Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated ...Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated by β- hexosaminidase activity as a method to evaluate TCMIs at nonclinical stage. Methods: RBL-2H3 cells were used to study the degranulation by co-culture with positive control C48/80 and some TCMIs through morphological and ultra-structure observation, β-hexosaminidase activity detection. RBL-2H3 cell degranulation test was established to detect β-hexosaminidase activity caused by 17 kinds of TCMIs and their ingredients. The cytotoxicity effect of some TCMIs on both RBL 2H3 and BRL cells was measured by CCK-8 assay. Results: Toluidine blue staining and ultra-structure of electronic microscope observation of treated RBL-2H3 cells showed degranulation morphologically. Detection of β-hexosaminidase activity in the supernatant of treated cells showed some TCMIs had elevated enzyme release rates. Further analysis of the ingredients and compound in Tanreqing Injection and Shengmai Injection showed Scutellaria baicalensis Georgi in Tanreqing Injection, Red ginseng and Fructus Schisandrae Chinensis in Shengmai Injection were responsible to the degranulation of RBL-2H3 cells. Osmotic pressures and pH influenced RBL-2H3 degranulation. High Osmotic pressure of Tanreqing Injection and low pH of chlorogenic acid at 2.5 and 5.0 mmol/L congcentration might be responsible to high β-hexosaminidase activity. Most of the TCMIs inducing degranulation had cytotoxicity effect for both RBL-2H3 and BRL cells, but some TCMIs inducing degranulation had no cytotoxicity effect. Conclusion: Some TCMIs can induce degranulation of RBL-2H3 cells;RBL-2H3 cell degranulation test can be used in non-clinical stage to detect the risk causing anaphylactoid reactions. Osmotic pressures and pH influenced RBL-2H3 degranulation, and they should be measured before testing. The mechanism of degranulation caused by some TCMIs is cytotoxic, and some are non-cytotoxic and may be through exicytosis.展开更多
目的通过建立体外抑制肥大细胞脱颗粒的药效学模型,探讨丹皮酚抗Ⅰ型变态反应作用机制。方法用MTT比色法检测不同浓度的丹皮酚对RBL-2H3肥大细胞增殖的影响。通过ELISA法,明确体外RBL-2H3细胞活化分泌组胺及TNF-α动力学特征。检测不同...目的通过建立体外抑制肥大细胞脱颗粒的药效学模型,探讨丹皮酚抗Ⅰ型变态反应作用机制。方法用MTT比色法检测不同浓度的丹皮酚对RBL-2H3肥大细胞增殖的影响。通过ELISA法,明确体外RBL-2H3细胞活化分泌组胺及TNF-α动力学特征。检测不同浓度药物对RBL-2H3肥大细胞分泌组胺及TNF-α的影响。结果丹皮酚对RBL-2H3细胞增殖有抑制作用,IC50值为0.22 mg/mL。RBL-2H3细胞活化脱颗粒,分泌组胺的释放率在前30 m in上升较快,30 m in后上升进入平台期;TNF-α分泌的量在1h达高峰。丹皮酚抑制RBL-2H3细胞释放组胺和TNF-α作用不弱于色甘酸钠0.1,0.5 mg/mL。丹皮酚浓度的对数与其对RBL-2H3细胞释放组胺和TNF-α的抑制率呈线性相关(P=0.000<0.01)。结论丹皮酚呈剂量依赖性抑制RBL-2H3细胞分泌组胺和TNF-α。展开更多
