Research progress of fibroblast growth factor in nervous system diseases / 浙江大学学报·医学版

Fibroblast growth factors (FGF) are a group of structurally related polypeptides which constitute an elaborate signaling system with their receptors. Evidence accumulated in the years suggests that the FGF family plays a key role in the repair of central nervous system injury. The main protective mechanisms include activating the expression of PI3K-Akt, peroxisome proliferator-activated receptor (PPARγ) and other signals; inhibiting NF-κB-mediated inflammatory response, oxidative stress and apoptosis; regulating neuronal differentiation and neuronal excitability as well as participating in protection of neurovascular units and nerve function repair. This paper comprehensively summarizes the latest research progress in FGF signaling related to diseases of the central nervous system such as cerebral infarction, cerebral hemorrhage, traumatic brain injury, Alzheimer's disease, Parkinson's disease, epilepsy and depression, aiming to provide scientific basis and reference for the development of innovative FGF drugs for the prevention and treatment of neurological diseases.

A novel mesenchymal stem cell-based regimen for acute myeloid leukemia differentiation therapy

Currently the main treatment of acute myeloid leukemia (AML) is chemotherapy combining hematopoietic stem cell transplantation. However, the unbearable side effect of chemotherapy and the high risk of life-threatening infections and disease relapse following hematopoietic stem cell transplantation restrict its application in clinical practice. Thus, there is an urgent need to develop alternative therapeutic tactics with significant efficacy and attenuated adverse effects. Here, we revealed that umbilical cord-derived mesenchymal stem cells (UC-MSC) efficiently induced AML cell differentiation by shuttling the neutrophil elastase (NE)-packaged extracellular vesicles (EVs) into AML cells. Interestingly, the generation and release of NE-packaged EVs could be dramatically increased by vitamin D receptor (VDR) activation in UC-MSC. Chemical activation of VDR by using its agonist 1α,25-dihydroxyvitamin D3 efficiently enhanced the pro-differentiation capacity of UC-MSC and then alleviated malignant burden in AML mouse model. Based on these discoveries, to evade the risk of hypercalcemia, we synthetized and identified sw-22, a novel non-steroidal VDR agonist, which exerted a synergistic pro-differentiation function with UC-MSC on mitigating the progress of AML. Collectively, our findings provided a non-gene editing MSC-based therapeutic regimen to overcome the differentiation blockade in AML.

Progress of cellular growth factors in neurorehabilitation and neuroplasticity / 中国康复理论与实践

@#With potent biological activities, cellular growth factors are polypeptide factors that primarily stimulate cell growth and proliferation. They participate in the regulation of not only normal physiological functions such as human embryonic development and cell growth, but also neurorehabilitation and neuroplasticity in pathological processes such as nerve injury and recovery. Specifically, cellular growth factors have been shown to promote neuron survival, facilitate nerve regeneration and regulate synaptic plasticity, promote cell differentiation/vascular regeneration and modulate the microenvironment, promote nerve fiber myelination and improve nerve conduction. This review summarized current knowledge on the roles and various growth factors in neurorehabilitation and neuroplasticity, providing an update on potential clinical application of cellular growth factors in the field of neural rehabilitation.

Evidence for lung repair and regeneration in humans: key stem cells and therapeutic functions of fibroblast growth factors / 医学前沿

Regeneration carries the idea of regrowing partially or completely a missing organ. Repair, on the other hand, allows restoring the function of an existing but failing organ. The recognition that human lungs can both repair and regenerate is quite novel, the concept has not been widely used to treat patients. We present evidence that the human adult lung does repair and regenerate and introduce different ways to harness this power. Various types of lung stem cells are capable of proliferating and differentiating upon injury driving the repair/regeneration process. Injury models, primarily in mice, combined with lineage tracing studies, have allowed the identification of these important cells. Some of these cells, such as basal cells, broncho-alveolar stem cells, and alveolar type 2 cells, rely on fibroblast growth factor (FGF) signaling for their survival, proliferation and/or differentiation. While preclinical studies have shown the therapeutic benefits of FGFs, a recent clinical trial for acute respiratory distress syndrome (ARDS) using intravenous injection of FGF7 did not report the expected beneficial effects. We discuss the potential reasons for these negative results and propose the rationale for new approaches for future clinical trials, such as delivery of FGFs to the damaged lungs through efficient inhalation systems, which may be more promising than systemic exposure to FGFs. While this change in the administration route presents a challenge, the therapeutic promises displayed by FGFs are worth the effort.

