數(shù)、擴增倍數(shù)以及細胞存活率，同時觀察細胞回輸患者后的不良反應(yīng)。 結(jié)果：細胞在培養(yǎng)的第2天即可觀察到效應(yīng)細胞增殖；細胞擴增培養(yǎng)周期為13．46±1．20d；擴增培養(yǎng)前外周血分離所得的PBMCs細胞數(shù)為6．60±2．23×10~6，擴增培養(yǎng)至細胞成熟收獲后所得的效應(yīng)細胞數(shù)為2．99±0．65×10~9，擴增倍數(shù)為488．06±191．25；臺盼藍染色檢測細胞存活率為97．71±1．07％；內(nèi)毒素及細菌、真菌、支原體、外來病毒檢測均為陰性�；颊呋剌斝�(yīng)細胞后無明顯的不良反應(yīng)。 結(jié)論：在本研究體系下體外誘導(dǎo)擴增培養(yǎng)腫瘤患者自體外周血致敏淋巴細胞效率較高，操作簡便，生物安全性良好。 目的：觀察35例惡性腫瘤病人外周血淋巴細胞在體外經(jīng)擴增培養(yǎng)后細胞表型的變化。 方法：體外大規(guī)模誘導(dǎo)和擴增惡性腫瘤病人外周血淋巴細胞及單個核細胞，然后應(yīng)用流式細胞術(shù)測定擴增前后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~-細胞在淋巴細胞中所占百分比的變化。 結(jié)果：經(jīng)體外擴增培養(yǎng)后，CD3~+，CD3~+CD8~+，，CIK(cytokine-induced killercells)細胞及其亞型CD3~+CD16~-CD56~+、CD3~+CD16~+CD56~-、CD3~+CD16~+CD56~+，CD45RO~+細胞及其亞型CD8~+CD45RO~+，CD8~+CD28~-，CD25~+，CD29~+，CD3~+HLA-DR~-，CD8~+HLA-DR~+和CD8~+HLA-DR~-細胞比例較培養(yǎng)前明顯增加(P0．01)；而CD19~+，CD3~+CD4~+，NK(natural killer cells)細胞及其亞型CD3~-CD16~-CD56~+、CD3~-CD16~+CD56~-、CD3~-CD16~+CD56~+，CD45RA~+細胞及其亞型CD4~+CD45RA~+、CD8~+CD45RA~+，CD4~+CD45RO~+，CD28~+細胞及其亞型CD8~+CD28~+，CD4~+CD25~+，CD4~+CD29~+，CD4~+HLA-DR~+和CD4~+HLA-DR~-細胞比例則明顯的降低(P0．01)；HLA-DR~+細胞和CD3~+HLA-DR~+細胞培養(yǎng)前后細胞比例變化無統(tǒng)計學(xué)意義(P值分別為0．137和0．423)。 結(jié)論：經(jīng)體外擴增培養(yǎng)后，所得細胞主要是以CD3~+細胞為主，CD4~+T細胞比例明顯下降，CD8~+T細胞比例顯著增高，調(diào)節(jié)性T細胞(Treg)細胞和CD19~+B細胞的比例極低，活化性T細胞的比例與培養(yǎng)前無顯著差異。在增多的CD8~+T細胞中，效應(yīng)性T細胞(T_E)并未明顯增加，但記憶性T細胞(T_M)卻顯著增多。NK細胞比例下降，但CIK細胞的比例卻明顯增多。從對培養(yǎng)后細胞表型的分析可知，理論上，培養(yǎng)后細胞不僅具有直接的殺傷腫瘤細胞的細胞毒活性，回輸患者體內(nèi)后經(jīng)抗原刺激后還具有活化為效應(yīng)細胞的能力。 目的：①研究人外周血淋巴細胞在體外經(jīng)擴增培養(yǎng)后細胞在不同保存條件下存活率的變化；②初步研究體外擴增培養(yǎng)細胞殺傷腫瘤細胞的活力。 方法：①按照本課題第一部分中的方法體外擴增3例惡性腫瘤患者外周血淋巴細胞，收獲細胞后計算效應(yīng)細胞濃度，然后將效應(yīng)細胞分別放置于4℃和25℃下保存，于細胞收獲后的不同時間點應(yīng)用碘化丙啶(PI)對兩種存放條件下的細胞進行染色并應(yīng)用流式細胞儀器檢測計算細胞存活率；②按照本課題第一部分中的方法體外擴增3例患者外周血淋巴細胞，按照本課題第二部分中的方法檢測擴增后細胞中NK細胞、CIK細胞的百分比，然后應(yīng)用MTT法檢測效應(yīng)細胞的細胞毒活性。 結(jié)果：①患者1效應(yīng)細胞初始存活率為98．8％，細胞濃度為7．4×10~7／mL，在4℃保存條件下其存活率明顯降低的時間點為102h，而在25℃保存條件下其存活率明顯下降的時間點為48h；患者2效應(yīng)細胞初始存活率為95．5％，細胞濃度為5．8×10~7／mL，在4℃保存條件下其存活率明顯下降的時間點為60h，在25℃保存條件下其存活率明顯降低的時間點為24h；患者3效應(yīng)細胞初始存活率為97．1％，細胞濃度10．4×10~7／mL，在4℃保存條件下其存活率明顯降低的時間點為84h，在25℃保存條件下其存活率明顯下降的時間點為24h。②患者1效應(yīng)細胞中NK細胞的百分比為2．4％，CIK細胞的百分比為53．9％，其12：1、25：1和50：1三個效靶比效應(yīng)細胞殺傷腫瘤細胞的殺傷率分別為64．9％、70．8％和83．4％；患者2效應(yīng)細胞中NK細胞的百分比為0．6％，CIK細胞的百分比為69．5％，其12：1、25：1和50：1三個效靶比效應(yīng)細胞殺傷腫瘤細胞的殺傷率分別為71．6％、75．1％和88．6％；患者3效應(yīng)細胞中NK細胞的百分比為11．6％，CIK細胞的百分比為41．7％，其12：1、25：1和50：1三個效靶比效應(yīng)細胞殺傷腫瘤細胞的殺傷率分別為62．9％、67．3％和74．8％。 結(jié)論：①4℃條件比25℃條件更適合與保存效應(yīng)細胞，效應(yīng)細胞初始存活率低者以及細胞濃度較高者其細胞存活率下降的比較明顯；②效應(yīng)細胞具有較強的殺傷腫瘤細胞的細胞毒活性，且該活性與CIK細胞在效應(yīng)細胞中所占的百分比的呈正相關(guān)。
[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 鹵 1.20 days .
The number of PBMCs isolated from peripheral blood before amplification culture was 6.60 鹵 2.23 脳 10 ~ 6 , the number of effector cells obtained after amplification was 2.99 鹵 0.65 脳 10 ~ 9 , and the amplification factor was 488.06 鹵 191.25 ;
The survival rate of trypan blue stain was 97.71 鹵 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
本文編號：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 鹵 1.20 days .
The number of PBMCs isolated from peripheral blood before amplification culture was 6.60 鹵 2.23 脳 10 ~ 6 , the number of effector cells obtained after amplification was 2.99 鹵 0.65 脳 10 ~ 9 , and the amplification factor was 488.06 鹵 191.25 ;
The survival rate of trypan blue stain was 97.71 鹵 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
本文編號：2134679
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