WO2023061512A1 - 体外细胞因子风暴模型及其构建方法和应用 - Google Patents
体外细胞因子风暴模型及其构建方法和应用 Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2301—Interleukin-1 (IL-1)
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- C12N2501/25—Tumour necrosing factors [TNF]
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Definitions
- the invention belongs to the field of biotechnology, and in particular relates to an in vitro cytokine storm model and its preparation method and application.
- Cytokine Release Syndrome also known vividly as Cytokine Storm, refers to the rapid and massive production of various cytokines in body fluids after the body is infected with microorganisms, such as TNF- ⁇ , IL -1, IL-6, IL-12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , MCP-1 and IL-8, etc., is a serious life-threatening syndrome, which marks an uncontrolled and A dysfunctional immune response, involving persistent activation and expansion of lymphocytes and macrophages, secreting large amounts of cytokines, may lead to systemic inflammatory responses, multiorgan failure, methemoglobinemia, acute respiratory distress syndrome, and other diseases.
- CAR-T cell therapy is also prone to cytokine storms.
- cytokine storms may also occur during immune checkpoint inhibitor therapy, but the probability is extremely low.
- microorganisms are commonly used to infect animals, or to stimulate macrophage secretion after tumor killing (Liu Y, et al,. Gasdermin E–mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome. Sci Immunol. 2020 Jan 17; 5 (43):eaax7969.) to simulate the endogenous storm of cytokines in animals. In this case, the concentration of cytokines is not confirmed enough, and the secretion concentration of factors after each tumor cell killing is unstable, so the animal body is also unstable.
- H 2 O 2 , LPS and TNF- ⁇ can damage human umbilical vein endothelial cells in vitro, and successfully established a human umbilical vein endothelial cell injury model , and within a certain concentration range, each injury factor has a concentration- and time-dependent injury degree to human umbilical vein endothelial cells.
- the present invention provides an in vitro cytokine storm model and its construction method and application, which solves the problem that the cell model of in vitro cytokine storm in the prior art is usually modeled by single cytokine treatment, and the obtained model is relatively simple and difficult to Shortcomings in meeting the ever-increasing demands of the medical field, and simulations are not very good.
- the present invention adopts the following technical solutions:
- the construction system of the cytokine storm model in vitro contains TNF- ⁇ factor, IL-1 ⁇ factor, IL-6 factor and vascular endothelial cells.
- the vascular endothelial cells are human umbilical vein endothelial cells.
- the culture medium is endothelial cell culture medium.
- the second technical solution provided by the present invention is: an in vitro cytokine storm model formed by the above-mentioned body.
- the in vitro cytokine storm model is formed by the construction system of any one of the aforementioned in vitro cytokine storm models.
- the third technical solution provided by the present invention is: the preparation method of the above-mentioned in vitro cytokine storm model.
- vascular endothelial cells were co-incubated with TNF- ⁇ factor, IL-1 ⁇ factor, and IL-6 factor.
- the concentration of any one of TNF- ⁇ factor, IL-1 ⁇ factor, and IL-6 factor is 100-400ng/ml; the incubation time is 24-168h.
- the concentration of any one of TNF- ⁇ factor, IL-1 ⁇ factor, and IL-6 factor is 100-200 ng/ml; the incubation time is 120-168 h.
- the concentrations of TNF- ⁇ factor, IL-1 ⁇ factor and IL-6 factor are the same; the incubation temperature is 37° C.; the incubation medium is endothelial cell medium.
- the fourth technical solution provided by the present invention is: the application of the in vitro cytokine storm model as described in the second technical solution in screening anti-cytokine release syndrome drugs.
- the vascular endothelial cells are human umbilical vein endothelial cells (HUVEC, Human Umbilical Vein Endothelial Cell).
- the culture medium for the incubation can be selected according to the routine in this field, and in some ways, it is Endothelial Cell Medium (ECM).
- ECM Endothelial Cell Medium
- the method further includes the step of separating the prepared in vitro cytokine storm model from the culture medium.
- the separation can be a conventional separation method in the art, such as replacing liquid.
