CN117417886A - 一种负载肿瘤抗原的树突状细胞激活的t淋巴细胞的培养方法 - Google Patents
一种负载肿瘤抗原的树突状细胞激活的t淋巴细胞的培养方法 Download PDFInfo
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Abstract
本发明提供了一种负载肿瘤抗原的树突状细胞激活的T淋巴细胞的培养方法，属于DC‑T细胞培养技术领域。本发明提供的培养方法，包括如下步骤：(1)将肿瘤细胞抗原与树突状细胞在诱导培养基Ⅰ中共孵育，得到负载肿瘤抗原的树突状细胞；(2)将所述负载肿瘤抗原的树突状细胞与T淋巴细胞在诱导培养基Ⅱ中共孵育，诱导得到负载肿瘤抗原的树突状细胞激活的T淋巴细胞。本发明提供的培养方法提高了负载肿瘤抗原的DC‑T细胞对肿瘤细胞的杀伤作用，提高了对小鼠体内肿瘤的抑制作用。
Description
技术领域
本发明涉及DC-T细胞培养技术领域，尤其涉及一种负载肿瘤抗原的树突状细胞激活的T淋巴细胞的培养方法。
背景技术
树突状细胞(也称DC细胞)是由加拿大学者Steinman于1973年发现的，是功能最强的抗原提呈细胞，因其成熟时伸出许多树突样或伪足样突起而得名。DC起源自骨髓多能造血干细胞，分化主要有两条途径：①髓样干细胞在GM-CSF的刺激下分化为DC，称为髓样DC(myeloid dendritic cells,MDC)，也称DC1，与单核细胞和粒细胞有共同的前体细胞；②来源于淋巴样干细胞，与T细胞和NK细胞有共同的前体细胞，称为淋巴样DC(Lymphoiddendritic cells,LDC)或浆细胞样DC(plasmacytoid dendritic cells,pDC)，即DC2。因为树突状细胞能够刺激T细胞并启动早期免疫反应，因此，可以用于肿瘤的免疫治疗，例如DC疫苗。
人体内大部分DC处于非成熟状态，表达低水平的共刺激因子和粘附因子，体外激发同种混合淋巴细胞增殖反应的能力较低，但未成熟DC具有极强的抗原吞噬能力，在摄取抗原(包括体外加工)或受到某些因素刺激时即分化为成熟DC，而成熟的DC表达高水平的共刺激因子和粘附因子。DC在成熟的过程中，由接触抗原的外周组织迁移进入次级淋巴器官，与T细胞接触并激发免疫应答。
DC作为目前发现的功能最强的APC，能够诱导特异性的细胞毒性T淋巴细胞(cytotoxic T lymphocyte，CTL)生成。研究表明，应用肿瘤相关抗原或抗原多肽体外冲击致敏DC，回输或免疫接种于载瘤宿主，可诱发特异性CTL的抗肿瘤免疫反应。但是，目前所得的负载肿瘤抗原DC-T细胞在肿瘤治疗中还存在一定的局限定，因此有必要对肿瘤相关抗原冲击致敏DC过程和负载肿瘤抗原的DC刺激T淋巴细胞的过程进行研究，从而提高负载肿瘤抗原的DC-T细胞对肿瘤的抑制作用。
发明内容
本发明的目的在于提供一种负载肿瘤抗原的树突状细胞激活的T淋巴细胞的培养方法，对肿瘤相关抗原冲击致敏DC过程和负载肿瘤抗原的DC刺激T淋巴细胞的过程进行优化，从而提高负载肿瘤抗原的DC-T细胞对肿瘤的治疗作用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种负载肿瘤抗原的树突状细胞激活的T淋巴细胞的培养方法，包括如下步骤：
(1)将肿瘤细胞抗原与树突状细胞在诱导培养基Ⅰ中共孵育，得到负载肿瘤抗原的树突状细胞；
(2)将所述负载肿瘤抗原的树突状细胞与T淋巴细胞在诱导培养基Ⅱ中共孵育，诱导得到负载肿瘤抗原的树突状细胞激活的T淋巴细胞；
