[Histone H3K27me3 in the regulation of skeletal muscle development].

Histone methylation is a modification which occurs in the N-terminal peptide chains of the histone nucleosome. The 4th, 9th, 27th, 36th and 79th lysines in N-terminal peptide chain of histone H3 are hot spots for this modification, including mono-, di-, and tri-methylation. H3K27me3 is the tri-methylation modification on histone H3 lysine 27, which mainly functions as a transcriptional repressor regulating skeletal muscle development. Studies have shown that H3K27me3 can finely regulate skeletal muscle proliferation, including the level and duration of skeletal muscle development by specifically binding to myogenic regulatory factors (e.g., MyoD, MyoG, etc.), cell cycling regulators, and epigenetic regulators including lncRNA and miRNA. In this review, we introduce the types and mechanisms of histone methylation and de-methylation of H3K27. We also summarize how H3K27me3 functions in the proliferation and differentiation of skeletal muscle cell. This review will contribute to the comprehension of the function of H3K27me3 in regulating skeletal muscle development and provide reference for further improving our understanding of mammalian muscle.

[Effects of RNA interference on the porcine NHEJ pathway repair factors on HR efficiency].

Non-homologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells. It inhibits the efficiency of homologous recombination (HR) by competing for DSB targets. To improve the efficiency of HR in porcine fetal fibroblasts (PFFs), several RNA interference (RNAi) systems were designed to knockdown NHEJ key molecules, such as polynucleotide kinase/phosphatase (PNKP), DNA ligase IV (LIG4) and NHEJ1. The results show that siRNA significantly knocked down LIG4, PNKP and NHEJ1 expression. Suppression of PNKP dramatically increased the efficiency of single-strand annealing (SSA), double-strand DNA (dsDNA) and single-strand DNA (ssODN) mediated homology-directed repair (HDR) by 55.7%, 37.4% and 73.1% after transfected with the SSA-GFP reporter, HDR-GFP system or ssODN-GFP system, respectively; whereas knockdown of LIG4 and NHEJ1 repair factors significantly increased dsDNA or ssODN-mediated HDR efficiency by 37.5% and 76.9%, respectively.

[Placental developmental defects in cloned mammalian animals].

The cloning technique, also called somatic cell nuclear transfer (SCNT), has been successfully established and gradually applied to various mammalian species. However, the developmental rate of SCNT mammalian embryos is very low, usually at 1% to 5%, which limits the application of SCNT. Placental developmental defects are considered as the main cause of SCNT embryo development inhibition. Almost all of SCNT-derived mammalian placentas exhibit various abnormalities, such as placental hyperplasia, vascular defects and umbilical cord malformation. Mechanistically, these abnormalities result from failure of establishment of correct epigenetic modification in the trophectoderm genome, which leads to erroneous expression of important genes for placenta development-related, particularly imprinted genes. Consequently, aberrant imprinted gene expression gives rise to placental morphologic abnormalities and functional defects, therefore decreases developmental competence of cloned embryos. Currently, although numerous methods that can improve the developmental ability of SCNT-derived embryos have been reported, most of them are unable to substantially enhance the success rate of SCNT due to failure to eliminate the placental development defects. In this review, we summarize placental abnormalities and imprinted gene expression in mammalian cloning, and propose directions for the future research aiming to improve the cloning efficiency.

Establishment of porcine Xist knockout model using CRISPR/Cas9 system.

Somatic cell nuclear transfer technique has great applications in livestock breeding, production of genetically modified animals, rescue of endangered species and treatment of human diseases. However, the currently low efficiency in animals cloning, an average of less than 5%, greatly hindered the rapid development of this technique. Among many factors which affect the efficiency of cloning pigs, X chromosome inactivation is an important one. Moreover, Xist gene is closely related to X chromosome inactivation, suggesting that it may directly or indirectly affects cloning efficiency. In this study, multiple sgRNAs were designed based on the CRISPR/Cas system, and two sites (Target 3 and Target 4) whose mutation efficiency were 1% and 3% at the cellular level were selected. We successfully knocked out Xist with 100% efficiency by microinjecting sgRNAs for Target 3 and Target 4 in embryo. Finally, 6 cloning piglets were born including two Xist-fully-knockout piglets. The follow-up studies on increasing cloning efficiency can be carried out based on the Xist-knockout model.

