The aim of this study was to establish IPEC-J2 cell lines with CD13 gene knockout by CRISPR/Cas9 genome editing technique and to reveal the role of cluster of differentiation 13 (CD13) in the porcine intestinal cells infected by pathogenic microorganisms. In this study, two guide RNAs (g3 and g5) were designed corresponding to the 2nd exon region of CD13 gene. The g3 and g5 were cloned into the pX330-GFP and pX330-RFP vector backbones, respectively. After 48 hours of transfection, cells with dual fluorescent labeling were sorted out by flow cytometry. Single cell clones were cultured, amplified by PCR, and then sequenced to obtain CD13 gene knockout positive cells. The results of PCR and sequencing of the obtained 20 cell clones showed that a total of 7 clones had fragment deletions, including one clone with a single allele fragment deletion, 3 clones with biallelic heterozygous fragment deletions, and 3 clones with biallelic homozygous fragments deletions. The protein expression levels of the biallelic homozygous fragment knockout cells were detected, and the results showed that CD13 protein was hardly expressed in the homozygous knockout cells, which indicated that IPEC-J2 cell lines with the CD13 knockout was successfully constructed. In this study, we obtained the CD13 knockout cell lines, which provided the foundation for studying the role of CD13 in the porcine intestinal cells infected by pathogenic microorganisms.

To understand the structure, function and transcriptional regulation mechanism of Nrf2 gene, six healthy Dapulian pigs, about 90 kg, were selected as experimental animals. The full cDNA sequence and promoter region of Nrf2 gene were cloned by RACE (rapid amplification of cDNA end) and Genome Walking techniques. Biological characteristics of Nrf2 gene and its promoter region were analyzed by bioinformatics softwares. The results showed that the full cDNA sequence of Nrf2 was 2 358 bp, containing 87 bp sequence of 5'UTR, 495 bp sequence of 3'UTR, and 1 776 bp sequence of coding region (CDS) encoded 591 amino acids. The protein molecular weight was 66.402 7 ku, and the isoelectric point (pI) was 4.66. The secondary structure was mainly consisted of α-helix. 2 091 bp DNA sequence of 5' flanking region of Nrf2 gene in Dapulian pig was obtained by the Genome Walking technique. The predicted 5' flanking sequence of the Nrf2 gene contained typical TATA-box, excluding the CpG island. A series of pGL3-Basic expression vectors with different promoter lengths were established. The promoter relative luciferase activities were measured by the dual-luciferase assay system after transient transfection into 293T cells. The results revealed that the region of -903—-710 bp had the highest activity, suggesting that the region of -903—-710 bp existed positive regulatory region or enhancer. Several transcriptional factor binding sites were predicted in this region by bioinformatics methods, which might be involved in Nrf2 gene transcriptional regulation. The complete cDNA sequence and promoter region of Nrf2 gene were successfully obtained. It was found that the region of -903—-710 bp was the core promoter. This study provides a theoretical basis for the genetic improvement of antioxidant stress in pigs.

The aim of this study was to explore the expression profile of HMGA1 gene in different tissues and at different ages in different pig breeds. Additionally, the association between the polymorphisms of the coding region and 3'UTR region and backfat thickness in pigs were analyzed. In this study, real-time quantitative PCR was used to detect the difference in mRNA expression of HMGA1 gene in 7 tissues of Large White, and in backfat of Large White and Minzhu at 60-, 150- and 210-day-old. The SNPs in the coding region and 3'UTR region of HMGA1 were detected by DNA re-sequencing in 576 Large White×Minzhu F2 generation resource populations. Then the association analysis between SNPs and backfat thickness (6-7 ribs) were performed with the slaughter weight as a covariate. The results showed that HMGA1 mRNA was widely expressed in various tissues of Large White, and significant difference was showed among different tissues (P<0.05). The expression of HMGA1 mRNA was the highest in the lung, but it was slightly expressed in the heart and muscle tissues. At 60- and 150-day-old, the expression of HMGA1 gene in backfat of Minzhu was significantly higher than that in the Large White (P<0.05), but the difference was not significant at 210-day-old between pig breeds(P>0.05). Thirteen single nucleotide polymorphism (SNP) sites were detected in the coding region of HMGA1, including 2 missense mutations sites:g.3261G>A and g.5818G>A, and the g.3261G >A was significantly associated with backfat thickness (P<0.05). Three SNPs were detected in the 3'UTR region as targets for the binding of microRNAs (miRNAs), the g.7217G>C and g.7280T>C were significantly associated with the backfat thickness(P<0.05). The above results indicate that HMGA1 gene has an effect on the backfat deposition of pigs, and the g.3261G>A, g.7217G>C and g.7280T>C loci can be used as potential molecular markers for the breeding of backfat thickness in pigs, which lay a foundation for the molecular breeding of pigs.

