Bovine Fibroblasts Research Articles

Correction of a Disease Mutation using CRISPR/Cas9-assisted Genome Editing in Japanese Black Cattle

Isoleucyl-tRNA synthetase (IARS) syndrome is a recessive disease of Japanese Black cattle caused by a single nucleotide substitution. To repair the mutated IARS gene, we designed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to create a double-strand break near the mutation site. CRISPR/Cas9 and donor DNA that contained a synonymous codon for the correct amino acid and an Aequorea coerulescens Green Fluorescent Protein (AcGFP) cassette with a piggyBac transposase recognition site at both ends were introduced into bovine fetal fibroblast (BFF) cells isolated from a homozygous mutant calf. Recombinant cells were enriched on the basis of expression of AcGFP, and two cell lines that contained the repaired allele were subcloned. We generated somatic cell nuclear transfer (SCNT) embryos from the repaired cells and transferred 22 blastocysts to recipient cows. In total, five viable fetuses were retrieved at Days 34 and 36. PiggyBac transposase mRNA was introduced into BFF cells isolated from cloned foetuses and AcGFP-negative cells were used for second round of cloning. We transferred nine SCNT embryos to recipient cows and retrieved two fetuses at Day 34. Fetal genomic DNA analysis showed correct repair of the IARS mutation without any additional DNA footprint.

PhiC31-based Site-Specific Transgenesis System for Production of Transgenic Bovine Embryos by Somatic Cell Nuclear Transfer and Intracytoplasmic Sperm Injection.

The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection. In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) approaches. PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique. The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein (EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection (ICSI) method.

Pro-oxidative Mitochondrial Metabolism of Bovine Arterial Wall Fibroblasts in Pulmonary Hypertension Syndrome can be Reversed by PTBP1 Silencing and Histone Deacetylase Inhibition

Pulmonary arterial hypertension (PH) is a serious complex disease often leading to heart failure and early death of patients. We and others have previously demonstrated that pulmonary adventitial fibroblasts from chronically hypoxic hypertensive calves (PH-Fibs) exhibit a highly proliferative, migratory, pro-inflammatory, and apoptosis-resistant phenotype similar to cancer cells. As in many cancer cells this is accompanied by a constitutive metabolic shift manifesting increased glycolysis, mainly due to increased expression ratio of pyruvate kinase M2/1 isoforms, lowered mitochondrial respiration and enhanced superoxide production[1], we focused on the regulation of an altered mitochondrial function in PH-Fibs by miR-124 and polypyrimidine tract binding protein 1 (PTBP1). Measuring membrane potential, respiratory parameters, Complex I function and redox status, we found that either miR-124 addition or PTBP1 silencing is capable to rescue mitochondrial function and superoxide production of PH-Fibs towards control fibroblasts (CO-Fibs) levels. Moreover, further regulation of the mitochondrial metabolism switch in PH-Fibs by class I histone deacetylase (HDAC) was proven. Our data reveal novel mechanisms and possible approaches how to reverse mitochondrial metabolic reprogramming of PH-Fibs which can result in potential future therapeutic employment.

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine

Accumulating evidence indicates the absence of paternally derived miRNAs, piwiRNAs, and proteins may be one important factor contributing to developmental failure in somatic cell cloned embryos. In the present study, we found microRNA-449b (miR-449b) was highly expressed in sperm. Target gene predictions and experimental verification indicate that several embryonic development-related genes, including CDK6, c-MYC, HDAC1 and BCL-2, are targets of miR-449b. We therefore investigated the role of miR-449b using somatic cell nuclear transfer (SCNT) embryo model. Bovine fetal fibroblasts, expressing miR-449b through a doxycycline (dox) induced expression system were used as nuclear donor cells for SCNT. The results showed that miR-449b expression in SCNT embryos significantly enhanced the cleavage rate at 48 h after activation and the levels of H3K9 acetylation at the 2-cell to 8-cell stages, meanwhile, significantly decreased the apoptosis index of blastocysts. In addition, we verified miR-449b could regulate the expression levels of CDK6, c-MYC, HDAC1 and BCL-2. In conclusion, the present study shows that miR-449b expression improves the first cleavage division, epigenetic reprogramming and apoptotic status of bovine preimplantation cloned embryos.