Epigenetic modification: A novel insight into diabetic wound healing.

Wound healing is an intricate and fine regulatory process. In diabetic patients, advanced glycation end products (AGEs), excessive reactive oxygen species (ROS), biofilm formation, persistent inflammation, and angiogenesis regression contribute to delayed wound healing. Epigenetics, the fast-moving science in the 21st century, has been up to date and associated with diabetic wound repair. In this review, we go over the functions of epigenetics in diabetic wound repair in retrospect, covering transcriptional and posttranscriptional regulation. Among these, we found that histone modification is widely involved in inflammation and angiogenesis by affecting macrophages and endothelial cells. DNA methylation is involved in factors regulation in wound repair but also affects the differentiation phenotype of cells in hyperglycemia. In addition, noncodingRNA regulation and RNA modification in diabetic wound repair were also generalized. The future prospects for epigenetic applications are discussed in the end. In conclusion, the study suggests that epigenetics is an integral regulatory mechanism in diabetic wound healing.

Vedolizumab subcutaneous formulation maintenance therapy for patients with IBD: a systematic review and meta-analysis.

Background: The application of vedolizumab (VDZ) subcutaneous (SC) formulation has brought more convenience and hope to patients with moderate-to-severe inflammatory bowel diseases (IBDs) in the coronavirus disease 2019 context. Objective: This study aimed to systematically evaluate all previous studies that used VDZ SC formulation for maintenance therapy in patients with IBD. Design: Systematic review and meta-analysis. Data Sources and Methods: The search was conducted using the subject and free terms related to 'Vedolizumab', 'Subcutaneous', and 'IBD', in Embase, PubMed, Web of Science, Cochrane, and at ClinicalTrials.gov databases between 2008 and 2022. The methodological quality of randomized controlled trials (RCTs) and cohort studies was assessed using the Cochrane Handbook of Systematic Reviews and the Newcastle-Ottawa Scale, respectively. The endpoints included efficacy, safety, and immunogenicity. Results: A total of 60 studies and 2 completed clinical registry trials were retrieved, of which 3 RCTs with high methodological quality, and 3 cohort studies with large heterogeneity were included in the meta-analysis. In the RCT study design, patients with ulcerative colitis (UC) under different conditions after treated with VDZ SC were significantly distinct than those for placebo (PBO) in clinical remission, endoscopic remission, and biochemical remission. In Crohn's disease (CD), the aforementioned parameters were slightly higher than those for PBO, but there was not statistically significant in endoscopic remission and the efficacy of anti-tumor necrosis factor-naive patients. The clinical remission, endoscopic remission, and biochemical remission in patients with UC after VDZ SC treatment were similar to those after intravenous (IV) treatment. The risk ratios in patients experiencing adverse events (AEs) and serious AEs after VDZ SC and PBO treatments were 86% and 89% in UC, and 96% and 80% in CD, respectively. Compared with IV, safety was not statistically different. The risk of developing anti-VDZ antibody after VDZ SC treatment was only 20% of that after PBO in patients with UC, but it was 9.38 times in CD. Conclusion: VDZ SC treatment maintained the clinical efficacy of IV induction in patients with IBD without increasing the safety risk, and the efficacy was more pronounced in patients with UC. Immunogenicity might be a potential factor for the decrease in efficacy rate in patients with IBD. Registration: INPLASY 2022120115.

Effects of the Oat Hay Feeding Method and Compound Probiotic Supplementation on the Growth, Antioxidant Capacity, Immunity, and Rumen Bacteria Community of Dairy Calves.

