Human keratinocyte growth factor-1 (hKGF-1), a member of the fibroblast growth factor (FGF) family, plays crucial roles in organ development, cell proliferation, wound healing, and tissue repair, representing one of the most effective and specific growth factors for skin repair. However, obtaining recombinant hKGF-1 remains challenging due to its universally low expression efficiency in vitro. This study employs the Bombyx mori baculovirus expression system to establish a technological platform that utilizes the economically important insect Bombyx mori as a bioreactor for high-efficiency and low-cost expression and production of recombinant human keratinocyte growth factor 1 (hKGF-1) protein, ultimately achieving high-level expression of hKGF-1 in Bombyx mori ovary cell line (BmN). In this study, we optimized the hKGF-1 sequence based on the codon preference of baculovirus. By fusing hKGF-1 with polyhedrin (highly expressed in this system) and adding extra promoters and enhancers, we significantly improved the expreesion level of hKGF-1 in Bombyx mori cells. The results demonstrated that the aforementioned strategies significantly enhanced the expression level of hKGF-1 in Bombyx mori cells. SDS-PAGE and Western blotting results revealed that the highest hKGF-1 expression (accounting for 8.7% of total cellular protein) was achieved when the Polh promoter was combined in tandem with the P6.9 promoter and hKGF-1 was fused with a 15-residue polyhedrin fragment for co-expression. The optimal harvest time was determined to be 120 h post transfection. This study achieved the efficient expression of hKGF-1 in Bombyx mori cells, establishing an ideal technological platform for the industrial utilization of recombinant hKGF-1. The developed methodology not only provides valuable technical references for the production of other growth factors and complex proteins, but also demonstrates significant implications for employing silkworms as bioreactors for recombinant human protein expression.
Bombyx/metabolism* ; Animals ; Baculoviridae/metabolism* ; Humans ; Fibroblast Growth Factor 7/biosynthesis* ; Recombinant Proteins/genetics* ; Cell Line ; Genetic Vectors/genetics*

Bombyx mori nucleopolyhedrovirus (BmNPV) is extremely harmful to the silk industry. The polyhedrin, which encodes the polyhedrin (Polh), can be expressed at ultra-high levels and form occlusion bodies in the nucleus, embedding the progeny virus within it. However, the detailed mechanism by which polyhedrin is transported into the host cell nucleus remains unknown. Clarifying the nuclear import mechanisms of viral proteins can help us develop better prevention and treatment measures against baculoviruses. This study employed molecular cloning, co-immunoprecipitation, and immunofluorescence to analyze in detail the expression pattern of the highly expressed polyhedrin in the very late stage of the virus, and further revealed that the host protein importin α participates in the nuclear import of polyhedrin through protein interactions. This study provides a reference for further elucidating the nuclear import mechanisms of the baculovirus proteins including polyhedrin that can enter the nucleus.
Nucleopolyhedroviruses/metabolism* ; Active Transport, Cell Nucleus ; Animals ; Bombyx/virology* ; alpha Karyopherins/metabolism* ; Cell Nucleus/metabolism* ; Viral Structural Proteins/metabolism* ; Occlusion Body Matrix Proteins

Alzheimer's disease(AD)is a severe threat to human health and one of the three major causes of human death.Double-stranded RNA-dependent protein kinase(PKR)is an interferon-induced protein kinase involved in innate immunity.In the occurrence and development of AD,PKR is upregulated and continu-ously activated.On the one hand,the activation of PKR triggers an integrated stress response in brain cells.On the other hand,it indirectly upregulates the expression of 3-site amyloid precursor protein cleaving enzyme 1 and facilitates the accumulation of amyloid-β protein(Aβ),which could activate PKR activator to further activate PKR,thus forming a sustained accumulation cycle of Aβ.In addition,PKR can promote Tau phosphorylation,thereby reducing microtubule stability in nerve cells.Inflammation in brain tissue,neurotoxicity resulted from Aβaccumulation,and disruption of microtubule stability led to the progression of AD and the declines of memory and cognitive function.Therefore,PKR is a key molecule in the development and progression of AD.Effective PKR detection can aid in the diagnosis and prediction of AD progression and provide opportunities for clinical treat-ment.The inhibitors targeting PKR are expected to control the activity of PKR,thereby controlling the progression of AD.Therefore,PKR could be a target for the development of therapeutic drugs for AD.