This study aimed to predict and validate the key miRNAs that regulated SCD1 gene expression in ovine subcutaneous adipocytes. miRNAs targeting SCD1 were predicted by bioinformatics softwares of TargetScan, mirDB, StarBase and miRanda. The dual luciferase reporter system was used to verify the target relationships between SCD1 gene and miRNAs. Fluorescent quantitative PCR and Western-blotting techniques were used to detect the effect of miRNAs on SCD1 expression at mRNA and protein levels. The prediction results indicated that SCD1 gene was targeted by both miR-200c and miR-429. Dual luciferase reporter system assay demonstrated that there were target relationships between the 2 miRNAs and SCD1 gene. Fluorescent quantitative PCR analysis showed that miR-200c and miR-429 significantly inhibited SCD1 mRNA expression (P<0.05), and miR-200c was more effective. Western-blotting results showed that miR-200c and miR-429 reduced SCD1 protein level (P<0.01). Microscopical observation showed that miR-200c and miR-429 inhibited ovine subcutaneous lipid droplet formation. In conclusion, miR-200c and miR-429 can down-regulate SCD1 gene expression, thus inhibit ovine lipogenesis.

The aim of this study was to identify the promoter of miR-150 gene and explore the effect of reproductive hormone on its transcription regulation in sheep. The miR-150 promoter region of ovary in female sheep (Small-tail Han sheep) and the reproduction-related transcription factor binding site were predicted by biological softwares, then recombinant plasmids were constructed and the dual luciferase reporter gene was used to detect the activity of miR-150 promoter. Moreover, the miR-150 promoter recombinant plasmid was transfected and miR-150 promoter activity was detected by adding FSH, LH and E₂ in serum-free medium. The miR-150 promoter region and transcription initiation site were respectively predicted using Promoter 2.0 and PROMO, and transcription factor binding sites associated with reproduction were obtained. The predicted length of the sheep miR-150 promoter fragment was 582 bp, and the miR-150 promoter plasmid was successfully constructed. The optimal transfection efficiency (liposomes:plasmid DNA=1:1) of the sheep granulosa cells and the optimal transfection proportion (50:1) of the plasmid were determined. Thus, the predicted region with significant promoter activity was verified. In addition, the activity of the miR-150 promoter was significantly down-regulated in the FSH, LH and E₂ treatment groups compared with the control group (hormone-free group) (P<0.05). The results indicated that reproductive hormone could regulate the expression of miR-150 by affecting the miR-150 promoter activity, which provided a new idea for studying the mechanism of miR-150 regulating ovine follicular development.

The study aimed to explore the effects of melatonin on the periodic expression of let-7 family and its target genes in skin of Inner Mongolia Cashmere goat. In this study, six Inner Mongolia Hanshan Cashmere goats were selected and divided equally and randomly into 2 groups, melatonin implantation group and non-implantation group. Taking the skin tissue of the goats as experimental material in every months of the year. The expression changes of 11 members in let-7 family were quantified by reverse transcription-real time polymerase chain reaction (qRT-PCR) in the skin tissue. The expression changes of the target genes of let-7 family were analyzed using the RNA-Seq technique. The results showed that:1) During the whole hair follicle cycle, melatonin up-regulated the expression of let-7b, let-7e and let-7a-3p, down-regulated the expression of let-7f, let-7g, let-7c, let-7a-5p, let-7d and miR-98. 2) In April, melatonin up-regulated the expression of "let-7g, let-7i and miR-98", down-regulated the expression of target genes such as PTGS2 and PLCG1. It down-regulated the expression of "let-7a-5p, let-7b and let-7c", up-regulated the expression of target genes such as TGIF2 and RBL1. 3) In June, melatonin down-regulated the expression of "let-7g, let-7i and miR-98", up-regulated the expression of target genes such as PTGS2, PLCG1 and SRC; It up-regulated the expression of "let-7a-5p, let-7b and let-7c", down-regulated the expression of target genes such as GDF5, TGIF2 and RBL1. 4) The target genes of let-7 family were mainly involved in VEGF, TGF-β, Oxytocin and NF-kappa B signaling pathways, and played an important role in cell processes, hormone regulation and regulation of transcription factors. In summary, MT may affect the expression of the corresponding target genes by changing the expression level of members in the let-7 family. It plays an important role in the periodic growth of skin hair follicles. These results provided a theoretical basis for further revealing the molecular mechanism of melatonin mediating miRNAs to affect cashmere growth.