This study aimed to investigate the effects of the oat hay feeding method and compound probiotics (CMP) on the growth, health, serum antioxidant and immune indicators, rumen fermentation, and bacteria community of dairy calves from 3 to 5 months of age. Forty-eight female Holstein calves (80 ± 7 days of age, 93.71 ± 5.33 kg BW) were selected and randomly divided into four groups. A 2 × 2 factorial design was adopted for the experiment, with the factors of the oat hay feeding method (fed as free-choice or 16.7% in the diet) and compound probiotics (CMP) inclusion (0.15% or 0%) in the pelleted starter. The results showed that, compared with giving oat hay as free-choice, feeding a diet of 16.7% oat hay increased the pelleted starter intake at 1-84 d (p < 0.05), with an average daily gain (ADG) at 61-84 d (p = 0.02); adding CMP to the pelleted starter did not significantly affect body weight, and reduced the fecal index (p < 0.05). Feeding 16.7% oat hay increased the concentration of IgA, IgG, and IgM (p < 0.01), while adding CMP increased the catalase (p < 0.01) and decreased the concentration of malondialdehyde (p < 0.01) in serum. Feeding 16.7% oat hay increased the ruminal concentration of propionic acid (p < 0.05) and isobutyric acid (p = 0.08), and decreased the ruminal pH (p = 0.08), the concentration of acetic acid (p < 0.05), and the ratio of acetic acid to propionic acid (p < 0.01). Feeding 16.7% oat hay reduced the relative abundance of ruminal Firmicutes, Unidentified-Bacteria, Actinobacteria, Prevotella, NK4A214-group, Olsenella, and Actinobacteriota (p < 0.05); adding CMP increased the relative abundance of ruminal Prevotella, Rikenellaceae-RC9-gut-group, Ruminococcus, NK4A214-group, and Ruminococcus (p < 0.05), and decreased the abundance of Desulfobacterora, Prevotella-7, and Erysipelotricaceae-UCG-002 (p < 0.05). In conclusion, feeding a diet of 16.7% oat hay increased the pelleted starter intake and average daily gain, while slightly reducing the ruminal pH values; adding CMP to the pelleted starter resulted in reduced diarrhea incidence, increased serum antioxidant capacity and immunity, as well as ruminal richness and diversity of microorganisms in dairy calves from 3 to 5 months of age.

[Differential expression profile of microRNA between hyperplastic scar and normal skin].

OBJECTIVE: To explore the miRNA differential expression profiles of hyperplastic scar and normal skin so as to further elucidate the pathogenesis of hyperplastic scar and search for new therapeutic targets. METHODS: The total RNA was extracted from 5 human hyperplastic scar and normal skin tissues by Trizol. The specimens were collected from the First Affiliated Hospital of Nanchang University from November 2010 to May 2011, and purified by mirVana(TM) miRNA Isolation Kit and then labeled and hybridized by miRNA Complete Labeling and Hyb Kit. The images of hybridization were analyzed by the Feature Extraction (v10.7) software and the microarray results confirmed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In hyperplastic scar, 92 miRNA genes were up-regulated and 13 down-regulated. The most significantly up-regulated miRNAs were hsa-miR-564 and hsa-miR-936, etc. while hsa-miR-451, hsa-miR-223, hsa-miR-363 and hsa-miR-29b-1* became significantly down-regulated. The findings of RT-PCR on hsa-miR-21 and hsa-miR-451 of regulation were in a high concordance with the microarray results. CONCLUSION: Distinct differences of miRNA expression between human hyperplastic scar and normal skin, it may be closely correlated with the formation, development and evolution of hyperplastic scar.

Impairment characteristics of static balance and plantar load distribution of patients undergoing tibial cortex transverse distraction for diabetic foot ulcers.

OBJECTIVE: Tibial cortex transverse distraction (TCTD) has been recently reported for the treatment of diabetic foot ulcers. Herein, we explored the characteristics of the impairments in static balance and plantar load distribution in patients. METHODS: We performed a retrospective study of 21 patients with diabetic foot ulcers who underwent TCTD, who were regularly followed up for > 1 year after surgery, and 20 healthy individuals (control group). A pressure platform was used to assess the standing balance functions of the lower extremities and the plantar load distribution. RESULTS: One patient underwent amputation because of severe infection. In patient group, center of pressure (COP) ellipse sway area, COP path length and angle θ were all larger, compared with those of control group (250.15 ± 98.36 mm2 vs. 135.67 ± 53.21 mm2, 145.15 ± 67.43 mm vs. 78.47 ± 34.15 mm, 39.75 ± 17.61° vs. 22.17 ± 14.15°), with statistically significant differences (P < 0.01). The average plantar load and backfoot load of the unaffected side was significantly larger than that of the affected side (58.4 ± 5.5% vs. 41.6 ± 5.5%, 45.3 ± 6.4% vs. 36.5 ± 5.6%), but they were similar for the two feet of members of the control group. CONCLUSIONS: Although TCTD may represent an appropriate method for the treatment of diabetic foot ulcers, postoperative impairments in static balance and plantar load distribution remain in the long term. These potential long-term problems should be taken into account in further rehabilitation planning. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic III.

