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1.
Chinese Journal of Biologicals ; (12): 1483-1490, 2023.
Artículo en Chino | WPRIM | ID: wpr-1005873

RESUMEN

@#Objective To develop quality control methods for the key quality properties of recombinant monoclonal antibodies against proprotein convertase subtilisin/Kexin 9(PCSK9). Methods According to the corresponding requirements of Chinese Pharmacopoeia(Volume Ⅲ,2020 edition)and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use(ICH),the PCSK9 monoclonal antibody was identified for the identity by reducing trypsin peptide fingerprint mapping analysis,identified for the specificity by ELISA,determined for the charge heterogeneity by imaging capillary isoelectric focusing electrophoresis(icIEF),detected for the variant purity by reducing/non-reducing capillary electrophoresis-sodium dodecyl sulfonate(CE-SDS),measured for the content of monomers,high molecular weight substances and low molecular weight substances by size-exclusion chromatography high performance liquid chromatography(SEC-HPLC),analyzed for the glycotype with N-glycan labeled with 2AB after carbohydrate cutting by Waters ACQUITY UPLC system with fluorescence detector,determined for the biological potency with HepG2 cell line and low-density lipoprotein(LDL),and detected for the content of polysorbate 20 by the liquid phase detection system(CAD detector). Results PCSK9 monoclonal antibody sample had characteristic peptide segments,and the peptide fingerprint was consistent with that of reference substance. The sample contained specific antibodies;The relative area percentages of main peak,acid peak and alkaline peak of the sample were(49. 27 ± 0. 38)%,(46. 44 ± 0. 35)% and(4. 28 ± 0. 04)%,respectively. The relative area percentages of“heavy chain + light chain”peak,non-glycosyl main peak and low molecular weight substance peak were(94. 16 ± 0. 82)%,(4. 11 ± 0. 76)% and(0. 85 ± 0. 20)%,respectively. The relative area percentages of main peak,nonglycosyl main peak and low molecular weight substance peak were(94. 27 ± 0. 22)%,(2. 85 ± 0. 08)% and(2. 88 ± 0. 15)%,respectively. The relative area percentages of monomer,high molecular weight substance and low molecular weight substance were(98. 30 ± 0. 03)%,(0. 75 ± 0. 01)% and(0. 96 ± 0. 02)%,respectively. The relative percentages of G0F,G1F,G2F and non-fucosylated(G0 + G1 + G2 + Man5)were(39. 31 ± 0. 54)%,(34. 69 ± 0. 41)%,(9. 09 ± 0. 14)% and(12. 61 ± 0. 50)%,respectively. The relative biological potency was(101. 64 ± 3. 61)% of the reference. The content of polysorbate 20 was(0. 012 ± 0. 000 3)%. Conclusion According to the key quality attribute of monoclonal antibody against PCSK9,the corresponding key quality control methods have been established,which can ensure the safety,effectiveness and quality controllability of the product.

2.
JOURNAL OF RARE DISEASES ; (4): 6-16, 2023.
Artículo en Inglés | WPRIM | ID: wpr-1005062

RESUMEN

Familial hypercholesterolemia (FH) is a group of autosomal co-dominant genetic diseases mainly characterized by abnormal low-density lipoprotein related metabolism. It is one of the most common inherited diseases in children and one of the most serious lipid metabolism diseases which results in various life-threatening cardiovascular diseases and the complications. In recent years, the treatment protocols for FH have diversified thanks to the deeper understanding of the disease in China and abroad and the development of new lipid-lowering drugs. However, the current awareness and diagnosis rate of FH are very low. The treatment of the disease is much inadequate. This paper summarizes the clinical characteristics, diagnosis, screening strategy, and treatment of FH hoping to enhance the understanding and awareness of the disease in the society.

3.
Chinese Journal of Biologicals ; (12): 1391-1396, 2023.
Artículo en Chino | WPRIM | ID: wpr-998396

RESUMEN

@#Dyslipidemia is a causal risk factor of atherosclerotic cardiovascular disease(ASCVD),and lipid-lowering therapies play a major role in preventing and managing ASCVD. Proprotein convertase subtilisin/kexin type 9(PCSK9)promotes atherosclerosis by increasing low-density lipoprotein cholesterol(LDL-C)and inflammatory response,while PCSK9 inhibitors can target to reduce PCSK9 levels and have high lipid lowering efficiency. Especially on the basis of statin or ezetimibe treatment,it can also bring more clinical benefits. With the in-depth study,PCSK9 inhibitor has become the research focus in recent years. This paper reviewed the development progress of PCSK9 inhibitors,in order to provide references for the clinical application of this class of drugs.