- the cytokine release syndrome is cytokine release syndrome induced by TNF- ⁇ , IL-1 ⁇ , and IL-6.
- the fifth aspect of the present invention relates to a method for preparing an anti-cytokine release syndrome drug, specifically comprising the following steps
- the components with inhibitory effect on the cytokine storm in vitro are screened through the cytokine storm model in vitro; the effective components obtained from the screening are used to prepare anti-cytokine release syndrome medicines.
- the active ingredient is used in the pharmaceutical process, referring to the existing pharmaceutical process.
- the in vitro cytokine storm model of the present invention is obtained by jointly treating vascular endothelial cells with three factors of TNF- ⁇ , IL-1 ⁇ and IL-6, and can produce an in vitro cytokine storm with high cell apoptosis rate and significantly decreased cell viability
- This model provides a good basis for the study of multifactor-related inflammatory responses.
- the cell viability of the in vitro cytokine storm model is significantly reduced, which satisfies the need for higher therapeutic effects of in vitro research drugs.
- Fig. 1 is the fluorescent photogram of blank control group processing 24h, 48h and 72h;
- Fig. 2 is three-factor treatment group and processes 24h fluorescent photogram
- Fig. 3 is that three factor treatment groups are processed 48h fluorescent photograms
- Fig. 4 is that three factor treatment groups are processed 48h fluorescent photograms
- Figure 5 shows the apoptosis rate of HUVEC cells treated with three factors (100 ng/ml each) at different time points.
- Figure 6 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 72 hours
- Figure 7 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 24 hours
- Figure 8 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 96 hours
- Figure 9 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 120 h;
- Figure 10 shows the cell viability of HUVEC cells treated with three factors at different concentrations for 168 hours.
- the first aspect of this part describes the scheme of the present invention:
- the construction system of the cytokine storm model in vitro contains TNF- ⁇ factor, IL-1 ⁇ factor, IL-6 factor and vascular endothelial cells.
- the construction system is a composition preparation system of the storm model, and the materials in the construction system interact, and the storm model can be formed through the construction system.
- the construction system is the early stage system in the process of preparing the storm model, which is mainly used to characterize the material composition of the preparation of the storm model.
- other solutions having the same structure as the construction system should equally fall within the scope of the present invention.
- the construction system also has a culture medium; the vascular endothelial cells are human umbilical vein endothelial cells.
- the culture medium is endothelial cell culture medium.
- the second technical solution provided by the present invention is: an in vitro cytokine storm model formed by the above-mentioned body.
- the in vitro cytokine storm model is formed by the construction system of any one of the aforementioned in vitro cytokine storm models. Compared with other single-factor or multi-factor models, the storm model of the present invention has better simulation effect.
- the third technical solution provided by the present invention is: the preparation method of the above-mentioned in vitro cytokine storm model.
- vascular endothelial cells were co-incubated with TNF- ⁇ factor, IL-1 ⁇ factor, and IL-6 factor.
- the concentration of any one of TNF- ⁇ factor, IL-1 ⁇ factor, and IL-6 factor is 100-400ng/ml; the incubation time is 24-168h.
- the concentration of any one of TNF- ⁇ factor, IL-1 ⁇ factor, and IL-6 factor is 100-200 ng/ml; the incubation time is 120-168 h.
- the concentrations of TNF- ⁇ factor, IL-1 ⁇ factor and IL-6 factor are the same; the incubation temperature is 37° C.; the incubation medium is endothelial cell medium.
- the fourth technical solution provided by the present invention is: the application of the in vitro cytokine storm model as described in the second technical solution in screening anti-cytokine release syndrome drugs.
- the vascular endothelial cells are human umbilical vein endothelial cells (HUVEC, Human Umbilical Vein Endothelial Cell).
- the culture medium for the incubation can be selected according to the routine in this field, and in some ways, it is Endothelial Cell Medium (ECM).
- ECM Endothelial Cell Medium
- the method further includes the step of separating the prepared in vitro cytokine storm model from the culture medium.
- the separation can be a conventional separation method in the art, such as replacing liquid.
