[Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats].

Objective: To prepare the chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and to explore its effects on wound healing of full-thickness skin defects in rats. Methods: The research was an experimental research. Chitin nanofibers were prepared by acid hydrolysis and alkaline extraction method, and then mixed with hyaluronic acid and collagen to prepare chitin/hyaluronic acid/collagen hydrogels (hereinafter referred to as hydrogels). Besides, the hydrogels loaded with mouse adipose-derived stem cells were prepared. Thirty male 12-week-old guinea pigs were divided into negative control group, positive control group, and hydrogel group according to the random number table, with 10 guinea pigs in each group. Ethanol, 4-aminobenzoic acid ethyl ester, or the aforementioned prepared hydrogels without cells were topically applied on both sides of back of guinea pigs respectively for induced contact and stimulated contact, and skin edema and erythema formation were observed at 24 and 48 h after stimulated contact. Adipose-derived stem cells from mice were divided into normal control group cultured routinely and hydrogel group cultured with the aforementioned prepared hydrogels without cells. After 3 d of culture, protein expressions of platelet-derived growth factor-D (PDGF-D), insulin-like growth factor-Ⅰ (IGF-Ⅰ), and transforming growth factor ß1 (TGF-ß1) were detected by Western blotting (n=3). Eight male 8-week-old Sprague-Dawley rats were taken and a circular full-thickness skin defect wound was created on each side of the back. The wounds were divided into blank control group without any treatment and hydrogel group with the aforementioned prepared hydrogels loaded with adipose-derived stem cells applied. Wound healing was observed at 0 (immediately), 2, 4, 8, and 10 d after injury, and the wound healing rate was calculated at 2, 4, 8, and 10 d after injury. Wound tissue samples at 10 d after injury were collected, the new tissue formation was observed by hematoxylin-eosin staining; the concentrations of interleukin-1α (IL-1α), IL-6, IL-4, and IL-10 were detected by enzyme-linked immunosorbent assay method; the expressions of CD16 and CD206 positive cells were observed by immunohistochemical staining and the percentages of positive cells were calculated. The sample numbers in animal experiment were all 8. Results: At 24 h after stimulated contact, no skin edema was observed in the three groups of guinea pigs, and only mild skin erythema was observed in 7 guinea pigs in positive control group. At 48 h after stimulated contact, skin erythema was observed in 8 guinea pigs and skin edema was observed in 4 guinea pigs in positive control group, while no obvious skin erythema or edema was observed in guinea pigs in the other two groups. After 3 d of culture, the protein expression levels of PDGF-D, IGF-I, and TGF-ß1 in adipose-derived stem cells in hydrogel group were significantly higher than those in normal control group (with t values of 12.91, 11.83, and 7.92, respectively, P<0.05). From 0 to 10 d after injury, the wound areas in both groups gradually decreased, and the wounds in hydrogel group were almost completely healed at 10 d after injury. At 4, 8, and 10 d after injury, the wound healing rates in hydrogel group were (38±4)%, (54±5)%, and (69±6)%, respectively, which were significantly higher than (21±6)%, (29±7)%, and (31±7)% in blank control group (with t values of 3.82, 3.97, and 4.05, respectively, Pvalues all <0.05). At 10 d after injury, compared with those in blank control group, the epidermis in wound in hydrogel group was more intact, and there were increases in hair follicles, blood vessels, and other skin appendages. At 10 d after injury, the concentrations of IL-1α and IL-6 in wound tissue in hydrogel group were significantly lower than those in blank control group (with tvalues of 8.21 and 7.99, respectively, P<0.05), while the concentrations of IL-4 and IL-10 were significantly higher than those in blank control group (with tvalues of 6.57 and 9.03, respectively, P<0.05). The percentage of CD16 positive cells in wound tissue in hydrogel group was significantly lower than that in blank control group (t=8.02, P<0.05), while the percentage of CD206 positive cells was significantly higher than that in blank control group (t=7.21, P<0.05). Conclusions: The hydrogel loaded with mouse adipose-derived stem cells is non-allergenic, can promote the secretion of growth factors in adipose-derived stem cells, promote the polarization of macrophages to M2 phenotype in wound tissue in rats with full-thickness skin defects, and alleviate inflammatory reaction, thereby promoting wound healing.

