CN112704729A - 一种细胞因子颗粒 - Google Patents
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Abstract
本发明公开了一种细胞因子颗粒，所述颗粒包括细胞因子和PSAP蛋白，所述细胞因子包括有红细胞介素、集落刺激因子、淋巴因子、单核因子、非淋巴细胞、非单核‑巨噬细胞产生的细胞因子、干扰素、转化生长因子‑β、趋化因子，编辑后突变细胞因子,通过PCR扩增编辑后的基因组DNA,获得含突变位点的PCR片股。本发明的制作的细胞因子颗粒比细胞因子刺激作用更强，整体原料成本较低，制作方法简单，同时治疗肿瘤、感染、造血功能障碍、自身免疫性疾病等，具有非常广阔的应用前景。
Description
技术领域
本发明涉及生物技术领域，尤其是一种细胞因子颗粒。
背景技术
细胞因子(cytokine，CK)是由多种组织细胞(主要为免疫细胞) 所合成和分泌的小分子多肽或糖蛋白。细胞因子能介导细胞间的相互作用，具有多种生物学功能，如调节细胞生长、分化成熟、功能维持、调节免疫应答、参与炎症反应、创伤愈合和肿瘤消长等。最初，人们不清楚细胞因子的本质，便根据其生物学活性进行命名，结果导致同一细胞因子有多种名称。后来，人们认识到细胞因子主要由白细胞合成，主要介导白细胞间的相互作用，于是将这些因子统一命名为白细胞介素(interleukin，IL)，并按发现的先后顺序冠以阿拉伯数字进行命名，如IL-l、IL一2、IL-3等。自1957年Lssac发现干扰素以来，迄今已经发现了200多种细胞因子。人们将所有白细胞介素、干扰素、肿瘤坏死因子、造血因子、生长因子、趋化因子等统称为细胞因子。现代基因工程和细胞工程技术的快速发展，为发现更多的细胞因子和研究其结构与功能提供了技术条件，细胞因子的研究成果为临床上预防、诊断、治疗疾病提供了科学基础，特别是利用细胞因子治疗肿瘤、感染、造血功能障碍、自身免疫性疾病等，具有非常广阔的应用前景。
现有的细胞因子治疗效果差，不便于广泛的推广使用，针对这些问题，在这里我们提出一种细胞因子颗粒及其应用及其制备方法。
发明内容
本发明的目的在于提供一种细胞因子颗粒，解决了背景技术中所提出的问题。
为解决上述问题，本发明提供如下技术方案：一种细胞因子颗粒，所述颗粒包括细胞因子和PSAP蛋白，所述细胞因子包括有红细胞介素、集落刺激因子、淋巴因子、单核因子、非淋巴细胞、非单核-巨噬细胞产生的细胞因子、干扰素、转化生长因子-β、趋化因子，编辑后突变细胞因子,通过PCR扩增编辑后的基因组DNA,获得含突变位点的PCR片股。
作为本发明的进一步优选方式，所述集落刺激因子包括有粒细胞、巨噬细胞、SCF、EPO。
作为本发明的进一步优选方式，淋巴因子主要由淋巴细胞产生，包括T淋巴细胞、B淋巴细胞和NK细胞，还包括有有IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、IL-10、IL-12、IL-13、IL-14、IFN-γ、TNF-β、GM-CSF和神经白细胞素。
作为本发明的进一步优选方式，单核因子主要由单核细胞或巨噬细胞产生，如IL-1、IL-6、IL-8、TNF-α、G-CSF和M-CSF。
作为本发明的进一步优选方式，非淋巴细胞、非单核-巨噬细胞产生的细胞因子主要由骨髓和胸腺中的基质细胞、血管内皮细胞、成纤维细胞等细胞产生，如EPO、IL-7、IL-11、SCF、内皮细胞源性 IL-8和IFN-β。
作为本发明的进一步优选方式，S1，用灭菌剪刀细细剪碎，连同生理盐缓冲液溶液转移至15ml离心管中，1000rpm离心2min，倒掉上清，加3ml胰酶重悬沉淀，置于37℃水浴中消化20min；
S2，20min后，1000rpm离心5min，倒掉上清，加入10ml B27 培养基重新悬浮，置于35℃细胞培养皿中。
作为本发明的进一步优选方式，所述生理盐缓冲液百分浓度在 32-68％，选择NaCl、KCl、Na2HPO4和KH2PO4,溶于蒸馏水中，通过按照1.4:1.6:0.7：0.3的比例进行充分混合而成，PH控制在7.2。
与现有技术相比，本发明的有益效果如下：
