目的采用成簇的规律性间隔短回文重复序列及其相关蛋白9(clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9,CRISPR/Cas9)系统构建基因敲除细胞文库,以人结肠腺癌细胞Caco-2为研究对象构建稳定...目的采用成簇的规律性间隔短回文重复序列及其相关蛋白9(clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9,CRISPR/Cas9)系统构建基因敲除细胞文库,以人结肠腺癌细胞Caco-2为研究对象构建稳定表达Cas9蛋白的细胞系。方法包装重组Cas9慢病毒并感染Caco-2细胞,经杀稻瘟菌素及2次有限稀释法筛选Caco-2/Cas9单克隆细胞,并进行PCR和Western blot鉴定。用同时表达GFP和带有GFP向导RNA(single guide RNA,sgRNA)的慢病毒感染Caco-2/Cas9单克隆细胞,经嘌呤霉素筛选后,采用流式细胞术检测结果计算Caco-2细胞株的敲除效率,CCK-8法检测其细胞增殖活性。结果共获得5株Caco-2/Cas9单克隆细胞系,分别命名为Caco-2/Cas9-2、Caco-2/Cas9-3、Caco-2/Cas9-4、Caco-2/Cas9-5和Caco-2/Cas9-6,均可扩增出392 bp的Cas9基因条带,Cas9蛋白在细胞中稳定表达,敲除效率分别为91.27%、20.30%、24.13%、11.33%、12.27%、8.89%;Caco-2/Cas9-4、Caco-2/Cas9-5、Caco-2/Cas9-63株单克隆细胞及未感染Caco-2细胞的增殖活性分别为2.07、1.75、1.46和1.40。Caco-2/Cas9-5、Caco-2/Cas9-6单克隆细胞与未感染Caco-2细胞比较,差异均无统计学意义(t分别为1.92和0.37,P均>0.05)。结论筛选出1株具有Cas9高酶切活性且细胞增殖活性较未感染Caco-2细胞无显著变化的Caco-2/Cas9单克隆细胞系,即Caco-2/Cas9-6单克隆细胞,为进一步构建高覆盖率敲除细胞文库奠定了基础,也为筛选病毒感染有关基因和其他特定功能基因提供了平台。展开更多
Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant ...Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant and fibrinolytic activities,and relevant mechanisms have been confirmed through in vitro experiments.However,whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown.This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.Methods:The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3.Then,the protein bands of EPF3 incubated with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and homogenate of Caco-2 cells(HC2C)were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability.Finally,the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.Results:EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid.It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3.EPF3 was stable in SIF and HC2C and unstable in SGF.The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent.The efflux ratio was less than1.2 during transport,and the transport behavior was not affected by inhibitors.EPF3 could reversibly reduce the expression of tight junction-related proteins,including zonula occludens-1,occludin,and claudin-1 in Caco-2 cells.Conclusion:EPF3 could play a thrombolytic and antithrombotic role in zebrafish.It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction.This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation.Please cite this article as:Zhong WL,Yang JQ,Liu H,Wu YL,Shen HJ,Li PY,Du SY.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong(Pheretima vulgaris Chen)and its transport mechanism in Caco-2 monolayer through cell bypass pathway.J Integr Med.2025;23(4):415–428.展开更多
基金supported by grants from the Fundamental Research Funds for the Central Universities(No.2020-JYB-ZDGG-032)National Natural Science Foundation of China(No.82104352)。
文摘Objective:EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen,an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome.Its composition,anticoagulant and fibrinolytic activities,and relevant mechanisms have been confirmed through in vitro experiments.However,whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown.This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.Methods:The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3.Then,the protein bands of EPF3 incubated with simulated gastric fluid(SGF),simulated intestinal fluid(SIF),and homogenate of Caco-2 cells(HC2C)were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability.Finally,the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.Results:EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid.It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3.EPF3 was stable in SIF and HC2C and unstable in SGF.The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent.The efflux ratio was less than1.2 during transport,and the transport behavior was not affected by inhibitors.EPF3 could reversibly reduce the expression of tight junction-related proteins,including zonula occludens-1,occludin,and claudin-1 in Caco-2 cells.Conclusion:EPF3 could play a thrombolytic and antithrombotic role in zebrafish.It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction.This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation.Please cite this article as:Zhong WL,Yang JQ,Liu H,Wu YL,Shen HJ,Li PY,Du SY.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong(Pheretima vulgaris Chen)and its transport mechanism in Caco-2 monolayer through cell bypass pathway.J Integr Med.2025;23(4):415–428.
