Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

In vitro development of bovine nuclear transfer embryos reconstructed with mammary gland epithelial cells at different passages

In the present study, the effect of passage of nuclear donor cells on the in vitro development of nuclear transfer (NT) embryos was investigated using colostrum‐derived mammary gland epithelial (MGE) cells at different passages (3–30 passages) to find reliable passages for the efficiency of cloning. Development of NT embryos to the blastocyst stage was affected by the number of passages of MGE cells (P < 0.05). Nuclear transfer embryos reconstructed with MGE cells at 3–7 passages showed a significantly higher blastocyst development (31.3–48.5%) than those with the cells at 10–30 passages (2.5–12.5%, P < 0.05). No difference in the proportion of the MGE cells with normal diploid was observed among passage of 3, 15 and 30 (P > 0.05). The use of MGE cells at early passages for nuclear donor cells may be advantageous for the production of NT embryos.     

The research development of endothelial progenitor cells

Endothelial progenitor cell (EPC) is a kind of directional cell that is able to differentiate to endothelial cell. The role of EPC is associated not only with vasculogenesis during embryonic development but also with physiological organ maintenance and angiogenesis during postnatal and adult period. There is a good clinical therapeutic prospective use for EPC in the treatment of ischemia diseases and inhibition of tumor angiogenesis.     

Promoting angiogenesis of protein kinase D1 in vitro and in vivo

AIM: To explore the effect of protein kinase D1 (PKD1) on promoting angiogenesis in vitro and in vivo, and to furnish a new idea on targeting PKD1 for the treatment of ischemic heart disease such as myocardial infarction. METHODS: The culture, isolation and identification of endothelial progenitor cells (EPCs) were performed in vitro. The effects of PKD1 and its specific blocking agent CID755673 on expression levels of vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR) in EPCs were determined. The rat model of myocardial infarction was established, the intervention effects of PKD1 and CID755673 on morphology, changes of microvessels and endothelial cells, and the expression of VEGF and KDR in the impaired myocardial tissue were analyzed. RESULTS: PKD1 significantly upregulated the expression of VEGF and KDR in EPCs in vitro. Meanwhile, the structure of myocardial tissue was more regular and clear, the cytomembrane of endothelial cells were more smooth and integrity, the pericytes were visible, and the expression of VEGF and KDR was significantly increased in PKD1 treatment group in vivo.CONCLUSION: PKD1 has the ability of angiogenesis obviously, which might be mediated by VEGF.     

Soluble Klotho protein suppresses THP-1-derived foam cell formation

AIMTo investigate the role of soluble Klotho protein in THP-1-derived foam cell formation. METHODSTHP-1 monocytes were induced into macrophages by treatment with 160 nmol/L phorbol myristate acetate for 48 h, and then were divided into 6 groups: negative control group (THP-1-derived macrophages), positive control group [THP-1-derived foam cells induced by oxidized low-density lipoprotein (ox-LDL) for 48 h], and 25, 50, 100 and 200 μg/L soluble Klotho protein groups (THP-1-derived macrophages pretreated with soluble Klotho protein at the indicat?ed concentraions for 2 h and then induced by ox-LDL for 48 h). Lipid droplets in cytoplasm were observed by oil red O staining. The cholesterol outflow rate was detected by scintillation counting technique. The content of intracellular total cholesterol, free cholesterol and cholesterol ester was detected by enzyme fluorescence analysis. The expression of acyl-coenzyme A:cholesterol acyltransferase 1 （ACAT1） and ATP-binding cassette transport?er A1 (ABCA1) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTSOil red O staining and lipid mass quantification showed that THP-1-derived foam cell formation was dose-dependently suppressed by soluble Klotho protein. The cholesterol efflux rate of THP-1-derived foam cells was increased by soluble Klotho protein in a dose-dependent manner (P<0.05). In addition, soluble Klotho protein decreased the expression of ACAT1 and increased the expression of ABCA1 in a dose-dependent manner (P<0.05). CONCLUSION The soluble Klotho protein inhibits THP-1-derived foam cell formation in a dose-dependent manner by down-regulating the expression of ACAT1 and up-regulating the expression of ABCA1.     

