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Endotoxin contamination is a threat to the safety of pharmaceutical products, especially parenteral drugs. Any sterile and/or pyrogen-free pharmaceutical product requires regulatory specifications to ensure safe patient use. This study covers the performance evaluation study of an endotoxin quantitation commercial kit by recombinant Factor C (rFC), Endozyme II® Go, for 0.9% sodium chloride injection. The samples were spiked with endotoxin solutions between 0.0005 and 10 EU/mL and tested by the rFC kit to evaluate precision, accuracy, detection and quantification limits, linearity, and robustness. Each of the six points was assayed at least five times.The relative standard deviation for precision testing ranged from 1.9 to 8.3%. The recovery accuracy values of endotoxin were between 61% and 125% for the range from 0.005 to 10 EU/mL. The results demonstrated that the rFC method allows endotoxin quantification with accuracy, precision, specificity, and linearity for the range of 0.005 and 10 EU/mL for 0.9% sodium chloride injection. (AU)
A contaminação por endotoxinas é uma ameaça à segurança dos produtos farmacêuticos, especialmente dos medicamentos parenterais. Qualquer produto farmacêutico estéril e/ou livre de pirogênios requer especificações regulatórias para garantir a segurança de uso para o paciente. Este estudo abrange o estudo de avaliação de desempenho empregando o kit comercial Endozyme II® Go para quantificação de endotoxina, por Fator C recombinante (FCr), em amostras de cloreto de sódio 0,9% para uso parenteral. As amostras foram fortificadas com cinco concentrações distintas de soluções de endotoxina na faixa entre 0,0005 e 10 UE/mL. Cada um dos cinco níveis foi testado pelo menos cinco vezes para avaliação dos critérios de precisão, exatidão, limites de detecção e quantificação, linearidade e robustez. O desvio padrão relativo para os testes de precisão variou de 1,9 a 8,3%. Os valores de recuperação de endotoxina para o parâmetro exatidão estiveram compreendidos entre 61% e 125%. Os resultados demonstraram que o método por FCr permite a quantificação de endotoxinas com exatidão, precisão, especificidade e linearidade para a faixa de 0,005 e 10 UE/mL em amostras de cloreto de sódio 0,9% para uso parenteral. (AU)
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In Vitro Techniques , Endotoxins , Saline Solution , Sodium ChlorideABSTRACT
Objective To study the clinical effect of levosimendan combined with recombinant human brain natriuretic peptide(rhBNP)on patients with acute heart failure.Methods A total of 100 patients with acute heart failure in the hospital from December 2019 to December 2021 were selected as the research sub-jects.According to different treatment options,the subjects were divided into the control group,levosimendan group,rhBNP group and combined treatment group,with 25 cases in each group.The control group received traditional conventional diuretic,tube expansion and other treatment;the levosimendan group was treated with levosimendan on the basis of the control group;the rhBNP group was treated with rhBNP on the basis of the control group;the combined treatment group was treated with levosimendan and rhBNP on the basis of the control group.The improvement of New York Heart Association(NYHA)classification,death,rehospitaliza-tion rate,6-minute walking distance,improvement of serological indicators and adverse reactions were recor-ded in each group.Results Before treatment,there was no significant difference in baseline data between the groups(P>0.05).On the 1 st and 3 rd day after treatment,the improvement of NYHA classification in the combined treatment group was better than that in the other groups(P<0.05),and the improvement of NY-HA classification in the levosimendan group and rhBNP group was better than that in the control group(P<0.05).The readmission rate within 6 months after treatment in the combined treatment group was lower than that in the other groups(P<0.05).At 5 and 9 days after treatment,the 6-minute walking distance in the combined treatment group was longer than that in the other groups(P<0.05).At 9 days after treatment,the left ventricular ejection fraction(LVEF)in the combined treatment group was higher than that in the other groups(P<0.05),and the level of N-terminal B-type natriuretic peptide(NT-proBNP)in the combined treatment group was lower than that in the other groups(P<0.05).No significant difference was found in the comparison of the occurrence of adverse reactions among the four groups(P>0.05).Conclusion The combina-tion of levosimendan and rhBNP in the treatment of patients with acute heart failure is superior to traditional treatment and monotherapy in early clinical improvement,and dose not increase the incidence of adverse reactions.
