广灵驴成纤维细胞系的建立及其相关生物学特性研究

试验旨在建立广灵驴成纤维细胞库,以期在细胞水平上对广灵驴进行保护。本研究以1月龄广灵驴耳缘皮肤组织为试验材料,采用组织块贴壁法进行成纤维细胞培养,建立了广灵驴耳缘皮肤组织成纤维细胞系,并对其相关生物学特性进行了鉴定。结果发现,试验所建立的广灵驴成纤维细胞系大多数细胞呈长梭形,部分细胞呈三角状或星型。在培养过程中,广灵驴原代成纤维细胞在贴壁4 d时开始有细胞从组织边缘游离出来,贴壁14 d后,细胞汇合率达到80%,可以进行第一次传代培养;细胞生长态势良好,生长曲线呈典型的S型曲线;细胞冻存复苏后活率有所下降,但生长状态良好。细胞分裂中期染色体数2n=62,说明成功建立了广灵驴成纤维细胞系。通过本方法建立的广灵驴成纤维细胞系为后续广灵驴的相关研究奠定了基础。     

PDL1Ig gene-modified BMSCs induce immune tolerance in rat liver transplantation

AIM: To investigate the effects of bone marrow mesenchymal stem cells(BMSCs) modified by programed death ligand-1 immunoglobulin(PDL₁Ig) gene on immune rejection of orthotopic liver transplantation in rats. METHODS: Rat BMSCs were cultured and identified. The protein expression of PDL₁Ig in the BMSCs 72 h after infection with pAdEasy-1/PDL₁Ig was detected by Western blot. Mixed lymphocyte reaction was used to detect the inhibitory effect of BMSCs on the viability of T-lymphocytes in peripheral blood. The male Wistar rats were used as donors(n=40), and the male SD rats were used as recipients(n=40). The rat model of orthotopic liver transplantation was established by improved cuff method for observing acute rejection. The rats were randomly divided into control group, BMSCs treatment group, BMSCs/GFP treatment group and BMSCs/PDL₁Ig treatment group with 10 pairs each. Five rats were executed at the 7th day and the remains were used for measuring the survival time. RESULTS: The expression of PDL₁Ig in the BMSCs was detected after pAdEasy-1/PDL₁Ig infection. The effect of BMSCs/PDL₁Ig on the viability of the lymphocytes was stronger than that of BMSCs/GFP. The level of IL-4 in BMSCs/PDL₁Ig group was significantly higher than that in the other 3 groups, while the levels of IFN-γ and IL-2 were significantly decreased. The liver function in BMSCs/PDL₁Ig group was significantly improved and the levels of ALT, AST and TBil were almost recovered to normal at the 7th day after transplantation. Severe rejection reaction was observed in control group, and rejection reactions were decreased with different degrees in BMSCs treatment group and BMSCs/GFP treatment group. Much slighter rejection reaction and significantly longer survival time were showed in BMSCs/PDL₁Ig group than those in the other 3 groups. CONCLUSION: PDL₁Ig-modified BMSCs inhibit the rejection of liver transplantation in rats and induce the immune tolerance, and the effect is better than that of BMSCs alone.     

Tanshinone IIA prevents high glucose-induced human umbilical vein endothelial cell apoptosis

AIM: To investigate the effect of tanshinone IIA on the apoptosis of human umbilical vein endothelial cells(HUVECs) after high glucose treatment.METHODS: The cell viability was determined by MTT assay. The cell apoptotic rate was examined by flow cytometry with Annexin V-FITC/PI double staining. The expression of Bcl-2 and Bax, and the release of mitochondrial cytochrome C(Cyt C) were analyzed by Western blotting.RESULTS: Tanshinone IIA significantly inhibited high glucose-induced decrease in cell viability and increased the cell apoptosis. Additionally, after tanshinone IIA treatment, Bax expression and the release of mitochondrial Cyt C were significantly inhibited, while Bcl-2 expression was increased.CONCLUSION: Tanshinone IIA prevents high glucose-induced endothelial cell apoptosis via mitochondria-dependent pathway.     

Effect of simvastatin on function of mouse pancreatic beta cells and its mechanisms

AIM: To observe the influence of simvastatin on insulin secretion function of mouse pancreatic beta cell line MIN6 and to explore its possible mechanisms.
METHODS: MIN6 cells were randomly divided into normal control group and low-, middle-and high-concentration simvastatin treatment groups, which were cultured for 48 h with high-glucose DMEM containing 15% fetal bovine serum plus 0, 2, 5 and 10 μmol/L simvastatin, respectively. The insulin secretion of MIN6 cells was measured by radioimmunoassay. The content of ATP in MIN6 cells was measured by biochemiluminescence method. The mRNA and protein expression levels of inwardly rectifying potassium channel 6.2 (Kir6.2), voltage-dependent calcium channel 1.2 (CaV1.2) and glucose transporter 2 (GLUT2) were detected by real-time fluorescence quantitative PCR and Western blotting, respectively.
RESULTS: Compared with normal control group, middle-and high-concentration simvastatin treatment markedly decreased the synthesis and secretion of insulin in MIN6 cells (P<005). The content of ATP in MIN6 cells was markedly decreased in simvastatin treatment groups (P<005). The mRNA expression level of Kir6.2 in MIN6 cells was significantly up-regulated in simvastatin treatment groups (P<001), while the mRNA expression levels of CaV1.2 and GLUT2 were significantly down-regulated in middle-and high-concentration simvastatin treatment groups (P<001). The protein expression of Kir6.2 was significantly increased but that of CaV1.2 was significantly decreased in middle-and high-concentration simvastatin treatment groups (P<001), and the protein expression level of GLUT2 was markedly decreased in high-concentration simvastatin treatment group (P<001).
CONCLUSION: Simvastatin inhibits insulin synthesis and secretion in mouse pancreatic beta cell line MIN6 via suppressing ATP production, up-regulating the expression of Kir6.2 and down-regulating the expression of CaV1.2 and GLUT2.     