The aim of this study was to screen the signaling pathways and candidate genes related to goat reproduction of hypothalamus tissue of Henan Huai goats under different illumination conditions by transcriptome sequencing and bioinformatics analysis. The transcriptome sequencing of 8 infecund ewes hypothalamus tissue (4 in each group, one year old) treated with natural light (8 h light:16 h dark) and artificial light (16 h light:8 h dark) were performed by using a second generation high throughput sequencing platform(NGST), and PE150 sequencing strategy. The total number of unigene assembled was compared to the reference genome sequence, differentially expressed genes(DEGs) analysis, Go and KEGG enrichment analysis and candidate gene screening were performed. The relative expression of candidate genes was analyzed by Real-time PCR. The results showed that about 440 million reads were obtained by RNA-Seq, and the average reads number per sample was about 52.49 million. Four hundred and forty-eight differentially expressed genes were obtained by DESeq analysis between natural and artificial illumination groups, which were enriched in 241 signaling pathways in KEGG database, including 5 reproductive-related signaling pathways. Three candidate genes of tachykinin family(TACR1,TACR2 and TACR3) were significantly enriched in the calcium signaling pathway (chx04020). The results of Real-time PCR analysis showed that the sequencing results of the transcriptome were reliable. These results suggest that calcium signaling pathway (chx04020) and TACR1,TACR2 and TACR3 genes may play the important role in goat reproduction.

The aim of this study was to analyze the expression level of miRNAs in Hu sheep ovaries at different developmental stages and to understand the expression of miRNAs and their regulatory mechanism. In this study, RNA-seq was used to identify miRNAs in ovarian tissues of 1 month and 8 months old Hu sheep. Bioinformatics methods were used to predict target genes of differentially expressed miRNAs. GO and KEGG functional annotations were performed on target genes to screen miRNAs and biological processes involved in ovarian development. The results showed that the numbers of known miRNAs expressed in the 6 samples were 2 186, 2 091, 2 217, 2 136, 2 176 and 2 088, respectively. The number of novel miRNAs was 613, 567, 668, 640, 662 and 450, respectively. 701 novel differentially expressed miRNAs and 38 known differentially expressed miRNAs were identified. GO and KEGG analysis of genes targeting to differentially expressed miRNAs revealed that they mainly were enriched in vascular endothelial growth factor pathway and ovarian steroidogenesis. The qRT-PCR verification result of 3 differential miRNAs was consistent with the sequencing results. Target genes of oar-let-7b, oar-miR-29a, oar-miR-134-3p, oar-miR-541-3p were enriched in the biological processes associated with ovarian development such as TGF-beta signaling pathway and ovarian follicular growth. The target genes of oar-miR-148a and oar-miR-136 regulate granulosa cell proliferation and steroidogenesis. It's inferred that they are involved in the regulation of ovarian development in sheep.

In order to improve the detection rate of returning estrous cows via pedometer, the effects of season, parity and time of returning estrus on activity of breeding cows were investigated. The returning estrus situation and activity change of 214 multiparous cows after artificial insemination were monitored by using pedometer, and the returning estrous cows were confirmed by rectal examination. Afterwards, the activity before and during returning estrus of returning estrous cows were statistically analyzed. Statistical analysis result showed that the proportion of returning estrous cows in the first, second, third and fourth estrous cycle after artificial insemination was 48.7%, 29.9%, 16.2%, 5.1%, respectively. The activity before and during returning estrus showed a downtrend with the increase of parity and time of returning estrus as a whole. Simultaneously, the duration of returning estrus also gradually decreased with the prolongation of pregnancy, and the activity of cows during returning estrus was significantly decreased in summer (P<0.05). Cow's reproduction should be paid more attention during the first and second estrous cycle after artificial insemination. In addition to parity, seasons and time of returning estrus must be considered comprehensively, the duration of returning estrus should also be paid special attention when the threshold is set, so that the automatic monitoring of returning estrous cows can be carried out scientifically by using activity parameter.