4.
Chinese Critical Care Medicine ; (12): 807-812, 2023.
Artículo en Chino | WPRIM | ID: wpr-992030

RESUMEN

Objective:To investigate the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on platelet activation in sepsis.Methods:① Clinical trial: a prospective study was conducted. Patients with sepsis and septic shock aged ≥ 18 years old who met the diagnostic criteria of Sepsis-3 admitted to the department of intensive care medicine of the Affiliated Hospital of Binzhou Medical College from January to October in 2021 were selected as subjects. Healthy subjects in the same period were taken as healthy control group. Platelet count (PLT) in the first routine blood test after admission was recorded. Venous blood was taken 1 day after diagnosis, and serum PCSK9 level was determined by enzyme-linked immunosorbent assay (ELISA). The differences of PCSK9 level and PLT between the two groups were compared, and subgroup analysis was conducted based on PLT for patients with sepsis. The correlation between PCSK9 level and PLT in septic patients was analyzed by Pearson correlation method. ② Animal experiment: 80 male C57BL/6 mice were randomly divided into control group, sepsis model group [lipopolysaccharide (LPS) group], PCSK9 inhibitor pretreatment group (PCSK9 inhibitor+LPS group) and PCSK9 inhibitor control group (PCSK9 inhibitor group), with 20 mice in each group. The mouse model of sepsis was reproduced by intraperitoneal injection of LPS 12 mg/kg, and the control group and PCSK9 inhibitor group were intraperitoneally injected with the same amount of sterile normal saline. PCSK9 inhibitor+LPS group and PCSK9 inhibitor group were pretreated with PCSK9 inhibitor 5 mg/kg intraperitoneal injection for 7 days before injection of LPS or normal saline, respectively, and the control group and LPS group were injected with an equal amount of sterile normal saline. The lung tissues were taken for pathological and immunohistochemical observation 24 hours after modeling. Blood was taken from the heart for determining PLT. Platelet activation was detected by flow cytometry. The expression level of platelet-activation marker CD40L was detected by Western blotting.Results:① Clinical trial: there were 57 cases in the sepsis group and 27 cases in the healthy control group. Serum PCSK9 level in the sepsis group was significantly higher than that in the healthy control group (μg/L: 232.25±72.21 vs. 191.72±54.92, P < 0.05), and PLT was significantly lower than that in the healthy control group [×10 9/L: 146.00 (75.50, 204.50) vs. 224.00 (194.00, 247.00), P < 0.01]. Subgroup analysis showed that the serum PCSK9 level in the thrombocytopenia patients ( n = 20) was significantly higher than that in the non-thrombocytopenia patients ( n = 37; μg/L: 264.04±60.40 vs. 215.06±72.95, P < 0.01). Correlation analysis showed a significant negative correlation between serum PCSK9 levels and PLT in septic patients ( r = -0.340, P = 0.010). ② Animal experiment: there were no significant pathological changes in lung tissue in the control group and PCSK9 inhibitor group under light microscope, and no significant differences in PLT, platelet activation and plasma CD40L protein expression was found between the two groups. In the LPS group, a large number of inflammatory cells were infiltrated in the pulmonary interstitium, the alveolar structure was damaged obviously, the alveolar septum was widened, the alveolar cavity was extensively bleeding, the capillary dilatation with bleeding and platelet aggregation were found, the PLT was significantly decreased, the platelet activation and the expression level of CD40L protein in plasma were significantly increased. The infiltration of inflammatory cells in lung tissue of mice in the PCSK9 inhibitor+LPS group was reduced to a certain extent, the thickening of alveolar septa was reduced, the platelet aggregation in lung tissue was decreased as compared with the LPS group, the PLT was significantly increased (×10 9/L: 515.83±46.60 vs. 324.83±46.31, P < 0.05), the platelet activation and the expression level of CD40L protein in plasma were significantly decreased [positive expression rate of platelet activation dependent granule surface facial mask protein CD62P: (12.15±1.39)% vs. (18.33±2.74)%, CD40L protein (CD40L/β-actin): 0.77±0.08 vs. 1.18±0.10, both P < 0.05]. Conclusion:PCSK9 level has a certain effect on promoting platelet activation in sepsis, and inhibition of PCSK9 level may have potential research value in improving adverse outcomes caused by sepsis thrombocytopenia.

5.
Rev. colomb. cardiol ; 29(6): 663-675, dic. 2022. tab, graf
Artículo en Español | LILACS-Express | LILACS | ID: biblio-1423797

RESUMEN

Resumen: La hiperlipidemia es altamente prevalente y contribuye de forma sustancial a la enfermedad cardiovascular aterosclerótica, que es una de las principales causas de morbilidad y mortalidad en Colombia. La reducción del colesterol LDL (c-LDL) produce una disminución del riesgo de enfermedad cardiovascular aterosclerótica y de eventos cardiovasculares adversos. La terapia dirigida a la proproteína convertasa subtilisina/kexina tipo 9 (PCSK9; su sigla en inglés) ha surgido como una herramienta novedosa para el tratamiento de la hiperlipidemia. Inclisiran es un ARN pequeño de doble hebra, que actúa inhibiendo la transcripción de PCSK-9 en los hepatocitos, lo que conduce a una reducción marcada y sostenida del c-LDL. En contraste con otras terapias hipolipemiantes, como estatinas, ezetimibe y anticuerpos monoclonales (MAbs; su sigla en inglés) e inhibidores de PCSK9, inclisiran propone un régimen de dosificación infrecuente de dos o tres veces al año. Su efecto prolongado representa una ventaja frente al incumplimiento del tratamiento, que es una de las principales causas por las que no se alcanzan los objetivos de c-LDL con la terapia estándar. Esta revisión tiene como objetivo presentar y discutir los datos científicos actuales con relación a la eficacia, tolerabilidad y seguridad del inclisiran en el tratamiento de la hipercolesterolemia.