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Objective To optimize the immune scheme of SARS-CoV-2 RBD recombinant protein vaccine based on P.pastoris,and investigate the effect of different adjuvants on neutralizating antibody(NAb)titer,in order to provide reference for the continuous optimization research of SARS-CoV-2 vaccine.Methods The RBD protein was selected and the corresponding gene fragment was synthesized,which was constructed into the pPICZαA plasmid,and the plasmid was integrated into the genome of P.pastoris after linear transforma-tion for recombinant expression.The obtained recombinant protein vaccine was combined with different adju-vants to immunize mice to evaluate its immunogenicity.Results Both the target proteins wtRBD and Delta RBD were able to achieve satisfactory overexpression through the P.pastoris system.Compared with the 42 d interval,the IgG antibody titer at the 28 d interval increased by 1.8 times(44 923 vs.80 507).After 3 doses of immunization at an interval of 28 d,the geometric mean titer of NAb for Delta variant was 2.5 times higher than that at an interval of 42 days(2 191 vs.891).After immunization with Delta RBD recombinant protein vaccine combined with aluminum adjuvant,the NAb geometric mean titer for Delta variants reached 32 255(2 167-88 084).When using 5 μg or 30 μg Delta RBD immunization,the NAb titers of the aluminum adju-vant+CpG adjuvant group were about 10 times higher than those of the aluminum adjuvant group alone.Af-ter the third immunization,there was no significant difference in Delta RBD specific IgG titers between the 5 μg antigen group and the 30 μg antigen group(P＞0.05).Conclusion Both wtRBD and Delta RBD prepared based on P.pastoris could be used as effective antigens,with three doses of vaccine administered at a 28 day in-terval being the most effective.The combined immunization of Delta RBD recombinant protein with aluminum adjuvant+CpG adjuvant could obtain higher titers of NAb to exert immune effects on SARS-CoV-2 and its va-riants,providing some reference for the continuous optimization research of SARS-CoV-2 vaccines.
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Objective To investigate the prognostic value of echocardiography indicators combined with se-rum recombinant human arginase 1(ARG1)and glucose-6-phosphate dehydrogenase(G6PD)in children with sepsis.Methods A total of 116 children with sepsis admitted to the hospital from May 2022 to June 2023 were enrolled in the study as the sepsis group.According to the severity of sepsis,the children were further divided into general sepsis group(52 cases),severe sepsis group(38 cases)and septic shock group(26 cases).Ac-cording to the prognosis of the children,the children with sepsis were divided into good prognosis group(84 cases)and poor prognosis group(32 cases).A total of 116 healthy children who underwent physical examina-tion in the hospital during the same period were enrolled as the control group.The left ventricular ejection fraction(LVEF),left ventricular end-diastolic diameter(LVEDD),left ventricular end-diastolic volume(LV-EDV)and early diastolic mitral flow peak velocity(E)were detected by using color Doppler ultrasound.Ser-um ARG1 and G6PD levels were detected by using enzyme-linked immunosorbent assay(ELISA).The echo-cardiographic indexes and serum ARG1 and G6PD levels were compared between the sepsis group and the con-trol group,and among sepsis children with different disease severity and different prognosis.The receiver op-erating characteristic(ROC)curve was used to analyze the predictive value of echocardiographic indexes com-bined with serum ARG1 and G6PD for poor prognosis in children with sepsis.Results Compared with the control group,the sepsis group had significant reductions in LVEF,E,and G6PD(P＜0.05)and significant increases in LVEDD,LVEDV,and ARG1(P＜0.05).With the aggravation of sepsis,the levels of LVEF,E,and G6PD in children with sepsis gradually decreased(P＜0.05),while the levels of LVEDD,LVEDV,and ARG1 gradually increased(P＜0.05).Compared with the good prognosis group,the poor prognosis group had significantly lower levels of LVEF,E,and G6PD(P＜0.05)and significantly higher levels of LVEDD,LV-EDV,and ARG1(P＜0.05).ROC curve analysis showed that the AUC of echocardiographic indexes com-bined with serum ARG1 and G6PD in predicting poor prognosis of children with sepsis was 0.971,and the sensitivity and specificity were 84.4％and 83.2％,respectively.Conclusion The levels of LVEF,E,and G6PD in children with sepsis significantly decreases,and the levels of LVEDD,LVEDV,and ARG1 signifi-cantly increases.Echocardiographic parameters combined with serum ARG1 and G6PD have high predictive value for poor prognosis in children with sepsis.
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In recent years, recombinant human urokinase (rhPro-UK) has been widely used in the treatment of a variety of thromboembolic diseases, with significant efficacy and no obvious adverse reactions. In addition, it has a wide range of applications in many new technology fields. This article focuses on the application of rhPro-UK in the treatment of acute myocardial infarction, cerebrovascular disease, lower extremity deep vein thrombosis, arterial thrombosis and other diseases. rhPro-UK has demonstrated good thrombolytic efficacy and safety in these diseases, especially in patients with acute myocardial infarction, and adjuvant PCI therapy can significantly increase myocardial reperfusion, improve cardiac function, and do not increase the risk of bleeding. For cerebrovascular disease, rhPro-UK can significantly improve the degree of neurological deficit and has a high safety profile. In the treatment of lower extremity deep vein thrombosis, rhPro-UK has shown superior thrombolytic efficacy and safety compared with urokinase. For arterial thrombosis and biological stents, the use of rhPro-UK has also achieved some efficacy, but more research is needed to confirm its efficacy and safety. In addition, ultrasound-mediated drug-loaded thrombolysis systems also have potential applications in rhPro-UK therapy. Future research on rhPro-UK will focus more on the development of new technologies.
