Establishment of "Chinese way" for trauma and burn management based on the engineered growth factors research and application / 中华烧伤杂志

Innovation and translation application are important topics that have been discussed repeatedly in national community of science and technology in recent years. We do a systemic review about the research and development history of growth factors, their application in trauma and burn management in China, and the conception and experience about the establishment of "Chinese way" for trauma and burn management in the process of constructing a disciplinary system for wound treatment with Chinese characteristics. It is our hope that these precious experiences will provide references and inspiration to our peers, especially the young generation in their research.

The therapeutic effect of rh-aFGF carbomer 940 gel on wound healing in type I diabetic SD rat models / 中国药理学通报

Aim To study the therapeutic effect of re-combinant human acidic fibroblast growth factor (rh-aFGF) carbomer 940 gel in the treatment of skin wound healing in type I diabetic rats. Methods Two types of skin trauma models, namely, full-thickness wound and scalded wound,were established in a model of type I diabetes mellitus using STZ-induced SD rats. The rats were divided into control group, vehicle group,90 AU rh-aFGF gel group and 270 AU rh-aFGF gel group in each skin wound models. The wound area and wound healing rate were used to evaluate the thera-peutic effect. The growth of fibroblasts, fibrocytes, collagen fibers and vessel capillaries in the wound was observed using HE staining and analysed by semi-quantitative score. Results The rh-aFGF carbomer gel significantly reduced the traumatic area as well as promoted the wound healing rate of the skin trauma model of SD rats of type I diabetes mellitus (P <0.05). HE staining showed that rh-aFGF carbomer gel significantly promoted the pathological score of fibro-blasts and collagen fibers(P<0.05). Conclusions rh-aFGF carbomer gel might play a protective role in micro-environment of wound and rh-aFGF, which could benefit for proliferation of fibroblasts and colla-gen, therefore promoting the healing process of skin wound in SD rats with type I diabetes mellitus, and it might be expected to be a new preparation for the treat-ment of chronic trauma in diabetes mellitus.

Expression of oleosin-rhFGF9 fusion protein in Carthamus tinctorius and determination of hair regeneration and wound repair potential in mice / 中国中药杂志

The expression of fibroblast growth factor 9 (FGF9) recombinant fusion protein in Carthamus tinctorius was used to identify its effect on hair regrowth and wound repair system in mice, providing a basis for C. tinctorius as a plant bioreactor, and establishing a foundation for commercial applications of FGF9 fusion protein in hair regrowth and wound repair. The identified pOTBar-oleosin-rhFGF9 plasmid was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method, and the oleosin-rhFGF9 gene was transformed into safflower leaves by A. tumefaciens mediated method. Transgenic safflower seedlings were then obtained by tissue culture. After basta screening, transgenic T₃ safflower seeds were obtained by grafting method, PCR verification and propagation. The expression of oleosin-rhFGF9 was detected by Western blot, and the content of oleosin-rhFGF9 fusion protein was 0.09% by using ELISA quantitative method. It was observed that 60 μg·L⁻¹ transgenic safflower oil had better effect on promoting NIH/3T3 cells proliferation in a certain dose-dependent manner. Sixty C57BL/6 mice were used to establish alopecia model and wound model respectively, and then were randomly divided into control group (treated with PBS or saline), negative group (treated with wild type safflower seed oil bodies, 60 g·L⁻¹), positive group (treated with FGF9, 0.054 g·L⁻¹), low dose group (treated with transgenic safflower oil bodies, 10 g·L⁻¹) and high dose group (treated with transgenic safflower oil bodies, 60 g·L⁻¹). The skin of all above-mentioned mice models were coated with soft adhesive manner every other day, 100 μL/time. After 15 days, the mice skin was cut and embedded for histological analysis. The hair regrowth experimental results showed that the hair of mice grew well, and the mice in high dose group had bushy hair, with significant effect on regeneration hair number as compared with the positive group. The healing was obvious in wound experiment, with significant healing effect in positive group, high dose group and low dose group as compared to blank control group. Furthermore, high dose group remarkably showed a better and higher healing effect than the positive group at day 5. Oleosin-rhFGF9 was successfully transformed into safflower, and T₃ transgenic safflower oil bodies expressed oleosin-rhFGF9 fusion protein were obtained, with the role of promoting hair regeneration and wound repair in mice.