Experimental study on accelerated healing of jaw fracture using gelatin sponge compound growth factor

To explore the role and mechanism of drug delivery systems using growth factor combined with gelatin sponge on accelerating the healing of jaw fracture and to seek better treatment of accelerating the maxillofacial fracture. About 100 μg recombinant human bone morphogenetic protein (BMP)-2 was completely dissolved in 1 mL recombinant bovine basic fibroblast growth factor (bFGF), and the solution (40 μL) was dropped in gelatin sponge (0.5 cm×0.5 cm×1.0 cm). Then, it was freeze dried and prepared into bFGF/BMP/gelatin sponge delivery systems. The mandibular fracture model on two sides were prepared in 12 New Zealand rabbits and randomly divided into two groups. The left side was the control group, which was only fixed with titanium plates. The right side was the experimental group, in which bFGF/BMP/gelatin sponge delivery systems were put under the titanium plates. General observation, X-ray, and histological examination were taken at 2, 4, and 12 weeks after surgery. After 2 weeks, more fibrous tissues were seen between the fracture ends in the experimental group than in the control group. After 4 weeks, fibrous fracture callus were seen in the fracture gap in the experimental group. The ingrowths of fibrous tissue and blood vessels were seen in the control group. The fracture healing of the experimental group was significantly faster than the control group at 2 and 4 weeks. After 12 weeks, the experimental and control groups all healed completely. bFGF/BMP/gelatin sponge can accelerate and improve fracture healing; thus, it has better clinical application prospect.

Efficacy of two kinds of eye drops in stabilizing tear film and relieving dry eye after cataract surgery

Objective To compare the efficacy of recombinant bovine basic fibroblast growth factor (rb-bFGF) eye drops and hypromellose 2910 dextran 70 and glycerol eye drops in maintaining the tear film stability and relieving dry eye symptoms after age-related cataract surgery. Methods The data of 131 eyes of 100 patients with age-related cataract were selected, and they were divided randomly into the rb-bFGF group, 64 eyes of 50 cases; and the hydroxyl indican group, 67 eyes of 50 cases. All patients underwent phacoemulsification combined with intraocular lens implantation. After surgery, rb-bFGF group were treated with rb-bFGF eye drops and the hydroxyl indican group were treated with hydroxyl indican eye drops. All patients were followed up for 3 months, and efficacy were compared between the two groups. Results The schirmer I test (SIT) in rb-bFGF group 3 months after surgery was (14.47±4.69)mm and the break-up time (BUT) was(13.96±2.84)s, in hydroxyl indican group was (12.03±4.76) mm and (12.35±3.27) s. The difference were significant between the two groups (P=0.004, 0.003). The scores of cornea fluorescein staining (CFS) and dry eye symptoms in rb-bFGF group were significantly lower than those in hydroxyl indican group(P=0.011, 0.000). Conclusion Both rb-bFGF eye drops and hydroxyl indican eye drops can prolong BUT, improve tear film stability, relieve dry eye symptoms, and promote the repair and regeneration of corneal epithelial cells after age-related cataract surgery. Rb-bFGF eye drops have better effects. Key words: Cataract, age-related; Phacoemulsification; Lens, intraocular; Eye drops, recombinant bovine basic fibroblast growth factor (rb-bFGF), hypromellose 2910, dextran 70 and glycerol

Effect of POU5F1 Expression Level in Clonal Subpopulations of Bovine Fibroblasts Used as Nuclear Donors for Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) success is partially hindered by the low epigenetic reprogramming efficiency of the donor cell. Previous studies suggest cellular heterogeneity among donor nuclei in regard to reprogramming potential, which precludes comparison among different strategies to increase cloning success. In this context, we evaluated the effect of using clonal cell populations (CPs) of bovine adult fibroblasts established by single-cell plating in SCNT. Different CPs were evaluated in regard to proliferation rate, senescence level, and chromosome stability, as well as for POU5F1 (POU class 5 homeobox 1) mRNA expression levels. In total, 9 of 24 CPs (37.5%) were successfully expanded in vitro up to the fourth passage and shown to proliferate following cryopreservation, at which time cell analyses were performed. The use of a CP with low senescence level, normal karyotype, and highest POU5F1 expression levels did not improve embryo development rates or quality following SCNT. As previously suggested, this study supports the notion that levels of POU5F1 expression in the donor nucleus do not impact the SCNT results. Notably, the single-cell seeding approach used herein to isolate CPs may be extended to the evaluation of additional predictor markers of reprogrammability success for SCNT in future experiments.