This experiment was conducted to investigate the variation of digestive enzyme activities in the simulated gastric and small intestinal digestion for pigs, which will provide a reference to develop the simulated digestion method for determining nutrients digestibility for pigs. In experiment 1, a single factor completely randomized design was used to investigate the variation of digestive enzyme activities depending on digestion time when the simulated gastric and small intestinal fluid were injected once during the simulated digestion for corn, soybean meal, wheat bran and corn-soybean meal. The digestion time of 0, 1, 2, 3 and 4 h was arranged for gastric digestion phase. The specific and total activities of pepsin in the digestive fluid were determined. The digestion time of 0, 2, 4, 6, 8, 12 and 16 h was arranged for the small intestine digestion phase. The specific and total activities of trypsin, chymotrypsin and amylase in the digestive fluid were determined. In experiment 2, the digestive enzymes were supplemented at 4 h of small intestine digestion phase according to the results of experiment 1. The digestive enzyme activities were determined at 4, 6 and 8 h of small intestine digestion phase after supplementing digestive enzymes. Each in vitro digestion time was considered as a treatment, and each treatment contained 5 replicates with 1 digestion tube per replicate. The results showed that:1) When the simulated gastric fluid was mixed with corn, soybean meal, wheat bran or corn-soybean meal, the specific activity of pepsin in the digestive fluid was rapidly reduced (accounting for 28.8% to 59.1% of initial activity). However, from 1 to 4 h of the simulated digestion, the specific and total activities of pepsin were increased with the quadratic curve(P<0.05). The final values were 78.0% to 96.1% relative to initial specific activity and 87.2% to 102.7% relative to initial total activity of simulated gastric fluid, respectively. In the small intestine digestion phase, different extent of reduction of activities of amylase, trypsin and chymotrypsin in simulated digestive fluid was observed (P<0.05). The quadratic or non-linear curve variation of amylase and chymotrypsin activity in digestive fluid was observed with different substrates(P<0.05). The reduction of trypsin activity in digestive fluid with quadratic curve was observed (P<0.05). The greatest reduction of activity was observed in trypsin. 2) After the digestive enzymes were supplemented in simulated small intestinal digestion, the specific activities of amylase, trypsin and chymotrypsin in the digestive fluid could be recovered to the initial value, but they still decreased with the digestion time(P<0.05), among them, the trypsin activity still decreased rapidly. In conclusion, in the process of simulated digestion, there is no need to supplement pepsin in the gastric digestion stage; the digestive enzyme needs to be supplemented in the small intestine digestion stage.

The aim of this study was to investigate the effect of broussonetia papyrifera L. (Paper mulberry) silage on growth performance, biochemical indexes of serum and fatty acid composition in the longissimus dorsi muscle of Dorper×Thin-tailed Han crossbred sheep. Ninety-six healthy sheep weighted (26±2.5) kg on 3 months old were allocated into 4 groups with 24 sheep per group, 12 male sheep and 12 female sheep in each group. The sheep in control group (A) were fed the diet without Paper mulberry silage, the sheep in experimental group (B, C and D) were fed the diets with 15%, 30% and 45% (dry matter basis) Paper mulberry silage, respectively. The experiment lasted for 70 d. The results showed that:1) Paper mulberry silage was very palatable to sheep, increased dry matter intake. The average daily gain (ADG) and dry matter intake (DMI) of D group were significantly higher than that of A and B groups (P<0.05); 2) Compared with control group, Paper mulberry silage promoted the immunity and antioxidation capacity of sheep, the content of IgA, IgG and IgM in serum of D group were significantly higher than those of A and B groups (P<0.05), the total antioxidant capacity (T-AOC) and activity of catalase (CAT) of serum in D group were significantly higher than those in A group (P<0.05), the content of malondialdehyde (MDA) of D group was significantly lower than that of A group (P<0.05); 3) The addition of Paper mulberry silage to the diet increased the content of n-3 polyunsaturated fatty acids (n-3 PUFA) in the longissimus dorsi muscle of sheep, and the content of n-3 PUFA of B,C,D groups was significantly higher than that of A group (P<0.05). The results showed that Paper mulberry silage supplementation might improve the growth performance, immunity and antioxidant capacity of sheep, was good for the health of sheep, meanwhile, the content of n-3 PUFA in the longissimus dorsi muscle of sheep increased and the fatty acids composition in muscle was improved. D group had the most significant effects in the 3 silage addition groups.