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To establish and optimize a method for the detection of recombinant human midkine (rhMK) activity and verify its methodology, cell counting kit-8 (cck-8) method was used to measure the proliferation activity of rat knee chondrocytes. The specificity, accuracy, precision, linearity and robustness of the method were also verified in this study. The established method was proven to have good specificity because the buffer of rhMK and recombinant human interleukin-1 receptor antagonist have no obvious active effect; the recoveries of the samples with relative activities of 50%, 75%, 100%, 125%, 150% were in the range of 80.0% to 124.0% by statistical analysis, the relative standard deviations (RSD) of relative potency were all within 20%, the linear correlation coefficient, R2 ≥ 0.98, suggesting that the accuracy, precision and linearity of the method were good; the robustness correlation coefficient, R2 ≥ 0.92 and the ratio of maximum to minimum of sigmoidal dose-response were no less than 1.5, indicating that robustness of the methods was good. In conclusion, a bioactivity measurement method for rhMK was established and fully validated in this study and it provides a reliable method for the bioactivity analysis of rhMK routine samples during the development. This study was approved by the Animal Care and Use Committee of Shanghai Model Organisms Center, Inc. (approval number: 2019-0008-06).
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Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.
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OBJECTIVE@#To investigate the effect of bone cement containing recombinant human basic fibroblast growth factor (rhbFGF) and recombinant human bone morphogenetic protein-2 (rhBMP-2) in percutaneous kyphoplasty(PKP)treatment of osteoporotic vertebral compression fracture(OVCF).@*METHODS@#A total of 103 OVCF patients who underwent PKP from January 2018 to January 2021 were retrospectively analyzed, including 40 males and 63 females, aged from 61 to 78 years old with an average of (65.72±3.29) years old. The injury mechanism included slipping 33 patients, falling 42 patients, and lifting injury 28 patients. The patients were divided into three groups according to the filling of bone cement. Calcium phosphate consisted of 34 patients, aged(65.1±3.3) years old, 14 males and 20 females, who were filled with calcium phosphate bone cement. rhBMP-2 consisted of 34 patients, aged (64.8±3.2) years old, 12 males and 22 females, who were filled with bone cement containing rhBMP-2. And rhbFGF+rhBMP-2 consisted of 35 patients, aged (65.1±3.6) years old, 14 males and 21 females, who were filled with bone cement containing rhbFGF and rhBMP-2. Oswestry disability index (ODI), bone mineral density, anterior edge loss height, anterior edge compression rate of injured vertebra, visual analog scale (VAS) of pain, and the incidence of refracture were compared between groups.@*RESULTS@#All patients were followed for 12 months. Postoperative ODI and VAS score of the three groups decreased (P<0.001), while bone mineral density increased (P<0.001), anterior edge loss height, anterior edge compression rate of injured vertebra decreased first and then slowly increased (P<0.001). ODI and VAS of group calcium phosphate after 1 months, 6 months, 12 months were lower than that of rhBMP-2 and group rhbFGF+rhBMP-2(P<0.05), bone mineral density after 6 months, 12 months was higher than that of rhBMP-2 and group calcium phosphate(P<0.05), and anterior edge loss height, anterior edge compression rate of injured vertebra of group rhbFGF+rhBMP-2 after 6 months and 12 months were lower than that of group rhBMP-2 and group calcium phosphate(P<0.05). There was no statistical difference in the incidence of re-fracture among the three groups (P>0.05).@*CONCLUSION@#Bone cement containing rhbFGF and rhBMP-2 could more effectively increase bone mineral density in patients with OVCF, obtain satisfactory clinical and radiological effects after operation, and significantly improve clinical symptoms.