- the cytokine release syndrome is cytokine release syndrome induced by TNF- ⁇ , IL-1 ⁇ , and IL-6.
- the fifth aspect of the present invention relates to a method for preparing an anti-cytokine release syndrome drug, specifically comprising the following steps
- the components with inhibitory effect on the cytokine storm in vitro are screened through the cytokine storm model in vitro; the effective components obtained from the screening are used to prepare anti-cytokine release syndrome medicines.
- the active ingredient is used in the pharmaceutical process, referring to the existing pharmaceutical process.
- HUVEC human umbilical vein endothelial cells
- human TNF- ⁇ , IL-1 ⁇ and IL-6 all purchased from Beijing Tongli Haiyuan, and recombinant human IL-1 ⁇ Protein product number GMP-TL513; recombinant human IL-6 protein product number GMP-TL512; recombinant human TNF- ⁇ protein product number GMP-TL303
- cytokines are ready-to-use, and diluted to 100ng/ml with cell culture medium.
- the HUVEC cells in the control group were added with the normal culture medium, and the HUVEC cells in the three-factor treatment group were cultured with the three-factor medium, and cultured in the culture plate. 95% CO 2 , 5% O 2 ), and after 24h, 48h, and 72h of culture for the corresponding time, fluorescent staining was used to detect apoptotic bodies.
- the concentration of each cytokine was 100ng/ml to treat HUVEC cells for 24h, 48h, and 72h (that is, the length of co-incubation), and set up a control group at the same time, and use the fluorescent staining method to detect apoptotic bodies.
- 2Put Hoechst 33258 dye solution (Standard Reagent, P4403, 1ml Hoechst 33258 dye solution into 99ml PBS, keep away from light) on a magnetic stirrer and stir for more than 15min.
- the apoptosis rate at different time points showed a downward trend, the highest at 24h and the lowest at 72h.
- p ⁇ 0.001 at 24h and 72h p ⁇ 0.05 at 48h; compared with 48h and 24h, and compared with 72h and 48h, the apoptosis rate has a significant difference, p ⁇ 0.05; p ⁇ 0.001 compared with 72h and 24h.
- Human TNF- ⁇ , IL-1 ⁇ , and IL-6 were combined to treat HUVEC cells at a concentration of 50ng/ml, 100ng/ml, and 200ng/ml for 72 hours, and a control group was set up to observe cell viability.
- Human TNF- ⁇ , IL-1 ⁇ , and IL-6 were used to treat HUVECs in combination.
- concentrations of the three factors were the same, and the different experimental groups were set at 100ng/ml, 200ng/ml, and 400ng/ml, respectively, and the treatment time points were 24h, 96h, 120h, 168h, processing method and detection method are the same as embodiment 2.
- the results are shown in Figure 7-10.
- the cell viability reflects the cell death rate, that is, the lower the cell viability, the higher the cell death rate.
- Figure 7 is the cell viability when combined treatment for 24h. Compared with the blank control group, p ⁇ 0.001, the difference is extremely significant; 200ng/ml compared with the blank control group, p ⁇ 0.001, the difference is extremely significant; 400ng/ml compared with the blank control group, the difference is not significant, 100ng/ml Compared with the 400ng/ml treatment group, p ⁇ 0.05, the difference is significant, the other 100ng/ml and 200ng/ml, 200ng/ml and 400ng/ml, the difference is not significant.
- Figure 8 is the cell viability when combined treatment for 96h.
- 100ng/ml compared with the blank control group the difference is extremely significant, p ⁇ 0.001, 200ng/ml compared with the blank control group, the difference is significant, p ⁇ 0.05, 400ng/ml compared with the blank control group, the difference is significant, p ⁇ 0.05;
- Fig. 9 is the cell viability of joint treatment for 120h. Compared with the blank control group, 100ng/ml, 200ng/ml and 400ng/ml were significantly different, p ⁇ 0.001; in addition, 100ng/ml and 200ng/ml, 100ng/ml and 400ng/ml, 200ng/ml and 400ng /ml showed no significant difference.
- Fig. 10 is the cell viability of combined treatment for 168h.