本发明的制作的细胞因子颗粒比细胞因子刺激作用更强，整体原料成本较低，制作方法简单，同时治疗肿瘤、感染、造血功能障碍、自身免疫性疾病等，具有非常广阔的应用前景。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本实用发明提供一种技术方案：一种细胞因子颗粒，所述颗粒包括细胞因子和PSAP蛋白，所述细胞因子包括有红细胞介素、集落刺激因子、淋巴因子、单核因子、非淋巴细胞、非单核-巨噬细胞产生的细胞因子、干扰素、转化生长因子-β、趋化因子，编辑后突变细胞因子,通过PCR扩增编辑后的基因组DNA,获得含突变位点的PCR 片股。
集落刺激因子包括有粒细胞、巨噬细胞、SCF、EPO。
淋巴因子主要由淋巴细胞产生，包括T淋巴细胞、B淋巴细胞和 NK细胞，还包括有有IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、IL-10、 IL-12、IL-13、IL-14、IFN-γ、TNF-β、GM-CSF和神经白细胞素。
单核因子主要由单核细胞或巨噬细胞产生，如IL-1、IL-6、IL-8、 TNF-α、G-CSF和M-CSF。
非淋巴细胞、非单核-巨噬细胞产生的细胞因子，主要由骨髓和胸腺中的基质细胞、血管内皮细胞、成纤维细胞细胞产生，包括EPO、 IL-7、IL-11、SCF、内皮细胞源性IL-8和IFN-β。
具体包括以下步骤方法，S1，用灭菌剪刀细细剪碎，连同生理盐缓冲液溶液转移至15ml离心管中，1000rpm离心2min，倒掉上清，加3ml胰酶重悬沉淀，置于37℃水浴中消化20min；
S2，20min后，1000rpm离心5min，倒掉上清，加入10ml B27 培养基重新悬浮，置于35℃细胞培养皿中。
生理盐缓冲液百分浓度在32-68％，选择NaCl、KCl、Na2HPO4和 KH2PO4,溶于蒸馏水中，通过按照1.4:1.6:0.7：0.3的比例进行充分混合而成，PH控制在7.2。
实施例1
红细胞介素3份、集落刺激因子1.5份、淋巴因子2份、单核因子1份、非淋巴细胞0.5份、非单核-巨噬细胞产生的细胞因子2份、干扰素0.8份、转化生长因子-β1.5份、趋化因子2.5份。
生物解剖显微镜下取出脊柱C4-C6区，用灭菌剪刀细细剪碎，连同PBS溶液转移至15ml离心管中，1000rpm离心5min，倒掉上清，加3ml胰酶重悬沉淀，置于37℃水浴中消化20min；20min后，1000 rpm离心5min，倒掉上清，加入10ml B27培养基重新悬浮，置于 35mm细胞培养皿中，选取患有感染疾病的白兔进行测试；放于37℃， 5％CO2恒温培养箱中孵育；第二天加神经生长因子，浓度为26μg/ml，待细胞贴壁良好后，将培养培养基更换为不含神经生长因子的普通B27培养基，于36℃，24小时后，去除培养基，用PBS液体冲洗， 4％多聚甲醛固定，利用抗SV1抗体染色，通过免疫荧光实验，镜下观察感觉神经元轴突的变化。
实施例2
红细胞介素2份、集落刺激因子1份、淋巴因子0.6份、单核因子1份、非淋巴细胞0.5份、非单核-巨噬细胞产生的细胞因子2.2 份、干扰素0.8份、转化生长因子-β1.5份、趋化因子3份。
生物解剖显微镜下取出脊柱C4-C6区，用灭菌剪刀细细剪碎，连同PBS溶液转移至15ml离心管中，1000rpm离心5min，倒掉上清，加3ml胰酶重悬沉淀，置于37℃水浴中消化20min；20min后，1000 rpm离心5min，倒掉上清，加入10ml B27培养基重新悬浮，置于 35mm细胞培养皿中，选取患有肿瘤疾病的白鼠进行测试；放于37℃， 5％CO2恒温培养箱中孵育；第二天加神经生长因子，浓度为26μg/ml，待细胞贴壁良好后，将培养培养基更换为不含神经生长因子的普通 B27培养基，于36℃，24小时后，去除培养基，用PBS液体冲洗， 4％多聚甲醛固定，利用抗SV1抗体染色，通过免疫荧光实验，镜下观察感觉神经元轴突的变化。
实施例3
红细胞介素1份、集落刺激因子3份、淋巴因子0.8份、单核因子2份、非淋巴细胞0.5份、非单核-巨噬细胞产生的细胞因子3份、干扰素0.8份、转化生长因子-β1.5份、趋化因子3份。