Abstract: Hyperlipidemia is a highly prevalent condition and contributes substantially to atherosclerotic cardiovascular disease (ASCVD), which is one of the main causes of morbidity and mortality in Colombia. The reduction of LDL cholesterol (LDL-C) decreases the risk of ASCVD and adverse cardiovascular events. Targeted therapy for the proprotein convertase subtilisin/kexin type 9 (PCSK-9) has emerged as a novel tool for the treatment of hyperlipidemia. Inclisiran is a small double-stranded small interfering RNA that acts by blocking PCSK-9 transcription in hepatocytes, leading to a marked and sustained reduction in circulating LDL-C levels. In contrast to other lipid-lowering therapies such as statins, ezetimibe and monoclonal antibodies (MAbs) PCSK-9 inhibitors, Inclisiran proposes an infrequent dosing regimen of twice or three times a year. Its prolonged effect represents an advantage over non-compliance of the treatment, which is one of the main reasons why LDL-C goals are not achieved with standard therapy. This review aims to present and discuss current scientific data regarding the efficacy, tolerability and safety of Inclisiran in the treatment of hypercholesterolemia.

6.
Chinese Journal of Laboratory Medicine ; (12): 260-267, 2022.
Artículo en Chino | WPRIM | ID: wpr-934364

RESUMEN

Objective:To explore the postprandial plasma low-density lipoprotein cholesterol (LDL-C) changes by various detection methods.Methods:A total of 85 subjects admitted to the Second Xiangya Hospital of Central South University from November 2017 to May 2019 were included. Serum samples were collected from fasting and the 2 nd hour and the 4 th hour after breakfast. Serum lipid levels were measured with enzymatic assays and nuclear magnetic resonance spectroscopy (NMRS), and proprotein invertase subtilisin/kexin type 9 (PCSK9) levels were measured with enzyme-linked immunosorbent assays. The differences of blood lipid components at different time points were compared by Friedman two-way rank analysis of variance and Wilcoxon signed rank test, and the correlation between PCSK9 level and lipoprotein particles was analyzed by Spearman correlation. Results:Measured by enzymatic assays, compared with the fasting state, LDL-C decreased at the 2 nd hour and the 4 th hour after the meal (2.58[2.09, 3.12], 2.47[1.92, 3.02], 2.37[1.82, 2.80] mmol/L, P<0.001). Measured by NMRS, the concentration of LDL particles (1 086[830, 1 239], 1 083[848, 1 213], 1 061[814, 1 213] nmol/L, P=0.417) did not change significantly, and cholesterol in LDL particles were 2.13 (1.56, 2.54), 2.16 (1.68, 2.50), 2.06 (1.58, 2.50) mmol/L, respectively ( P=0.047),and postprandial cholesterol in LDL particles in the 2 nd hour and in the 4 th hour did not change significantly compared with fasting ( P>0.05). while the concentration of large LDL particles (185.2[150.6,221.6], 173.0[144.8,220.3], 178.1[144.0,233.6] nmol/L, P=0.001), and the cholesterol level in large LDL particles (0.49[0.39, 0.57], 0.47[0.38, 0.57], 0.46[0.37, 0.58]mmol/L, P<0.001) decreased after the meal. The PCSK9 level also decreased significantly after the meal (299[233, 397], 257[208, 342], 251[215, 340] ng/ml, P<0.001). There was an independent positive correlation between the decrease of PCSK9 levels and the increase of remnant cholesterol detected by MNRS after the meal ( r=0.232, P=0.035). Conclusions:The postprandial LDL-C level measured by NMRS and enzymatic assays is not consistent. The decrease of LDL-C measured by enzymatic assays is not caused by the clearance of LDL particles, but by the redistribution of cholesterol in each LDL subfraction.