The helicase family is a class of enzymes that are essential to all living organisms. The main function of the helicase family is gene unwapping. The DEAD/H-box family RNA helicase is a kind of important helicase. DEAD-box RNA helicase DDX21 has been proved that it may regulate innate immunity of host cells. Newcastle disease virus (NDV) is a kind of pathogen which is harmful to poultry industry. NDV can regulate the innate immunity of the host by various means. In order to verify how DDX21 regulates NDV replication, this study used CRISPR/Cas9 system to establish a DDX21 knockout HeLa cell line, and to verify its effect on NDV infection. Six sgRNAs were designed to construct knockout vectors for DDX21 gene and electrotransfected into HeLa cells. After drug screening and subcloning, the knockout efficiency was identified by PCR and Western blot, and the replication efficiency of NDV in wild type and DDX21 knockout cells was compared. The results showed that only one DDX21 heterozygote knockout cell line was obtained because DDX21 is critical for cell growth. Western blot results showed that the expression of DDX21 was significantly inhibited. The infection ability of NDV to knockout cells was significantly lower than that of wild type cells, indicating that DDX21 played a positive role in regulating NDV replication. The research above laid a foundation for the study of DDX21 regulating antiviral innate immunity.

In present study we investigated the effects of porcine pseudorabies virus (PRV) infection on proliferation and heat shock protein 27,70 and 90 expression of host cells in vitro. PK-15 cells were infected with different amounts of PRV-YY strain after the titer was determined by Reed-Muench method; cell proliferation was monitored by an iCELLigence Real-Time Labeled Cell Function Analyzer after infection; levels of viral nucleic acid and the mRNA and protein expression of HSP27, 70 and 90 in PRV-infected cells were detected using Real-time fluorescence quantitative PCR (qPCR) and Western blot assays, respectively. Results were as follows:The cell indexes were decreased when cells were infected with 80 TCID₅₀ and 100 TCID₅₀ of virus (10⁶ TCID₅₀·0.1 mL^-1) within the time of detection. Sixty hours after infection, cell indexes of all treatment groups, except the group of 10 TCID₅₀ virus infection, were significantly lower than that of the control group (P<0.01). Levels of viral nucleic acid reached a peak after cells were infected with 10 TCID₅₀virus for 60 h. The transcription of HSP70 and 90 mRNA increased significantly at 0 h after infection (P<0.01), and significantly decreased at 6 and 12 h after infection (P<0.01), respectively; and the transcription of HSP27 increased significantly at 0 (P<0.05) and 3 h after infection (P<0.01), and expression of its protein significantly decreased at 48 h post of infection (P<0.01). The change trends of the three HSP proteins were similar to those of their mRNAs after PRV infection other than partial time points. The results indicate that PRV-YY infection can inhibit the proliferation of PK-15 cells, and induce rapid stress response in PK-15 cells with increasing expression of HSP27, 70 and 90. The rapid response of the three HSPs in host cells upon PRV infection may play important roles in protecting cells from virus infection.

The aim of this study was to investigate the dynamic changes of Dectin-1, TLR-2 and TLR-4 pattern recognition receptors and cytokines in skin lesions of mice infected with Microsporum gypseum. M. gypseum from giant panda were subcutaneously inoculated into the C57BL/6 mice. The skin tissues with pathological changes were collected on the 3rd, 7th and 14th day, then the spore colonization sites and pathological damages were observed by PAS staining and HE staining. The transcription of pattern recognition receptors mRNA were detected by real-time PCR at the 3rd, 6th and 12th hour. The transcription of cytokines mRNA were detected by real-time PCR at the 1st, 3rd, 7th and 14th day. The results showed that M. gypseum infected mice resulted in a small subcutaneous abscess, a large number of inflammatory cells infiltrated, and accompanied by thickening of the stratum corneum. The transcription of the pattern recognition receptors Dectin-1, TLR-2 and TLR-4 were increased (P<0.01), the transcription levels of cytokines IL-6, IL-1β, IL-23, TGF-β, IL-17A, IL-17F and IL-22 were increased (P<0.01). The pattern recognition receptor can recognize the surface pattern recognition molecules of M. gypseum and activate the Th17 pathway, and differentiate the mature Th17 cells into secret cell cytokines IL-22, IL-17A and IL-17F, thereby exerting resistance to M. gypseum.

