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.

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.

Effect of Human Cytomegalovirus Infection on Nerve Growth Factor Expression in Human Glioma U251 Cells

Objective To explore the change of endogenic nerve growth factor （NGF） expression in human glioma cells infected with human cytomegalovirus （HCMV）. Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD 169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group （P〈0.05）. Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

Adenovirus-mediated human β-nerve growth factor gene transfer has a protective effect on cochlear spiral ganglion after blast exposure

Objective:To study whether adenovirus-mediated human β-nerve growth factor (Ad-hNGFβ) gene has any protective effect on blast hearing impairment. Methods:Deafness was induced by blast exposure (172.0 dB) in 30 healthy guinea pigs. On day 7 of blast exposure, Ad-hNGFβ was infused into the perilymphatic space of 20 animals as the study group (hNGFβ group), and artificial perilymph fluid (APF) was infused into the perilymphatic space of the other 10 animals as the control group. At weeks 1, 4 and 8 after blast exposure, the animals were sacrificed and the cochleae were removed for immunohistochemical and HE stainings. Results: Expression of Ad-hNGFβ protein was detected in each turn of the cochlea at the 1st week, with almost equal intensity in all turns. At the 4th week, the reactive intensity of the expression of Ad-hNGFβ protein decreased. At the 8th week, no expression was detectable. The results of HE staining showed that the amount of spiral ganglions in hNGFβ group was significantly greater than that of the control group at week 4 (P<0.01). Conclusion: Ad-hNGFβ can be expressed at a high level and for a relatively long period in the blast impaired cochlea, suggesting that Ad-hNGFβ has a protective effect on cochlear spiral ganglion cells after blast exposure and the efficient gene transfer into cochlea had been achieved without toxicity.

EFFECTS OF TRANSFORMING GROWTH FACTOR β AND RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN 2 ON HUMAN PERIODONTAL LIGAMENT FIBROBLASTS

Objective To evaluate the effects of transforming growth factor β(TGF-β) and recombinant human bone morphogenetic protein 2 (rhBMP2) on human periodontal ligament fibroblasts (HPDLFs). Methods HPDLFs were done primary culture to detect the distinct concentrations of TGF-P and rhBMF2 on its proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) synthesis and formation of the minerali-zed nodules, respectively. Results TGF-β (5～100ng/ml) significantly stimulated the proliferation of HPDLFs. The ALP activity of HPDLFs was evaluated evidently by 5ng/ml TGF-β. TGF-β( 0. 5 ～ 100ng/ml) had no effects on OC synthesis and formation of the mineralized nodules of HPDLFs. rhBMP2 (0. 25～2mg/ ml) had no remarkable effect on the proliferation of HPDLFs. The ALP activity, OC synthesis and forma-tion of the mineralized nodules of HPDLFs were significantly stimulated by 0. 5～ 2mg /ml rhBMP2. Conclusion The effects of TGF-β and rhBMP2 on HPDLFs are dose-dependent. TGF-P can stimulate HPDLFs to express the early marker of osteoblastic phenotype, and it lacks the ability to promote maturation of the osteogenic phenotype. rhBMP2 can not only stimulate the expression but also promote the maturation of osteoblas-tic phenotype of HPDLFs.

Hepatocyte growth factor gene transfer effects on the femoral and intramuscular nerve in a canine model of lower limb ischemia