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Male , Female , Humans , Middle Aged , Aged , Bone Cements/therapeutic use , Fractures, Compression/complications , Retrospective Studies , Spinal Fractures/complications , Osteoporotic Fractures/etiology , Kyphoplasty/adverse effects , Vertebroplasty/adverse effects , Calcium Phosphates/therapeutic use , Treatment Outcome , Recombinant Proteins , Transforming Growth Factor beta , Fibroblast Growth Factor 2 , Bone Morphogenetic Protein 2ABSTRACT
BACKGROUND:Previous studies have shown that human dental pulp stem cells have good osteogenic differentiation potential and are potential seed cells in bone tissue engineering,and the effect of recombinant human growth hormone on the proliferative osteogenic differentiation of human dental pulp stem cells is still unclear. OBJECTIVE:To explore the effect of recombinant human growth hormone on the proliferation and osteogenic differentiation of human dental pulp stem cells. METHODS:Human dental pulp stem cells were isolated and cultured by tissue block culture method.After screening according to the drug concentration gradient,recombinant human growth hormone containing 10,100,250,500,1 000 μg/L was selected as the experimental group,and 0 μg/L without recombinant human growth hormone was selected as the control group.CCK-8 detection reagents were used on days 1,3,5,and 7 after the drug intervention to detect the proliferation of human dental pulp stem cells.Different concentrations(10,100,250,500,and 1 000 μg/L)of recombinant human growth hormone were added to the osteogenesis induction solution to intervene in human dental pulp stem cells.Alkaline phosphatase activity was detected by alkaline phosphatase staining and semi-quantitative analysis on day 7 of mineralization induction.The mRNA expression levels of osteogenic gene type I collagen,osteocalcin and Runt-related transcription factor 2 were detected by fluorescence quantitative RT-qPCR.Alizarin red staining was performed on day 14 of mineralization induction to detect osteogenic mineralized nodules. RESULTS AND CONCLUSION:(1)CCK-8 assay results showed that from the third day of intervention,the 100,250,500,1 000 μg/L recombinant human growth hormone group could promote the proliferation of human dental pulp stem cells compared with the control group(P<0.01).(2)The alkaline phosphatase activity of human dental pulp stem cells in the 100,250,and 500 μg/L recombinant human growth hormone group was significantly increased compared with the control group(P<0.01).The number of alizarin-stained mineralized nodules in human dental pulp stem cells in the 100,250 μg/L recombinant human growth hormone group was significantly increased compared with the control group(P<0.01).Compared with the control group,the mRNA expression of type I collagen and osteocalcin increased in the 250 μg/L recombinant human growth hormone group(P<0.05,P<0.01).mRNA expression of Runt-associated transcription factor 2 increased in the 100 and 250 μg/L recombinant human growth hormone groups(P<0.01).(3)According to the above results,recombinant human growth hormone at a concentration of 250 μg/L is a more suitable concentration to promote the proliferation and osteogenic differentiation of human dental pulp stem cells.
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BACKGROUND:Collagen is the most abundant extracellular matrix component,which is closely related to the structure and function of the extracellular matrix of skeletal muscle,but the effect and mechanism of recombinant human collagen(rhC)produced by bioengineering technology on the extracellular matrix of skeletal muscle are unclear. OBJECTIVE:To investigate the effect of rhC supplementation on the remodeling of skeletal muscle extracellular matrix after eccentric exercise,thereby revealing the possible mechanism by which rhC improves the injury of skeletal muscle extracellular matrix and promote the recovery after exercise. METHODS:A total of 104 healthy male C57 mice aged 8 weeks old were randomly divided into control group(normal saline),low-dose rhC group(0.2 g/kg),medium-dose rhC group(1.0 g/kg),and high-dose rhC group(2.0 g/kg).Two mice in each group were selected after continuous 7 days of intragastric intervention,and organs were dissected for hematoxylin-eosin staining to determine inflammatory infiltrates.On the 8th day,the remaining mice were subjected to eccentric exercise.The structural changes of the skeletal muscle extracellular matrix were observed under scanning electron microscopy immediately(0),24,48,and 96 hours after eccentric exercise.Meanwhile,grip strength,creatine kinase activity,and protein levels of matrix metalloproteinases 2,9,14 and tissue inhibitor of metalloproteinase-2 in skeletal muscle were detected by western blot assay. RESULTS AND CONCLUSION:Hematoxylin-eosin staining results indicated that short-term rhC supplementation showed no significant effects on the morphology of the heart,liver,spleen and kidney.After one-time eccentric exercise,the recovery rate of grip strength in the medium-and high-dose rhC groups was significantly increased(P＜0.01).The trend of creatine kinase changes was consistent in all groups and there was no significant difference between groups.The recovery process of the extracellular matrix in the low-dose rhC group was faster than that in the control group,and the muscle tract membrane in the medium-and high-dose rhC groups was more complete.The protein level of matrix metalloproteinase 9 in the high-dose rhC group was significantly decreased(P＜0.05).The protein levels of matrix metalloproteinase 14 in the medium-and high-dose rhC groups were significantly decreased(P＜0.05).The protein levels of matrix metalloproteinase 2 in the medium-and high-dose rhC groups were significantly decreased(P＜0.05).Tissue inhibitor of metalloproteinase-2 protein levels in the medium-and high-dose rhC groups were significantly increased(P＜0.05).The ratio of matrix metalloproteinase 2 to tissue inhibitor of metalloproteinase-2 in each rhC group was significantly decreased(P＜0.05).To conclude,pre-supplementation of 1.0 and 2.0 g/kg rhC for 7 days can inhibit extracellular matrix degradation in skeletal muscle after exercise by modulating matrix metalloproteinases and matrix metalloproteinase inhibitors,thereby promoting recovery of skeletal muscle strength in mice.