[Clinical analysis of 4 children with hereditary hypercholesterolemia].

Objective: To investigate the clinical characteristics of hereditary hypercholesterolemia in childhood. Methods: The clinical data including general conditions, clinical manifestations, laboratory tests, and genetic testing results of 4 children with hereditary hypercholesterolemia who admitted to Henan Children's Hospital from January 2020 to December 2020 were retrospectively analyzed. Results: There were 4 female children aged 5.5,1.5,6.3,3.1 years, all presented with skin xanthoxoma as the chief complaint. Plasma total cholesterol (range 11.8 to 20.9 mmol/L) and low density lipoprotein-cholesterol (range 8.2 to 13.7 mmol/L) were significantly elevated. The serum ß-glutamate levels in case 1 (241.2 µmol/L) and case 2 (164.2 µmol/L) increased significantly. Genetic analysis revealed compound heterozygous variants of ABCG8 gene in case 1 and ABCG5 gene in case 2 who were diagnosed with sitosterolemia. Case 3 and 4 who all had family history of hypercholesterolemia and compound heterozygous variants of LDLR gene were diagnosed with familial hypercholesterolemia. After diet treatment, the blood lipids returned normal and the skin xanoma subsided in case 1 and 2. In case 3 and 4, the blood lipids gradually decreased after diet and rosuvastatin treatment. Conclusions: Xanthomatosis is the common clinical manifestation of sitosterolemia and familial hypercholesterolemia. Family history, blood plant sterol profile, genetic variation, and changes in blood lipids after early dietary treatment are helpful for disease identification.

miR-182 selectively targets HOXA10 in goat endometrial epithelium cells in vitro.

Proper HOXA10 expression was essential for endometrial receptivity what was crucial for successful embryo implantation in mammalian. This study confirmed that miR-182 regulated the expression levels of HOXA10 by binding to its 3' UTR, selectively downregulated HOXA10 in goat endometrial epithelium cells (gEECs) but not stromal cell (gESCs) in vitro. However, HOXA10 and miR-182 both up-expressed in the goat endometrium at gestational day 15 (D15) compared with gestational day 5 (D5), suggesting that there were some other factors regulated the expression of HOXA10 during the development of goat endometrium in vivo. What's more, HOXA10 gene silencing (HOXA10-siRNA) resulted in gEECs apoptosis in vitro, and it regulated the protein levels of oestrogen receptor a (ERa), progesterone receptor B (PRb), insulin-like growth factor 1 receptor (IGF1R), BCL-2, pleiotrophin (PTN), AKT and p-JNK in gEECs. Furthermore, HOXA10 might regulate the protein levels of endometrial receptivity biomarker genes, including vascular endothelial growth factor (VEGF), osteopontin (OPN), cyclooxygenase-2 (COX-2) and prolactin receptor (PRLR) in gEECs. In conclusion, miR-182 targeted HOXA10 selectively in EECs in vitro, and HOXA10 played an important role in maintaining the function of EECs in dairy goats.

Polymorphism and DNA methylation in the promoter modulate KISS1 gene expression and are associated with litter size in goats.