BACKGROUND： Recent advancements in gene therapy have provided new methodology for treating ischemia in lower extremities. Gene transfer of angiogenic factors to ischemic tissues may promote local proliferation of new vessels and form collateral circulation. OBJECTIVE： To observe histopathological changes in the femoral and intramuscular nerve three months after intramuscular injection of hepatocyte growth factor （HGF） into the peripheral skeletal muscle in a canine model of lower limb ischemia. DESIGN： Randomized occlusion modelled and verification animal study. SETTING： Experimental Center, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA. MATERIALS： This study was performed at Animal Experimental Center, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from September to November 2006. A total of eight male mongrel dogs, weighing 12–15 kg and 1.5–3 years of age, were selected for this study. This experimental study was in accordance with local ethics standards. Recombinant plasmid carrying HGF （pUDKH） and occlusion model plasmid （pUDK） were provided by the Third Laboratory of Radiation Medical Institute, Academy of Military Medical Sciences of PLA. METHODS： Grouping and model establishment： under anesthesia, complete vascular occlusion models were established on the left lower extremities. The experimental dogs were randomly divided into a model group and a pUDKH treatment group, with four dogs in each group. Dogs in the pUDKH group were injected with 0.15 mg/kg pUDKH. Ten minutes later, intramuscular injections were performed at three spots into the peripheral skeletal muscle of the left hind limb, as well as lateral injections at two spots. The injection volume at each spot was 0.2 mL. Dogs in the model group were injected with pUDK, and dosage and injection method were identical to the treatment group. MAIN OUTCOME MEASURES： Histopathological changes in the femoral nerve, as well as internal and external intramuscular nerve tissues in the hind limb of dogs three months after plasmid injection under optic microscope. RESULTS： （1） Histopathological changes in the femoral nerve： tiny nerves from the femoral nerve to the intramuscular nerve exhibited marked degeneration in the model group. The degenerating features included neurites, myelin sheaths, and Schwann cell nuclei. Neuropathy in the pUDKH treatment group was not detected. （2） Histopathological changes of the intramuscular nerve： large and irregular vacuoles were present on several longitudinal sections of intramuscular nerve fibers in the model group, as well as annular-shaped blank regions on transverse sections of peripheral neurites. In the pUDKH treatment group, large, blank regions were present in several segments of partial nerve fibers of the longitudinal intramuscular nerve region, but only a few nerve fibers exhibited annular-shaped blank regions on the transverse section of peripheral neurites. CONCLUSION： Local pUDKH injection may relieve or block femoral and intramuscular nerve tissue injury in a canine mocel of lower limb ischemia.

Antitumor effect of recombinant human endostatin combined with cisplatin on rats with transplanted Lewis lung cancer

Objective: To observe the antitumor effect and mechanism of recombinant human endostatin(Endostar) injection in tumor combined with intraperitoneal injection of cisplatin on subcutaneous transplanted Lewis lung cancer in rats. Methods: A total of 30 C57 rats were selected, and the monoplast suspension of Lewis lung cancer was injected into the left axilla to prepare the subcutaneous transplanted tumor models in the axilla of right upper limb. The models were randomly divided into Groups A, B, and C. Medication was conducted when the tumor grew to 400 mm3. Group A was the control group without any interventional treatment. Group B was injected with Endostar 5 mg.kg-1.d for 10 d. Group C was given the injection of Endostar 5 mg.kg-1.d combined with intraperitoneal injection of cisplatin 5 mg.kg-1.d for 10 d. All the rats in three groups were executed the day after the 10-d medication and the tumor was taken off for measurement of volume and mass changes and calculation of antitumor rate, after which the vascular endothelial growth factor(VEGF) concentration in rats' plasma was determined by ELISA. The tumor tissues were cut for the preparation of conventional biopsies. After hematoxylin-eosin staining, the pathologic histology was examined to observe the structures of tumor tissues, VEGF score and microvessel density(MVD) in each group. Results: The volume and mass of tumor in Groups B and C were significantly lower than Group A(P < 0.05) while the tumor volume and mass in Group C were significantly lower than Group B(P < 0.05). The antitumor rate in Group C was significantly higher than Group B(P < 0.05), but the tumor VEGF score, MVD and plasma VEGF level in Group C were significantly lower than Groups A and B(P < 0.05). In Group B, the tumor VEGF score, MVD and plasma VEGF level were significantly lower than Group A(P < 0.05). The microscopic image of Group C showed that its number of active tumor cells and the blood capillary around tumor was significantly smaller than that of Groups A and B, and meanwhile atrophy and liquefactive necrosis were seen in local tumor. Conclusions: Endostar injection combined with intraperitoneal injection of cisplatin is effective in reducing tumor VEGF score and MVD of transplanted tumor tissues in rats with Lewis lung cancer to obstruct the nutrient supply of tumor cells and kill tumor cells, so that the inhibition of tumor cell proliferation and metastasis can be achieved with a remarkable effect.