Cloning and expression analysis of tocopherol cyclase gene in Carthamus tinctorius / 中国中药杂志

The tocopherol cyclase was one of the key enzymes in plant vitamin E biosynthesis pathway. According to the study of Carthamus tinctorius transcriptome data,the Tocopherol cyclase gene was obtained using RT-PCR techniques and named CtTC . Bioinformatics analysis showed theopen reading frame （ORF）of CtTC was 1 524 bp. The putative protein contained 507 amino acids with a predicted molecular mass of 62.9 kDa and theoretically isoelectric point was 5.01.Signal peptide analysis showed that it was a non secretory protein, and there was no signal peptide. The subcellular localization showed that the CtTC protein was located in the chloroplast. The expression of CtTC gene in safflower seeds at different development stages was determined by quantitative real-time PCR, it was found that the highest expression level of CtTC gene was detected in 50 DAF.Quantitative RT-PCR analysis suggested that expression of CtTC is induced and strengthened by drought stresses. This research provided a candidate gene for metabolic engineering of vitamin E and resisting stress.

Full-length cDNA cloning of flavonol synthase genes of Carthamus tinctorius and construction plant expression vector / 中国中药杂志

Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids.

The role of cell apoptosis mediated by endoplasmic reticulum stress (ERS) of deep tissue injury of pressure ulcer of rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To observe the the expression of endoplasmic reticulum stress (ERS) related factors in deep tissue injury (DTI) at pressure ulcer rat and to investigate the ERS mechanism of DTI in muscle tissue and protective effect of 4-phenylbutyric acid (4-PBA) in local tissue.</p><p><b>METHODS</b>Fifty male SD rats were randomly devided into control group, model group, experimental group NS group and PBA group, the experimental groups were divided into 4 d, 7 d, 14 d and 21 d group according to the observation time (n = 5). Rats in the PBA group were administrated with gastric perfusion of 4-PBA after the modeling; the NS group was given normal saline of the same quantity. Using HE staining to observe morphologic character. The expression of glucose regulated protein 78 (GRP78), CHOP, Caspase 12 were detected by immunohistochernical staining. Cell apoptosis was detected by TUNEL assay.</p><p><b>RESULTS</b>HE staining results showed that each group demonstrated compression injury compared with control group: cellular swelling, ompaction of nuclear, and apoptosis in muscle tissue. The new muscle fiber in 4-PBA group fused faster than those in NS group. The number of TUNEL positive cells peaked at 4 day after compression, then got decreased on day 7 in muscle tissue, apoptosis positive cells were diminished after 4-PBA treatment. The immunohistochemical staining results showed that the expression of protein GRP78, CHOP, Caspase 12 peakd 4 d after modeling and decreased gradually. The GRP78, CHOP, Caspase 12 protein expression were significantly higher than those of PBA group at all time points (P < 0.05).</p><p><b>CONCLUSION</b>Cell apoptosis induced by endoplasmic reticulum stress took part in deep tissue injury resulting of pressure ulcer, which mechanism might be related to reducing apoptosis mediated by CHOP, Caspase 12.</p>

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.

ISSR-PCR analysis in different species and populations of Rhizoma Curcumae / 中国中药杂志