- 100ng/ml, 200ng/ml and 400ng/ml compared with the blank control group the difference is extremely significant, p ⁇ 0.001; in addition, 100ng/ml and 200ng/ml, 100ng/ml and 400ng/ml compared, the difference is significant, p ⁇ 0.05; compared with 200ng/ml and 400ng/ml, the difference is not significant.
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Abstract
提供了体外细胞因子风暴模型及其构建方法和应用。所述体外细胞因子风暴模型的构建体系,含有TNF-α因子、IL-1β因子、IL-6因子和血管内皮细胞。还提供了体外细胞因子风暴模型构建方法,该方法是将血管内皮细胞与TNF-α因子、IL-1β因子、IL-6因子共同孵育。还提供了体外细胞因子风暴模型在筛选抗细胞因子释放综合征药物中的应用。还提供了体外细胞因子风暴模型在制备筛选抗细胞因子释放综合征药物中的应用。本发明的体外细胞因子风暴模型通过TNF-α、IL-1β和IL-6三因子联合处理血管内皮细胞得到,可制得细胞凋亡率高、细胞活力亦明显下降的体外细胞因子风暴模型,对多因子相关的炎症反应的研究提供了较好的基础。
Description
本发明属于生物技术领域,具体涉及一种体外细胞因子风暴模型及其制备方法和应用。
细胞因子释放综合征(Cytokine Release Syndrome,CRS),也被形象地称为细胞因子风暴(Cytokine Storm)是指机体感染微生物后引起体液中多种细胞因子迅速大量的产生,如TNF-α、IL-1、IL-6、IL-12、IFN-α、IFN-β、IFN-γ、MCP-1和IL-8等,是一种严重危及生命的综合症状,标志着一种不受控制和功能失调的免疫反应,涉及淋巴细胞和巨噬细胞的持续激活和扩增,分泌大量的细胞因子,可能导致全身性炎症反应、多器官衰竭、高铁血红蛋白血症、急性呼吸窘迫综合征等疾病。除了细菌、病毒等微生物感染以外,进行CAR-T细胞治疗也易出现细胞因子风暴,此外,进行免疫检查点抑制剂治疗时也可能会出现细胞因子风暴,但概率极低。
现有技术中常用微生物感染动物,或者进行肿瘤杀伤后刺激巨噬细胞分泌(Liu Y,et al,.Gasdermin E–mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome.Sci Immunol.2020Jan 17;5(43):eaax7969.)来模拟细胞因子动物内源性风暴,这样的话细胞因子浓度不够确认,由于每次肿瘤细胞杀伤后的因子分泌浓度不稳定,所以动物体内也不稳定。
而体外细胞因子风暴模型的报道有:
(1)娄振凯等(H
2O
2、LPS和TNF-α诱导人类脐静脉内皮细胞损伤的实验研究)的文章中分别采用不同浓度的过氧化氢(H
2O
2)、脂多糖(LPS)和肿瘤坏死因子(TNF‐α)刺激人脐静脉内皮细胞,孵育不同时间,研究表明H
2O
2、LPS和TNF-α能体外损伤人类脐静脉内皮细胞,成功建立人类脐静脉内皮细胞损伤模型,且在一定浓度范围内各损伤因子对人类脐静脉内皮细胞 损伤程度呈浓度和时间依赖性。
(2)Speciale,A等(Silibinin as potential tool against SARS-Cov-2:In silico spike receptor-binding domain and main protease molecular docking analysis,and in vitro endothelial protective effects.Phytotherapy Research.2021;35:4616–4625.)探讨了水飞蓟属植物水飞蓟宾在体外对细胞因子(TNF-α)诱导的人脐静脉内皮细胞(HUVECs)炎症和功能障碍的作用。