7.
Chinese Critical Care Medicine ; (12): 614-619, 2022.
Artículo en Chino | WPRIM | ID: wpr-956020

RESUMEN

Objective:To investigate the prognostic value of proprotein convertase subtilisin/kexin type 9 (PCSK9) and blood lipid indexes in patients with sepsis.Methods:Patients with sepsis or septic shock who were ≥ 18 years old and met the Sepsis-3.0 diagnostic criteria admitted to the department of critical care medicine of Binzhou Medical University Hospital from January to October 2021 were enrolled. Healthy adults at the same period were selected as healthy control group. Baseline characteristics, acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) and sequential organ failure assessment (SOFA) score were recorded. Venous blood samples were collected within 24 hours after diagnosis, and serum PCSK9 was determined by enzyme-linked immunosorbent assay (ELISA) at 1, 3 days and 5 days. Meanwhile, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG) and lipoprotein A were detected. The differences of each index between sepsis group (28-day death group and survival group) and healthy control group were compared. Meanwhile, the indexes of patients with different severity and 28-day prognosis in sepsis group were compared. Receiver operator characteristic curve (ROC curve) was drawn to evaluate the predictive value of PCSK9 and blood lipid for the prognosis of sepsis. Multivariate Logistic regression was used to analyze the influencing factors for the prognosis of sepsis, and the Kaplan-Meier survival curve at 28th day was drawn.Results:There were 50 patients in sepsis group (including 19 patients with sepsis, 31 patients with septic shock) and 27 patients in healthy control group. In the sepsis group, 19 patients died and 31 patients survived within 28 days. The serum PCSK9 in the sepsis group was significantly higher than that in the healthy control group [μg/L: 223.09 (198.47, 250.82) vs. 188.00 (165.27, 214.90), P < 0.01], and HDL-C, LDL-C, TC and lipoprotein A were significantly lower than those in the healthy control group [HDL-C (mmol/L): 0.82±0.35 vs. 1.45±0.24, LDL-C (mmol/L): 1.53 (1.14, 2.47) vs. 2.89 (2.55, 3.19), TC (mmol/L): 2.03 (1.39, 2.84) vs. 4.24 (3.90, 4.71), lipoprotein A (g/L): 8.80 (5.66, 17.56) vs. 27.03 (14.79, 27.03), all P < 0.01]. PCSK9 in the sepsis death group was higher than that in the survival group [μg/L: 249.58 (214.90, 315.77) vs. 207.01 (181.50, 244.95), P < 0.01], and the HDL-C, LDL-C and TC were lower than those in the survival group [HDL-C (mmol/L): 0.64±0.35 vs. 0.93±0.30, LDL-C (mmol/L): 1.32±0.64 vs. 2.08±0.94, TC (mmol/L): 1.39 (1.01, 2.23) vs. 2.69 (1.72, 3.81), all P < 0.01]. With the progression of the disease, the PCSK9 in the sepsis death group and the survival group was significantly lower than that within 1 day of diagnosis (all P < 0.05). ROC curve analysis showed that PCSK9 had higher predictive value of 28-day death than HDL-C, LDL-C, TC [area under ROC curve (AUC) and 95% confidence interval (95% CI): 0.748 (0.611-0.885) vs. 0.710 (0.552-0.868), 0.721 (0.575-0.867), 0.702 (0.550-0.854)]. Multivariate Logistic regression analysis showed that PCSK9 was an independent risk factor affecting the 28-day prognosis of sepsis (β value was 1.014, P = 0.020). Kaplan-Meier survival curve analysis showed that when PCSK9 ≥ 208.97 μg/L, with the increase of PCSK9, the 28-day survival rate of sepsis patients decreased significantly. Conclusions:PCSK9, HDL-C, LDL-C and TC can all predict the 28-day prognosis of patients with sepsis. The prognostic value of PCSK9 is the highest. PCSK9 is an independent risk factor affecting the prognosis of sepsis. In the early stage of the disease, PCSK9 may have a good predictive value for the prognosis of sepsis. When PCSK9 ≥ 208.97 μg/L, the 28-day survival rate decreased significantly.

8.
Chinese Journal of Nephrology ; (12): 583-590, 2021.
Artículo en Chino | WPRIM | ID: wpr-911886

RESUMEN

Objective:To evaluate the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on lipid homeostasis and cellular injury of podocytes, and to clarify its mechanism.Methods:Twelve-week old C57BL/6 wild-type mice ( n=10) and PCSK9 knockout ( PCSK9 KO) mice ( n=10) were selected as the animal models. The renal tissues were taken after perfusion through heart. Mouse podocytes were transfected with PCSK9 siRNA to downregulate PCSK9 expression. BODIPY 493/503 staining was performed for evaluating lipid accumulation, and standard transmission electron microscopy (TEM) was used to observe the foot process of podocytes, the shape of mitochondria and lipid droplet in podocytes. TUNEL staining was carried out to evaluate cell apoptosis in glomerulus. The parameters about mitochondria function (key enzymes such as PGC-1α, CPT-1 and Acox-1) and apoptosis were quantified through qPCR and western blotting. Results:The lipid accumulation in glomerulus of PCSK9 KO mice were more serious than controls. The expression of PGC-1α protein and PGC-1α, CPT-1 and Acox-1 mRNA in PCSK9 KO mouse kidney tissues were decreased than controls (all P<0.05), and mitochondria swelling and cristae disappearance in podocytes of PCSK9 KO mice were observed. In PCSK9 KO group, the foot process of podocytes partially fused and disappeared, and the apoptosis index increased compared with the control group ( P<0.05). In vitro, compared with the control group, the lipid accumulation was more significant, transcription level of key enzymes related to mitochondrial function was decreased, mitochondrial structure was damaged and the apoptosis index was increased in cultured podocyte PCSK9 siRNA group (all P<0.05). Conclusions:PCSK9 is involved in the lipid homeostasis of podocytes. The decrease of PCSK9 results in the increase of intracellular lipid accumulation, accompanied by the mitochondrial structure damage and disfunction of podocytes, and leads to cell apoptosis.

