Efficacy of recombinant human epidermal growth factor plus sodium hyaluronate eye drops in diabetic dry eye post-cataract surgery

BACKGROUND Dry eye syndrome(DES)after diabetic cataract surgery can seriously affect the patient’s quality of life.Therefore,effective alleviation of symptoms in patients with this disease has important clinical significance.AIM To explore the clinical effect of recombinant human epidermal growth factor(rhEGF)plus sodium hyaluronate(SH)eye drops on DES after cataract surgery in patients with diabetes.METHODS We retrospectively evaluated 82 patients with diabetes who experienced DES after cataract surgery at Tianjin Beichen Hospital,Affiliated Hospital of Nankai University between April 2021 and April 2023.They were classified into an observation group(42 cases,rhEGF+SH eye drops)and a control group(40 cases,SH eye drops alone),depending on the different treatment schemes.The therapeutic efficacy,dry eye symptom score,tear film breakup time(TFBUT),basic tear secretion score[assessed using Schirmer I test(SIt)],corneal fluorescein staining(FL)score,tear inflammatory markers,adverse reactions during treat-ment,and treatment satisfaction were compared between the two groups.RESULTS Therapeutic efficacy was higher in the observation group compared with the control group.Both groups showed improved TFBUT and dry eye,as well as improved SIt and FL scores after treatment,with a more pronounced improvement in the observation group.Although no marked differences in adverse reactions were observed between the two groups,treatment satisfaction was higher in the observation group.CONCLUSION rhEGF+SH eye drops rendered clinical benefits to patients by effectively ameliorating dry eye and visual impairment with favorable efficacy,fewer adverse reactions,and high safety levels.Thus,this treatment should be promoted in clinical practice.

Clinical Observation of Recombinant Bovine Basic Fibroblast Growth Factor Eye Gel Combined with Tobramycin Dexamethasone Eye Drops in the Treatment of Dry Eye Syndrome in Patients after Cataract Surgery

Objective:To evaluate the therapeutic effect of recombinant bovine basic fibroblast growth factor(rbFGF)eye gel combined with tobramycin-dexamethasone(TOB-Dex)eye drops on dry eye syndrome(DES)after cataract surgery.Methods:86 patients with DES after cataract surgery,admitted from November 2021 to November 2023,were randomly divided into groups.The observation group included 43 patients treated with rbFGF eye gel combined with TOB-Dex eye drops.The reference group included 43 patients treated with TOB-Dex eye drops alone.Multiple indicators,including total effective rate and clinical symptom scores,were compared between the two groups.Results:The total effective rate in the observation group was higher than in the reference group(P<0.05).Before treatment,there were no differences in clinical symptom scores,serum factors,or disease severity scores between the two groups(P>0.05).Three weeks after treatment,the observation group had lower clinical symptom scores,serum factors,and disease severity scores compared to the reference group(P<0.05).The adverse reaction rate in the observation group was lower than in the reference group(P<0.05).Conclusion:rbFGF eye gel combined with TOB-Dex eye drops can improve the clinical efficacy for patients with DES after cataract surgery,alleviate disease symptoms,reduce inflammatory responses,and have fewer adverse reactions.

Pregnant Women with Severe Factor VII Deficiency Undergoing Cesarean Section Managed with a Short-Term Regimen of Recombinant Factor VIIa

To editor:Factor VII(FVII)is a determining factor in activating the exogenous coagulation pathway;F7 gene mutation decreases the FVII number or function.Factor VII deficiency(FVIID)is characterized by an isolated prolongation of the prothrombin time(PT)with a normal activated partial thromboplastin time(aPTT).Factor assay and genotyping for FVII can be done to confirm the diagnosis.1,2 Patients may present with menorrhagia,and bleeding tendencies may vary from slight gingival bleeding,epistaxis,and ecchymosis,to severe bleeding,such as gastrointestinal and intracranial bleeding.