过敏是人和养殖动物常见的疾病,而且发生的频率呈现逐渐增加的趋势。为了深入了解过敏反应的机制,本研究利用CRISPR/Cas9基因编辑技术,构建钙调磷酸酶A beta(calcineurin A beta,CnAβ)基因敲除的大鼠RBL-2H3细胞株,并探讨CnAβ基因对RB...过敏是人和养殖动物常见的疾病,而且发生的频率呈现逐渐增加的趋势。为了深入了解过敏反应的机制,本研究利用CRISPR/Cas9基因编辑技术,构建钙调磷酸酶A beta(calcineurin A beta,CnAβ)基因敲除的大鼠RBL-2H3细胞株,并探讨CnAβ基因对RBL-2H3细胞生长及脱颗粒的影响。选取大鼠CnAβ基因第一外显子为敲除靶点,设计并合成3个单导向RNA(single guide RNA,sgRNA),构建pX459-CnAβ-sgRNA质粒,并用脂质体3000将构建好的质粒转染到RBL-2H3细胞内;利用嘌呤霉素对转染细胞进行筛选,通过DNA测序验证获得CnAβ基因敲除的RBL-2H3细胞株;并检测CnAβ基因缺失对细胞增殖和脱颗粒的影响。结果表明,成功构建了CnAβ单基因敲除的大鼠RBL-2H3细胞株;CnAβ基因缺失对RBL-2H3细胞增殖、细胞的颗粒形成以及颗粒含量无显著影响,但显著抑制由细胞表面受体介导的RBL-2H3细胞脱颗粒作用。该研究结果有助于深入了解动物过敏性疾病的发生机制,为动物过敏性疾病的预防提供了理论基础。展开更多
文摘Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated by β- hexosaminidase activity as a method to evaluate TCMIs at nonclinical stage. Methods: RBL-2H3 cells were used to study the degranulation by co-culture with positive control C48/80 and some TCMIs through morphological and ultra-structure observation, β-hexosaminidase activity detection. RBL-2H3 cell degranulation test was established to detect β-hexosaminidase activity caused by 17 kinds of TCMIs and their ingredients. The cytotoxicity effect of some TCMIs on both RBL 2H3 and BRL cells was measured by CCK-8 assay. Results: Toluidine blue staining and ultra-structure of electronic microscope observation of treated RBL-2H3 cells showed degranulation morphologically. Detection of β-hexosaminidase activity in the supernatant of treated cells showed some TCMIs had elevated enzyme release rates. Further analysis of the ingredients and compound in Tanreqing Injection and Shengmai Injection showed Scutellaria baicalensis Georgi in Tanreqing Injection, Red ginseng and Fructus Schisandrae Chinensis in Shengmai Injection were responsible to the degranulation of RBL-2H3 cells. Osmotic pressures and pH influenced RBL-2H3 degranulation. High Osmotic pressure of Tanreqing Injection and low pH of chlorogenic acid at 2.5 and 5.0 mmol/L congcentration might be responsible to high β-hexosaminidase activity. Most of the TCMIs inducing degranulation had cytotoxicity effect for both RBL-2H3 and BRL cells, but some TCMIs inducing degranulation had no cytotoxicity effect. Conclusion: Some TCMIs can induce degranulation of RBL-2H3 cells;RBL-2H3 cell degranulation test can be used in non-clinical stage to detect the risk causing anaphylactoid reactions. Osmotic pressures and pH influenced RBL-2H3 degranulation, and they should be measured before testing. The mechanism of degranulation caused by some TCMIs is cytotoxic, and some are non-cytotoxic and may be through exicytosis.
文摘目的通过建立体外抑制肥大细胞脱颗粒的药效学模型,探讨丹皮酚抗Ⅰ型变态反应作用机制。方法用MTT比色法检测不同浓度的丹皮酚对RBL-2H3肥大细胞增殖的影响。通过ELISA法,明确体外RBL-2H3细胞活化分泌组胺及TNF-α动力学特征。检测不同浓度药物对RBL-2H3肥大细胞分泌组胺及TNF-α的影响。结果丹皮酚对RBL-2H3细胞增殖有抑制作用,IC50值为0.22 mg/mL。RBL-2H3细胞活化脱颗粒,分泌组胺的释放率在前30 m in上升较快,30 m in后上升进入平台期;TNF-α分泌的量在1h达高峰。丹皮酚抑制RBL-2H3细胞释放组胺和TNF-α作用不弱于色甘酸钠0.1,0.5 mg/mL。丹皮酚浓度的对数与其对RBL-2H3细胞释放组胺和TNF-α的抑制率呈线性相关(P=0.000<0.01)。结论丹皮酚呈剂量依赖性抑制RBL-2H3细胞分泌组胺和TNF-α。