(3)Chen,Tielong等(Quercetin inhibits TNF-αinduced HUVECs apoptosis and inflammation via downregulating NF-kB and AP-1 signaling pathway in vitro,Medicine:September 18,2020-Volume 99-Issue 38-p e22241)报道槲皮素通过下调NF-kB和AP-1信号通路抑制TNF-a诱导的HUVECs细胞凋亡和炎症反应。
由于本领域目前仅有使用单一因子在体外制备细胞因子风暴模型,如使用TNF-α,所得模型较为简单,并不能满足医药领域对于特定细胞因子诱导的炎症进行研究的模型需求,更没有多因子联合造模的报道,故急需一种制备多因子诱导的体外细胞因子风暴模型。
发明内容
针对上述问题,本发明提供体外细胞因子风暴模型及其构建方法和应用,解决了现有技术中体外细胞因子风暴的细胞模型通常是以单一细胞因子处理进行造模,且所得模型较为简单,难以满足医药领域日益变多的需求的缺陷,且模拟效果不是很好。
为了解决上述问题,本发明采用如下技术方案:
体外细胞因子风暴模型的构建体系,含有TNF-α因子、IL-1β因子、IL-6因子和血管内皮细胞。
一些方式中,还具有培养基;所述血管内皮细胞为人脐静脉内皮细胞。
一些方式中,所述培养基为内皮细胞培养基。
本发明提供的技术方案之二为:由上述体形成的体外细胞因子风暴模型。
体外细胞因子风暴模型,由前述任一所述体外细胞因子风暴模型的构建体系形成。
本发明提供的技术方案之三为:由上述体外细胞因子风暴模型的制备方法。
体外细胞因子风暴模型构建方法,将血管内皮细胞与TNF-α因子、IL-1β因子、IL-6因子共同孵育。
一些方式中,TNF-α因子、IL-1β因子、IL-6因子中任一浓度为100-400ng/ml;孵育时长为24-168h。
一些方式中,更具体为,TNF-α因子、IL-1β因子、IL-6因子中任一浓度为100-200ng/ml;孵育时长为120-168h。
一些方式中,TNF-α因子、IL-1β因子、IL-6因子三者浓度相同;孵育的温度为37℃;孵育的培养基为内皮细胞培养基。
本发明提供的技术方案之四为:如技术方案之二所述的体外细胞因子风暴模型在筛选抗细胞因子释放综合征药物中的应用。
前述体外细胞因子风暴模型在制备筛选抗细胞因子释放综合征药物中的应用。
在本发明一具体实施方案中,所述血管内皮细胞为人脐静脉内皮细胞(HUVEC,Human Umbilical Vein Endothelial Cell)。
本发明中,所述孵育的培养基可按本领域常规选择,一些方式中为内皮细胞培养基(Endothelial Cell Medium,ECM)。
在本发明一较佳实施方案中,所述方法在孵育结束后,还包括将制得的体外细胞因子风暴模型与培养液分离的步骤,所述分离可为本领域常规的分离方法,例如换液。
一些情况中,所述细胞因子释放综合征为由TNF-α、IL-1β和IL-6引发的细胞因子释放综合征。
本发明第五方面涉及抗细胞因子释放综合征药物制备方法,具体包括下述步骤
通过体外细胞因子风暴模型筛选对体外细胞因子风暴具有抑制作用的组分;将筛选所得有效组分用于制备抗细胞因子释放综合征药物。
前段将有效组分用于制药过程一种方式参考现有制药工艺。
本发明的有益效果是:
本发明所述的体外细胞因子风暴模型通过TNF-α、IL-1β和IL-6三因子联合处理血管内皮细胞得到,可制得细胞凋亡率高、细胞活力亦明显下降的体外细胞因子风暴模型,对多因子相关的炎症反应的研究提供了较好的基础。所述体外细胞因子风暴模型的细胞活力显著降低,满足体外研究药物治疗效果更高的需求。
图1为空白对照组处理24h、48h和72h的荧光拍照图;