所述所述诱导培养基Ⅰ以RPMI-1640完全培养基为基础培养基，含有如下浓度的刺激因子：rhGM-CSF 400～600U/mL、rhIL-4800～1200U/mL、rhIL-15800～1200U/mL、TNF-α80～120U/mL。
优选的，所述肿瘤细胞抗原按照制备前肿瘤细胞的数量计，与树突状细胞的混合细胞数之比为5～10：1。
优选的，步骤(1)中所述共孵育的时间为4～5d。
优选的，所述诱导培养基Ⅱ以RPMI-1640完全培养基为基础培养基，含有如下浓度的成分：rhIL-2800～1200U/mL、rhIL-6800～1200U/mL、TNF-α800～1200U/mL。
优选的，所述负载Hep-2肿瘤抗原的树突状细胞与T淋巴细胞的混合细胞数之比为9～10:1。
优选的，步骤(2)中所述共孵育的时间为3～4d。
优选的，所述肿瘤为喉癌。
本发明提供了一种负载肿瘤抗原的树突状细胞激活的T淋巴细胞的培养方法，本发明通过对肿瘤相关抗原冲击致敏DC过程的诱导培养基成分和负载肿瘤抗原的DC刺激T淋巴细胞过程的诱导培养基成分进行优化，提高了负载肿瘤抗原的DC-T细胞对肿瘤细胞的杀伤作用，提高了对小鼠体内肿瘤的抑制作用。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
以喉癌肿瘤为例
(1)制备Hep-2肿瘤细胞冻融抗原
收集处于对数生长期的Hep-2肿瘤细胞，用生理盐水洗3次，然后用RPMI-1640完全培养基重悬，浓度为2×107个/mL。在-80℃/37℃下反复冻融3次，然后2000rpm离心45min取上清，过滤除菌，得到Hep-2肿瘤细胞冻融抗原。4℃保存备用。
(2)制备树突状细胞
取健康人新鲜外周抗凝血，采用Ficoll不连续密度梯度离心法分离获得单个核细胞，低渗裂解红细胞，PBS洗去血小板，以含20％PBS的RPMI-1640完全培养基调整细胞浓度至1×107个/mL，取5mL加入T-25塑料培养瓶，37℃、5％CO2条件下培养2h，培养2h后吸去未贴壁细胞和培养基，然后加入含有rhGM-CSF 500U/mL、rhIL-41000U/mL、rhIL-151000U/mL、TNF-α100U/mL的RPMI-1640完全培养基(诱导培养基Ⅰ)继续在相同条件下培养，每2d换液一次，培养4d。得到待诱导的树突状细胞(DC)。
(3)DC的诱导和鉴定
取待诱导的贴壁DC(5×105个/瓶)，每瓶加入1mL Hep-2肿瘤细胞冻融抗原，浓度为5×106个/mL(以肿瘤细胞冻融前的浓度计)。同时以未加Hep-2肿瘤细胞冻融抗原的作为对照，继续培养4d后，收集悬浮细胞，即为负载肿瘤抗原的DC。取部分细胞进行观察和小鼠抗人CD83McAb以S-P免疫细胞化学染色法进行鉴定。观察鉴定结果表明，诱导组和对照组的DC在形态上无明显区别，均具有树突状突起且大部分悬浮生长。染色结果显示，阳性率达到80％以上，已经分化成熟。
(4)制备T淋巴细胞以及体外诱导
取上述健康人新鲜外周抗凝血，同步骤(2)采用Ficoll不连续密度梯度离心法分离获得单个核细胞，然后用含20％PBS的RPMI-1640完全培养基调整单个核细胞的浓度至1×108个/mL，注入T细胞尼龙毛柱，37℃温育1h后用RPMI-1640完全培养基冲洗，收集含T淋巴细胞的冲洗液。然后与上述制备的负载肿瘤抗原的DC按照10:1的细胞数量比混合，置于含有rhIL-21000U/mL、rhIL-61000U/mL、TNF-α1000U/mL的RPMI-1640完全培养基(诱导培养基Ⅱ)中孵育，37℃、5％CO2孵育72h，得到负载Hep-2肿瘤抗原的DC激活的T淋巴细胞(AgDC-T细胞)。