The FGF metabolic axis / 医学前沿

Members of the fibroblast growth factor (FGF) family play pleiotropic roles in cellular and metabolic homeostasis. During evolution, the ancestor FGF expands into multiple members by acquiring divergent structural elements that enable functional divergence and specification. Heparan sulfate-binding FGFs, which play critical roles in embryonic development and adult tissue remodeling homeostasis, adapt to an autocrine/paracrine mode of action to promote cell proliferation and population growth. By contrast, FGF19, 21, and 23 coevolve through losing binding affinity for extracellular matrix heparan sulfate while acquiring affinity for transmembrane α-Klotho (KL) or β-KL as a coreceptor, thereby adapting to an endocrine mode of action to drive interorgan crosstalk that regulates a broad spectrum of metabolic homeostasis. FGF19 metabolic axis from the ileum to liver negatively controls diurnal bile acid biosynthesis. FGF21 metabolic axes play multifaceted roles in controlling the homeostasis of lipid, glucose, and energy metabolism. FGF23 axes from the bone to kidney and parathyroid regulate metabolic homeostasis of phosphate, calcium, vitamin D, and parathyroid hormone that are important for bone health and systemic mineral balance. The significant divergence in structural elements and multiple functional specifications of FGF19, 21, and 23 in cellular and organismal metabolism instead of cell proliferation and growth sufficiently necessitate a new unified and specific term for these three endocrine FGFs. Thus, the term "FGF Metabolic Axis," which distinguishes the unique pathways and functions of endocrine FGFs from other autocrine/paracrine mitogenic FGFs, is coined.

Development and psychometric test of oncology missed nursing care self-rating scale / 肿瘤研究与临床

Objective@#To develop a scientific oncology missed nursing care self-rating scale suitable for hospital culture background in China, and to test its validity and reliability.@*Methods@#The initial scale was formulated by literature review, qualitative interview and delphi expert consultation. A total of 388 clinical nurses from Shanxi Province were selected by convenience sampling. Item analysis was used to filtrate items. The validity was evaluated by using evaluation scale of construction and content validity, and the reliability was tested by using internal consistency, split-half reliability and retest reliability.@*Results@#Oncology missed nursing care self-rating scale consisted of 33 items and 4 dimensions including nursing assessment, nursing plan, basic nursing and nursing intervention. Exploratory factor analysis (EFA) revealed that 4 factors accounted for 63.664% of the accumulated variances. The scale-level content validity index (S-CVI) was 0.904. The Cronbach α coefficient was 0.948, the spilt-half reliability coefficient was 0.786, and the retest reliability coefficient was 0.833.@*Conclusion@#Oncology missed nursing care self-rating scale has good validity and reliability, which can be used as a measuring tool for oncology missed nursing care.

Effects of Acidic Fibroblast Growth Factor on Hippocampal Astrocytes Injury Induced by Gentamicin:Role of Mitogen-Activated Protein Kinase Signal Pathway / 中国康复理论与实践

Objective To explore the mechanism of the protection of acidic fibroblast growth factor (aFGF) for hippocampal astrocytes from injury induced by gentamicin. Methods Hippocampal astrocytes were isolated from newborn (24 hours) Sprague-Dawley rats, puri-fied, and identified with glial fibrillary acidic protein (GFAP) immunofluorescence. The third generations were cultured for 3 days and divid-ed into 3 groups:control group was cultured routinely, injury group was cultured with 2.0 g/L gentamicin for 24 hours, and protection group was cultured with 4.25μg/L aFGF for 24 hours and then cultured with 2.0 g/L gentamicin for 24 hours. Western blotting was adopted to de-tect the expressions of P38, extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK)1/2. Results Hippocampal as-trocytes were culturated successfully with the purity above 95%. The ERK1 increased in the injury group compared with the control group (P0.05). Conclusion The mitogen-activated protein kinase signal pathway, especially P38 and ERK1, may associate with the protection of aFGF for hippocampal astrocytes from injury induced by gentamicin.