Combination of a 2940 nm Er:YAG laser with recombinant bovine basic fibroblast growth factor (rb-bFGF) and light-emitting diode-red light (LED-RL) for the treatment of striae alba: A pilot study.

Striae distensae (SD) are a common dermatologic problem that plagues many people. Although there are many therapeutic modalities have been used to treat SD, effective method has been disappointing for striae Alba. To evaluate the clinical and histopathologic efficacy and safety of the 2940-nm erbium yttrium aluminum garnet (Er:YAG) ablative fractional laser (AFL) with recombinant bovine basic fibroblast growth factor (rb-bFGF) and light-emitting diode-red light (LED-RL) for the treatment of striae alba. Thirty volunteers with striae distensae alba were enrolled. The subjects completed treatments with the 2940-nm Er:YAG AFL 6 times at 4-week intervals. Following this treatment, the subjects were required to spray rb-BFGF for 1week at home. They then received LED-RL once every 7days for three sessions between the two laser treatments. Two independent investigators evaluated clinical improvement at pretreatment and 1, 3, 6, and 12months post-treatment, patients also provided self-assessments of clinical improvement. Two biopsies were obtained from two subjects, both of the same sites of striae alba, one before the first treatment and one 6months after the last session. All 30 subjects demonstrated clinical improvement after treatment. Skin biopsies after treatment showed an increase in epidermal thickness, dermal thickness, and collagen and elastin density when compared to that at the baseline. The combination of the 2940-nm Er:YAG laser with rb-bFGF and LED-RL for the treatment of striae alba was a safe and effective approach for improving the appearance of striae alba.

Physicochemical properties of ionic and non-ionic biocompatible hydrogels in water and cell culture conditions: Relation with type of morphologies of bovine fetal fibroblasts in contact with the surfaces

Cationic, anionic and non-ionic hydrogels having acrylamide polymer backbones were synthesized via free radical polymerization with N,N-methylenebisacrylamide (BIS) as crosslinker. The chemical structures of the hydrogels were characterized by Fourier Transform Infrared Spectroscopy (FTIR). Physicochemical properties such as swelling kinetic, maximum swelling capacity, volume phase transition temperature (VPTT) and wettability (static water contact angle) of hydrogels swollen in aqueous and cell culture medium, at room and cell culture temperatures were studied. In order to correlate the surface properties of the hydrogels and cellular adhesivity of bovine fetal fibroblasts (BFFs), cellular behaviour was analyzed by inverted fluorescence optical microscopy and atomic force microscopy (AFM). MTT assay demonstrated that the number of viable cells in contact with hydrogels does not significantly change in comparison to a control surface. Flattened and spindle-shaped cells and cell spheroids were the adopted morphologies during first days of culture on different hydrogels. Cell spheroids were easily obtained during the first 5days of culture in contact with PNIPAM-co-20%HMA (poly (N-isopropylacrylamide-co-20%N-acryloyl-tris-(hydroxymethyl)aminomethane)) hydrogel surface. After 15days of culture all hydrogels showed high adhesion and visual proliferation. According to obtained results, non-ionic and hydrophilic surfaces with moderated wettability induce the formation of BFFs cell spheroids. These hydrogel surfaces could be used in clinical and biochemical treatments at laboratory level to cell growth and will allow generating the base for future biotechnologic platform.