9.
Chinese Journal of Experimental Traditional Medical Formulae ; (24): 44-51, 2021.
Artículo en Chino | WPRIM | ID: wpr-906204

RESUMEN

Objective:To observe the expression of hepatocyte nuclear factor 1<italic>α</italic> (HNF1<italic>α</italic>), proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein cholesterol (LDLR) in hypercholesterolemia rat liver, and investigate the mechanism of Shuangyu Tiaozhi Decoction regulating cholesterol metabolism and attenuating hypercholesterolemia. Method:After providing a high-fat diet for 4 weeks, 40 SD rats were selected, 8 of which were randomly selected as normal group and fed a normal diet, and the remaining 32 rats were fed a high-fat diet. The rats successfully established as hypercholesterolemic model, were randomized into 4 groups: model group, low dose of Shuangyu Tiaozhi decoction group (7.8 g·kg<sup>-1</sup>), high dose of Shuangyu Tiaozhi decoction group (15.6 g·kg<sup>-1</sup>), and simvastatin group (4 mg·kg<sup>-1</sup>), with 8 rats in each group. The drugs were continuously given for 8 weeks. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) were measured. The pathomorphological changes in liver were observed by hematoxylin and eosin (HE) staining. The immunohistochemistry was used to detect PCSK9 and LDLR expression in liver. The mRNA and protein expression levels of HNF1<italic>α</italic>, PCSK9 and LDLR were determined by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) and Western blot. Result:Compared with normal group, the TC, TG, LDL-C levels in model group were significantly increased (<italic>P</italic><0.01), the morphology showed obvious liver steatosis. The mRNA and protein expression of HNF1<italic>α</italic> and PCSK9 were increased (<italic>P</italic><0.05), the mRNA and protein expression of LDLR was decreased (<italic>P</italic><0.05). Compared with model group, the serum TC, TG, LDL-C levels were significantly lowered in the Shuangyu Tiaozhi decoction high-dose group (<italic>P</italic><0.01), the serum TC, LDL-C levels were significantly lowered in the Shuangyu Tiaozhi decoction low-dose group and simvastatin group (<italic>P</italic><0.05,<italic>P</italic><0.01), while no significant effect was observed on the serum HDL-C levels in each treatment group. The liver steatosis decreased in each treatment group. The mRNA and protein expression of HNF1<italic>α</italic> was obviously decreased in each treatment group (<italic>P</italic><0.05,<italic>P</italic><0.01), the mRNA and protein expression of PCSK9 was obviously decreased in Shuangyu Tiaozhi decoction low and high-dose groups (<italic>P</italic><0.05,<italic>P</italic><0.01), the mRNA expression of PCSK9 was significantly increased in the simvastatin group (<italic>P</italic><0.01), while the protein expression showed a downward trend. The LDLR mRNA levels were significantly increased in each treatment group (<italic>P</italic><0.01), the LDLR protein expression was significantly increased in Shuangyu Tiaozhi high-dose group (<italic>P</italic><0.01), and showed an upward trend in Shuangyu Tiaozhi low-dose group and simvastatin group. Results of immunohistochemistry showed PCSK9 expression was weakly positive, the expression of LDLR was strongly positive in each treatment group. The therapeutic effect of Shuangyu Tiaozhi decoction high-dose group was more remarkable than simvastatin group, while there was no obvious difference between the Shuangyu Tiaozhi decoction low-dose group and simvastatin group. Conclusion:Shuangyu Tiaozhi decoction may reduce the blood lipid levels through HNF1<italic>α</italic>/PCSK9/LDLR signaling pathway, play an active role on regulating cholesterol metabolism and alleviating high-fat diet-induced hypercholesterolemia.

10.
Journal of Zhejiang University. Medical sciences ; (6): 229-238, 2021.
Artículo en Inglés | WPRIM | ID: wpr-879961

RESUMEN

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.