Effect of recombinant human endostatin onradiotherapy for esophagus cancer

Objective:To investigate the effect of radiotherapy plus recombinant human endostatin（RHendostatin） on esophageal cancer and its mechanism.Methods:A total of SO nudemice were equally randomized into control group,radiotherapy group,and combined therapy group Ⅰ,Ⅱ,and Ⅲ after inoculating with Ecal09 cell suspension（1×107 cells/mL）.On the day of grouping,control group and radiotherapy group were injected normal saline,while radiotherapy group and 3 combined therapy groups received radiotherapy;besides,combined therapy group Ⅰ,Ⅱ,and Ⅲ was injected RH-endostatin of 2.5,5,10 mg/kg respectively.After 3-week therapy,the tumors of each group were collected and microvessel density and VEGF expression in tumors were determined.In vitro,Eca109 cells were divided into control group,radiotherapy group,and combined therapy group.Forty-eight hours after treatment cell cycle distribution and apoptosis rate were detected,and the activity of VEGF signal paths was semiquantitatively analyzed.Results:Since the 6th day of treatment,the relative tumor proliferation rate of combined therapy group Ⅱ was lower than radiotherapy group（P<0.05） and 40%since the 15 th day.Average microvessel density and EGFR expression in combined therapy group Ⅱ were lower than radiotherapy group（P<0.05）.In vitro,the cell percentage in S and G2/M phase of combined therapy group cells was lower than that in radiotherapy group cells,while the apoptosis rate and the expression of VEGF,AKT,p-AKT,ERK1/2 and p-ERKl/2 in combined group were higher than that in radiotherapy group（P<0.05）.Conclusions:RH-endostatin promotes the efficacy of radiotherapy on esophageal cancer,which may be partly realized by inhibiting the activity of VEGF related signal paths.

Fresh human amniotic membrane effectively promotes the repair of injured common peroneal nerve

Suture and autologous nerve transplantation are the primary therapeutic measures for completely severed nerves. However, imbalances in the microenvironment and adhesion of surrounding tissues can affect the quality of nerve regeneration and repair. Previous studies have shown that human amniotic membrane can promote the healing of a variety of tissues. In this study, the right common peroneal nerve underwent a 5-mm transection in rats. Epineural nerve repair was performed using 10/0 non-absorbable surgical suture. The repair site was wrapped with a two-layer amniotic membrane with α-cyanoacrylate rapid medical adhesive after suture. Hindlimb motor function was assessed using footprint analysis. Conduction velocity of the common peroneal nerve was calculated by neural electrical stimulation. The retrograde axoplasmic transport of the common peroneal nerve was observed using fast blue BB salt retrograde fluorescent staining. Hematoxylin- eosin staining was used to detect the pathological changes of the common peroneal nerve sputum. The mRNA expression of axon regeneration-related neurotrophic factors and inhibitors was measured using real-time polymerase chain reaction. The results showed that the amniotic membrane significantly improved the function of the injured nerve;the toe spread function rapidly recovered, the nerve conduction velocity was restored, and the number of fast blue BB salt particles were increased in the spinal cord. The amniotic membrane also increased the recovery rate of the tibialis anterior muscle and improved the tissue structure of the muscle. Meanwhile, mRNA expression of nerve growth factor, growth associated protein-43, collapsin response mediator protein-2, and brain-derived neurotrophic factor recovered to near-normal levels, while Lingo-1 mRNA expression decreased significantly in spinal cord tissues. mRNA expression of glial-derived neurotrophic factor did not change significantly. Changes in mRNA levels were more significant in amniotic-membrane-wrapping-treated rats compared with model and nerve sutured rats. These results demonstrate that fresh amniotic membrane wrapping can promote the functional recovery of sutured common peroneal nerve via regulation of expression levels of neurotrophic factors and inhibitors associated with axonal regeneration. The study was approved by the Committee on Animal Research and Ethics at the Affiliate Hospital of Zunyi Medical University, China (approval No. 112) on December 1, 2017.