Using carbon nanotubes to deliver genes to hard-to-transfect mammalian primary fibroblast cells

The insertion of DNA into primary mammalian cells is an important step towards gene therapy and production of transgenic animals. Recently, carbon nanotubes have been explored as an efficient novel non-viral system for delivering genes to cells due to their unique structure and properties. However, their potential for transfecting primary bovine cells in non-toxic concentrations has not been tested. The purpose of this study was to evaluate the cytotoxicity of carboxylic acid-functionalized multiwalled carbon nanotubes (COOH-MWCNTs) and their use to deliver plasmid DNA encoding the gene of green fluorescent protein to bovine primary fibroblast cells. Flow-cytometry cell viability results have shown the non-toxic nature of COOH-MWCNTs at low concentrations. The frequency shifts in Raman spectroscopy showed that the plasmid DNA connects to the nanomaterial. Fluorescence imaging, flow-cytometry and PCR analysis confirmed that the COOH-MWCNT nanovectors delivered pDNA into primary fibroblast cells successfully. The results show that COOH-MWCNTs can be attractive alternatives for delivery of DNA into hard-to-transfect primary bovine cells.

Hypoxia enhances secretion of matrix metalloproteinases in bovine dermal fibroblasts: an in vitro approach to bovine digital dermatitis

ABSTRACTDermal fibroblasts are of great importance in skin homeostasis. They are responsible for the synthesis and remodelling of extracellular proteins by the way of synthesis of some specific kind of Matrix Metalloproteinases (MMPs). Imbalance between MMPs and their tissue inhibitors (TIMPs) are known to involve ulcerative wounds in many tissues, including skin. In veterinary medicine, bovine digital dermatitis (BDD) is an ulcerative skin disorder of dairy cattle. To date, Koch’s postulate has not yet been fulfilled for the pathogenesis. In present study, we tested in vitro role of hypoxia if it evokes a response in MMP-1, MMP-2 and MMP-13 enzymes and their inhibitors in bovine dermal fibroblasts. Dermal fibroblasts were exposed to hypoxia in various time periods (6, 12 and 24 h). Immunoblotting revealed 6-, 12- and 24-h hypoxia, which resulted in 43.5%, 75% and 65% increase in MMP-1, 76%, 61% and 55% increase in MMP-2 and 80%, 78% and 55% increase in MMP-13 enzymes levels, respectively. We observed slight increases in TIMP-1 and TIMP-2 enzymes, but differences between treatment and control groups did not show any statistical significance. Our findings indicate that hypoxia induces MMP-1, MMP-2 and MMP-13 enzymes and may contribute to pathogenesis of BDD through in the development of in vivo lesions.

Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-azacytidine and cultured in differentiation medium containing BMP2, BMP4 or follicular fluid.

This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 μM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 μM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.

Genome-wide methylation analysis reveals differentially methylated loci that are associated with an age-dependent increase in bovine fibroblast response to LPS

BackgroundDifferences in DNA methylation are known to contribute to the development of immune-related disorders in humans but relatively little is known about how methylation regulates immune function in cattle. Utilizing whole-transcriptome analyses of bovine dermal fibroblasts, we have previously identified an age and breed-dependent up-regulation of genes within the toll-like receptor 4 (TLR4) pathway that correlates with enhanced fibroblast production of IL-8 in response to lipopolysaccharide (LPS). Age-dependent differences in IL-8 production are abolished by treatment with 5-aza-2-deoxycytidine and Trichostatin A (AZA-TSA), suggesting epigenetic regulation of the innate response to LPS. In the current study, we performed reduced representation bisulfite sequencing (RRBS) on fibroblast cultures isolated from the same animals at 5- and 16-months of age to identify genes that exhibit variable methylation with age. To validate the role of methylation in gene expression, six innate response genes that were hyper-methylated in young animals were assessed by RT-qPCR in fibroblasts from animals at different ages and from different breeds.ResultsWe identified 14,094 differentially methylated CpGs (DMCs) that differed between fibroblast cultures at 5- versus 16-months of age. Of the 5065 DMCs that fell within gene regions, 1117 were located within promoters, 1057 were within gene exons and 2891 were within gene introns and 67% were more methylated in young cultures. Transcription factor enrichment of the promoter regions hyper-methylated in young cultures revealed significant regulation by the key pro-inflammatory regulator, NF-κB. Additionally, five out of six chosen genes (PIK3R1, FES, NFATC1, TNFSF13 and RORA) that were more methylated in young cultures showed a significant reduction in expression post-LPS treatment in comparison with older cultures. Two of these genes, FES and NFATC1, were similarly down-regulated in Angus cultures that also exhibit a low LPS response phenotype.ConclusionsOur study has identified immune-related loci regulated by DNA methylation in cattle that may contribute to differential cellular response to LPS, two of which exhibit an identical expression profile in both low-responding age and breed phenotypes. Methylation biomarkers of differential immunity may prove useful in developing selection strategies for replacement cows that are less susceptible to severe infections, such as coliform mastitis.