Chronic Toxicity of a Novel Recombinant Human Granulocyte Colony-stimulating Factor in Rats

Objective To assess the severity and reversibility of the chronic toxicity of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and the dose-effect relationship.Methods A total of 100 Sprague-Dawley rats (equal numbers of male and female) were randomly divided into five groups (20 rats in each group):four groups were treated with rhG-CSFa at 500,100,10,1 μg/kg,respectively,and one group was treated with vehicle only to serve as the control.The rats were received subcutaneous injections of rhG-CSFa or vehicle daily for 13 weeks.During the course of the chronic toxicity study,the physical status,body weight,and food consumption were monitored.Half of the rats in each group (n=10) were sacrificed after the last rhG-CSFa administration,and the other half were sacrificed at five weeks after the last rhG-CSFa administration.Urinalyses,blood biochemistry,hematological analysis,histopathological examination,and immunological tests were performed for each of the rats.Results The hematological analyses revealed that the mean white blood cells count,neutrophils count,and neutrophils percentage were increased in male rats at the dose of 10 μg/kg or higher,and these were related with the biological activity of rhG-CSFa.Some small abnormalities were observed in the spleen of a few rats when used highest dose (500 μg/kg,a dosage of 200 folds higher than the normal clinical dosage),but these abnormalities were recovered within 5-week recovery period.No other rhG-CSFa-related abnormalities were observed in this chronic toxicity study.Conclusion No significant toxicity and immunogenicity are observed with rhG-CSFa administration to rats in the chronic toxicity studies.

Pharmacokinetic Study of a Novel Recombinant Human Granulocyte Colony-stimulating Factor in Rats

Objective To study the pharmacokinetics of a novel recombinant human granulocyte colonystimulating factor (rhG-CSFa) in rats and to determine the proteolytic rates of rhG-CSFa in the whole blood and serum of rats in vitro. Methods The pharmacokinetics of rhG-CSFa and conventional (wild type,WT) granulocyte colonystimulating factor (G-CSF) were investigated in Sprague-Dawley rats which received either intravenous or subcutaneous injection of rhG-CSFa or WT G-CSF at three different doses (20,50,or 100 μg/kg). The blood samples of rats were collected at multiple time points (from 0.08 to 12 h) and the concentrations of rhG-CSFa and WT G-CSF in serum were determined with a sandwich enzyme-linked immunosorbent assay (ELISA). For the study of proteolytic rates in vitro,the concentrations of rhG-CSFa or WT G-CSF were determined at 3-minute intervals after addition of the respective drug to rat’s whole blood or serum. Results Pharmacokinetic analysis of serum rhG-CSFa or WT G-CSF levels indicated that,at each dose tested,for either route of drug administration,the area under concentration-time curve values and the maximum serum concentration of rhG-CSFa were higher than those of WT G-CSF,and the serum half life of rhG-CSFa was longer than that of WT G-CSF. Subsequent in vitro whole blood and serum stability study showed that the rates of drug degradation in WT G-CSF were 1.8 folds and 1.5 folds higher than those in rhG-CSFa,respectively. Conclusion rhG-CSFa has better serum and whole blood stability in vitro and higher bioavailability in vivo as compared to WT G-CSF.

Clinical observation of recombinant human nerve growth factor in the treatment of neurotrophic keratitis

AIM:To characterize changes of corneal nerve morphology and tear indices in patients with neurotrophic keratitis(NK)treated with recombinant human nerve growth factor(rhNGF).METHODS:In a prospective observational study,six patients(nine eyes)were locally treated with rhNGF.Visual acuity,corneal fluorescein staining score,the heights of the tear river,lipid layer thickness(LLT),tear ferning(TF)test,conjunctival impression cytology(CIC)examination,the densities of cornea subbasal nerve fibers were determined before and after treatment.RESULTS:Compared with baseline,there was a significant difference in corneal fluorescence staining scores(P<0.01);all patient corneal epithelial defects recovered completely within 8wk,but there was no significant improvement in the height of the tear river(P=0.202).LLT was significantly increased when compared with baseline(P=0.042);however,the function of conjunctival goblet cells and mucin content did not significantly improve using the TF test and CIC examination(P=0.557,P=0.539).After 8wk of treatment,the average corneal subbasal nerve fiber density increased significantly(P<0.01),as did the number of corneal nerve fiber branches(P=0.001).CONCLUSION:RhNGF can increase the density of corneal subbasal nerve fibers,promote the healing of persistent corneal epithelial defects and corneal ulcers in patients with NK,also improving tear function partially.