同时按照上述步骤(4)将步骤(3)制备的未负载Hep-2肿瘤抗原的DC与T淋巴细胞共孵育，得到未负载Hep-2肿瘤抗原的DC激活的T淋巴细胞(DC-T细胞)。
用含10％PBS的RPMI-1640完全培养基重悬AgDC-T细胞、DC-T细胞，与处于对数生长期的Hep-2肿瘤细胞(5×103个/孔)，按照细胞数量比10:1加入96孔板中(共0.2ml/孔)；同时设置T淋巴细胞与Hep-2肿瘤细胞混合孵育(10:1，浓度同上)的孔，作为对照，每组设置5个重复。37℃、5％CO2孵育72h，然后加入5mg/mL MTT溶液0.2ml/孔继续培养4h，离心沉淀、去上清、无水乙醇溶解，用酶联免疫检测仪在570nm下测光密度值(OD)，计算各组对靶细胞的杀伤率，结果如表1所示。
杀伤率(％)＝[1-(效靶细胞OD值-效应细胞OD值)]/靶细胞OD值*100％
其中，效靶细胞OD值指的是AgDC-T细胞或DC-T细胞组测定的OD值；效应细胞OD值指的是T淋巴细胞组测定的OD值；靶细胞OD值指的是Hep-2肿瘤细胞单独培养的OD值。
表1AgDC-T细胞对Hep-2肿瘤细胞的杀伤率
| 组别 | 杀伤率/％ |
| AgDC-T细胞 | 89.62％ |
| DC-T细胞 | 10.06％ |
可见，本发明制备的AgDC-T细胞对Hep-2肿瘤细胞具有明显的体外杀伤作用。
对比例1
本对比例是按照传统诱导培养基对树突状细胞和T淋巴细胞进行培养和诱导，具体的是将实施例1中的两种诱导培养基中的细胞因子进行替换：
将其中的诱导培养基Ⅰ替换成含有rhGM-CSF 1000U/mL、rhIL-41000U/mL的RPMI-1640完全培养基；将诱导培养基Ⅱ替换成含有TNF-α1000U/mL的RPMI-1640完全培养基。其他操作同实施例1。培养得到负载Hep-2肿瘤抗原的DC激活的T淋巴细胞后，对其对Hep-2肿瘤细胞的杀伤率进行测定，结果其对Hep-2肿瘤细胞的杀伤率为72.15％，显著低于实施例1培养的AgDC-T细胞。
实验例1
取实施例1和对比例1培养至对数生长期的AgDC-T细胞和处于对数生长期的Hep-2肿瘤细胞，用PBS洗涤2次后用PBS(pH＝7.2)重悬，检测细胞活性大于95％后，调整细胞浓度5×106个/mL，备用。
取雌性裸鼠BALA/C，4～6周龄，体重在(20±2)g之间，将其中的30只随机分为3组，实验组、对照组和空白对照组，每组10只。剔除背部左右两侧的被毛，分别作如下处理：
实验组：于背部右侧皮下接种0.2mL实施例1培养的AgDC-T细胞(1×106个/mL)，3天后，于背部左侧皮下接种0.2mLHep-2肿瘤细胞(1×106个/mL)。
对照组：于右侧皮下接种0.2mL对比例1培养的AgDC-T细胞(1×106个/mL)，3天后，于背部左侧皮下接种0.2mL Hep-2肿瘤细胞(1×106个/mL)。
空白对照组：于右侧皮下接种0.2mL生理盐水，3天后，于背部左侧皮下接种0.2mLHep-2肿瘤细胞(1×106个/mL)。
以上3组处理完后每天统计肿瘤发生情况，结果：空白对照组与接种Hep-2肿瘤细胞的第7天发现1只裸鼠发生肿瘤，第15天所有裸鼠都发现肿瘤，肿瘤发生率为100％。对照组观察至第10天有1只裸鼠发生肿瘤，观察至第15天有3只裸鼠发生肿瘤，肿瘤发生率为30％。实验组观察至第20天暂未发现肿瘤，肿瘤发生率为0。可见，实施例1培养的AgDC-T细胞对于预防肿瘤发生具有更好的效果。
实验例2
按照实验例1的方法准备AgDC-T细胞和Hep-2肿瘤细胞，并按照空白对照组的处理方式，15天后得到携带肿瘤鼠。
将肿瘤鼠分为3组，实验组、对照组和空白组，每组10只。分别作如下处理：
