Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

Age-related differences in clinical characteristics of Kawasaki disease

This study aimed to examine and summarize clinical characteristics of Kawasaki disease (KD) at different ages to further strengthen clinicians understanding of children with KD, improving the level of diagnosis, and reducing coronary artery complications of KD. A total of 398 patients with KD who were diagnosed between January 2016 and December 2017 were reviewed retrospectively. These participants were allocated into three groups according to age: group A (<1 year, n=62), group B (≥1 and <5 years, n=286), and group C (≥5 years, n=50). Clinical manifestations, laboratory results, and echocardiographic findings were compared among the groups. Most (71.86%) patients with KD were aged 1-5 years. The prevalence of cervical lymphadenopathy was lowest in group A. The duration of fever before admission was longest in group A. The rate of cervical lymphadenopathy and laboratory data were different among the groups. Group A had higher frequencies of gastrointestinal involvement, neurological symptoms, and redness at the Bacillus Calmette-Guerin inoculation site than the other groups. Infants aged <1 year with KD often have a longer duration of fever before admission, a lower prevalence of cervical lymphadenopathy, and a higher prevalence of gastrointestinal and neurological symptoms.

Predictive value of serum BNP and Hcy levels for therapeutic effect and prognosis of coronary artery bypass grafting／ / 心血管康复医学杂志

To analyze and evaluate predictive value of preoperative changes of serum levels of brain natri‐uretic peptide (BNP) and homocysteine (Hcy) levels for therapeutic effect and prognosis of coronary artery bypass grafting (CABG ) in patients undergoing CABG .Methods : Clinical data of 115 patients undergoing CABG in our hospital were collected .Preoperative serum levels of BNP and Hcy were compared among patients with different disease severity , therapeutic effect and prognosis .Influence of serum BNP and Hcy levels on therapeutic effect and prognosis of CABG were analyzed .Results : Compared with medium and mild disease group , there were significant rise in preoperative serum levels of BNP [ (151.86 ± 22.57) pg／L vs.(82.57 ± 10.26) pg／L vs.(283.51 ± 32.47) pg／L] and Hcy [ (18.37 ± 4.51) μmol／L vs.(12.74 ± 2. 04) μmol／L vs.(31.56 ± 5.17) μmol／L] in severe disease group , and those of medium disease group were significantly higher than those of mild disease group , P＝0.001 all ;compared with effective group and ineffective group , there were significant reductions in preoperative serum levels of BNP [ (227. 49 ± 24. 52) pg／L vs.(308.26 ± 34.12) pg／L vs.(90.13 ± 10. 75) pg／L] and Hcy [ (29. 12 ± 5. 83) μmol／L vs.(46.15 ± 7.49) μmol／L vs.(19.03 ± 3.77) μmol／L] in markedly effective group , and those of effec‐tive group were significantly lower than those of ineffective group , P＝ 0.001 all.Compared with stenotic graft group and smooth graft group , there were significant rise in preoperative serum levels of BNP [ (271. 47 ± 25.18) pg／L vs .(92.41 ± 11.06) pg／L vs.(312. 54 ± 35.06) pg／L] and Hcy [ (33.08 ± 6. 14) μmol／L vs.(20. 05 ± 3. 68) μmol／L vs.(50.21 ± 7.75 ) μmol／L ] in death group , and those of stenotic graft group were significantly higher than those of smooth graft group , P＝0.001 all .Spearman correlation analysis indicated that serum BNP and Hcy levels were significant positively correlated with severity of CHD and prognosis of CABG ( r＝0. 624～0.814 , P＜0.05 or ＜0. 01).Multifactor Logistic regression analysis indicated that serum BNP and Hcy levels were independent risk factors for prognosis after CABG in CHD patients (OR＝ 5.133 , 1. 803 , P＝ 0.001 both ).Conclusion : The higher preoperative levels of BNP and Hcy before CABG are , the worse therapeutic effects are , and the higher risk of adverse prognosis of vascular stenosis and death are .