Effects of epidermal growth factor on the growth of human gastric cancer cell and the implanted tumor of nude mice

AIM: Epidermal growth factor (EGF) plays an important role in the regulation of gastrointestinal tissue growth and development, and it can stimulate epithelial proliferation, cell differentiation and growth. It has been established that the EGF can promote gastric cytoprotection and ulcer healing. But the potential ability of EGF to regulate the gastric cancer growth is unknown. This study is to investigate the influence of EGF on human gastric cancer cell and the implanted tumor growth of nude mice. METHODS: The cell growth rates of human gastric adenocarcinoma cell lines MKN-28, MKN-45, SGC-7901 and normal human gastric epithelial cells 3T3 were assessed when incubated with recombinant human EGF (rhEGF, 0.05, 0.1, 0.5, 1.0, 10, 50, 100 mg.L(-1)) using MTT method. The cells of MKN-28, MKN-45, SGC-7901 (gastric cancer tissue 1.5mm(3)) were implanted in the BALB/cA nude mice for 10 days.The EGF was given intraperitoneally (15, 30, 60 microg.kg(-1)) for 3 weeks. The body weights of the tumor-bearing animals and their tumor mass were measured afterwards to assess the mitogenic effect of rhEGF in the nude mice. RESULTS: Within the concentration range of 0.05-100mg.L(-1), rhEGF could increase the cell growth of normal 3T3 cells (cell growth rate 100% vs 102.8%, P【0.05), but partially restrain the gastric cancer cell growth. The latter effect was related to cell differentiation. In 15-60 microg/kg rhEGF groups, the mean implanted tumor mass of MKN-28 cell were 1.75 g, 1.91 g, 2.08 g/NS group 1.97 g (P】0.05), the mean tumor mass of SGC-7901 cell were 1.53 g, 1.07 g, 1.20 g/NS group 1.07 g (P】0.05), and for MKN-45 cell, the tumor mass were respectively 1.92 g, 1.29 g, 1.77 /NS group 1.82 g (P】0.05). So rhEGF had no obvious effect on implanted MKN-28, SGC-7901 and MKN-45 tumor growth. CONCLUSION: EGF has no stimulating effect on the human gastric cancer cell growth neither in vitro nor in vivo.

Artificial nerve graft constructed by coculture of activated Schwann cells and human hair keratin for repair of peripheral nerve defects

Studies have shown that human hair keratin(HHK) has no antigenicity and excellent mechanical properties. Schwann cells, as unique glial cells in the peripheral nervous system, can be induced by interleukin-1β to secrete nerve growth factor, which promotes neural regeneration. Therefore, HHK with Schwann cells may be a more effective approach to repair nerve defects than HHK without Schwann cells. In this study, we established an artificial nerve graft by loading an HHK skeleton with activated Schwann cells. We found that the longitudinal HHK microfilament structure provided adhesion medium, space and direction for Schwann cells, and promoted Schwann cell growth and nerve fiber regeneration. In addition, interleukin-1β not only activates Schwann cells, but also strengthens their activity and increases the expression of nerve growth factors. Activated Schwann cells activate macrophages, and activated macrophages secrete interleukin-1β, which maintains the activity of Schwann cells. Thus, a beneficial cycle forms and promotes nerve repair. Furthermore, our studies have found that the newly constructed artificial nerve graft promotes the improvements in nerve conduction function and motor function in rats with sciatic nerve injury, and increases the expression of nerve injury repair factors fibroblast growth factor 2 and human transforming growth factor B receptor 2. These findings suggest that this artificial nerve graft effectively repairs peripheral nerve injury.

Chemical and enzymatic synthesis and doning of human epidermal growth factor gene

The human epidermal growth factor gene was synthesized with DNA synthesizer andby enzymes.Computer assisted to design the gene sequence and the restriction enzyme sites,andto elhninate the repeated and self-complementary sequences.So that multiple doning and gene ex-pression were available for the designed gene sequence.The whole gene was formed by annealing8 chemically synthesized oligodeoxynucleotide fragments,enzymatically filling up the 4 twenty-basespaces and ligating the nicks,and was inserted into EcoR I and HInc Ⅱ sites ofplasmid pUC12.The recombinant plasmid was transformed into E.coli JM103 and 200 whitedones were obtained by X-gal and ampicilin screening.The positive clones were isolated andcharacterized by restriction enzyme analysis and DNA sequencing.