<p><b>OBJECTIVE</b>To study the genetic diversity and genetic relationship in different species and populations of Curcuma by ISSR-PCR marker technique.</p><p><b>METHOD</b>Eighty populations and 37 samples of Curcuma including C. phaeocaulis, C. kwangsiensis and C. wenyujin were studied by ISSR-PCR markers. The systematic diagram of Similar coefficient and genetic distance were set up by POPGEN32 software and clustered by UPGMA method.</p><p><b>RESULT</b>A total of 65 loci were scored by 5 primers, among which 34 were polymorphic loci. The percentage of polymorphic loci was 52.3%. Genetic similarity coefficient changed from 0.6864 to 0.9997. Nei's gene diversity index (H), and Shannon information index (I) were 0.1521 and 0.2338. The inner genetic diversity of Curcuma species was lower than the outer.</p><p><b>CONCLUSION</b>The genetic variation of different populations Curcuma was big. The inherited differentiation of inner populations was low. Different populations of Curcuma were related to character of species and geological distribution.</p>

Studies on chemical constituents from herbs of Taraxacum mongolicum / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the chemical constituents of the herbs of Taraxacum mongolicum.</p><p><b>METHOD</b>The chemical constituents were isolated by various column chromatographic methods and their structures elucidated mainly by NMR and MS evidences.</p><p><b>RESULT</b>Forty-four components were obtained and identified were as artemetin (1), quercetin (2), quercetin-3', 4', 7-trime-thyl ether (3), luteolin (4), luteolin-7-O-beta-D-glucopyranoside (5), luteolin-7-O-beta-D-galactopyranoside (6), genkwanin (7), isoetin (8), hesperetin (9), genkwanin-4'-O-beta-D-lutinoside (10), hesperidin (11), quercetin-7-O-[beta-D-glucopyranosyl (1-->6) -beta-D-glucopyranoside (12), quercetin-3, 7-O-beta-D-diglucopyranoside (13), isoetin-7-O-beta-D-glucopyranosyl- 2'-O-alpha-L-arabinopyranoside (14), isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-glucopyranoside (15), isoetin-7- O-beta-D-glucopyranosyl-2'-O-beta-D-xyloypyranoside (16), caffeic acid (17), furulic acid (18), 3-O-caffeoylquinic acid (19), 3, 5-di-O-caffeoylquinic acid (20), 3, 4-di-O-caffeoylquinic acid (21), 4, 5-di-O-caffeoylquinic acid (22), 1-hydroxymethyl-5-hydroxy-phenyl-2-O-beta-D-glucopyranoside (23), p-hydroxybenzoic acid (24), p-coumaric acid (25), 3, 5-dihydroxylbenzoic acid (26), gallic acid (27), gallicin (28), syringic acid (29), 3, 4-dihydroxybenzoic acid (30), caffeic acid ethyl ester (31), esculetin (32), rufescidride (33), mongolicumin A [6, 9, 10-trihydroxy-benzoxanthene-1, 2-dicarboxylic acid] (34), mongolicumin B [1 l-hydroxy-2-oxo-guaia-1 (10), 3, 5-trien-8, 12-lactone] (35), isodonsesquitin A (36), taraxacin (37), sesquiterpene ketolactone (38), taraxasteryl acetate (39), phi-taraxasteryl acetate (40) and lupenol acetate (41), palmitic acid (42), beta-sitosterol (43), and stigmasterol (44).</p><p><b>CONCLUSION</b>Four compounds (14, 15, 34 and 35) were new compounds, compounds 1, 3, 6-13, 20-22, 30 and 31 were isolated from this genus for the first time, while compounds 18, 23-29, 32 and 37-42 were obtained from this species for the first time.</p>

Effect and mechanism of action of non-mitogenetic human acidic fibroblast growth factor on the mitogenic activity of the mammary tumor cell / 药学学报