Effect of Estradiol-17β on protein synthesis and degradation rates in fused bovine satellite cell cultures

Although androgenic and estrogenic steroids are widely used to enhance muscle growth and increase feed efficiency in feedlot cattle, their mechanism of action is not well understood. Further, in vivo studies indicate that estradiol (E2) affects muscle protein synthesis and/or degradation, but in vitro results are inconsistent. We have examined the effects of E2 treatment on protein synthesis and degradation rates in fused bovine satellite cell (BSC) cultures. Additionally, to learn more about the mechanisms involved in E2-enhanced muscle growth, we have examined the effects of compounds that interfere with binding of E2 or insulin-like growth factor (IGF)-1 to their respective receptors on E2-induced alterations in protein synthesis and degradation rates in BSC cultures. Treatment of fused BSC cultures with E2 results in a concentration-dependent increase (P < 0.05) in protein synthesis rate and a decrease (P < 0.05) in protein degradation rate. The pure estrogen antagonist ICI 182 780 suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation in fused BSC cultures. The G-protein coupled receptor (GPR)-30 agonist G1 does not affect either synthesis or degradation rate, which establishes that GPR30 does not play a role in E2-induced alterations in protein synthesis or degradation. JB1, a competitive inhibitor of IGF-1 binding to the Type 1 insulin-like growth factor receptor (IGFR-1), suppresses (P < 0.05) E2-induced alterations in protein synthesis and degradation. In summary, our data show that E2 treatment directly alters both protein synthesis and degradation rates in fused BSC cultures via mechanisms involving both the classical estrogen receptor (ER) and IGFR-1.     

大白鼠乳腺上皮细胞的体外培养与鉴定

 试验选取泌乳期大白鼠乳腺组织,采用机械剪切结合胰酶消化的方法,在体外进行乳腺上皮细胞原代分离培养及鉴定。结果成功培养出大鼠原代乳腺上皮细胞,经过角蛋白免疫组化鉴定染色后,呈现阳性反应。显微镜下观察,细胞呈鹅卵石样;经多次传代,细胞增殖仍然长势很好。     

Effect of fucoxanthin on growth and apoptosis of HSC-T6 cells

AIM: To explore the effect of fucoxanthin (Fu) on the growth and apoptosis of HSC-T6 cells. METHODS: HSC-T6 cells were divided into blank control group, negative control group and drug groups (treated with different concentrations of Fu). The cell viability was detected by CCK-8 assay at 24 h, 48 h and 72 h after Fu treatment. The cell cycle distribution and apoptotic rate were analyzed by flow cytometry. The protein expression of Bcl-2 and Bax were detected by Western blot. RESULTS: Compared with blank control group, the viability of HSC-T6 cells was inhibited by Fu at concentrations of 15~75 μmol/L in a dose- and time-dependent manner (P < 0.01). The cell ratio of G₁ phase was significantly decreased (P < 0.01) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.01) in 60 μmol/L Fu group after 24 h. The cell ratio of G₁ phase was significantly decreased (P < 0.05) and the cell ratio of S phase and G₂ phase was significantly increased (P < 0.05) in 15 μmol/L and 30 μmol/L Fu groups in a dose-dependent manner after 48 h. The early cell apoptotic rates and total cell apoptotic rates were significantly increased in the Fu treatment groups in a dose-dependent manner (P < 0.05). The protein expression of Bax was significantly increased in the Fu treatment groups and the protein expression of Bcl-2 was significantly decreased in 30 μmol/L and 60 μmol/L Fu groups (P < 0.05).CONCLUSION: Fu inhibits the growth of HSC-T6 cells possiblely via arresting the cell cycle at S phase and G₂ phase. The apoptosis of HSC-T6 cells induced by Fu might be via down-regulating the protein expression of Bcl-2 and up-regulating the protein expression of Bax.     

PK15细胞全悬浮摇瓶培养工艺初探

PK15细胞是一种被广泛应用于兽用疫苗生产的细胞,然而现今生物制品行业快速发展,传统贴壁培养PK15细胞已经不能满足生产需要.目前通过全悬浮培养技术来培养细胞、病毒已经是发展趋势,但是由于生产用细胞多数为贴壁细胞,难以实现悬浮培养,所以,目前该项技术在疫苗生产上还未广泛应用,技术也还未成熟.故本文探讨PK15细胞在无血...     

BHBA对HT22细胞中ACE表达的影响

生酮饮食(KD)已被证实具有神经保护作用,但其机制尚不明确。本试验以小鼠海马神经元HT22为细胞模型,利用2 mmol/Lβ-羟基丁酸酯(BHBA)对HT22细胞进行处理,通过荧光定量PCR,免疫蛋白印迹和信号通路阻断试验,观察BHBA对HT22细胞ACE表达的影响,并探讨参与调控的信号通路。结果表明,2 mmol/L BHBA可通过激活HT22细胞内GPR109A受体和ERK1/2信号通路进而增加HT22细胞中ACE的表达。