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Objective:To investigate the effect of different administration methods of recombinant human prourokinase (rhPro-UK) during emergency percutaneous coronary intervention (PCI) on myocardial perfusion and prognosis of patients with acute ST segment elevation myocardial infarction (STEMI).Methods:The clinical data of 132 patients with STEMI who underwent emergency PCI in the Military Hospital of the 71st Army Group of the Chinese People′s Liberation Army from August 2017 to August 2022 were analyzed retrospectively. Among them, 66 patients treated with rhPro-UK injection after the guide wire passed through the coronary artery lesion, balloon dilation and stent placement were included in group A. The other 66 patients treated with rhPro-UK injection once after the guide wire passed through the coronary artery lesion were included in group B. The two groups were compared in terms of PCI conditions, target vessel perfusion status [corrected TIMI frame count (CTFC) and blood flow (thrombolysis in myocardial infarction, TIMI) grade], myocardial perfusion status [TIMI myocardial perfusion grade (TMPG), ST segment regression rate (STR) at 90 min after operation and the incidence of no reflow/slow flow (NR/SF)], cardiac function indicators [left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV) and left ventricular ejection fraction (LVEF)], the incidence of major adverse cardiovascular events (MACE), and the incidence of bleeding events.Results:There were no statistically significant differences between the two groups in terms of the distribution of culprit blood vessels, intubation methods, and number of stents implanted ( P>0.05). After treatment, the proportion of TIMI blood flow grade 3 in the group A was higher than that in the group B:99.97% (64/66) vs. 87.88% (58/66). CTFC of the two tgroups decreased, and CTFC of group A was lower than that of group B: (23.49 ± 4.27) frames vs. (27.14 ± 4.83) frames ( P<0.05). The proportion of TMPG grade 3 in group A was significantly higher than that in group B: 95.45% (63/66) vs. 83.33% (55/66)( P<0.05). STR in group A was significantly higher than that in group B: 95.45% (63/66) vs. 83.33% (55/66)( P<0.05). The incidence of NR/SF in group A was lower than that in group B: 3.03% (2/66) vs. 14.29% (10/66)( P<0.05). There were no statistically significant differences in LVEDV or LVESV between the two groups before and after treatment ( P>0.05). After 1 month of treatment, LVEF of the two groups increased, and LVEF of group A was higher than that of group B: (71.08 ± 6.38) % vs. (66.24 ± 6.49) % ( P<0.05). After treatment, the incidence of MACE in group A was lower than that in group B: 6.06%(4/66) vs. 13.64%(9/66) ( χ2 = 2.13, P = 0.144). There was no statistically significant difference in the incidence of bleeding events between the two groups ( P>0.05). Conclusions:For patients with STEMI undergoing PCI, fractional injection of rhPro-UK can better improve myocardial perfusion, reduce the incidence of MACE, and effectively improve the prognosis, compared with one-time injection of rhPro-UK.
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To characterize human antibodies against low-calcium response V(LcrV)antigen of Yersinia pestis,the mono-clonal antibodies were screened and assayed.Antibody gene was derived from peripheral blood mononuclear cells of the vaccin-ees immunized by plague subunit vaccine in phase Ⅱb clinical trial.Human ScFv antibody library was constructed by phage dis-play.After panning library by using recombinant LcrV antigen,antibody variable genes were sequenced and converted into IgG1 format to evaluate its binding specificity and relevant parameters.An anti-plague human ScFv antibody library was estab-lished contained 7.54× 108 independent clones.After panning by LcrV antigen,3 human antibodies named as RV-B4,RV-D1 and RV-E8,respectively,were identified.Using indirect enzyme-linked immunosorbent assay(ELISA)and Western blot(WB),the specific bindings of the mAbs to LcrV antigen were confirmed.The dissociation constant(KD)of them to LcrV is 2.1 nmol/L,1.24 nmol/L and 42 nmol/L,respectively.Minor protective efficacy was found among 3 human antibodies in Y.pestis 141-infected mice.Three anti-LcrV monoclonal antibodies generated from immunized vaccinees were binding specific antibod-ies and could not block plague infection in mice.These antibodies are the potential candidate reagents for basic research of plague immunity and the application of plague diagnosis.