Mesenchymal stem cell-derived exosomes: An emerging therapeutic strategy for normal and chronic wound healing.

Skin wound healing is a complex biological process. Mesenchymal stem cells (MSCs) play an important role in skin wound repair due to their multidirectional differentiation potential, hematopoietic support, promotion of stem cell implantation, self-replication, and immune regulation. Exosomes are vesicles with diameters of 40-100 nm that contain nucleic acids, proteins, and lipids and often act as mediators of cell-to-cell communication. Currently, many clinical scientists have carried out cell-free therapy for skin wounds, especially chronic wounds, using exosomes derived from MSCs. This review focuses on the latest research progress on the mechanisms of action associated with the treatment of wound healing with exosomes derived from different MSCs, the latest research progress on the combination of exosomes and other biological or nonbiological factors for the treatment of chronic skin wounds, and the new prospects and development goals of cell-free therapy.

Identification of differentially expressed miRNAs associated with thermal injury in epidermal stem cells based on RNA-sequencing.

Current research indicates that epidermal stem cells (EpSCs) play an important role in promoting wound healing, but the mechanism of action of these cells during wound repair following thermal damage remains unclear. In the present study, the trypsin digestion method was used to isolate human EpSCs and the cells were incubated in a 51.5°C water tank for 35 sec to construct a thermal injury model. The differentially expressed miRNAs were identified using high-throughput sequencing technology, and bioinformatic methods were used to predict their target genes and signaling pathways that may be involved in wound repair. A total of 33 miRNAs including, hsa-miR-1973, hsa-miR-4485-3p, hsa-miR-548-5p, hsa-miR-212-3p and hsa-miR-4461 were upregulated, whereas 21 miRNAs including, hsa-miR-4520-5p, hsa-miR-4661-5p, hsa-miR-191-3p, hsa-miR-129-5p, hsa-miR-147b and hsa-miR-6868-3p were downregulated following thermal injury of the human EpSCs. The bioinformatic analysis indicated that the differentially expressed miRNAs are involved in biological processes such as cell proliferation and differentiation, cell growth apoptosis, cell adhesion and migration. The results showed that there is a differential expression pattern of miRNAs after thermal injury of human EpSCs and these differences are involved in the regulation of the wound healing process. These findings provide new clues for further study of the wound healing mechanism and targeted therapy.

Tibial cortex transverse distraction in treating diabetic foot ulcers: what are we concerned about?

OBJECTIVE: To assess the effect and complications of tibial cortex transverse distraction (TCTD) in treating diabetic foot ulcers and draw attention to the concerning issues of this procedure. METHODS: This case series included 30 patients with diabetic foot ulcers from four centers. The ulcers had not healed after >6 months. The patients then underwent TCTD combined with other procedures (debridement, vacuum sealing drainage, and others). All patients were followed up for >12 months postoperatively. RESULTS: Three patients underwent amputation because of aggravated infections. Tibial fractures occurred in two patients after surgery, and the fractures healed after 3 months of plaster fixation. Pin-site infections occurred in five patients, and the infected pin site healed after the patients underwent pin removal and dressing changes for 3.3 ± 2.1 weeks. The ulcers of the other 27 patients healed by 13.5 ± 8.2 weeks postoperatively, and the postoperative visual analog scale score was significantly lower than the preoperative score. CONCLUSIONS: Although TCTD can be performed as an adjuvant treatment for diabetic foot ulcers, the effect should not be exaggerated and the complications should not be ignored. Further research is needed to propose a standard operative procedure and avoid postoperative complications such as tibial fractures.

Study on the association between estrogen receptor gene (ESR) and reproduction traits in Landrace pigs.