Midazolam inhibits proliferation of human pharyngeal squamous cell carcinoma FaDu cells via down-regulation of p300 expression

AIM：To explore the mechanisms by which midazolam inhibits the proliferation of human pharyngeal squamous cell carcinoma FaDu cells. METHODS：Cultured FaDu cells were treated with different concentrations of midazolam, and p300 gene was silenced by siRNA. MTT assay and BrdU incorporation assay were used to evaluate the proliferation of the cells. The mRNA and protein levels of p300 and the expression of cell cycle-related proteins were detected by RT-PCR and Western blotting. RESULTS：Midazolam inhibited the proliferation of FaDu cells, and attenuated the mRNA and protein levels of p300. Knockdown of p300 inhibited the proliferation of FaDu cells, and led to up-regulation of p21 and p27 proteins and down-regulation of p-Rb protein. CONCLUSION: Midazolam inhibits the proliferation of human pharyngeal squamous cell carcinoma FaDu cells through down-regulating p300 expression.     

Advance in islet β cell development and regeneration

Islet β cells excrete insulin and play a crucial role in glucose homeostasis. Decreased β cell mass or/and β cell dysfunction are one of the fundamental characteristics of diabetes. The advance in understanding β cell development and regeneration provides new approaches to diabetes treatment. This review focuses on the molecular mechanism about β cell development and the sources for β cell regeneration.     

scRNA-seq of ovarian follicle granulosa cells from different fertility goats reveals distinct expression patterns

The new technology of high-throughput single-cell RNA sequencing (10 × scRNA-seq) was developed recently with many advantages. However, it was not commonly used in farm animal research. There are few reports for the gene expression of goat ovarian follicle granulosa cells (GCs) during different developmental stages. In the current investigation, the gene expression of follicle GCs at different stages from two populations of Ji'ning grey goats: high litter size (HL; ≥3/L; 2 L) and low litter size (LL; ≤2 /L; 2 L) were analysed by scRNA-seq. Many GC marker genes were identified, and the pseudo-time showed that GCs developed during the time course which reflected the follicular development and differentiation trajectory. Moreover, the gene expression difference between the two populations HL versus LL was very clear at different developmental stages. Many marker genes differentially expressed at different developmental stages. ASIP and ASPN were found to be highly expressed in the early stage of GCs, INHA, INHBA, MFGE8 and HSD17B1 were identified to be highly expressed in the growing stage of GCs, while IGFBP2, IGFBP5 and CYP11A1 were found to be highly expressed in late stage. These marker genes could be used as reference genes of goat follicle GC development. This investigation for the first time discovered the gene expression patterns in goat follicle GCs in high- or low-fertility populations (based on litter size) by scRNA-seq which may be useful for uncovering the oocyte development potential.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Effect of pachyman polysaccharides on Th17/Treg balance in systemic lupus erythematosus patients

AIM: To investigate the immunomodulatory effect of pachyman polysaccharides (PPS) on T helper 17 cell (Th17)/regulatory T cell (Treg) balance in the peripheral blood of systemic lupus erythematosus (SLE) patients. METHODS: The CD4⁺ T cells were isolated from the peripheral blood samples obtained from 45 SLE patients and 35 healthy controls enrolled in our study using magnetic bead separation method. The proportions of Th17 and Treg cells were measured by flow cytometry. The CD4⁺ T cells from SLE patients and healthy controls were treated with PPS. The cytoto-xicity of PPS was evaluated by detecting cell viability with MTT assay. The contents of interleukin-17 (IL-17), IL-6, IL-10 and transforming growth factor-β (TGF-β) were measured by ELISA. The expression of retinoid-related orphan receptor γt (RORγt) and forkhead box protein P3 (Foxp3) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTS: The Th17 cells were significantly elevated, while Treg cells were obviously decreased in the SLE patients compared with the healthy control group (P<0.05). Compare with control group, the contents of IL-17 and IL-6 were decreased, while the contents of IL-10 and TGF-β were increased (P<0.05). The expression of RORγt at mRNA and protein levels was down-regulated and the expression of Foxp3 was up-regulated (P<0.05). The ratio of Th17/Treg was decreased in 100 μg/L nontoxic PPS-treated CD4⁺ T cells isolated from the SLE patients (P<0.05). CONCLUSION: PPS treatment inhibits Th17 cells and elevates Treg cells in the CD4⁺ T cells isolated from SLE patients, which may have a therapeutic effect on SLE patients.     

Effects of JAK-STAT signaling pathway,IL-1β and IL-6 on injury of PC12 cells with X-ray irradiation

AIM: To investigate the role of JAK-STAT pathway, IL-1β and IL-6 in the PC12 cells with X-ray irradiation.METHODS: The PC12 cells were irradiated with X-ray at doses of 2, 4 and 8 Gy. After 24 h, the levels of IL-1β and IL-6 were detected by ELISA. The protein levels of p-JAK1, p-JAK2, p-STAT1, p-STAT3 and p-STAT5 were measured by Western blot.RESULTS: Compared with control group, the levels of IL-1β and IL-6 increased. The protein levels of p-JAK1, p-JAK2, p-STAT1, p-STAT3 and p-STAT5 increased with the doses of X-ray exposed.CONCLUSION: JAK-STAT signaling pathway, IL-1β and IL-6 play a role in the injury of PC12 cells with X-ray irradiation.