Successful Management of Wound Dehiscence after Total Knee Arthroplasty by Topically Using Recombinant Basic Fibroblast Growth Factor

Wound complications are estimated to be affected in about 9% of TKA patients, which may increase the risk of deep periprosthetic infection and results in re-operation, joint fusion, or amputation. Here we have reported a female patient who suffered wound rupture due to early post-operation mobilization and weight-bearing. The wound dehiscence was successfully managed by applying recombinant basic fibroblast growth factor-2 and anti-infective treatment without removing prosthetic joint.

Preparation and analysis of immunocompetence of recombinant fusion protein of the immunodominant region in chlamydial protease-like activity factor from Chlamydophila pneumoniae and its application in serodiagnosis

To clone the gene coding the immunodominant region in the chlamydial protease-like activity factor （CPAF） from Chlaroydophila pneumoniae, to analyze immunoeoropetenee of the expressed protein, and to evaluate its value in serodiagnosis, the CPAF immunodominant region gene was amplified, ligated into a pGEX6p-2 vector, and then the expressed recombinant protein was purified with glutathione Stransferase （GST） agarose gel FF after renaturation, then identified by SDS-PAGE and Western blot. A new indirect ELISA was developed with the purified protein as coating antigen. The immunogenicity of the recombinant protein was evaluated by immunization to New Zealand rabbits, and its immunoreactivity was analyzed by reacting with anti-C, pneumoniae antibody. 300 clinical sera samples were respectively detected by mieroimmunofluorescenee （MIF） as reference method and the indirect ELISA, and the difference between the two methods was analyzed. Cross-reactivity against Chlamydia trachomatis was investigated with the indirect ELISA to detect anti-C, trachomatis positive antisera. The results indicated that a 51.3 kDa recombinant protein was obtained. Western blot assay proved that the recombinant protein could merely specifically react with human anti- C. pneurnoniae antisera. The titers of the specific IgG antibodies in the immunized New Zealand rabbits were above 1 ： 16 000. Anti- C. pneumoniae IgG positive and negative reference sera were detected with the indirect ELISA, and the concordance rate of negative and positive results were both 100% （40/40）. The sensitivity and specificity of the indirect ELISA in comparison with MIF were 93.8% （45/48） and 100% （252/252） separately by detecting 300 clinical sera samples, and the concordance rate between the two methods was 99.0%. No cross reaction against C. trachomatis was found with the indirect ELISA to detect anti-C, trachomatis positive antisera. In conclusion, the prepared recombinant protein of the CPAF immunodominant region shows excellent immunocompetence and can be used to develop a new indirect ELISA as a method to detect anti-C, pneumoniae antibody for diagnosis of C. pneumoniae infection.

Anti-apoptotic effects of recombinant human granulocyte colony-stimulating factor in focal cerebral ischemic rats

The neuroprotective effects of granulocyte colony-stimulating factor in cerebral ischemia/reperfusion injury are currently contentious. The present study examined the effects of subcutaneous injection of recombinant human granulocyte colony-stimulating factor （50 pg/kg） over 5 days in a model of cerebral ischemia/reperfusion with intraluminal filament occlusion in rats. The results indicated that recombinant human granulocyte colony-stimulating factor reduced brain infarct volume following cerebral ischemia/reperfusion injury in rats, down-regulated the expression of caspase-3 mRNA （a key protease for apoptosis in the cerebral ischemia zone）, lowered the rate of neuronal apoptosis in the cerebral ischemia zone, and notably ameliorated neurological function. These results indicate that recombinant human granulocyte colony-stimulating factor has anti-apoptotic effects on neurons following focal cerebral ischemia/reperfusion injury, and exerts neuroprotective effects.