To study the potential use of estrogen receptor gene (ESR) as a genetic marker to improve the reproductive traits of pigs, the genotypes of the ESR PCR product digested by Pvu II were determined in 2,239 litters from 612 Landrace sows. The data of the first, second, and later parities were separately evaluated. Although the frequency of the B allele was much lower than that of the A allele, likelihood ratio test showed that the gene frequencies were in Hardy-Weinberg equilibrium. The effects of ESR of different parities were not equal. In summary, the sows with the BB genotype showed better performance for total number of piglets born (TNB) and number of piglets born alive (NBA), but had a lower average piglet weight at birth (AWB). It was concluded that ESR could be used as a marker for the selection of litter size in the Landrace population.

Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus.

Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I collagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve fibers regenerated. Partial nerve fiber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats.

Genetic effects of IGF-1 gene on the performance in Landrace x Lantang pig resource population.

A Landrace x Lantang resource population (LL-SCAU) including 216 F2 pigs was founded by F2 design for analysis of IGF-1 gene polymorphism by PCR-RFLP. The least square means of divergent IGF-1 genotypes for the measured traits were estimated with the fixed model. The genetic effects of IGF-1 gene were estimated with a mixed model and the additive and dominant effects of IGF-1 gene were accordingly calculated. The results of the fixed model and the mixed model showed that IGF-1 locus significantly affected average daily gain after weaning. Average daily gain after weaning for AA genotype on IGF-1 locus was 20.58 g (P = 0.0347) higher than for AB. The additive and dominant effects attributed to IGF-1 were 1.78 g and -18.81 g respectively. IGF-1 locus also significantly affected carcass composition. Bone percentages for AA and AB on IGF-1 locus were lower than for BB by 5.22% (P = 0.0008) and 5.19% (P = 0.0007) respectively, and the additive and dominant effects were -2.61% and -2.58% respectively. Amount of carcass lean for AA on IGF-1 locus was less than for AB by 0.45 kg (P = 0.0264), and the additive and dominant effects were 0.16 kg and 0.61 kg respectively. Skin and fat percentage for AA and AB on IGF-1 locus was higher than for BB by 8.81% (P = 0.0206) and 7.64% (P = 0.0431) respectively, and the additive and dominant effects were 4.41% and 3.24% respectively. The genetic analysis of IGF-1 gene showed that IGF-1 locus significantly affected average daily gain after weaning, bone percentage, carcass lean, skin and fat percentage. The estimated additive and dominant effects showed that only the additive effect of IGF-1 locus on skin and fat percentage was much higher than dominant effect, and IGF-1 gene can be used as a major gene for effective selection of these traits.

[Founding and genetic analysis of Landrace x Lantang pig resource population for QTL mapping].

With F2 design, 16 Chinese Lantang sows crossbred with eight highly improved Landrace boars to establish a resource population including 40 F1 sows, eight F1 boars, and 232 F2 pigs. Genetic analysis of the resource population showed that the 32 performance traits displayed some degree of variation, and coefficients of variation of the majority of economic traits exceeded 10%. The variance component analysis revealed that the ratios of additive genetic variance to phenotypic variance of the majority of economic traits were high. Of the 22 microsatellite DNA markers, only 12 microsatellite DNA markers are polymorphisic in this populations. The average heterozygosity of these markers and PIC were 0.53 and 0.46 respectively. The results showed that these markers can provide enough information for QTL mapping. In conclusion,the F2 pigs were sufficiently segregated, and were capable of serving as a resource population for QTL mapping.

[Advances in the research of the role of MicroRNAs in wound healing].

MicroRNAs are endogenous noncoding RNA molecules with 19-22 nucleotides in length. MicroRNAs can post-transcriptionally regulate gene and (or) protein expression by binding to their target messenger RNAs (mRNAs), leading to mRNA degradation or suppression of translation. As a huge family that regulates gene expression, microRNAs has recently been shown to not only participate in the normal healing processes of wounds but also closely related to pathologic wound healing, and formation of hypertrophic scars and keloids. This review focuses on the biogenesis of microRNA and its role in wound healing.

[Effect of tetrandine on gene expression of collagen type I, collagen type III and TGF-beta1 in scar tissue's of rabbits ear].