Inflammatory damage of bovine mammary epithelial cells (MAC-T) was induced by different concentrations of angiotensin Ⅱ (Ang Ⅱ), and the changes of angiotensin-converting enzyme 2 (ACE 2) and the interaction with Ang Ⅱ were explored. The research includes:Detecting the secretion or release of inflammatory factors in cell supernatant by ELISA; Western blot analysis of protein expression changes of ACE 2 and ACE, and correlation analysis; The protective effect of ACE 2 active protein on Ang Ⅱ induced injury. Results were as follows:1) The levels of pro-inflammatory factors TNF-α, IL-6 and IL-8 in the supernatant of bovine MAC-T cells in the Ang Ⅱ treatment were significantly (P<0.05) or very significantly increased (P<0.01), the anti-inflammatory factor IL-10 content was significantly reduced (P<0.05); 2) Treatment with different concentrations of Ang Ⅱ, the expression of ACE 2 protein was decreased, and the 10^-6 mol·L^-1 Ang Ⅱ treatment group was significantly decreased (P<0.05); the expression of ACE protein was reversed. There was a significant negative correlation between ACE 2 and Ang Ⅱ concentrations; 3) After adding exogenous ACE 2 active protein, the results showed that the concentration of TNF-α, IL-6 and IL-8 in the supernatant of the cells decreased to some extent, and the concentration of IL-10 increased. The imbalance of the ACE 2/ACE axis is the main cause of inflammatory injury induced by Ang Ⅱ. The conclusion showed that high level of Ang Ⅱ can activate inflammatory factors to promote inflammatory response and is the main mediator of inflammatory response. ACE 2 can inhibit the up-regulation of inflammatory response by degrading Ang Ⅱ.

This experiment was conducted to investigate the effect of bisphenol A (BPA) exposure during pregnancy on the genotoxicity of adult offspring females and gene expression levels of DNA methyltransferase (DNMT). One hundred and twenty pregnant mice were randomly divided into six groups, twenty mice in each group. The pregnant mice were administered with BPA at the doses of 0, 2.5, 5, 10, 20, 40 mg·(kg·d)^-1, from the pregnancy day 0.5 to day 17.5. Ten adult offspring females (56 d) randomly selected from each group were sacrificed after anesthesia and the blood, uterus and ovaries were collected. Then the uterine and ovarian index were calculated; the ovarian histomorphology were observed after hematoxylin-eosin staining (HE). The activity of follicle stimulating hormone (FSH), luteinizing hormone (LH) and the contents of estradiol (E₂), progesterone (P₄) in serum were detected by radioimmunoassay. The contents of ovarian estrogen receptor α (ERα) and estrogen receptor β (ERβ) were detected by ELISA. The mRNA relative transcription levels of ERα, progesterone receptor (PgR), DNMT1, DNMT3A, and DNMT3B in ovaries were detected by real-time quantitative PCR (RT-qPCR). The results showed that:1) Compared with the control group, BPA significantly reduced the serum levels of FSH, LH, E₂ and P₄; 2) BPA significantly increased the ovarian ERα and ERβ levels; 3) BPA significantly reduced the mRNA relative transcript levels of ovarian ERα, PgR and DNMT in the adult offspring females (P<0.05 or P<0.01); 4) The ovarian index were increased in the adult offspring mice exposed to BPA, but the ovarian parenchyma was atrophied and the number of primary and secondary follicles were increased. Our results reveal that BPA exposure to during pregnancy can promote the production of ovarian follicles in offspring females, significantly increase the contents of ovarian hormone receptor, inhibit the transcriptional activity of ovarian DNMT, and reduce the contents of reproductive hormone and activity, causing reproductive damage to adult offspring females.