<p><b>AIM</b>To compare the effects of the non-mitogenetic human acidic fibroblast growth factor (nmhaFGF) and the human acidic fibroblast growth factor (haFGF) on the proliferation and MAPK signal transduction pathway of the malignant tumor cell and to study the clinical safety of nmhaFGF.</p><p><b>METHODS</b>The mammary tumor cells (MCF-7) were treated with haFGF and nmhaFGF separately. The mitogenic activities of both haFGF and nmhaFGF were detected by MTT method and the cell cycle was analyzed by flow cytometer (FCM). The expression levels of the signal proteins, Grb2 (growth factor receptor bound 2) and ERK1/2 (extracellular signal-regulated kinase 1/2), were detected by semi-quantitative Western blotting method.</p><p><b>RESULTS</b>The mitogenic activity of nmhaFGF was obviously lower than that of haFGF. The activity of nmhaFGF was weaker than that of the haFGF. The ratio of G1/G0, G2/M of haFGF was markedly lower than that of nmhaFGF and control group, and was reverse in S phase. The expression levels of both Grb2 and ERK1/2 of the nmhaFGF treated group were lower than that of the haFGF treated group and approaching the control group.</p><p><b>CONCLUSION</b>The mitogenic activity of the nmhaFGF decreased remarkably. Its mechanism probably via down-regulation of the expression of the signal moleculars, MAPK-ERK1/2 and Grb2.</p>

Research of the feeding strategy in the fermentation of recombinant human fibreblast growth factor mutant / 生物工程学报

Human acidic fibreblast growth factor (haFGF) was a kind of cell growth factor with wide bio-activity on cell from mesectoderm and neuro-ectoderm.In this paper, the effect of acetate concentration on the growth and expression of recombinant human acidic fibroblast growth factor mutant system E.coli BL21(DE3)/pET3C-haFGF was investigated. Four fed-batch modes: batch-fed, batch-DO static balance, DO static balance-glucose starvation, and pH-static state were investigated. The accumulation of acetate during the fermentation course was effectively inhibited. The OD600nm value was about 22, after purification, the soluble rhaFGF yielded 450mg/L. During the fermentation, no special ways such as pure oxygen, pressure were adopted, thus the established process would be easily scaled up for industry purpose.

Development on Plant Seed Oil Body Expression System for Recombinant Proteins Production / 中国生物工程杂志

Plant transgenic system secures a safe,economical and reliable supply of recombinant proteins.Plant oilbody expression system simplifies the downstream purification steps and reduces capital investment based on the nature of oleosin including high expression and easy extraction.The structures and characteristics of seed oil body and oleosin were reviewed.And the research progress and industry of the seed oil body expression system,as a new bioreactor,to produce valuable recombinant proteins were discussed.The benefits and questions of the oil body expression system were also set forth.New medicine haFGF based on the oilbody system is being developed,and its biological activity is being analyzed.As a new resource for medicine protein,oilbody expression system will be perfected and applied broadly.

Selection of bFGF Mimic Peptide by Phage Display / 中国生物工程杂志

Objective: To obtain the bFGF mimic peptide binding to FGFR via phage display, and to provide the base for developing peptide agonist of bFGF. Methods: Using Balb/c 3T3 cells as the target cells and COS-7 cells as the subtractive panning, the phage display heptapeptide library was biopanned for 4 rounds to obtain the single phage clones. The affinity and the specificity of the clones were assessed by ELISA. DNA sequencing was applied to further analyze the positive clones. Results: Twelve positive clones were selected from the enriched phages. A group of hydrophobic peptides containing a conserved motif, PR, was identified. Conclusion: Two bFGF mimic heptapeptides binding to FGFR were selected, which may be used as the candidates for bFGF agonist.

Protective effect of non-mitogenic haFGF on retinal injury induced by N-methyl-N-nitrosourea in Sprague-Dawley rats / 药学学报

<p><b>AIM</b>To study the effect of non-mitogenic human acidic fibroblast growth factor (nm-haFGF) on retinal injury induced by N-methyl-N-nitrosourea (MNU) in Sprague-Dawley rats and its mechanism.</p><p><b>METHODS</b>Female rats of 50-days-old were injected with MNU (60 mg x kg(-1)) intraperitoneally, and three doses of nm-haFGF (1.25 microg, 2.5 microg and 5 microg in one eye of each rat) were injected, separately, into vitreous body of one eye of each rat twice a day at 0 and 12 h after MNU treatment. 24 h later, apoptotic index of photoreceptor cells was detected by TUNEL labeling and the expressions of Bcl-2 and Bax were analyzed by Western blotting. At the 7th day, retinal injury was evaluated based on retinal thickness.</p><p><b>RESULTS</b>Compared with model group, apoptotic index of photoreceptor cells was significantly reduced in nm-haFGF groups at the dose of 1.25 microg and 2.5 microg in one eye of each rat at 24 h, and the total retinal thickness as well as the outer retinal thickness markedly increased 7 days after MNU, respectively. The expressions of Bcl-2 increased and that of Bax decreased adversely after being injected with different doses of nm-haFGF.</p><p><b>CONCLUSION</b>nm-haFGF partially suppressed retinal injury induced by MNU in Sprague-Dawley rats. The mechanism could be related to up-regulation of Bcl-2 and down-regulation of Bax.</p>