生物解剖显微镜下取出脊柱C4-C6区，用灭菌剪刀细细剪碎，连同PBS溶液转移至15ml离心管中，1000rpm离心5min，倒掉上清，加3ml胰酶重悬沉淀，置于37℃水浴中消化20min；20min后，1000 rpm离心5min，倒掉上清，加入10ml B27培养基重新悬浮，置于35mm细胞培养皿中，选取患有造血功能障碍的猴进行测试；放于 37℃，5％CO2恒温培养箱中孵育；第二天加神经生长因子，浓度为 26μg/ml，待细胞贴壁良好后，将培养培养基更换为不含神经生长因子的普通B27培养基，于36℃，24小时后，去除培养基，用PBS液体冲洗，4％多聚甲醛固定，利用抗SV1抗体染色，通过免疫荧光实验，镜下观察感觉神经元轴突的变化。
实验数据：
综上，本发明的制作的细胞因子颗粒比细胞因子刺激作用更强，同时治疗肿瘤、感染、造血功能障碍、自身免疫性疾病等，具有非常广阔的应用前景。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点,对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
Claims (7)
1.一种细胞因子颗粒，其特征在于：所述颗粒包括细胞因子和PSAP蛋白，所述细胞因子包括有红细胞介素、集落刺激因子、淋巴因子、单核因子、非淋巴细胞、非单核-巨噬细胞产生的细胞因子、干扰素、转化生长因子-β、趋化因子，编辑后突变细胞因子,通过PCR扩增编辑后的基因组DNA,获得含突变位点的PCR片股。
2.根据权利要求1所述的一种细胞因子颗粒，其特征在于，所述集落刺激因子包括有粒细胞、巨噬细胞、SCF、EPO。
3.根据权利要求1所述的一种细胞因子颗粒，其特征在于，所述淋巴因子主要由淋巴细胞产生，包括T淋巴细胞、B淋巴细胞和NK细胞，还包括有有IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、IL-10、IL-12、IL-13、IL-14、IFN-γ、TNF-β、GM-CSF和神经白细胞素。
4.根据权利要求1所述的一种细胞因子颗粒，其特征在于，所述单核因子主要由单核细胞或巨噬细胞产生，如IL-1、IL-6、IL-8、TNF-α、G-CSF和M-CSF。
5.根据权利要求1所述的一种细胞因子颗粒，其特征在于，所述非淋巴细胞、非单核-巨噬细胞产生的细胞因子，主要由骨髓和胸腺中的基质细胞、血管内皮细胞、成纤维细胞细胞产生，包括EPO、IL-7、IL-11、SCF、内皮细胞源性IL-8和IFN-β。
6.根据权利要求1所述的一种细胞因子颗粒的制备方法，其特征在于，具体包括以下步骤方法，S1，用灭菌剪刀细细剪碎，连同生理盐缓冲液溶液转移至15ml离心管中，1000rpm离心2min，倒掉上清，加3ml胰酶重悬沉淀，置于37℃水浴中消化20min；
S2，20min后，1000rpm离心5min，倒掉上清，加入10ml B27培养基重新悬浮，置于35℃细胞培养皿中。
7.根据权利要求6所述的一种细胞因子颗粒的制备方法，其特征在于，所述生理盐缓冲液百分浓度在32-68％，选择NaCl、KCl、Na2HPO4和KH2PO4,溶于蒸馏水中，通过按照1.4:1.6:0.7：0.3的比例进行充分混合而成，PH控制在7.2。
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| WO2020033863A1 (en) * | 2018-08-09 | 2020-02-13 | Bioverativ Therapeutics Inc. | Nucleic acid molecules and uses thereof for non-viral gene therapy |
| CN109593717A (zh) * | 2018-12-29 | 2019-04-09 | 广州和能生物科技有限公司 | 一种干细胞无血清培养基 |
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