The aim of the present study was to obtain a stable, spontaneous insulin-resistant adipocyte model by altering the gene sequence of glucose transporter 4 (GluT4) which resulted in the dysfunction of glucose transmembrane transport in 3T3-L1 cell line. In this study, the GluT4 coding region of 3T3-L1 was directionally cleaved by CRISPR/Cas9 protein, and the stably mutated cell lines were screened by flow cytometry and gene sequencing. The proliferation activities of the selected cell lines were detected by CCK-8 method. Subsequently, the cells were treated with adipogenic differentiation reagent and identified by oil red O staining. The glucose uptake of cells was detected under basal and insulin-stimulated state to determine the insulin resistant level. qPCR and Western blotting were used to detect the expression of essential genes in the process of adipogenic differentiation of insulin-resistant cells and control cells. The results showed that the 13 bp deletion in the 9th exon resulted in dysfunction of GluT4 protein in 3T3-L1 cells without affecting cell proliferation. Sever and stable spontaneous insulin resistance and impaired glucose uptake occurred as we expected. Gene transcription associated with adipogenic differentiation and lipid synthesis were significantly downregulated (P<0.05 or P<0.01) compared with controls. In summary, GluT4 dysfunction hinders insulin-stimulated glucose uptake, resulting in a spontaneous insulin resistance in adipocytes. This study verified the stability and effectiveness of the insulin resistance adipocyte model from multiple perspectives, provided a new material for studies related to adipose metabolism and insulin resistance.

The goal of the present study was to prepare 3 D printed canine tissue engineering meniscus scaffold and evaluate its performance, explore the effect of in vitro chondrogenic induction time on the synthesis of extracellular matrix of canine bone marrow mesenchymal stem cells (BMSCs)-scaffold construction. After preparation of polycaprolactone (PCL) meniscus scaffold using 3D printing technology, the morphology and microstructure of the scaffold were observed by gross and scanning electron microscopy. The compressive modulus of the scaffold was determined by biomechanical test. The compatibility of scaffold and cell was evaluated by CCK-8 cytotoxicity experiment. Canine BMSCs were seeded into scaffold and induced by cartilage in vitro for 7, 14, 21 and 28 d, respectively. The growth of the cells on the scaffold was observed by inverted phase contrast microscopy. The effects of different induction time in vitro on matrix synthesis were analyzed and compared by measuring the glycosaminoglycan (GAG) content and the expression of type Ⅱ collagen at different induction time. The results showed that the PCL scaffold prepared by 3D printing had high degree of reduction of the natural anatomical shape of the meniscus, uniform pores and good inter-well connectivity, certain mechanical compressive properties and slow degradation rate,the number of cells increased. During the in vitro induction of BMSCs-scaffold construction, the cells proliferated continuously. The synthesis of GAG and type Ⅱ collagen increased with the induction time. The synthesis amount at 21 d was significantly higher than other induction time (P<0.05). The results proved that the PCL meniscus scaffold prepared by 3D printing has good physical and chemical properties and cell compatibility. It is expected to be used as a meniscus tissue engineering scaffold, and in vitro induction time has an important effect on the differentiation of BMSCs-scaffold construction into cartilage.

In order to investigate the anti-inflammatory effect of thymol on endometritis, the inflammatory model was established in LPS-induced goat endometrial epithelial cells (gEECs)with thymol treantment. The secretion of NO and cytokines were measured by NO kit and ELISA kits respectively in LPS-induced gEECs. Gene transcription of IL-1β, TLR4, and NF-κB were detected by RT-PCR respectively in LPS-induced gEECs. The results showed that thymol (100, 50, 25 μg·mL^-1) inhibited the secretion of TNF-α, IL-1β, IL-6, NO, and PGE2 to a significant extent in LPS-induced gEECs (P<0.01). In addition, thymol markedly decreased gene transcription of IL-1β, TLR4, and NF-κB in LPS-induced gEECs (P<0.01). These results demonstrated that thymol exerted anti-inflammatory effect through inhibiting the level of inflammatory cytokines, which possibly involved in the inhibition of TLR4/NF-κB signaling pathway.