图2为三因子处理组处理24h荧光拍照图;
图3为三因子处理组处理48h荧光拍照图;
图4为三因子处理组处理48h荧光拍照图;
图5为HUVEC细胞使用三因子(各100ng/ml)药物处理不同时间点的细胞凋亡率。
图6为不同浓度三因子处理HUVEC细胞72h后的细胞活力;
图7为不同浓度三因子处理HUVEC细胞24h后的细胞活力;
图8为不同浓度三因子处理HUVEC细胞96h后的细胞活力;
图9为不同浓度三因子处理HUVEC细胞120h后的细胞活力;
图10为不同浓度三因子处理HUVEC细胞168h后的细胞活力。
本部分第一方面对本发明方案进行说明:
体外细胞因子风暴模型的构建体系,含有TNF-α因子、IL-1β因子、IL-6因子和血管内皮细胞。
该构建体系为风暴模型的组成制备体系,构建体系中的物质相互作用,通过该构建体系可以形成风暴模型。其中,构建体系为制备风暴模型过程中前期体系,主要为了表征制备风暴模型的物料组成。当然,其他具有与构建体系相同的方案,也应当等同落入本发明范围内。
一些方式中,构建体系还具有培养基;所述血管内皮细胞为人脐静脉内皮细胞。
一些方式中,所述培养基为内皮细胞培养基。
本发明提供的技术方案之二为:由上述体形成的体外细胞因子风暴模型。
体外细胞因子风暴模型,由前述任一所述体外细胞因子风暴模型的构建体系形成。相对其他单因子或多因子模型,本发明的风暴模型具有更好的模拟效果。
本发明提供的技术方案之三为:由上述体外细胞因子风暴模型的制备方法。
体外细胞因子风暴模型构建方法,将血管内皮细胞与TNF-α因子、IL-1β因子、IL-6因子共同孵育。
一些方式中,TNF-α因子、IL-1β因子、IL-6因子中任一浓度为100-400ng/ml;孵育时长为24-168h。
一些方式中,更具体为,TNF-α因子、IL-1β因子、IL-6因子中任一浓度为100-200ng/ml;孵育时长为120-168h。
一些方式中,TNF-α因子、IL-1β因子、IL-6因子三者浓度相同;孵育的温度为37℃;孵育的培养基为内皮细胞培养基。
本发明提供的技术方案之四为:如技术方案之二所述的体外细胞因子风暴模型在筛选抗细胞因子释放综合征药物中的应用。
前述体外细胞因子风暴模型在制备筛选抗细胞因子释放综合征药物中的应用。
在本发明一具体实施方案中,所述血管内皮细胞为人脐静脉内皮细胞(HUVEC,Human Umbilical Vein Endothelial Cell)。
本发明中,所述孵育的培养基可按本领域常规选择,一些方式中为内皮细胞培养基(Endothelial Cell Medium,ECM)。
在本发明一较佳实施方案中,所述方法在孵育结束后,还包括将制得的体外细胞因子风暴模型与培养液分离的步骤,所述分离可为本领域常规的分离方法,例如换液。
一些情况中,所述细胞因子释放综合征为由TNF-α、IL-1β和IL-6引发的细胞因子释放综合征。
本发明第五方面涉及抗细胞因子释放综合征药物制备方法,具体包括下述步骤
通过体外细胞因子风暴模型筛选对体外细胞因子风暴具有抑制作用的组分;将筛选所得有效组分用于制备抗细胞因子释放综合征药物。
前段将有效组分用于制药过程一种方式参考现有制药工艺。
本部分第二方面结合一些具体示例进行说明:
实验例1细胞因子处理时间对造模的影响
本研究采用HUVEC(人脐静脉内皮细胞)细胞,使用Sciencell ECM培养基进行培养,使用人源TNF-α、IL-1β和IL-6(均购自北京同立海源,其中重组人IL-1β蛋白货号GMP-TL513;重组人IL-6蛋白货号GMP-TL512;重组人TNF-α蛋白货号GMP-TL303)三因子联合,细胞因子采用现用现配,使用 细胞培养基稀释到100ng/ml。对照组HUVEC细胞加入正常培养的培养基,三因子处理组HUVEC细胞中加入三因子的培养基进行培养,在培养板中进行培养,培养流程按照HUVEC细胞的常规培养方式(培养条件为37℃,95%CO
2、5%O