Asunto(s)
Animales , Conejos , Sistemas CRISPR-Cas/genética , Mutación , Mutación Puntual , Proproteína Convertasa 9/metabolismo
11.
Artículo | IMSEAR | ID: sea-188638

RESUMEN

Background: Cucumber target leaf spot (TLS), caused by Corynespora cassiicola (C. Cassiicola), is a serious disease in cucumber (Cucumis sativus) production worldwide. Therefore, cultivating new varieties of TLS resistance of C. sativus is an important goal of cucumber breeding. Previous studies have shown that subtilisin-like protease (SUBP) plays an important role in response to C. Cassiicola infection in resistant plants. Objective: In this study, the full-length cDNA of the CsSUBP gene was cloned, and the prokaryotic expression vector was successfully constructed in order to study the effects of subtilisin. Futhermore, vital clues regarding CsSUBP gene involved in TLS resistance of C. sativus are gained from the bioinformatics assay. Method: The CsSUBP gene was identified by sequencing with the intermediate vector pMD18 by designing specific primers and PCR amplification techniques. The prokaryotic expression vector pET30a-CsSUBP was further constructed and identified by colony PCR and EcoR V and SalⅠ double digestion. Result: The primary structure of CsSUBP was predicted and analyzed by bioinformatics analysis. The results showed that CsSUBP was weakly acidic protein, N-terminal signal peptide region, including a Inhibitor_I9 domain domain. Conclusion: The pET30a-CsSUBP prokaryotic expression vector was constructed successfully. This study is convenient for the study of prokaryotic expression and its kinase activity.

12.
Chinese Journal of Cardiology ; (12): 367-373, 2019.
Artículo en Chino | WPRIM | ID: wpr-805167

RESUMEN

Objective@#To investigate the effect and mechanism of proprotein convertase subtilisin type 9 (PCSK9) on lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) mediated oxidized low-density lipoprotein (ox-LDL) uptake by mononuclear macrophage (THP-1) derived macrophages.@*Methods@#THP-1 monocyte was incubated with PMA for 48 hours to induce the differentiation into macrophages. Macrophages were pretreated with human recombinant PCSK9 protein for 1 hour and incubated with ox-LDL for 24 hours to induce foam cells. Oil red O staining was used to observe the accumulation of lipid in the control group (foam cells) and groups treated with different concentrations of recombinant PCSK9 protein, and the intracellular cholesterol content was measured by enzyme method, and mRNA and protein expressions of LOX-1 were detected by real-time PCR and Western blot. The uptake of Dil-labeled oxidized low density lipoprotein (Dil-ox-LDL) was observed by fluorescence microscopy in control group (macrophage), PCSK9 protein treated group and PCSK9 protein plus anti-LOX-1 antibody and IgG antibody treated group. mRNA and protein expression of toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX-2) were detected in control and PCSK9 protein treated group in the absence and presence of TLR4 inhibitor (TAK-242), NF-κB inhibitor (PDTC). In addition, reactive oxygen species (ROS) production was evaluated in the absence or presence of COX-2 inhibitor (NS-398) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (DPI). The mRNA and protein expression of LOX-1 in the control group (PCSK9 protein pretreated foam cells) and PCSK9 protein group in the absence or presence of TAK-242, PDTC, NS-398 and DPI respectively.@*Results@#(1) The total optical density of intracellular lipid droplets, total cholesterol level, cholesterol ester level and cholesterol ester/total cholesterol ratio as well as expression of LOX-1 were significantly higher in PCSK9 group than those in control group (all P<0.05). (2) The fluorescence intensity of Dil-ox-LDL was significantly higher in PCSK9 group and PCSK9+IgG antibody group than in the control group (all P<0.05). The fluorescence intensity was significantly lower in PCSK9+anti-LOX-1 antibody group than in PCSK9 group and PCSK9+IgG antibody group (all P<0.05). (3) The expressions of TLR4, NF-κB and COX-2 were significantly higher in PCSK9 group than in control group (all P<0.05). The expressions of TLR4, NF-κB and COX-2 were significantly lower in PCSK9+TAK-242 group and PCSK9+PDTC group than in PCSK9 group (all P<0.05). The ROS level was significantly higher in PCSK9 group than in the control group (P<0.05). The ROS levels were significantly lower in PCSK9+NS-398 and PCSK9+DPI groups than in PCSK9 group (all P<0.05). (4) The expressions of LOX-1 mRNA and protein were lower in respective PCSK9 protein plus TAK-242, PDTC, NS-398 or DPI group than in PCSK9 protein alone (all P<0.05).@*Conclusion@#PCSK9 may regulate LOX-1 mediated ox-LDL uptake by the THP-1 derived macrophage via TLR4/NF-κB/COX-2/ROS pathway.