实验组：于肿瘤鼠背部右侧注射0.2mL实施例1培养的AgDC-T细胞(1×106个/mL)，进行治疗。
对照组：于肿瘤鼠背部右侧注射0.2mL对比例1培养的AgDC-T细胞(1×106个/mL)，进行治疗。
空白组：于肿瘤鼠背部右侧注射0.2mL生理盐水。
观察肿瘤生长情况，记录肿瘤体积(mm3)，统计结果如表2。
表2不同处理下肿瘤体积(mm3)随治疗时间的变化
| 记录天数 | 空白组 | 对照组 | 实验组 |
| 第5天 | 2.30±1.0 | 1.5±1.1 | 0.9±0.02 |
| 第10天 | 7.2±2.5 | 6.2±2.6 | 5.3±1.3 |
| 第15天 | 15.2±8.4 | 13.8±3.8 | 11.4±2.6 |
| 第18天 | 25.4±11.2 | 21.3±3.9 | 18.6±5.5 |
| 第21天 | 46.8±13.5 | 39.8±10.3 | 26.7±7.4 |
| 第24天 | 66.3±20.8 | 48.4±12.2 | 35.2±10.1 |
| 第27天 | 110.4±21.1 | 76.8±15.9 | 46.6±13.5 |
| 第30天 | 167.5±29.7 | 110.6±21.6 | 65.8±16.9 |
| 第33天 | 228.3±31.8 | 159.5±22.9 | 85.3±23.5 |
| 第35天 | 282.2±56.4 | 220.8±36.5 | 110.7±36.5 |
| 第37天 | 356.4±63.5 | 289.3±45.8 | 145.8±33.8 |
| 第39天 | 426.8±66.4 | 364.8±52.3 | 198.4±47.1 |
由表2可知，实验组对于肿瘤的抑制效果要明显好于对照组和空白组，表明本发明实施例1培养的AgDC-T细胞具有更强的肿瘤杀伤作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种负载肿瘤抗原的树突状细胞激活的T淋巴细胞的培养方法，其特征在于，包括如下步骤：
(1)将肿瘤细胞抗原与树突状细胞在诱导培养基Ⅰ中共孵育，得到负载肿瘤抗原的树突状细胞；
(2)将所述负载肿瘤抗原的树突状细胞与T淋巴细胞在诱导培养基Ⅱ中共孵育，诱导得到负载肿瘤抗原的树突状细胞激活的T淋巴细胞；
所述所述诱导培养基Ⅰ以RPMI-1640完全培养基为基础培养基，含有如下浓度的刺激因子：rhGM-CSF 400～600U/mL、rhIL-4800～1200U/mL、rhIL-15800～1200U/mL、TNF-α80～120U/mL。
2.如权利要求1所述的培养方法，其特征在于，所述肿瘤细胞抗原按照制备前肿瘤细胞的数量计，与树突状细胞的混合细胞数之比为5～10：1。
3.如权利要求2所述的培养方法，其特征在于，步骤(1)中所述共孵育的时间为4～5d。
4.如权利要求1所述的培养方法，其特征在于，所述诱导培养基Ⅱ以RPMI-1640完全培养基为基础培养基，含有如下浓度的刺激因子：rhIL-2800～1200U/mL、rhIL-6800～1200U/mL、TNF-α800～1200U/mL。
5.如权利要求4所述的培养方法，其特征在于，所述负载Hep-2肿瘤抗原的树突状细胞与T淋巴细胞的混合细胞数之比为9～10:1。
6.如权利要求5所述的培养方法，其特征在于，步骤(2)中所述共孵育的时间为3～4d。
7.如权利要求1～6任一项所述的培养方法，其特征在于，所述肿瘤为喉癌。
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