Refolding with Simultaneous Purification of Recombinant Human Granulocyte Colony-stimulating Factor from Escherichia coli Using Strong Anion Exchange Chromatography

The urea denatured recombinant human granulocyte colony-stimulating factor (rhG- CSF) which was expressed in Escheriachia coli (E. coli) was refolded with simultaneous purification by strong anion exchange chromatography (SAX) in the presence of low concentration- of urea. The effect of urea concentration on this refolding process was investigated. The obtained refolded rhG-CSF has a high specific activity of 2.3×108 U/mg, demonstrating that the proteins were completely refolded during the chromatographic process. With only one step by SAX in 40 min, purity and mass recovery of the refolded and purified rhG-CSF were 97% and 43%, respectively.

A New Protocol for Solubilization, Refolding and Purification of Recombinant Human Granulocyte Colony-stimulating Factor in Inclusion Bodies

Recombinant human granulocyte colony-stimulating factor （rhG-CSF） in inclusion bodies was solubilized by 8 mol/L urea solution and subsequently precipitated by acetone to improve its purity. After that, the precipitates were solubilized by sodium hydroxide solution containing 2 mol/L urea. Then the solubilized rhG-CSF was passed through a size exclusion chromatography for refolding and extensive purification, and further purified by a weak anion exchange chromatography. The purity and mass recovery of refolded rhG-CSF were 96.5% and 75.6%, respectively. The bioactivity was 8.4x10^7 IU/mg.

Controlling malignant pericardial effusion by intrapericardial administration of recombinant mutant human tumor necrosis factor in patients with carcinoma

Objective: To evaluate the therapeutic efficacy of injecting recombinant mutant human tumor necrosis factor (rmhTNF) into pericardial cavity of carcinoma patients with malignant pericardial effusion. Methods: In 20 cases of malignant pericardial effusion, the intrapericardial catheter was inserted into pericardial cavity, and then rmhTNF of 1.5 × 107 U was infused. The infusion was repeated every 5-7 days with the total 4-6 times. If the effusion disappeared, rmhTNF was then used 2 more times and then the intrapericardial catheter was pulled out. Results: Of 20 patients, 14 were complete response (CR), 4 were partial response (PR) and 2 no change (NC). The disappearance of effusion in 6 cases lasted for more than 6 months. Conclusion: Injecting rmhTNF into pericardial cavity may be a better way to control malignant pericardial effusion and has mild side effects.

Effect of tumor necrosis factor-α on ventricular arrhythmias in rats with acute myocardial infarction in vivo

Acute myocardial infarction （AMI） is an acute cardiovascular emergency. This study was undertaken to assess the effect of tumor necrosis factor-a （TNF-a） on ventricular arrhythmias induced byAMI in rats in vivo. Two hundred and forty male Wistar rats were randomized into a sham- operation group, an AMI group, and a recombinant human tumor necrosis factor receptor：Fc fusion protein（rhTNFR：Fc） group. Acute anterior wall myocardial infarction was produced in the AMI group by ligating the left anterior descending coronary artery （LAD）, and there was no ligation but operation in the sham-operation group. The rhTNFR：Fc group was treated with rhTNFR：Fc（10 mg/kg）, a TNF-a antagonist, 24 hours before LAD ligation. The spontaneous and induced programmed electrical stimulation ventricular arrhythmias were recorded at baseline and 10 minutes, 20 minutes, 30 minutes, 60 minutes, 3 hours, 6 hours and 12 hours after ligation. At the same time the protein and mRNA expression levels of TNF-a among different groups were detected by histochemistry and real-time fluorescent quantitative PCR. Expression of TNF-a increased markedly from 10 minutes after infarction, peaked at 20-30 minutes, and returned to baseline gradually in the AMI group and rhTNFR：Fc group. The time- windows of spontaneous and induced ventricular arrhythmias were similar. Compared with the AMI group, the rhTNFR：Fc group showed a lesser expression of TNF-a protein and a lower incidence of ventricular arrhythmias （P〈0.05）. There was no obvious change in the sham-operation group. The expression of TNF-a induced by AMI could contribute to the onset of ventricular arrhythmias.