13.
Chinese Journal of Dermatology ; (12): 90-93, 2019.
Artículo en Chino | WPRIM | ID: wpr-734750

RESUMEN

Objective To investigate the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the pathogenesis of psoriasis by detecting the level of PCSK9 in the plasma of patients with psoriasis and evaluating its effect on the secretion of interferon gamma (IFN-γ) and interleukin-17A (IL-17A) by peripheral CD4+ T cells.Methods Totally,30 outpatients with psoriasis vulgaris and 30 healthy volunteers (controls) were enrolled from Hospital for Skin Diseases,Chinese Academy of Medical Sciences between February 2016 and December 2017.Of the 30 patients,16 were males,and 14 were females.Their age varied from 18 to 66 years,and the course of disease ranged from 1 month to 15 years.Peripheral venous blood samples were obtained from the patients and controls,and the plasma and was performed to measure mRNA expression of PCSK9 in the PBMC,and enzyme-linked immunosorbent assay (ELISA) to determine the concentration of PCSK9 in the plasma.Peripheral CD4+ T cells were isolated from the PBMC by magnetic bead method,and divided into 2 groups to be co-cultured with (experiment group) or without PCSK9 protein (control group).After 24-hour treatment,ELISA was conducted to detect the levels of IFN-γ and IL-17A in the culture supernatant.Statistical analysis was carried out by using two-sample t test for the comparison between the two groups,and Pearson correlation analysis for analyzing correlations between the plasma level of PCSK9 and psoriasis area and severity index (PASI) score in the patients with psoriasis.Results PCSK9 mRNA expression was undetected in the PBMC of the patients with psoriasis and controls.The plasma level of PCSK9 was significantly higher in the patients with psoriasis ([243.58 ± 11.91] μg/L) than in the healthy controls ([199.74 ± 31.09] μg/L,t =5.761,P < 0.001).After co-culture of the peripheral CD4+ T cells from patients with PCSK9 protein,the levels of IFN-γ and IL-17A both significantly increased ([6 150.00 ± 212.13] ng/L,[1 532.00 ± 11.31] ng/L,respectively) compared with the control group co-cultured without PCSK9 protein ([4 650.00 ± 212.13] ng/L,[698.5 ± 266.58] ng/L,respectively;t =7.071,4.418 respectively,both P < 0.05).IFN-γand IL-17A were undetected in the culture supernatant of CD4+ T cells from the healthy controls in the experiment group or control group.Conclusion The plasma level of PCSK9 increases in patients with psoriasis,which may be involved in the pathogenesis of psoriasis by activating peripheral CD4+ T cells.

14.
Chinese Traditional and Herbal Drugs ; (24): 2229-2234, 2019.
Artículo en Chino | WPRIM | ID: wpr-851176

RESUMEN

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the highligh of lipid-lowering therapy due to its effect on LDL-C in the cardiovascular field. PCSK9 inhibitor has become the hot spot in biological preparations in these years, but the properties of adverse reaction, high price and injection-methods of biologicals limit its development. Currently, it was reported that many traditional Chinese medicine and natural products had the functions of regulating PCSK9. This paper summarized the regulation and mechanisms of traditional Chinese medicine monomers, active ingredients, compounds and natural products on PCSK9 so as to provide references for the development of small-molecule inhibitors of PCSK9.

15.
Academic Journal of Second Military Medical University ; (12): 412-419, 2019.
Artículo en Chino | WPRIM | ID: wpr-837898

RESUMEN

Objective To explore the effect of aerobic exercise on blood lipid of hyperlipidemia rats and its mechanism. Methods A total of 120 healthy male SD rats aged 8 weeks were randomized into normal control group, high-fat diet (HF) group, SBC-115076 group and aerobic exercise group, with 30 rats in each group. The rats in the normal control group were fed with standard diet, and the rats in the other 3 groups were fed with HF to establish hyperlipidemia rat model. The rats in the SBC-115076 group were injected with proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor SBC-115076 (8 mg/kg) once a week for 8 weeks, and the rats in the aerobic exercise group underwent swimming without load 6 days a week for 8 weeks. After 8 weeks, the rats were sacrificed and the blood samples were collected to determine the levels of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Pathological changes of thoracic aorta were observed using H-E staining. The mRNA and protein expression levels of PCSK9, low-density lipoprotein receptor (LDLR) and sterol regulatory element binding protein (SREBP) in the hepatic tissues were detected by quantitative real-time PCR, Western blotting and immunofluorescence. Results Compared with the normal control group, the levels of serum TG, TC and LDL of the rats were significantly higher in the HF group, and the level of HDL was significantly lower (P<0.01). Compared with the HF group, the levels of serum TG, TC and LDL of the rats were significantly lower in the SBC-115076 and aerobic exercise groups, and the level of HDL was significantly higher (P<0.01). In the HF rats, the aortic tunica intima was thickened and endothelial cells were damaged and exfoliated. Compared with the HF group, the aortic intima thickening was reduced and endothelial damage was less in the aerobic exercise group. Compared with the normal control group, the mRNA and protein expression levels of PCSK9, SREBP1 and SREBP2 were significantly higher in the HF group, and the mRNA and protein expression levels of LDLR were significantly lower (P<0.01). Compared with the HF group, the mRNA and protein expression levels of PCSK9, SREBP1 and SREBP2 were significantly lower, and the mRNA and protein expression levels of LDLR were significantly higher (P<0.01). Conclusion Aerobic exercise can down-regulate the expression of TG, TC and LDL, up-regulate the expression of HDL, and alleviate the intimal thickening of aorta. The mechanism may be related to down-regulating the expression of PCSK9 and SREBPs, thus eliminating the inhibition of LDLR.