OBJECTIVE: To observe the effect of tetrandine on gene expression of collagen type I, collagen type III, transformation growth factor-beta1 and to investigate the inhibitory effect of tetrandine on the scar tissue hyperplasia in rabbits' ears. METHODS: After the scar model was formed on the rabbits' ears, the rabbits were divided into 4 groups to receive intro-lesion injection with saline, or prednisolone (Pre) or tetrandrine in low concentration (L-Tet, 1.0 mg/ml) or tetrandrine in high concentration (H-Tet, 7.5 mg/ml). The morphological changes of scar tissue were observed. The changes of fibroblasts quantity and collagen expression were observed with HE and Masson staining. Immunohistochemical study was used to observe the expression level of collagen type I and collagen type III and TGF-beta1. Collagen type I and collagen type III and TGF-beta1, and signal factor Smad 3 mRNA were detected with RT-PCR. RESULTS: (1) 24 days after injury, all the wounds healed completely with formation of red, tough and hypertrophic scar. HE and Masson staining showed significant increase of fibroblasts and collagen density with irregularly arrangement. (2) Compared with that in saline group, the scar in other groups became softer, lighter and thinner, especially in H-Tet group. (3) HE and Masson staining shows the scar in Tet and Pre groups contained less fibroblasts and lower collagen dentsity with comparatively regular arrangement than that in saline group (P < 0.01), especially in H-Tet group. (4) According to the immunohistochemical study, the expression of collage type I and III and TGF-beta was positive in all the groups, but the positive rate and the ratio of collagen density I to III decreased in the order of saline, L-Tet, H-Tet and Pre groups (P < 0.01). (5) PT-PCR detection results showed that the amplification bands brightness of collagen type I and III and TGF-beta1 and signal molecular Smad 3 mRNA in scar tissue were obviously different. Compared with that in saline group, the expression of collagen type I and III and TGF-beta1 and Smad 3 mRNA decreased in Tet and Pre groups (P < 0.01). H-Tet group showed the most obvious reduce in the expression of type I collagen and TGF-beta1 and Smad 3 mRNA. Conclusions Tetrandine can significantly suppress the expression of collagen type I and collagen type III and TGF-beta1 on hypertrophic scar of rabbit ears, and reduce signal factor Smad 3 mRNA' s expression. It may be one of the important mechanism for its inhibitory effect on scar hyperplasia.

[Effect of substance P combined with epidermal stem cells on wound healing and nerve regeneration in rats with diabetes mellitus].