Expression and Purification of Human Coagulation Factor X in Mammalian CHO-DG44 Cells

[Objectives]This study was conducted to obtain a Chinese hamster ovary cell line that stably expresses recombinant human coagulation factor X(rhFX),and to induce efficient expression of the target gene with different concentrations of methotrexate(MTX).[Methods]PCR was performed to obtain the rhFX gene,and a recombinant expression plasmid pOptiVEC-rhFX was constructed and subjected to double restriction endonuclease digestion and sequencing identification.CHO-DG44(DHFR-)cells were transfected by the liposome method,and the target protein was purified by affinity chromatography and detected by SDS-PAGE electrophoresis and Western blot.A cell line with efficient and stable expression of the target gene was obtained by increasing the concentration of MTX to select positive clones.[Results]PCR yielded a 1509 bp rhFX sequence,and the results of double digestion and sequencing showed that the constructed pOptiVEC-rhFX plasmid was correct.After transfection of cells,MTX significantly increased protein expression.When MTX reached 1.0μmol/L,the expression efficiency of the target protein was(9±0.27)μg/ml.The purity of the target protein purified by affinity chromatography was 93%,which could be used for subsequent experiments.The expression efficiency of rhFX in eukaryotic mammalian cells was improved by increasing MTX concentration,and an affinity chromatography purification process for the target protein was preliminarily established.[Conclusions]The results of this study provide data support for the expression and purification of rhFX,and will lay a solid foundation for the development of drugs related to rhFX.

人表皮生长因子及其在化妆品中的应用

通过文献调研 ,综述了新型生物功能性化妆品添加剂———人表皮生长因子 (humanEpidermalGrowthFactor,简称 :hEGF)的发现史、来源、结构与生物活性 ,以及hEGF与EGFR的作用机理 ;还综述了hEGF的 3种生产方法 ,重点介绍了物美价廉的基因重组hEGF的生产和表达特点。同时 ,引入从细胞层次营养肌肤的生物美容理念 ,展望了hEGF在化妆品中的广阔应用前景和含hEGF化妆品独特的生物美肤功能。旨在进一步指导实践 ,开发系列hEGF功能性化妆品。

Recombinant activated factor Ⅶ in hemophagocytic lymphohistiocytosis with disseminated intravascular coagulation

Hemophagocytic lymphohistiocytosis （HLH） is a lifethreatening disorder due to hyperinflammation resulting in infiltration of different organs with extensive hemophagocytosis. Severe coagulopathy was one of the main reasons for death in HLH. Over secretion of plasminogen activator by activated macrophages leads to hyperfibrinolysis. We reported a 36-year-old woman who was diagnosed as HLH probably secondary to lymphoma. Massive bleeding from gut and retroperitoneal area were not able to be controlled by conventional hemostatic treatments. This patient received one dose recombinant activated factor Ⅶ （rFVlla） 3.6 mg （70 μg/kg）. Hemostatic effect was achieved in 0.5 hour and lasted 24 hours. Prothrombin time （PT） and activated partial thromboplastin time （APTT） were quickly corrected to normal ranges.Fibrinogen level elevated from 0.5 g/L before using rFVIla to 1.8 g/L 20 hours after. Although dexamethasone and etopside were administrated to treat HLH, this patient died from septic shock after persistent neutropenia. This suggests that rFVlla may be effective in the management of intractable hemorrhage in patients with HLH.