Polymorphisms in the promoter region are likely to impact KISS1 gene transcription and reproductive traits. In this study, Guanzhong (GZ, n=350) and Boer (BE, n=196) goats were used to detect polymorphism in the promoter of the goat KISS1 gene by DNA sequencing. In the GZ goats, the g.1384G>A mutation was identified in the promoter of the goat KISS1 gene. Guanzhong goats were in Hardy-Weinberg disequilibrium at g.1384G>A locus (P<0.05). The 1384A allele was predicted to eliminate methylation, AHR-arnt heterodimers and AHR-related factors (AHRR) and myoblast determining factors (MYOD) transcription factor-binding sites. Statistical results indicated that the g.1384G>A SNP was associated with litter size in the GZ goats (P<0.05). Luciferase assay analysis suggested that the 1384A allele increased luciferase activity when compared to the 1384G allele. The RT-qPCR assay also demonstrated that the 1384A allele had greater amounts of KISS1 mRNA than the 1384G allele in homozygous individuals. Functional analysis suggested that this g.1384G>A SNP may be an important genetic regulator of KISS1 gene expression with effects on downstream processes that are modulated by KISS1 gene because of the changes of methylation and transcription factor-binding sites. Therefore, the current study provides evidence in goats for genetic markers that might be used in breeding programs.

Polymorphisms of PRLR and FOLR1 genes and association with milk production traits in goats.

We investigated the polymorphisms of PRLR and FOLR1 genes in Xinong Saanen, Guanzhong, and Boer goat breeds by DNA sequencing and PCR-RFLP. Two novel SNPs were identified: KC109741: g.62130C>T in the 3ꞌ-UTR of goat gene PRLR, and KC136296: g.7884A>C in exon 3 of goat gene FOLR1. In the three goat breeds, the polymorphism information content was 0.20-0.27 at the g.62130C>T locus. At the g.7884A>C locus, it was 0.36 in Boer goats. The three goat breeds were in Hardy-Weinberg disequilibrium at the g.62130C>T locus. The g.62130C>T SNP was found to be significantly associated with milk production traits in Xinong Saanen and Guanzhong breeds. These results are consistent with the regulatory function of PRLR in mammary gland development, milk secretion, and expression of milk protein genes; they extend the spectrum of genetic variation of the goat PRLR gene, which could be useful for breeding programs.

Molecular cloning and characterization of KISS1 promoter and effect of KISS1 gene mutations on litter size in the goat.

Kisspeptins, the product of the KISS1 gene, play an essential role in the regulation of reproductive functions, acting primarily at the hypothalamic level of the gonadotropic axis. We detected polymorphisms of the goat KISS1 gene in 723 individuals from three goat breeds (Xinong Saanen, Guanzhong, and Boer) by DNA pooling, PCR-RFLP, and DNA sequencing methods. We cloned the promoter sequence of this gene and found it to share high similarity with that of the bovine KISS1 promoter. Six TATA boxes were found in the goat KISS1 promoter region. Two novel SNPs (g.2124T>A and g.2270C>T) were identified in the intron 1 of the KISS1 gene of all three goat breeds. The three goat breeds were in Hardy-Weinberg disequilibrium at g.2124T>A and g.2270C>T loci. The g.2124T>A and g.2270C>T loci were closely linked in the three goat breeds (r2 > 0.33). The g.2124T>A and g.2270C>T SNPs were significantly associated with litter size, and the C1 female goats had a larger litter size than did those with the other genotypes. These results extend the spectrum of genetic variation of the goat KISS1 gene, which contributes to our knowledge of goat genetic resources for breeding programs.

Polymorphism identification in goat GNRH1 and GDF9 genes and their association analysis with litter size.

This study investigated the polymorphisms of GNRH1 and GDF9 genes in 641 goats of three breeds: Xinong Saanen, Guanzhong and Boer. Two allelic variants were identified in the GNRH1 gene (JN645280:g.3548A>G and JN645281:g.3699G>A) and one allelic variant was found in the GDF9 gene (JN655693:g.4093G>A). Furthermore, g.4093G>A was a missense mutation (p.Val397Ile of GDF9). Results of an association analysis indicated that SNPs g.3548A>G and g.4093G>A had significant effects on litter size (P < 0.05). The combination genotypes of SNPs g.3548A>G, g.3699G>A and g.4093G>A also affected litter size with the C5 genotype having the highest litter size in the first, third, fourth and average parity. Hence, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the GNRH1 and GDF9 genes could serve as genetic markers for litter size in goat breeding.

A new approach for cloning hLIF cDNA from genomic DNA isolated from the oral mucous membrane.

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.