Cell type\u2013specific extracellular matrix guided the differentiation of human mesenchymal stem cells in 3D polymeric scaffolds

The tissue microenvironment has profound effects on tissue-specific regeneration. The 3-dimensional extracellular matrix (ECM) niche influences the linage-specific differentiation of stem cells in tissue. To understand how ECM guides tissue-specific regeneration, we established a series of 3D composite scaffolds containing ECMs derived from different primary cells isolated from a single animal species and assessed their impact on the differentiation of human mesenchymal stem cells (hMSCs). Synthetic microfiber scaffolds (fiber mats) were fabricated by electrospinning tyrosine-derived polycarbonates (pDTEC). The bovine primary fibroblasts, chondrocytes and osteoblasts cultured on the fiber mats produced and assembled their ECMs, infiltrating the pores of the fibrous scaffold. The composite scaffolds were decellularized to remove cellular components, preserve ECM and minimally affect polymer integrity. Characterization of the ECMs derived from different primary cells in the composite scaffolds showed overlapping but distinct compositions. The chondrogenic and osteogenic differentiation of hMSCs on the different composite scaffolds were compared. Our results showed that ECM derived from chondrocytes cultured in synthetic fiber mats promoted the chondrogenic differentiation of hMSC in the presence or absence of soluble inducing factors. ECM derived from co-culture of osteoblasts and chondrocytes promoted osteogenic differentiation in hMSCs better than ECM derived from chondrocytes. This study demonstrated that decellularized ECMs derived from different cell types formed within synthetic fiber scaffolds guide the tissue-specific differentiation of hMSCs. These composite scaffolds may be developed into models to study the mechanisms of ECM-induced tissue regeneration.Graphical

In vivo and in vitro ageing results in accumulation of de novo copy number variations in bulls

We have identified de novo copy number variations (CNVs) generated in bulls as they age. Blood samples from eight bulls were collected and SNP arrayed in a prospective design over 30 months allowing us to differentiate de novo CNVs from constant CNVs that are present throughout the sampling period. Quite remarkably, the total number of CNVs doubled over the 30-month period, as we observed an almost equal number of de novo and constant CNVs (107 and 111, respectively, i.e. 49% and 51%). Twice as many de novo CNVs emerged during the second half of the sampling schedule as in the first part. It suggests a dynamic generation of de novo CNVs in the bovine genome that becomes more frequent as the age of the animal progresses. In a second experiment de novo CNVs were detected through in vitro ageing of bovine fibroblasts by sampling passage #5, #15 and #25. De novo CNVs also became more frequent, but the proportion of them was only ~25% of the total number of CNVs (21 out of 85). Temporal generation of de novo CNVs resulted in increasing genome coverage. Genes and quantitative trait loci overlapping de novo CNVs were further investigated for ageing related functions.

Bovine trophectoderm cells induced from bovine fibroblasts with induced pluripotent stem cell reprogramming factors.

Thirteen independent induced bovine trophectroderm (iBT) cell lines were established by reprogramming bovine fetal liver-derived fibroblasts after viral-vector transduction with either six or eight factors, including POU5F1 (OCT4), KLF4, SOX2, MYC, NANOG, LIN28, SV40 large T antigen, and hTERT. Light- and electron-microscopy analysis showed that the iBT cells had epithelial cell morphology typical of bovine trophectoderm cells. Reverse-transcription-PCR assays indicated that all of the cell lines expressed interferon-tau (IFNT) at passages 1 or 2. At later passages (≥ passage 8), however, immunoblot and antiviral activity assays revealed that more than half of the iBT cell lines had stopped expressing IFNT. Messenger RNAs specific to trophectoderm differentiation and function were found in the iBT cell lines, and 2-dimensional-gel analysis for cellular proteins showed an expression pattern similar to that of trophectoderm cell lines derived from bovine blastocysts. Integration of some of the human reprogramming factors, including POU5F1, KLF4, SOX2, MYC, NANOG, and LIN28, were detected by PCR, but their transcription was mostly absent in the iBT cell lines. Gene expression assessment of endogenous bovine reprogramming factor orthologs revealed endogenous bLIN28 and bMYC transcripts in all; bSOX2 and bNANOG in none; and bKLF4 and bPOU5F1 in less than half of the iBT cell lines. These results demonstrate that bovine trophectoderm can be induced via reprogramming factor expression from bovine liver-derived fibroblasts, although other fibroblast populations-e.g., derived from fetal thigh tissue-may produce similar results, albeit at lower frequencies.