Expression of human nerve growth factor βgene in central nervous system mediated by recombinant adeno-associated viruses type-2 vector

Background Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer’s disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor β (hNGFβ) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.Methods rAAV-2 containing hNGFβ gene was constructed. The ability of hNGFβ gene mediated by rAAV-2 vector (rAAV-2/hNGFβ) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFβ in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFβ. rAAV-2/hNGFβ and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFβ concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.Results After 48 hours, hNGFβ content in supernatant was up to (188.0±28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFβ at multiplicity of infection (MOI)1.0×106 vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFβ. Whole brain hNGFβ content in rAAV-2/hNGFβ transferred group was up to (636.2±140.6) pg/ml. hNGFβ content of BBB disruption in rAAV-2/hNGFβ infused group increased significantly compared to the control group (P<0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.Conclusion rAAV-2/hNGFβ successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.

Effects of Recombinant Human Growth Hormone on Corneal Healing, Epithelial Nerve Regeneration and Tear Inflammatory Factors in Rabbits

Objective:The goal of this study is to investigate the effects of recombinant human growth hormone(rh GH)on corneal healing,epithelial nerve regeneration and tear inflammatory factor levels in rabbits.Methods:After corneal epithelial injury models were established,fifty adult clean New Zealand white rabbits were randomly divided into two groups,normal saline was administered to the control group,while recombinant human growth hormone was administered to the observation group.The healing rate of corneal epithelial injury,the regeneration ability of corneal epithelial nerve and the level of inflammatory factors in tears were observed and compared between the two groups of rabbits before and 24,48,72 and 96 h after modeling.Results:There were significant differences in corneal epithelial healing rate,time and interaction between the two groups(P<0.05).The experimental group exhibited a superior healing rate of corneal epithelium at 24,48,72,and 96 h compared to the control group(P<0.05).There were significant differences in central cornea sensitivity between the two groups,along with variations in time and interaction(P<0.05).There was no significant difference in the central corneal sensitivity between the two groups before modeling and at 24,72 and 96 h after modeling(P>0.05),whereas the experimental group exhibited a higher central corneal sensitivity compared to the control group at 48 h after modeling(P<0.05).There were significant differences in IL-1α,TNF-α,IL-17a and IL-21 between the two groups(P<0.05).There were significant differences in IL-17a and IL-21 between the two groups(P<0.05).The experimental group exhibited a significant decrease in IL-1αlevels compared to the control group between 24 and 72 h after modeling(P<0.05),the experimental group exhibited a substantial increase in IL-17a levels compared to the control group at 72 h after modeling(P<0.05),and the level of TNF-αin the experimental group was significantly lower than that of the control group between 24 and 96 h after modeling.Conclusion:Recombinant human growth hormone aids in expediting the healing process of the healing of rabbit corneal epithelial injury,facilitating the restoration of epithelial nerve,and mitigating the inflammatory response.

Efficacy of rhNGF-loaded amniotic membrane transplantation for rabbit corneal epithelial and nerve regeneration