EGF-linker-PE40重组毒素的构建、表达及其意义

目的构建人表皮生长因子(EGF)-柔性连接肽(linker)-铜绿假单胞菌外毒素活性片段(PE40)重组毒素的表达载体并诱导表达,以探索其成为治疗肿瘤新型靶向药物的可能性。方法用PCR技术扩增出EGF和PE40基因,通过引物设计在两者之间加上19个中性氨基酸短肽(TS-(G4S)3-AC)的linker编码序列、克隆PCR产物,并构建pET22b(+)-EGF-linker-PE40表达质粒,将其转化E.coli BL21(DE3)后,诱导表达,SDS-PAGE及凝胶薄层扫描分析EGF-linker-PE40重组毒素的表达情况。结果 EGF-linker-PE40重组毒素在BL21(DE3)中可溶性表达,目的蛋白占细胞总蛋白的18%左右。结论应用基因工程技术,成功地构建了重组毒素EGF-linker-PE40的表达载体,并得到高效可溶性表达,为进一步研究其结构功能关系和肿瘤临床应用奠定了基础。

重组人酸性成纤维细胞生长因子在鼻腔泪囊吻合术中的应用

目的 探讨重组人酸性成纤维细胞生长因子在鼻腔泪囊吻合术中的应用。方法 对2016年3月~2021年12月46例慢性泪囊炎患者,行碘普罗胺泪囊造影,确定阻塞部位位于泪囊。选取手术治疗的慢性泪囊炎患者,随机分为两组,A组患者行经鼻内镜鼻腔泪囊吻合术联合应用重组人酸性成纤维细胞生长因子,B组患者经鼻内镜鼻腔泪囊吻合术,比较两组术后的临床疗效。结果 两组鼻腔泪囊吻合术的患者,经过术后2周、1个月、3个月、6个月对患者进行随访观察,发现A组患者在术后2周和1个月内吻合口周围痂皮较少,吻合口上皮化程度较快,在术后3个月和6个月吻合口无明显差异。结论 重组人酸性成纤维细胞生长因子在创面早期可促进肉芽组织生长,诱导毛细血管胚芽形成与再上皮化,加速鼻腔泪囊吻合口的上皮化的进程。

Four recombinant pluripotency transcriptional factors containing a protein transduction domain maintained the in vitro pluripotency of chicken embryonic stem cells

Long-term in vitro maintenance of embryonic stem cell (ESC) pluripotency enables the pluripotency and differentiation of ESCs in animals to be investigated. The ability to successfully maintain and differentiate chicken embryonic stem cells (cESCs) would provide a useful tool for avian biology research and would be a resource directly applicable to agricultural production. In this study, endogenous chicken pluripotency transcription factors, POUV, Sox-2, Nanog and Lin28 were cloned and expressed as recombinant proteins containing a nine consecutive arginine protein transduction domain (PTD). cESCs were cultured with these recombinant proteins to maintain cESC pluripotency in vitro. Cultured cESCs exhibited typical characteristics of pluripotency, even after six generations of rapid doubling, including positive staining for stage-specific embryonic antigen I, and strong staining for alkaline phosphatase. Expression levels of the pluripotency markers, POUV, Nanog, C-Myc, Sox-2 and Lin28 were the same as in uncultured stage X blastoderm cells, and most significantly, the formation of embryoid bodies (EBs) by 6th generation cESCs confirmed the ability of these cultured cESCs to differentiate into cells of all three embryonic germ layers. Thus, transcription factors could be translocated through the cell membrane into the intracellular space of cESCs by using a PTD of nine consecutive arginines and the pluripotency of cESCs could be maintained in vitro for at least six generations.

Effect of recombinant human basic fibroblast growth factor on angiogenesis during mandible fracture healing in rabbits

Objective: To investigate the effect of recombinant human basic fibroblast growth factor ( rhbFGF) on angiogenesis during mandible fracture healing in rabbit.Methods: Fifty adult white rabbits were used for animal model and randomly divided into a control group (25 rabbits) and an experimental group (25 rabbits). The membranous complex of rhbFGF and bovine type I collagen was prepared and implanted into the rabbit mandible fracture site under periosteum. The animals were sacrificed on 7, 14, 28, 56 and 84 days respectively after operation and the whole mandibles were harvested. The expression of factor Vm related antigen (F8-RA) in callus was examined with immimohistochemical staining.Results: The amounts of microvascular formation in calluses in the rhbFGF-treating group on days 7, 14, 28 and 56 were more than those of the control group (P < 0.01).Conclusions: The results indicated that rhbFGF could stimulate microvascular formation during mandible fracture healing in rabbits.