2)进行,培养对应时间后24h、48h、72h,使用荧光染色方法检测凋亡小体。
每种细胞因子浓度100ng/ml处理HUVEC细胞24h、48h、72h(即共同孵育的时长),同时设置对照组,使用荧光染色方法检测凋亡小体。
(1)实验设置:
A.空白对照组(HUVEC细胞不做处理)
B.三因子处理组(HUVEC+TNF-α+IL-1β+IL-6(各100ng/ml))
(2)荧光染色检测方法:
①配置固定液:甲醇:冰醋酸(3:1)混合,摇匀。现配现用。
②将Hoechst 33258染液(Standard Reagent,P4403,1ml Hoechst 33258染液加入99ml PBS中,避光保存)放至磁力搅拌器上搅拌15min以上。
③吸弃12孔板培养板孔内培养基,加PBS漂洗3次。
④加入现配的固定液,固定15min。
⑤吸弃固定液,加入新的固定液继续固定15min。吸弃后,将培养板自然晾干。(完全晾干)。
⑥吸取Hoechst 33258染液,1ml/孔。避光染色30min。
⑦吸弃后加入纯水洗去多余的染液,轻摇,重复3次。
⑧吸干孔内纯水,自然晾干,将培养板在避光的情况下转至荧光倒置显微镜下观察,并拍照进行凋亡率统计。
(3)实验结果:
凋亡小体荧光拍照图见图1-4(一个时间点只展示一张图,凋亡小体用圆 圈出,需说明:图片仅为培养物外观展示,每个视野拍照的细胞数及染色的凋亡小体数目都不同,具体细胞凋亡率统计见图5),其中A正常对照组未见凋亡细胞,B、三因子处理组有凋亡细胞出现。具体统计分析见图5,处理后24h凋亡率最高。*表示与空白对照组相比,p<0.05,***表示与空白对照组相比,p<0.001。不同时间点的细胞凋亡率呈下降趋势,24h的最高,72h的最低。与空白对照组相比,均具有差异显著性,其中24h和72h的p<0.001,48h的p<0.05;48h和24h相比以及72h和48h相比,细胞凋亡率具有差异显著性,p<0.05;72h和24h相比,p<0.001。
由于炎症反应常见的表现为多种细胞因子共同作用后相互影响的代谢通路造成细胞死亡,经上述三因子处理后,HUVEC随着时间的推移细胞死亡率增加,可知本实施例成功制备了TNF-α+IL-1β+IL-6三因子诱导的体外细胞因子风暴模型。
实验例2细胞因子的浓度对造模的影响
使用人源TNF-α、IL-1β、IL-6三因子联合处理,三因子设置的浓度为50ng/ml、100ng/ml、200ng/ml处理HUVEC细胞72h,同时设置对照组,观察细胞活力。
(1)实验设置:
A.空白对照组(HUVEC细胞不做处理)
B.50ng/ml处理组(HUVEC+TNF-α+IL-1β+IL-6(各50ng/ml))
C.100ng/ml处理组(HUVEC+TNF-α+IL-1β+IL-6(各100ng/ml))
D.200ng/ml处理组(HUVEC+TNF-α+IL-1β+IL-6(各200ng/ml))
(2)CCK-8检测细胞活力
①细胞接种于96孔板中,100μl/孔,培养第二天进行不同浓度药物处理,继续培养72h;
②吸出培养液,加入110μl混合的cck-8培养液(100μl培养液+10μl CCK-8;
③在培养箱内孵育2小时;
④用酶标仪测定在450nm处的吸光度。
(3)实验结果
具体结果见图6。图6结果显示,不同浓度的三因子处理HUVEC细胞,细胞活力下降,50ng/ml、100ng/ml、200ng/ml处理组与空白对照组相比,均具有显著性差异,***表示各处理组与对照组相比p<0.001,此外,50ng/ml和100ng/ml处理组相比,差异没有显著性;200ng/ml与50ng/ml相比具有差异显著性,p<0.05,200ng/ml与100ng/ml处理组相比,具有差异显著性,p<0.05。
实验例3细胞因子联合处理浓度和处理时间对造模的影响