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@#Objective To explore the application effect of recombinant human granulocyte colony stimulating factor(rhG-CSF)in the treatment of chemotherapy-induced oral mucositis in children.Methods Totally 60 children with chemotherapy-induced oral mucositis who underwent chemotherapy in the tumor surgery of our hospital from January 2020 to June 2022 were randomly divided into two groups,30 cases each.The control group was given physiological saline oral care,and the experimental group was given rhG-CSF oral care.Compare the intervention effect,oral mucositis grading,quality of life[short form of health survey(SF-36)]and nursing satisfaction of parents of the two groups.Results The total effective rate of the experimental group was higher than that of the control group(P<0.05).After 5 days of intervention,the score of oral mucositis in the experimental group was better than that in the control group(P<0.05).After 5 days of intervention,the SF-36 score of children in the two groups was higher than that before intervention,and that in the experimental group was higher than that in the control group(P<0.05).The parents'satisfaction in the experimental group was higher than that in the control group(P<0.05).Conclusion rhG-CSF oral care can effectively improve the intervention effect of chemotherapy-induced oral mucositis in children,reduce the grade of oral mucositis,improve the quality of life of children,and improve the nursing satisfaction of parents of children.
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@#Objective To investigate the feasibility of using quantitative PCR(qPCR)technology to detect large fragments of host cell residual DNA(HCD)in recombinant adeno-associated virus(rAAV)gene therapy products.Methods Four different serotypes of rAAV were extracted for the nucleic acids,two fragment sequences of 244 bp and 562 bp within the long terminal repeat sequence(LTR)in the genome of host cells HEK293 were specifically quantified by qPCR,and the proportion of HCD in the total nucleic acids was calculated.Results Large fragments of HCD in qPCR quantifiable range were detected in four different serotype rAAV products,with the abundance ranging from 0. 3% to 5. 4%. As the length of the detected fragment increased,the abundance of HCD fragments showed a decreasing trend.Conclusion qPCR technology can be used to determine the presence of large fragments of HCD in rAAV products.
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Objective@#To explore the effect of adenoassociated virus sense transfection up-regulating the expressionlevel of the growth and differential factor 11 ( GDF11) in vivo on aortic injury in type 2 diabetic mellitus rats(T2DM).@*Methods@#Nine-week-old male SD rats were randomly selected to establish a T2DM model by usinghigh -sugar and high-fat chow plus small-dose streptozotocin (STZ) combined induction. Both normal rats and diabetic model rats were randomly divided into five groups: blank control group ( Control group) , negative virus control group ( NC group), GDF11 adeno-associated virus group ( GDFl1 group), diabetic group ( DM group), anddiabetic + GDF11 adeno-associated virus group ( DM + GDFl1 group). After 8 weeks of feeding, the serum con-centrations of insulin ( INS ), advanced glyeosylation end products ( AGES), recombinant growth transforming fac.tor 11 ( GDF11 ), total cholesterol (T-CH0 ), triglycerides (TG), low-density lipoproteins (LDL-C), high-density lipoproteins ( HDL-C), asymmetric dimethylarginine ( ADMA), and malondialdehyde ( MDA) were assayed inthe rats ; periodic acid-schiff stain( PAS stain) was used to observe the sites of glycogen deposition, and hematoxylin-eosin staining ( HE) was used to observe vascular damage. Scanning electron microscopy was used to observethe damage of vascular endothelial cells and vascular elastic fibers, and protein blotting and immunohistochemistrwere used to detect the expression levels of vascular injury-related proteins. Protein blotting and immunohistochemistry were used to detect the expression levels of vascular injury-related proteins. @*Results@#The biochemical indexes showed that the serum concentrations of AGES, T-CHO, TG, LDL-C and MDA were higher in the DM groupthan those in the Control group (P <0. 05), the concentrations of INS, GDF11, HDL-C and ADMA were signifi.cantly lower than those in the Control group (P <0. 05 ), and the concentrations of AGE'S and HDL-C were not sig.nificantly lower in the DM + GDF1l group compared with the DM group ( P <0. 05). HDL-C was not significantlydilerent from the DM group, and several other data were improved ( P<0. 05 ). Pathological staining suggestedthat PAS staining in the DM group suggested that glycogen particles deposited in the endothelium and subendotheli.um of the aorta, HE staining observed thickening of the aortic mesentery, endothelial cells and elastic fibers brokeolf in an irregular alignment , and electron microscopy observed endothelial damage in the vasculature and elastic fibers broke off in the DM group, and these changes attenuated in the DM + GDFl1 group. Protein blotting and im.munohistochemistry indicated that the expression of endothelial cell-associated proteins decreased in the DM groug( P <0. 05), and mesenchymal markers elevated in the DM group ( P <0. 05 ), these proteins were regressed inthe DM + GDFl1 group, and the difference was statistically significant (P<0.05).@*Conclusion@#Inereasing theexpression level of GDFl1 in vivo can improve aortic vascular injury caused by diabetes mellitus, which is inferredthat it may be related to the inhibition of endothelial mesenchymal transition to protect the function of vascular endo.thelial cells and thus improve vascular injury.