Effect observation of honey and rb-bFGF in promoting the wound healing of oral mucosa

Objective To explore the efficacy of honey and recombinant bovine basic fibroblast growth factor (rb-bFGF) in promoting wound healing after the surgery of oral mucosa transplantation, and provide evidence for clinical nursing. Methods Eighty patients who underwent oral mucosa transplant surgery were selected and randomly divided into four groups by random number table method, with 20 cases in each group. Group A was the control group, the oral mucosa wounds were exposed to air and kept clean and dry; in group B, the oral mucosa wounds were treated with honey; in group C, the oral mucosa wounds were treated with rb-bFGF gel; and in Group D, the patients were treated with honey combined with rb-bFGF gel. The wound healing level, eating recovery time, and pain level of four groups were evaluated and compared. Results The comparison in wound healing degree among four groups seven days after surgery was statistically significant different (P<0.05) ; the wound healing level of group D was better than group B and C (P<0.05) ; the pain level of patients in four groups was significantly different (P<0.05) ; the pain level of group D was better than other three groups (P<0.05) ; the comparison of eating recovery time among four groups was significantly different (P<0.05) ; the eating recovery time of group D was better than group B and C (P<0.05) . Conclusions Both honey and rb-bFGFcan effectively promote the wound healing of oral mucosa. The combination of honey and rb-bFGF, can shorten the eating recovery time, relieve pain, and promote wound healing, so as to improve patients' quality of life. Key words: Honey; Recombinant bovine basic fibroblast growth factor; Oral mucosa; Wound healing

Knock-in Efficiency Depending on Homologous Arm Structure of the Knock-in Vector in the Bovine Fibroblasts

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.

Effectiveness of heparin eye drops in paraquat-induced ocular injury

Purpose: To evaluate the efficacy of heparin eye drops in the treatment of paraquat-induced ocular surface injury.Design and methods: In this retrospective study, we included 25 patients (31 eyes) with paraquat-induced ocular surface injury, who attended the Affiliated Hospital of Weifang Medical University between October 2008 and October 2013. The patients were split into two groups according to whether or not received heparin eye drops. The clinical data were compared between the two groups, i.e. clinical histories, results of examinations, treatments and outcomes.Results: Eleven patients (group A, 15 eyes) received prompt irrigation with 0.9% saline every two hours, 0.1% pranoprofen eye drops four times a day, 20% autologous serum every two hours, recombinant bovine basic fibroblast growth factor eye-gel two times a day, oral vitamin C 2.0 g and prednisone 30 mg daily. Fourteen patients (group B, 16 eyes) received additional treatment with heparin eye drops. Ten eyes in group A and seven eyes in group B developed a pseudomembrane on the ocular surface at significantly different rate (mean ± SD) of 1.20 ± 1.01 and 0.43 ± 0.51, respectively (t = 2.66, p = 0.01). Seven eyes among 10 had a pseudomembrane reoccurred in group A while none had a pseudomembrane reoccurred in group B (Fisher’s exact test, p = 0.01). No significant differences were seen in the duration of epithelial recovery between the two groups: 15.13 ± 5.13 days in group A and 16.81 ± 5.56 days in group B (t = 0.87, p = 0.39). After the treatment, mild corneal opacity and pannus were observed in five patients of group A and four patients of group B, without any significant difference between the two groups (p = 0.70).Conclusions: The paraquat-induced ocular surface injury observed in this case series was characterized by the formation of conjunctival pseudomembrane with good prognosis and mild complications. Heparin eye drops reduce the occurrence, especially the reoccurrence of pseudomembrane. Further studies are warranted.

Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity.

The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25kDa PEI and linear 58kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.