The objective of this study was to investigate the effect of Zhu Qin Extractive Fluid (ZQEF) on the proliferation and transcription of inflammatory factors in IPEC-J2 cells injured by lipopolysaccharide (LPS). Principal active component were identified by TLC and the content of baicalin was determined by HPLC in ZQEF. IPEC-J2 cells were randomly divided into control group, LPS model group and drug group Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅴ (concentration of ZQEF in each group was 10^-1, 10^-2, 10^-3, 10^-4, 10^-5 g·mL^-1, respectively), each group set up 5 holes, 10⁴ cells·hole^-1. The proliferative rate of cells were determined by MTT method, and the mRNA transcription changes of TFF3, TNF-α and IL-8 were measured by qRT-PCR. The results showed:the main therapeutic ingredients include baicalin, astragaloside and atractylenolide-Ⅰ, and the content of baicalin was 1.469 8% in ZQEF. Compared with the control group, the proliferative rate of cells highly decreased, and the mRNA transcription of TFF3, TNF-α and IL-8 extremely increased in LPS model group (P<0.01). Compared with the LPS model group, the proliferative rate of cells highly enhanced in drug group Ⅱ (10^-2g·mL^-1) and Ⅲ (10^-3 g·mL^-1) (P<0.01 and P<0.05 respectively), the mRNA transcription of TFF3 and TNF-α significantly down-regulated (P<0.01), and the mRNA transcription of IL-8 down-regulated in drug group Ⅱ, non-significantly (P>0.05). The results suggest that ZQEF could facilitate the proliferation of IPEC-J2 cells injured by LPS, inhibit the transcription of inflammatory factors, which may be connected with effect of repairing the intestinal mucosa.

This study aimed to investigate the effect of 1α, 25-(OH)₂D₃ on the formation of osteoclast (OC) in the co-culture system of chicken bone marrow mesenchymal stem cells (BMSCs) and bone marrow mononuclear macrophage cells (BMMs). In the study, tartrate-resistant acid phosphatase (TRAP), bone absorption function identification, and OC marker mRNA and protein expression detection were performed on cells treated with 1α, 25-(OH)₂D₃. The results showed that, 1α, 25-(OH)₂D₃ up-regulated the expression of receptor activator for nuclear factor-κB ligand (RANKL) and down-regulated the expression of osteoprotegerin (OPG) of BMSCs. Compared to the control group, the number of OC was increased in the co-culture system at day 5 treated by 1α, 25-(OH)₂D₃. Meanwhile, the bone resorption activity, the mRNA and protein expression of matrix metalloproteinase 9 (MMP-9), carbonic anhydrase Ⅱ (CAⅡ), cathepsin K and TRAP were significantly enhanced (P<0.01) by 1α, 25-(OH)₂D₃. Among them, 10^-8 mol·L^-11α, 25-(OH)₂D₃ had the most obvious effect. The results confirmed that 10^-8 mol·L^-1 1α, 25-(OH)₂D₃ could mediate the formation of OC, which showed bone resorption activity, in the co-culture system of BMSCs and BMMs from chicken. Our study lays a foundation for further research on the mechanism of poultry OC in bone, calcium and phosphorus metabolic diseases.

The objective of this study was to investigate the distribution of nutrient transporter related genes in different segments of small intestine of rabbit. Ten healthy white rex rabbits at 110 days old with similar weight were chose to slaughter. The intestinal segment samples were collected from duodenum, jejunum, ileum for detecting the mRNA abundance of oligopeptide transporter Pep Tl, amino acid transporter CAT1, B⁰AT, EAAT3, rBAT, glucose transporter SGLT1, GLUT2, GLUT5 and fatty acid transporter FATP4 by real-time PCR. The results showed that the abundance of oligopeptide transporter Pep T1 mRNA was the highest in duodenum, and lower in jejunum. The abundance of basic amino acid transporter CAT1, facultative amino acid transporter rBAT, and neutral amino acid transporter B⁰AT mRNA were the highest in ileum, and lower in jejunum. The acidic amino acid transporter EAAT3 mRNA expression was higher in jejunum and ileum. The expression of glucose transporter SGLT1 and GLUT5 mRNA was higher in duodenum and jejunum. The abundance of glucose transporter GLUT2 and fatty acid transporter FATP4 mRNA were the highest in jejunum, and lower in duodenum. The result indicate that the main part in intestine for transporting and absorbing oligopeptide, glucose and fatty acid is the first half of the small intestine, the main part for transporting and absorbing amino acid is the posterior segment of the small intestine.