Synergistic Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Platelet-Rich Plasma in Streptozotocin-Induced Diabetic Rats

BACKGROUND: Diabetic wounds are a major clinical challenge, because minor skin wounds can lead to chronic, unhealed ulcers and ultimately result in infection, gangrene, or even amputation. Studies on bone marrow derived mesenchymal stem cells (BMSCs) and a series of growth factors have revealed their many benefits for wound healing and regeneration. Platelet-rich plasma (PRP) may improve the environment for BMSC development and differentiation. However, whether combined use of BMSCs and PRP may be more effective for accelerating diabetic ulcer healing remains unclear. OBJECTIVE: We investigated the efficacy of BMSCs and PRP for the repair of refractory wound healing in a diabetic rat model. METHODS: Forty-eight rats with diabetes mellitus induced by streptozotocin were divided into four groups: treatment with BMSCs plus PRP, BMSCs alone, PRP alone, phosphate buffered saline. The rate of wound closure was quantified. A histopathological study was conducted regarding wound depth and the skin edge at 7, 14, and 28 days after surgery. RESULTS: Wound healing rates were significantly higher in the BMSC plus PRP group than in the other groups. The immunohistochemistry results showed that the expression of platelet/endothelial cell adhesion molecule 1, proliferating cell nuclear antigen, and transforming growth factor-beta1 increased significantly in the BMSC plus PRP group compared to the other treatment groups. On day 7, CD68 expression increased significantly in the wounds of the BMSC plus PRP group, but decreased markedly at day 14 compared to the controls. CONCLUSION: The combination of BMSCs and PRP aids diabetic wound repair and regeneration.

Construction of eukaryotic expression vectors of FGFR3 gene and their expressions in human leukemia K562 cell line / 吉林大学学报(医学版)

Objective To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3) MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN, and to detect their expressions in human chronic myeloid leukemia(CML)K562 cell line.Methods The full-length FGFR3 (fgfr3-WT)and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR). The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry. Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method, and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared with control (K562 MSCV)group,the expression level of FGFR3-WT in MSCV/puro-fgfr3-WT transfection (K562-WT)group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN transfection (K562-DN)group,but there was no expressions in control(K562 MSCV)group and K562-WT group.The flow cytometry results showed that the high expressions of FGFR3-WT were in 57.5% cells in K562-DN group and the high expressions of FGFR3-DN were in 41.5% cells in K562-DN group. Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.

Expression, purification and immunogenicity analysis of recombinant MPB64 using baculovirus expression system / 中国免疫学杂志

Objective:To express mycobacterium tuberculosis protein MPB 64 in baculovirus insect cell expression system ,and identify its immunogenicity.Methods:The target gene MPB64 was connected to pFastBac vector ,then the pFastBac-MPB64 plasmid which was harvested would transformed to DH10Bac competent,and the target gene was transposition into Bacmid by Tn7 transposase fragment,therefore Bacmid-MPB64 Shuttle vector was obtained.The shuttle vector was packaged by liposomes and transfected Sf 9 cells to harvest P1-generation virus ,then high titers of P4 generation virus was harvested by repeat transfected Sf 9 cells three times.The target protein MPB64 was purified from the supernatant by Q Sepharose FF and Ni affine chromatography ,which were used to immunize BALB/c mice.Antibody changes in serum would be detected ,and the proliferation of immunized mice spleen cells would be detected by MTT,detected the IFN-γsecretion by MPB64 stimulated spleen cells by ELISA method.Results: MPB64 successfully expressed in insect cells.The purity of target protein was over 90% and yield up to 35 mg/L after purification.Purified protein can effectively stimulate BALB/c mice to produce antibodies , increase the content of IFN-γmedium in mice spleen cells ,and significantly promoting proliferation in spleen cells between 0.2-100 μg/ml.Conclusion: MPB64 which has immunogenicity was successfully expressed in baculovirus insect cell expression system ,that open a new avenue for tuberculosis vaccine production.

Intramuscular transplantation of macroencapsulated islets for type 1 diabetes mellitus in mice / 中国组织工程研究

10.3969/j.issn.2095-4344.2013.25.011

Analysis of an acellular pigskin based nerve scaffold / 生物工程学报

A scaffold fabricated with lysine/nerve growth factor (NGF)/poly (lactic acid coglycolic acid) copolymer (PLGA) and acellular pigskin was evaluated in vitro as a potential artificial nerve scaffold. Properties of the scaffold such as microstructure, mechanical property, degradation behavior in PBS and water, Schwann cell adhesion property, and release of NGF were investigated. Results showed PLGA had permeated into the porous structure of acellular pigskin; its breaking strength was 8.308 MPa, breaking extensibility was 38.98%, elastic modulus was 97.27 MPa. The porosities of the scaffold ranged from 68.3% to 81.2% with densities from 0.62 g/cm3 to 0.68 g/cm3. At 4 weeks of degradation in vitro, maximum mass loss ratio was 43.3%. The release of NGF could still be detected on the 30th day, and its accumulative release rate was 38%. Lysine added into the scaffold neutralized the acidoid preventing degradation of PLGA to maintain a solution pH value. Schwann cells had grown across the scaffold after co-cultivation for 15 days. These in vitro properties of the pigskin based composite might indicate its potentiality to be an artificial nerve scaffold.