OBJECTIVE: To observe the effect of sensory neuropeptide substance P combined with epidermal stem cells (ESC) on wound healing and nerve regeneration in diabetic rats. METHODS: ESC that had been isolated from SD rats were identified and cultured in vitro, and they were inoculated onto nourishing layer of amniotic membrane to construct amniotic membrane-ESC. Four full-thickness skin wounds were produced on the back of each of 48 diabetic rats. The resulted 192 wounds were randomly divided into ESC + substance P group, ESC group, substance P group, and control group according to the lottery method, with 48 wounds in each group. Wounds in ESC + substance P group and ESC group were transplanted with amniotic membrane-ESC, and those in substance P group and control group were transplanted with amniotic membrane. After transplantation, 250 µL substance P in the concentration of 1 × 10(-7) mol/L was injected around and into the middle of the wounds in ESC + substance P group and substance P group, 2 times a day, and continued for 4 days, while 250 µL PBS solution was injected in the above-mentioned position in ESC group and control group as control, 2 times a day, and continued for 4 days. On post injury day (PID) 4, 7, 10, 14, 17, and 23, the wound healing rate (with 8 wounds at each time point) was observed and determined, and changes in wound tissue structure were observed with HE staining. On PID 4, 7, and 10, collagen distribution in wound tissue was observed with Masson staining, and type I and type III collagen deposition in wound tissue was respectively observed after immunohistochemical staining. The distribution of protein gene product 9.5 (PGP 9.5) and regeneration of substance P positive nerve fibers in wound tissue were observed with immunohistochemical staining on PID 14 and 23. Data were processed with one-way analysis of variance and t test. RESULTS: (1) The wound healing rate in ESC + substance P group reached 100.0% on PID 14, which was obviously earlier than that in ESC group, substance P group, and control group, healing was respectively observed on PID 17, 17, and 23. The wound healing quality in ESC + substance P group was better than that in the other three groups as shown by HE staining. (2) On PID 10, collagen that was darkly stained and widely distributed was observed in wound tissue of ESC + substance P group and substance P group, while collagen in the other two groups was lightly stained and narrowly distributed. Deposition quantity of type I collagen gradually increased, and that of type III collagen gradually decreased in the wounds of each group over time. On PID 4, 7, and 10, distribution amount of type I collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.72, 118.21, 26.71, P values all below 0.01) and control group (with t value respectively 44.37, 22.76, 30.32, P values all below 0.01), while there was no significance between ESC + substance P group and substance P group. On PID 4, 7, and 10, distribution amount of type III collagen in wound tissue of ESC + substance P group was significantly higher than that in ESC group (with t value respectively 32.27, 28.68, 14.51, P values all below 0.01) and control group (with t value respectively 35.68, 22.52, 22.24, P values all below 0.01). (3) A large amount of PGP 9.5 and regeneration of substance P positive nerve fibers, and some peripheral nerve fibers in deep wound extending to epidermis were observed in wound tissue of ESC + substance P group and substance P group. A small amount of PGP 9.5 and regeneration of substance P positive nerve fibers without peripheral nerve fibers extending to epidermis were observed in deep wound tissue of ESC group and control group. On PID 14, 23, ratios of area of PGP 9.5 positive nerve fiber in the wounds of ESC + substance P group were (3.86 ± 0.25)% and (7.03 ± 0.28)%, and they were significantly higher than those of ESC group [(1.48 ± 0.30)%, (3.01 ± 0.43)%, with t value respectively 23.95, 30.27, P values all below 0.01] and control group [(1.46 ± 0.23)%, (2.84 ± 0.29)%, with t value respectively 27.35, 40.32, P values all below 0.01]. On PID 14, 23, ratios of substance P positive nerve fiber area in the wounds of ESC + substance P group were (2.01 ± 0.14)% and (1.19 ± 0.11)%, which were obviously higher than those of ESC group [(0.85 ± 0.17)%, (1.34 ± 0.21)%, with t value respectively 20.50, 2.60, P < 0.05 or P < 0.01] and control group [(0.74 ± 0.15)%, (1.30 ± 0.17)%, with t value respectively 23.98, 2.41, P < 0.05 or P < 0.01]. CONCLUSIONS: Joint application of substance P and ESC can effectively promote healing of wound and nerve regeneration in diabetic rats.

[Experimental study on the differentiation of human induced pluripotent stem cells into epidermal-like stem cells].

OBJECTIVE: To investigate the feasibility of differentiation of human induced pluripotent stem cells (iPSCs) into epidermal-like stem cells. METHODS: (1) Human strain of iPSCs were plated on-to trophoblast of inactivated Fb strain of mouse embryos and cultured in complete medium of embryonic stem cells, iPSCs were subcultured by collagenase IV digestion method. The morphology and growth of iPSCs were observed under inverted phase contrast microscope, and the cells were stained with alkaline phosphatase (AKP). iPSCs were cultured in incomplete medium of embryonic stem cells to observe the ability of embryoid body formation. (2) Human iPSCs were inoculated onto 6-well plate covered with human amniotic membrane to culture as induction group. Other iPSCs were cultured on 6-well plate without human amniotic membrane as control group. Morphological changes in iPSCs in two groups were observed. Expressions of integrin beta1 and CK19 of iPSCs in two groups were determined by immunocytochemical staining. RESULTS: Human iPSCs showed a typical stem cell clone-like growth with a clear boundary, and they proliferated vigorously in complete medium of embryonic stem cells. These cells were AKP-positive. iPSCs formed embryoid body in trophoblast-free and suspension culture conditions. After 4 days of co-culture, stem cell clones were formed on the surface of amniotic membrane in induction group, and part of the cells were integrin beta1 and CK19 positive. Most of the cells died, and no integrin beta1 and CK19 positive cells were found in control group. CONCLUSIONS: Human iPSCs can be differentiated into epidermal-like stem cells by amniotic membrane induction, and it lays an experimental basis for providing new source of seed cells of skin tissue engineering.