16.
Tianjin Medical Journal ; (12): 765-770, 2018.
Artículo en Chino | WPRIM | ID: wpr-810921

RESUMEN

@#Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors can reduce the low density lipoprotein cholesterol (LDL-C) level in circulatory system by blocking PCSK9-low density lipoprotein receptor (LDLR) pathway to mediate the degradation of LDLR. At present, PCSK9 inhibitors have become the focus of cardiovascular lipid-lowering therapy. A variety of PCSK9 inhibitors have entered the clinical trial stage. This paper mainly reviews the molecular structure and mechanism of PCSK9, the classification of PCSK9 inhibitors, and recent clinical study of the monoclonal antibodies of PCSK9 inhibitors in order to evaluate the effectiveness and long-term safety of cardiovascular risk reduction.

17.
Tianjin Medical Journal ; (12): 220-224, 2018.
Artículo en Chino | WPRIM | ID: wpr-698011

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Atherosclerotic cardiovascular diseases(ASCVD)are the major causes of morbidity and mortality worldwide. Previous randomized controlled trials confirm that statin therapy can effectively reduce the level of low density lipoprotein cholesterol(LDL-C),all-cause and cardiovascular disease mortality in patients with and without ASCVD.However,there is no widespread use of lipid-lowering therapy to achieve the benefit in high risk patients with ASCVD and patients without ASCVD. Therefore, it is necessary to further elaborate the clinical benefits of statins and their combined use for lipid regulating therapy and increasing the beneficiaries.

18.
Journal of Shanghai Jiaotong University(Medical Science) ; (12): 339-342, 2018.
Artículo en Chino | WPRIM | ID: wpr-695666

RESUMEN

Coronary atherosclerotic heart disease (CHD) remains the leading cause of morbidity and mortality in the world, with elevated low density lipoprotein-cholesterol (LDL-C) levels as a major risk factor. Lower levels of LDL-C can effectively reduce the risk of CHD. To date, lipid-lowering medicines such as statins are effective in lowering LDL-C, but a proportion of patients do not achieve lipid reduction target with statins or are intolerant to statins. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are a new class of agents reducing LDL-C which gain more and more concerns. Through inhibitory effect on PCSK9 and increasing low-density lipoprotein receptors recycling, they can significantly reduce serum LDL-C levels. PCSK9 inhibitors are currently in phase Ⅲ of clinical trials, and the results showed that they had good lipid-lowering effects and tolerability. This review provided an overview of the latest advances and challenges about PCSK9 inhibitors.

19.
Journal of Shanghai Jiaotong University(Medical Science) ; (12): 212-216, 2018.
Artículo en Chino | WPRIM | ID: wpr-695643

RESUMEN

Elevated low density lipoprotein cholesterol (LDL-C) is a major risk factor for cardiovascular disease.Studies show that proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulation enzyme and serves a pivotal function in the degradation of low density lipoprotein receptor,which contributes to the decrease in hepatic cholesterol uptake and increase in circulating LDL-C.PCSK9 inhibitor can significantly elevate the surface of low density lipoprotein receptor of liver cells and bond more LDL-C to decrease the level of LDL-C.Thus PCSK9 has emerged as a popular target for the development of new cholesterol lowering drugs and therapeutic intervention of cardiovascular disease.In this article,the history,mechanism of action,metabolic effects of PCSK9 and the clinical outcomes of PCSK9 inhibitors will be briefly reviewed.

20.
Chinese Journal of Nephrology ; (12): 845-850, 2018.
Artículo en Chino | WPRIM | ID: wpr-711170

RESUMEN

Objectives To evaluate the role of PCSK9 (proprotein convertase subtilisin kexin type 9) on the lipid accumulation and kidney injury of C57BL/6 mice. Methods The 24 h urine of 12 weeks old wide type C57BL/6 mice and PCSK9 knockout (KO) mice were collected through a metabolic cage, followed by perfusion and sacrifice. Urinary microalbumin?to?creatinine ratio (UACr), total cholesterol and triglyceride in kidney tissues were measured by ELISA. BODIPY 493/503 staining and standard transmission electron microscopy (TEM) of kidney tissues was performed for evaluating lipid accumulation and podocyte foot effacement in the kidney. Kidney tissues were also evaluated by PAS stain and TUNNEL stain. PCSK9, podocin and nephrin were quantified through real?time PCR, and the Bcl?2, Bax and cleaved caspase 3 were evaluated by Western blotting. Results Total cholesterol and triglyceride contents were higher in the kidneys of PCSK9 KO mice than controls (P<0.05). The level of lipid accumulation in glomeruli and tubules through BODIPY 493/503 stain, and the amount of lipid drop in TEM were more serious in PCSK9 KO mice. UACr and podocyte foot process effacement were increased, and the transcription of podocin and nephrin were decreased in the kidneys of PCSK9 KO mice (all P<0.05). The expression of Bcl?2 was decreased, and Bax and cleavedcaspase 3 were increased in the kidney samples of PCSK9 KO mice. Conclusion PCSK9 might be reversely involved in lipid homeostasis and accumulation, resulting in injury and apoptosis in the kidneys of C57BL/6 mice.

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