AIM:To evaluate the efficacy of recombinant human nerve growth factor-loaded amniotic membrane(rh NGF-AM)on corneal epithelial and nerve regeneration in rabbit model.METHODS:Freshly prepared human amniotic membrane(AM)were immersed into PBS buffer containing 100 or 500μg/mL rh NGF for 15,30,and 60 min at 4℃.The in vitro release kinetics of rh NGF was measured with ELISA.For in vivo evaluation,the AM were immersed with 500μg/mL rh NGF for 30 min.Fifty-seven rabbits were selected to establish corneal epithelial defect model.In addition to the 19 rabbits in control group,38 rabbits received AM transplantation with or without rh NGF after the removal of central epithelium.Corneal epithelial defect area,sub-epithelial nerve fiber density,corneal sensitivity,rh NGF contents in resident AM and corneas were measured after the surgery.RESULTS:rh NGF was sustained release from the AM within 14 d in vitro,with the positive correlation with initial immersion concentration.The immersion of AM in 500μg/mL rh NGF for 30 min achieved the most stable release within 14 d.After transplantation in rabbit cornea,a high concentration of rh NGF in resident rh NGF-AM and cornea was maintained within 8 d.Corneal epithelial healing,nerve fiber regeneration and the recovery of corneal sensitivity were significantly accelerated after the rh NGF-AM transplantation when compared to simple AM transplantation(all P<0.05).CONCLUSION:Simple immersion of AM achieves the sustained release of rh NGF,and promotes corneal epithelial wound healing and nerve regeneration,as well as the recovery of corneal sensitivity in rabbit.

Repair effect of diabetic ulcers with recombinant human epidermal growth factor loaded by sustained-release microspheres

In this study the w/o/w extraction–evaporation technique was adopted to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loading recombinant human epidermal growth factor (rhEGF). The micro-spheres were characterized for morphology by transmission electron microscopy (TEM) and particle size distribution. The release performances, the proliferation effects and therapeutic effects of rhEGF-loaded PLGA microspheres were all studied. The results showed that these spherical micro-spheres had a narrow size distribution and a high drug encapsulation efficiency (85.6%). RhEGF-loaded microspheres enhanced the growth rate of fibroblasts and wound healing more efficiently than pure rhEGF. The number of the proliferating cell nuclear antigen (PCNA) in the epidermis layer with the mi-crosphere treatment was significantly larger than those of the control groups. Overall locally sustained delivery of rhEGF from biodegradable PLGA microspheres may serve as a novel therapeutic strategy for diabetic ulcer repair.

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.

Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

Transplantation of human umbilical cord blood mesenchymal stem cells to treat a rat model of traumatic brain injury

In the present study, human umbilical cord blood mesenchymal stem cells were injected into a rat model of traumatic brain injury via the tail vein. Results showed that 5-bromodeoxyuridine-labeled cells aggregated around the injury site, surviving up to 4 weeks post-transplantation. In addition, transplantation-related death did not occur, and neurological functions significantly improved. Histological detection revealed attenuated pathological injury in rat brain tissues following human umbilical cord blood mesenchymal stem cell transplantation. In addition, the number of apoptotic cells decreased. Immunohistochemistry and in situ hybridization showed increased expression of brain-derived neurotrophic factor, nerve growth factor, basic fibroblast growth factor, and vascular endothelial growth factor, along with increased microvessel density in surrounding areas of brain injury. Results demonstrated migration of transplanted human umbilical cord blood mesenchymal stem cells into the lesioned boundary zone of rats, as well as increased angiogenesis and expression of related neurotrophic factors in the lesioned boundary zone.

A comparative study on the transplantation of different concentrations of human umbilical mesenchymal cells into diabetic rats

AIM: To observe the effects of intravitreal injections of different concentrations of human umbilical mesenchymal stem cells on retinopathy in rats with diabetes mellitus.METHODS: Healthy and adult male Sprague-Dawley(SD) rats were randomly assigned to a normal control group(group A), a diabetic retinopathy(DR) blank control group(group B), a high-concentration transplantation group(group C), a low-concentration transplantation group(group D) and a placebo transplantation group(group E). The expression of nerve growth factor(NGF)protein in the retinal layers was detected by immunohistochemical staining at 2, 4, 6 and 8wk.RESULTS: The expression of NGF was positive in group A and most positive in the retinal ganglion cell layer. In groups B and E, the expression of NGF was positive 2wk after transplantation and showed an increase in all layers. However, the level of expression had decreased in all layers at 4wk and was significantly reduced at 8wk. In groups C and D, the expression of NGF had increased at 2wk and continued to increase up to 8wk. The level of expression in group C was much higher than that in group D.CONCLUSION: DR can be improved by intravitreal injection of human umbilical mesenchymal stem cells.High concentrations of human umbilical mesenchymal stem cells confer a better protective effect on DR than low concentrations.