Ri plasmid transformation and metabolism of ginseng products on HepG2 cells / 中国中药杂志

<p><b>OBJECTIVE</b>To study the Ri plasmid transformation and metabolism of ginseng products on HepG2 cells.</p><p><b>METHOD</b>The inhibitory effect of ginseng saponin on hepatoma HepG2 cells was studied. The hairy root-induced conditions were screened by orthogonal experimental design. The culture conditions were determined through hairy root biomass accumulation and saponin content. The effect of ginsenoside on HepG2 cells was determined by MTT assay.</p><p><b>RESULT AND CONCLUSION</b>The optimal ginseng hairy root inducing conditions: A = 0.6, infection time of 10 min, pre-incubation time for the 3 d. The best culture conditions: MS medium, pH 6.1, 24 degrees C. At those conditions the hairy root bio-accumulation and saponin content were higher. The results of ginseng saponins on the inhibitory effect of HepG2 cells showed that inhibition of ginseng saponins on HepG2 was the concentration positively related.</p>

Studies on identification and secondary metabolites of endophytic fungi strain E8 from Curcuma wenyujin / 中国中药杂志

<p><b>OBJECTIVE</b>To identify the endophyte strain E8 with high activity from Curcuma wenyujin and study its secondary metabolites.</p><p><b>METHOD</b>The strain E8 was identified by morphological observation and ITS sequence analysis. Manifold chromatographic methods were used to separate and purify the chemical constituents of fermentation broth from strain E8, and their structures were identified by physiochemical properties and spectral data.</p><p><b>RESULT</b>The strain E8 belongs to P. oxalicum. Four compounds were isolated from the fermentation broth of this strain and elucidated as chrysophanol, emodin, secalonic acid A and beta-sitosterol.</p><p><b>CONCLUSION</b>The endophyte P. oxalicum was isolated from medicinal plant Curcuma wenyujin for the first time. Four compounds were first isolated from endophytic fungus in C. wenyujin. Thus, microbial fermentation is a new access for these compounds production.</p>

Instantaneous expression aFGF-GFP fusion gene in safflower / 中国中药杂志

<p><b>OBJECTIVE</b>To study the instantaneous expression aFGF-GFP fusion gene in Carthamus tinctorius.</p><p><b>METHOD</b>Molecular biology methods were applied to construct aFGF and GFP fusion gene vector, it is transformed into C. tinctorius by Agrobacterium tumefaciens, forming the resistant callus, fluorescence microscopy was used for detection.</p><p><b>RESULT</b>aFGF gene and GFP gene were amplified by PCR reaction. It was successfully constructed plant fluorescence expression vector pCAMBIA1390: :35S: :aFGF-GFP, it was used to transform C. tinctorius, and the acquired resistance calli showed strong green fluorescence under UV light.</p><p><b>CONCLUSION</b>The expression of GFP in resistance C. tinrictorius calli is good, it is indicated that aFGF gene in plant cells has also been expressed.</p>

Properties and clinical application of collagen sponge / 中国组织工程研究

BACKGROUND: Collagen sponge is considered the most useful biomaterial owing to its excellent function and properties, easy processing, sterilization, and preservation and has been widely used in scientific research and clinical application. OBJECTIVE: To describe the properties of collagen sponge and to review the research progress of collagen sponge in clinical application in recent years. METHODS: A computer-based retrieval was performed by the first author to search for manuscripts published from January 2000 to August 2010 in Pubmeds and Elsevier databases and for manuscripts published from January 1993 to August 2010 in CNKI database using key words "collagen, collagen sponge, clinical application" in title and abstract items in English and Chinese language, respectively. RESULTS AND CONCLUSION: Collagen sponge, as a new biological material, has been widely applied in tissue engineering research in terms of hemostasis, wound healing, anti-infection, cartilage repair, and nerve repair. But at present, nearly all collagen is from animals and immunogenicity cannot be thoroughly eliminated. Researchers outside of China have synthesized recombinant human collagen using bioreactor and transgenic technology, but its efficacy and safety in clinical application needs further investigation and research.