使用人源TNF-α、IL-1β、IL-6三因子联合处理HUVEC,三因子浓度相同,不同实验组分别设置为100ng/ml、200ng/ml、和400ng/ml,处理时间点为24h、96h、120h、168h,处理方法和检测方法同实施例2。结果见图7-10。其中细胞活力反映细胞死亡率,即细胞活力越低,细胞死亡率越高。
图7为联合处理24h时的细胞活力。与空白对照组相比,p<0.001,差异极显著;200ng/ml与空白对照组相比,p<0.001,差异极显著;400ng/ml与空白对照组相比,差异不显著,100ng/ml与400ng/ml处理组相比,p<0.05,差异显著,另外100ng/ml和200ng/ml相比,200ng/ml和400ng/ml相比,差异不显著。
图8为联合处理96h时的细胞活力。100ng/ml与空白对照组相比,差异极显著,p<0.001,200ng/ml与空白对照组相比,差异显著,p<0.05,400ng/ml与空白对照组相比,差异显著,p<0.05;此外,100ng/ml和200ng/ml,100ng/ml和400ng/ml,200ng/ml和400ng/ml相比,均不具有差异显著性。
图9为联合处理120h细胞活力。100ng/ml、200ng/ml以及400ng/ml与空白对照组相比,均差异极显著,p<0.001;此外,100ng/ml和200ng/ml,100ng/ml和400ng/ml,200ng/ml和400ng/ml相比,均不具有差异显著性。
图10为联合处理168h细胞活力。100ng/ml、200ng/ml以及400ng/ml与空白对照组相比,差异极显著,p<0.001;此外,100ng/ml和200ng/ml,100ng/ml和400ng/ml相比,差异显著,p<0.05;200ng/ml和400ng/ml相比,差异不显著。
由上可知,三因子孵育处理的时间越长,细胞活力先上升后很快下降,且随时间延长而下降越多。可以发现一开始三种因子的协同促进内皮细胞活力升高的作用,随着时间的推移内皮细胞刚开始是刺激生长的状态,但到了一定的时间,其死亡率攀升,这也是与临床表征相似的。综合而言造模效果在处理168h时最好。
本领域的技术人员可以明确,在不脱离本发明的总体精神以及构思的情形下,可以做出对于以上实施例的各种变型。其均落入本发明的保护范围之内。本发明的保护方案以本发明所附的权利要求书为准。
Claims (9)
- 体外细胞因子风暴模型的构建体系,其特征在于,含有TNF-α因子、IL-1β因子、IL-6因子和血管内皮细胞。
- 根据权利要求1所述的体外细胞因子风暴模型的构建体系,其特征在于,还具有培养基;所述血管内皮细胞为人脐静脉内皮细胞。
- 根据权利要求2所述的体外细胞因子风暴模型的构建体系,其特征在于,所述培养基为内皮细胞培养基。
- 体外细胞因子风暴模型构建方法,其特征在于,将血管内皮细胞与TNF-α因子、IL-1β因子、IL-6因子共同孵育。
- 根据权利要求4所述的体外细胞因子风暴模型构建方法,其特征在于,TNF-α因子、IL-1β因子、IL-6因子中任一浓度为100-400ng/ml;孵育时长为24-168h。
- 根据权利要求5所述的体外细胞因子风暴模型构建方法,其特征在于,TNF-α因子、IL-1β因子、IL-6因子中任一浓度为100-200ng/ml;孵育时长为120-168h。
- 根据权利要求4-6任一所述的体外细胞因子风暴模型构建方法,其特征在于,TNF-α因子、IL-1β因子、IL-6因子三者浓度相同;孵育的温度为37℃;孵育的培养基为内皮细胞培养基。
- 体外细胞因子风暴模型,其特征在于,由权利要求1-3任一所述体外细胞因子风暴模型的构建体系形成或由权利要求4-6任一体外细胞因子风暴模型构建方法制备而得。
- 权利要求8中体外细胞因子风暴模型在制备筛选抗细胞因子释放综合征药物中的应用。权利要求8中体外细胞因子风暴模型在制备筛选抗细胞因子释放综合征药物中的应用方法,其特征在于,用于筛选抗细胞因子风暴的物质。
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