Human epidermal growth factor (hEGF) can be applied in the treatment of surgical trauma (burns, scalds), tissue repair, skin moisturizing, beauty, skincare, etc. However, the low expression and high cost limit the application of hEGF. In order to improve the expression level of hEGF and reduce the production cost, considering the high expression of polyhedrin, this study fused a partial sequence of polyhedrin with hEGF and expressed the fused sequence by using a silkworm baculovirus expression vector system. In view of the small molecular weight of hEGF, we connected hEGF genes in series and optimized the codons to construct multiple fusion expression vectors by fusing different partial sequences of polyhedrin at the N-terminus. The results showed that through the above strategy, the protein expression level of hEGF was significantly increased. The expression vector containing three concatenated hEGF genes with optimized codons and fused with the sequence encoding 25 or 35 residues at the N-terminus of polyhedrin showed the highest expression level.
Humans ; Epidermal Growth Factor/biosynthesis* ; Genetic Vectors/genetics* ; Recombinant Fusion Proteins/biosynthesis* ; Animals ; Bombyx/metabolism* ; Occlusion Body Matrix Proteins/genetics* ; Nucleopolyhedroviruses/genetics* ; Amino Acid Sequence

OBJECTIVE:To observe clinical effect and safety of Shenfu injection combined with linezolid in the treatment of severe pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). METHODS:In retrospective study,62 MRSA severe pneumonia patients selected from ICU of our hospital during Jun. 2012-Oct. 2015 were divided into observation group (25 cases)and control group(28 cases)according to medication plan. Based on routine treatment,control group was additionally given Linezolid injection 600 mg,ivgtt,bid. Observation group was additionally given Shenfu injection intravenously with initial dose of 60 mL,and then given continuous intravenous infusion of 60 mL at a rate of 20 mL/h,q12 h,on the basis of control group. Both groups were treated for consecutive 2 weeks. Clinical efficacy,bacteriological efficacy,cardiac function indexes and serum levels of inflammatory factors before and after treatment as well as the occurrence of ADR were compared between 2 groups. RESULTS:The clinical response rate of observation group was 88.00%,which was significantly higher than 60.71% of control group,with statistical significance (P<0.05). Bacterial clearance rate of observation group was 72.00%, and that of control group was 64.28%,there was no statistical significance between 2 groups(P>0.05). Before treatment,there was no statistical significance in cardiac function indexes and serum inflammatory factor levels between 2 groups(P>0.05). After treatment,LVEF,SV,CO and CI of 2 groups were increased significantly compared to before treatment,while the levels of TNF-α,IL-6,CRP and PCT were de-creased significantly;the indexes of observation group was significantly better than those of control group,with statistical signifi-cance (P<0.05). No severe ADR was found in 2 groups. CONCLUSIONS:Shenfu injection combined with linezolid effectively improves the cardiac function of patients with MRSA severe pneumonia,enhances the anti-inflammatory effect,and have a very significant clinic effect with good safety.