Morphological and distribution characteristics of sweat glands in hypertrophic scar and their possible effects on sweat gland regeneration / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In hypertrophic scar tissue, no sweet gland and hair follicle exist usually because of the dermal and epidermal damage in extensive thermal skin injury, thus imparing regulation of body temperature. This study was designed to reveal the morphological and distributional characteristics of the sweat glands in normal skin and hypertrophic scar obtained from children and adults, and to study the possible interfering effects of the scar on regeneration of the sweat gland after burn injury.</p><p><b>METHODS</b>Biopsies of hypertrophic scar were taken from four children (4 - 10 years) and four adults (35 - 51 years). Normal, uninjured full-thickness skin adjacent to the scar of each patient was used as control. Keratin 19 (K19) was used as the marker for epidermal stem cells and secretory portion of the sweat glands, and keratin 14 (K14) for the tube portion, respectively. Immunohistochemical and histological evaluations were performed.</p><p><b>RESULTS</b>Histological and immunohistochemical staining of skin tissue sections from both the children and adults showed K19 positive cells in the basement membrane of epidermis of normal skin. These cells were seen only single layer and arranged regularly. The secretory or duct portion of the eccrine sweat glands was situated in the dermis and epidermal layer. However, in the scar tissue, K19 positive cells were scant in the basal layer, and the anatomic location of the secretory portion of sweat glands changed. They were located between the border of the scar and reticular layer of the dermis. These secretory portions of sweat glands were expanded and were organized irregularly. But a few K14 positive cells were scattered in the scar tissues in cyclic form.</p><p><b>CONCLUSIONS</b>There are some residual sweat glands in scar tissues, in which the regeneration process of active sweat glands is present. Possibly the sweat glands could regenerate from adult epidermal stem cells or residual sweat glands in the wound bed after burn injury.</p>

Expression and Purification of Basic Fibroblast Growth Factor Mutant with Reduced Mitogenic Activity / 中国生物工程杂志

In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.

Expression and Purification of Basic Fibroblast Growth Factor Mutant with Reduced Mitogenic Activity / 中国生物工程杂志

In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.

Pharmacokinetic study of recombinant human acidic fibroblast growth factor in rabbits by skin external use / 药学学报

<p><b>AIM</b>To investigate the pharmacokinetic characteristics of recombinant human acidic fibroblast growth factor (rhaFGF) after external use in rabbits.</p><p><b>METHODS</b>125I-rhaFGF 180 U.cm-2 was daubed to normal skin and scathed skin in rabbits. The radioactivity and paper chromatography were used to determine the 125I-concentrations and distribution in plasma and organs at different times.</p><p><b>RESULTS</b>The plasma concentration of 125I-rhaFGF increased rapidily, and reach peak plasma level (73.03 pg.mL-1) thirty minutes after administration. Then the concentration of 125I-rhaFGF decreased quickly after thirty minutes, and approached to zero after three hours. Highest radioactivity accumulated in the skin, followed by kidney, lowest in the brain 96 h after administration.</p><p><b>CONCLUSION</b>rhaFGF can not be absorbed from the normal skin, whereas a small amount of rhaFGF can be absorbed through scathed skin. The t1/2 of rhaFGF in plasma was very short. Cumulative effect of rhaFGF was not observed. Absorbed rhaFGF showed high affinity to skin, and can be distributed to skin far from the site of administration.</p>