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Objective@#To evaluate the effectiveness of recombinant Mycobacterium tuberculosis fusion protein skin test (EC-ST) in screening for latent tuberculosis infection (LTBI) among HIV/AIDS patients, so as to provide insights into the applicability of EC-ST in LTBI screening among HIV/AIDS patients.@*Methods@#From April to June 2023, HIV/AIDS patients under management and treatment in Yangzhou City, Jiangsu Province, were selected as study subjects. Basic information was collected through questionnaire surveys. LTBI was screened by EC-ST and interferon-gamma release assay (IGRA). Taking IGRA results as the diagnostic standard, the positive rate, sensitivity, specificity and consistency rate of EC-ST, and the impact of CD4+T lymphocyte (CD4) counts on the screening effect of EC-ST were analyzed.@*Results@#A total of 523 HIV/AIDS patients were screened, including 458 males (87.57%) and 65 females (12.43%). The median age was 48.00 (interquartile range, 21.00) years. The positive rate of EC-ST was 7.27% and the positive rate of IGRA was 7.46%, with no statistically significant difference (P>0.05). The consistency rate of the two methods was 94.84%, and the Kappa value of 0.621 (95%CI: 0.489-0.752, P<0.05). The sensitivity of EC-ST was 64.10% and the specificity was 97.31%. Comparing the groups with CD4 counts <500 and ≥500 cells/μL, the consistency rates of the two methods were 95.32% and 94.44%, and the Kappa values were 0.568 and 0.650, respectively (both P<0.05). There were no statistically significant differences in the positive rates, sensitivity, and specificity of EC-ST (all P>0.05). Comparing the groups with CD4 counts <200 and ≥200 cells/μL, the consistency rates of the two methods were 96.55% and 94.62%, and the Kappa values were 0.648 and 0.619, respectively (both P<0.05). There were no statistically significant differences in the positive rates, sensitivity, and specificity of EC-ST (all P>0.05).@*Conclusion@#The effectiveness of EC-ST in screening for LTBI among HIV/AIDS patients is consistent with that of IGRA and is not affected by CD4 counts.
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【Objective】 To investigate the binding and carrying effects of human serum albumin (HSA) from various sources on sphingosine-1-phosphate (S1P). 【Methods】 Utilizing human plasma-derived HSA (pHSA) and recombinant HSA (rHSA) samples as the focal points of our investigation, LC-MS/MS technology was employed to meticulously compare and analyze the disparities in S1P content among the aforementioned samples. Subsequently, under physiological concentration conditions, S1P was directly introduced to HSA samples for loading processing, facilitating a comprehensive comparison of the binding efficacy of HSA from different sources to S1P. Within a serum-free culture setting, HSA samples from various sources were co-cultured with HUVEC cells. The alterations in S1P content within the cell culture supernatant across different treatment groups were meticulously analyzed, allowing for a nuanced comparison of the S1P carry effects exerted by HSA from different sources on cells.The interaction between HSA and S1P molecules from different sources was analyzed and their affinity was calculated using surface plasmon resonance (SPR) technology. Furthermore, leveraging AutoDock Vina software and the Molprophet platform, the molecular docking analysis of HSA and S1P was conducted, aiming to predict the key binding pocket domain of S1P within HSA. 【Results】 All pHSA samples exhibited detectable levels of S1P (ranging from 3.31±0.03 to 30.35±0.07 μg/L), with significant variations observed among pHSA samples from different manufacturers (P<0.001). Conversely, S1P was undetectable in all rHSA samples. Upon load treatment, the binding affinity of HSA from diverse sources to S1P demonstrated significant discrepancies (P<0.001), with rHSA exhibiting approximately double the average S1P loading compared to pHSA (ΔCrHSA=801.75±142.45 μg/L vs ΔCpHSA=461.94±85.73 μg/L; P<0.001, t=5.006). Co-culture treatment outcomes revealed a significant elevation in S1P concentration within the supernatant after 6 hours of co-culture across all HSA sample processing groups with HUVEC cells, while no changes were observed in the supernatant of the blank control group. Notably, significant differences in supernatant S1P concentration were observed among treatment groups at 6 h, 12 h, and 24 h (P<0.001). SPR analysis unveiled a stronger affinity of pHSA for S1P compared to rHSA (KDpHSA-S1P: 2.38E-06, KDrHSA-S1P: 3.72E-06). Molecular docking analysis and binding pocket prediction suggested that the key binding pocket of HSA and S1P may reside in the IB subdomain of the HSA molecule. 【Conclusion】 HSA from various sources exhibits distinct binding and carrying effects on S1P, which appear to be closely associated with the IB subdomain of the HSA molecule.