Objective To detect the expression of human epithelial growth factor receptor 2 (HER-2) in advanced lung adenocarcinoma with epithelial growth factor receptor (EGFR) mutation, and to explore the potential of HER-2 as a therapeutic target for drug resistance in patients with EGFR mutations. Methods HER-2 is commonly expressed in the advanced lung adenocarcinoma with EGFR mutations, mainly in the cell membrane. Results The overexpression rate of HER-2 protein in tissues of advanced lung adenocarcinoma with EGFR mutations was 33.3%(28/84). The overexpression rate of HER-2 protein in patients>50 years of age was 40.3%(27/67), which was significantly higher than that of patients ≤50 years of age [5.9 % (1/17)], the difference was statistically significant (χ2=7.227, P=0.007). The overexpression rate of HER-2 protein in patients with high pathological differentiation [44.4 % (8/18)] was higher than that in patients with poor pathological differentiation [30.3%(20/66)], but the difference was not statistically significant (χ2=1.273, P=0.259). The overexpression of HER-2 protein in patients with EGFR 21 exon mutation [40.5 % (17/42)] was significantly higher than that of EGFR19 exon mutation [25.0%(10/40)], but the difference was not statistical significance (χ2=2.222, P=0.136). Conclusions The overexpression rate of HER-2 protein in advanced lung adenocarcinoma patients with EGFR mutation is high, which is related to the age and tumor differentiation. HER-2 is expected to be a potential therapeutic target for drug resistance patients with EGFR mutations.

ObjectiveTo study the effect of angiotensin Ⅱ type 1 receptor antagonist (ARB) losartan on reducing the incidence of stroke in patients which suffer from hypertension and atrial fibrillation(AF).MethodsProspective randomized analysis was used to divide one hundred and eighty hypertemion patients with atrial fibrillation into two groups.ARB treatment group was treated with losartan (n =90) and beta-blockers treatment group was treated with metoprolol ( n =90),all patients were treated for three years and followed up.Blood pressure,pulse pressure,incidence of stroke and myocardial infarction and mortality of cardiovascular events were evaluated.ResultsAfter antihypertensive treatment,blood pressure was reduced in two groups,the pulse pressure in losartan group was reduced obviously( all P ＜0.01 ).The incidence of stroke and myocardial infarction and mortality of cardiovascular events in losartan group were 22.2％,10.0％ and 13.3％,respectively,lower than that in metoprolol group 70.0％,40.0％ and 44.4％ ( all P ＜0.01 ).ConclusionLosartan reduced the incidence of stroke in the hypertension patients with AF.

Objective To evaluate the blond-saving effect of low central venous pressure(CVP) combined with acute hypervolemic hemedilution(AHHD)in patients undergoing hepatic lobectomy.Methods sixty ASA I orⅡpatients of both sexes aged 32-48 yr weighing 47-72 kg undergoing hepatic lobectomy for primary malignant hepatonm under epidural combined with general anesthesia were randomly divided into 3 groups(n=20 each);group I control(C);group 1I AHHD and group Ⅲ low CVP+AHHD.Group C received crystalloid and coloid in a ratio of 1.5:1 during operation.In groupⅡ4% suecinylated gelatin was infused at 50 ml·kg-1·h-1 for 30 min after tracheal intubation (AHHD);while inⅢ group low CVP was induced and maintained by epidural administration of a mixture of 1.5% lidnoaine +O.2% bupivacaine 6-8 ml combined with intravenous infmion of propofol at 6 mg·kg-1·h-1 until 10 min after hepatic lobectomy was completed.then 4% succinylated gelatin was infused at 50 ml·kg-1·h-1 for 30 min.Blood glucose,Hb,Hct, WBC count,blood coagulation (PT,AVIT,Fib),shtmic-pyruvic transaminase (GPT) and renal function (BUN,Cr) were determined before operation (baseline),immediately before skin incision,immediately before and 10 min after liver lobe was removed,at the end of operation and 7 d after operation.Urine output,intraoperative blood loss and blood transfusion and complications were recorded.Results The glood glucose concentration.WBC count and GPT levd were significantly lower;the amount of fluid infused and urinary output before hepatic lobe resection and the percentage of the patients with allogeneic blood transfusion during operation were less;Hb,Hct and the amounl of fluid infused and urinary output after hepatic lobe resection were uigher in grolp Ⅲ than in group I and ⅡⅡⅡ.There were no significant differences in blood coagulation,renal function,the total amount of fluid infused and urine output among the 3 groups.No patient developed any complication.Conclusion The low CVP hefor combined with AHHD after hepatic resection can decrease intraoperative blood loss and allogeneic blood transfusior and is safe.