Mechanism of inhibitory effect of P7 on 3T3 cell proliferation induced by basic fibroblast growth factor / 药学学报

To investigate the mechanism of inhibitory effect of a novel bFGF antagonist peptide isolated from the phage display random heptapeptide library on cell proliferation induced by basic fibroblast growth factor. The effect of P7 on cell morphology was observed under an inverted microscope. Flow cytometry was applied to analyze the effect of P7 on cell cycle progress of bFGF-stimulated cells. The effect of P7 on bFGF-induced activation of MEK and Erk1/2 in MAPK pathway was detected by Western blotting. The results showed that no significant cell morphology change was observed in the range of detected concentrations of P7. Cell cycle analysis showed that P7 decreased S-phase cell population and arrested cell cycle at the G0/G1 phase of bFGF-stimulated cells. The results of MAP kinase activation assay indicated that P7 decreased bFGF-induced MEK and Erk1/2 phosphorylation in a dose-dependent manner. P7 inhibited proliferation of bFGF-stimulated Balb/c 3T3 cells possibly via cell cycle arrest at the G0/G1 phase and down-regulation of signal molecular activation in MAPK pathway.

Development of genetic transformation system and transformation of active fibroblast growth factor (aFGF) in Carthamus tinctorius / 中国中药杂志

<p><b>OBJECTIVE</b>To establish genetic transformation system of active fibroblast growth factor (aFGF) in Carthamus tinctorius.</p><p><b>METHOD</b>The culture condition was optimized by orthogonal experiment design with cotyledon of C. tinctorius as the explant. The aFGF was transferred into safflower through Agrobacterium-mediated transformation and screened under different concentrations of antibiotics, and then PCR was identified.</p><p><b>RESULT</b>It confirmed the optimal differentiation medium: MS + BA 1.0 mg x L(-1) + NAA 0.2 mg x L(-1), the optimal root medium: 1/4 MS + NAA 2.0 mg x L(-1) + IAA0.5 mg x L(-1). The bacteriostatic effect of the three antibiotics showed slight difference. From them Tim was selected with the concentration of 400 mg x L(-1). It showed the bacteriostatic effect and promoted also differentiation. The selective concentration of hyg was confirmed to be 6 mg x L(-1). The eight transformed plants were identified, the positive rate was 25%.</p><p><b>CONCLUSION</b>It was determined the best hormones and the ratios for the differentiation and rooting of the safflower by organogenesis. It was identified the optimal concentration of inhibitory antibiotics and selection antibiotics. The aFGF gene was cloned in a part of plant by PCR analysis. It is shown that the aFGF gene has been integrated into safflower genome.</p>

A rudimentary study of the acid fibroblast growth factor's plant expression vector construction and transformation tobacco / 生物医学工程学杂志

Acid fibroblast growth factor (aFGF) has great potential in clinical application, but it is very expensive. In order to reduce the cost of production and to make full use of the merits integrated with plant bioreator, we have explored the aFGF in transgenic Tobacco expression. AFGF gene was inserted into plant expression vector pBI121; the acquired plants contained aFGF gene expression vector pBI121-TOAB-aF. Using Agrobacterium-mediated gene transformation of Tobacco and using transgenic Tobacco containing kanamycin and cephalosporin culture medium, we obtained kanamycin resistant transgenic Tobacco plants. PCR detection, RT-PCR detection and Western blot detection confirmed that foreign genes were successfully expressed in Tobacco. These data could serve as a theoretical foundation on which to use the plant bioreactor for production of aFGF.

Cloning of keratinocyte growth factor 2 gene (KGF2) and its transformation to Brassica napus L / 生物工程学报

Recently, more research about the plant bioreactor expressing genes encoding human proteins was reported. In the present study, the cDNA of the human gene keratinocyte growth factor 2 (KGF2) was replaced with plant preferred codons by PCR, and the modified full-length cDNA was cloned into the plant expression vector pCAMBIA-YO containing the oil-body promoter. The fusion construct pCAMBIA-YO-KGF2 was transformed into Brassica napus by Agrobacterium tumefacien-mediated cotyledon transformation method. The transgenic seedlings were identified by PCR, Southern and western blot analysis all showed that KGF2 gene was successfully expressed in in transgenic Brassica napus.