Isolation and biological characteristics of multipotent mesenchymal stromal cells derived from chick embryo intestine

ABSTRACT

1. Over the past decade, rapid advancement in isolation methods for identifying markers of the once elusive intestinal stem cell (ISC) populations has laid the foundation for unravelling their complex interrelationships during homeostasis. Study on ISC in avian intestinal tissue might play a pivotal foundation for further studies on the epithelial-to-mesenchymal transition (EMT) in gastrointestinal disease and cell-based therapy as well as intestinal tissue engineering.

2. The following experiment isolated a population of fibroblast-like, plastic adhering cells derived from chick embryo intestine, showing a strong self-renewing and proliferative ability, which was maintained in vitro up to passage 25. The findings included growth characteristics, detected expression of cell surface markers and characterised the capability of these cells to differentiate towards the osteogenic, adipogenic, and chondrogenic cell lineages.

3. RT-PCR analysis showed that these cells from chick embryos expressed mesenchymal stromal cell markers CD44, CD90 and VIMENTIN as well as ISC-specific genes LGR5, MI1, SMOC2, BMI1, and HOPX. Immunofluorescence and flow cytometry confirmed this biology characterisation further.

4. In conclusion, cells were isolated from the intestine of 18-day-old chicken embryos that exhibited the biological characteristics of mesenchymal stromal cells as well as markers of intestinal stem cells. Our findings may provide a novel insight for in vitro cell culture and characteristics of ISCs in avian species, which may also indicate a benefit for obtaining cell source for intestinal tissue engineering as well as cell-based investigation for gastrointestinal disease and treatment.     

Therapeutic potential of amniotic fluid stem cells to treat bilateral ovarian dystrophy in dairy cows in a subtropical region

Amniotic fluid is a rich source of multipotent mesenchymal stem cells (MSCs). Amniotic fluid stem cells (AFSCs) have become a new source of stem cells; they have low immunogenicity and are easily harvested. For this reason, they may be useful in clinical tissue engineering. Moreover, AFSCs have anti‐inflammatory properties and can repair tissues. This study evaluated the utility of AFSC injection to treat bilateral ovarian dystrophy in Holstein‐Friesian cows. Bovine AFSCs (BAFSCs) were collected at slaughter from Holstein‐Friesian cows during the third or fourth month of pregnancy and cultured in vitro. The BAFSCs began to show a fibroblast‐like morphology. They were positive for β‐integrin, CD44, CD73, CD106 and Oct4 and negative for CD34 and CD45. After induction, the cells differentiated into mesodermal lineages. Bilateral ovarian dystrophy was confirmed by ultrasonography in 16 lactating cows. The subsequent experiment lasted 15 weeks. Serum was collected weekly to analyse progesterone concentrations, and weekly ultrasonography recorded ovarian changes. Each cow was equipped with an automatic heat detection system to facilitate oestrus observation and breeding records. The progesterone concentration of two cows in the treatment group (25%) significantly increased during weeks 10–15. On ultrasonography, the treatment group demonstrated mature follicles after BAFSCs injection, and foetuses were visualized approximately 40 days after artificial insemination (AI). Oestrus rates in the control and treatment groups were 0% (0/8) and 50% (4/8), respectively; pregnancy rates were 0% (0/8) and 25% (2/8), respectively. Calves were successfully delivered in both cases of pregnancy. These results show that BAFSCs can alleviate bovine ovarian dystrophy and restore fertility.     

东达山高寒草甸8种植物气孔特征沿土壤水分梯度的变化

植物叶片气孔密度和大小具有可塑性,在外界胁迫下而变化,是植物适应环境变化的重要机制。为揭示高寒草甸生境趋向干旱化过程中植物气孔特征的变化规律,选择藏东南东达山高寒草甸的8种优势或常见植物作为研究对象,设置10个土壤水分梯度,分析气孔特征的变化。结果表明,根据植物对土壤水分的依赖程度可划分为4种类型:大花嵩草为沼生型,随土壤干旱程度的增加气孔密度增加、气孔大小减小;鸭跖草、小大黄、矮生嵩草和珠芽蓼为湿生型,气孔密度和大小与土壤含水率之间的关系多呈现为单峰曲线格局;圆穗蓼和线叶嵩草为旱生型,气孔特征随土壤干旱程度增加的变化趋势与沼生型植物几乎相反;高山嵩草气孔特征与土壤含水率之间的相关性不显著,在土壤水分梯度上分布范围广,为广布型。每种植物生长都有适宜的土壤水分条件,过高或过低的土壤含水率都将胁迫植物的生长,因此植物气孔特征与土壤含水率之间的关系呈现多种变化格局。     

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.