ABSTRACT
Objective:To compare the immunogenicity of the prefusion (PreF) and postfusion (PostF) conformations of the respiratory syncytial virus (RSV) F protein.Methods:The expression of PreF and PostF recombinant proteins was analyzed by SDS-PAGE and Western blot. The binding affinity between F protein and its specific antibodies was detected by Octet. The binding antibodies and neutralizing antibodies in immune serum were detected after immunizing mice with PreF or PostF recombinant protein.Results:PreF protein was stable in the form of a trimer after modification with higher binding affinity with monoclonal antibodies such as D25, 8897, AM14, Palivizumab and Motavizumab. PostF protein lacked the antigenic site ? and showed a monomer conformation. Besides, it was unable to bind to D25, 8897 and AM14 antibodies. Animal experiments showed that AS01 adjuvant was better than aluminum adjuvant in inducing binding antibodies and neutralizing antibodies against RSV Long strains. The binding antibodies induced by PreF and PostF recombinant proteins had similar binding ability to PreF protein, while the binding antibodies induced by PostF recombinant protein showed stronger binding ability to PostF than to PreF.Conclusions:PreF has more epitopes and the trimer form of PreF recombinant protein after modification is more stable and can induce stronger neutralizing antibodies. Moreover, the immunopotentiating effect of AS01 adjuvant is better than that of aluminum adjuvant. Therefore, stabilization-based trimer structure modification of PreF and the development of adjuvants are crucial for the development of RSV vaccines.
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@#Objective To develop and verify an anion-exchange high-performance liquid chromatography(AEX-HPLC)method for the determination of empty capsid ratio of recombinant adeno-associated virus type 9(rAAV9).Methods AEXHPLC based on the differences in surface charge was used to establish a method for detecting the ratio of empty and full capsid rAAV9 by optimizing the elution gradient of mobile phase,pH,column temperature,flow rate,sample concentration,injection volume and detection wavelength of fluorescence detector. The specificity,linearity,limit of detection(LOD),limit of quantitation(LOQ),precision and accuracy of the method were verified to confirm the feasibility.Results Using a CIMac AAV full/empty-0. 1 mL column,20 mmol/L BIS-Tris propane(BTP)as mobile phase A and 20 mmol/L BTP+1 mol/L NaCl as mobile phase B,gradient elution was performed with pH of 9.0,column temperature of 20 ℃,flow rate of1 mL/min,sample concentration of 4×10~(12)vg/mL,injection volume of 10 μL,excitation wavelength of 280 nm and emission wavelength of 330 nm,which realised the baseline isolation and quantitative detection of empty and full capsid rAAV9. The verification results of the method showed that the preparation buffer had no interference with good specificity;rAAV9 showed a good linear relationship in the range of(1.6-8)×10~(12)vg/mL,r = 0. 993;the LOD was 5×10~(10)vg/mL,and the LOQ was 1×10~(11)vg/mL;the RSD of repeatability and intermediate precision were 2. 95% and 2. 10%,respectively;the accuracy rates were not less than 80%.Conclusion A highly sensitive and rapid AEX-HPLC method for determination of the ratio of empty capsid to full capsid rAAV9 was developed,which could be used for the analysis of empty capsid rate and quality control in gene therapy products.
ABSTRACT
@#Objective To explore a method for detecting the integrity of target nucleic acid in recombinant adeno-associated virus(rAAV),and establish a preliminary analysis algorithm.Methods The free DNA fragment of rAAV was digested,and the virus genome was extracted.Five pairs of overlapping primers were designed,using the orthogonal array method,which were detected by digital PCR respectively,with conventional conditions:20 μL reaction system with EveGreen and4 μL template,and digital PCR conditions:95 ℃ 5 min;95 ℃ 15 s,55 ℃ 30 s,72 ℃ 90 s for 45 cycles.The enhancement condition of ultra-long nucleic acid fragment was 25 μL reaction system with Mix B-2 000 bp and 2 μL template,and the quantitative analysis was performed by using the software attached to the instrument.Using the least square method,the number of full-length fragments was fitted and analyzed,and then the integrity distribution of target nucleic acid was interpreted.Results The fragments with the distance between primers of no more than 1 200 nt were amplified effectively,and a series of effective copies of fragments with a length of about 1 000 nt were obtained by systematic analysis.The copy number of common fragments was fitted and analyzed by the least square method.It was estimated that the full-length fragments in the sample were no more than 1 234 copies/μL,and there was a signficant difference(P < 0.05)between this value and the maximum measured value of 1 443 copies/μL,with the difference of approximately 16.9%.Conclusion A preliminary detection method for the integrity of target nucleic acid in rAAV has been developed,and a certain amount of incomplete target nucleic acids were analyzed in the test sample,laying a foundation for further in-depth research.