蛋氨酸三肽对奶牛乳腺上皮细胞酪蛋白和小肽转运载体基因表达量的影响

本试验旨在研究蛋氨酸三肽(Met-Met-Met)对奶牛乳腺上皮细胞(BMECs)酪蛋白和小肽转运载体基因表达量的影响。采用酶消化法培养的第3代奶牛乳腺上皮细胞为模型,各处理在培养基中分别添加0(对照)、40、50、60、70和80μg/mL的蛋氨酸三肽,每个处理5个重复,每个重复1个培养孔,分别培养细胞24、48和72h,检测奶牛乳腺上皮细胞的相对增殖率,整体试验重复2次,确定最佳培养时间;各处理在培养基中分别添加0(对照)、40、50、60、70和80μg/mL的蛋氨酸三肽,每个处理3个重复,每个重复1个培养孔,以最佳培养时间培养,用实时定量PCR法检测酪蛋白基因的表达量,确定适宜蛋氨酸三肽浓度,整体试验重复3次;以最佳培养时间和适宜蛋氨酸三肽浓度培养细胞,以未添加蛋氨酸三肽的培养基为对照,每个处理3个重复,每个重复1个培养孔,测定小肽转运载体基因的表达量,整体试验重复3次。结果表明:在培养基中添加蛋氨酸三肽培养奶牛乳腺上皮细胞24h时,相对增殖率最高;培养基中加入60μg/mL的蛋氨酸三肽培养细胞24h,αs1-酪蛋白和β-酪蛋白的基因表达量最高,同时发现奶牛乳腺上皮细胞中小肽转运载体1和小肽转运载体2基因表达量显著高于对照处理(P0.05)。综上所述,培养基中添加60μg/mL的蛋氨酸三肽能够提高奶牛乳腺上皮细胞酪蛋白和肽转运载体基因的表达量。     

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.     

Establishment of an in vitro culture model to study milk production and the blood–milk barrier with bovine mammary epithelial cells

This study attempted to establish a culture model to recreate the milk production pathway in bovine mammary epithelial cells (BMECs). BMECs were isolated from Holstein cows (nonlactating, nonpregnant, and parous) and were stored by cryopreservation. To separate the apical and basolateral compartments, BMECs were cultured on a cell culture insert with a collagen gel in the presence of bovine pituitary extract and dexamethasone to induce milk production and tight junction (TJ) formation. The culture model showed the secretion of the major milk components, such as β‐casein, lactose, and triglyceride, and formed less‐permeable TJs in BMECs. Moreover, the TJs were distinctly separated from the apical and basolateral membranes. Glucose transporter‐1, which transports glucose into the cytoplasm through the basolateral membrane, localized in the lateral membrane of BMECs. Toll‐like receptor‐4, which binds to lipopolysaccharide in the alveolar lumen in mastitis, localized in the apical membrane. Beta‐casein was mainly localized near the Golgi apparatus and the apical membrane. Moreover, milk components were almost secreted into the upper chamber of the cell culture insert. These findings indicate that this model has clear cell polarity as well as in vivo and is effective to study of milk production and the blood–milk barrier in lactating BMECs.     

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

Milk casein and fatty acid fractions in early lactation are affected by nutritional regulation of body condition score at the beginning of the transition period in primiparous and multiparous cows under grazing conditions

The objective was to evaluate the effect of body condition score (BCS) at 30 days before calving (−30 days) induced by a differential nutritional management, parity and week of lactation (WOL) on milk yield and composition, and milk casein and fatty acid composition. Primiparous and multiparous Holstein cows with high BCS (PH, n = 13; MH, n = 9) and low BCS (PL, n = 9; ML = 8) under grazing conditions were sampled at WOL 2 and 8 (before and after peak of lactation). Milk yield was greater in multiparous than in primiparous cows and tended to decrease from WOL 2 to 8 only in ML cows. Milk protein, fat and casein yields were greater in multiparous than in primiparous cows and decreased from WOL 2 to 8. Milk casein concentration in milk protein was greater in MH cows than in ML, PH and PL cows at WOL 2. Milk κ‐casein was greater, and β‐casein was less in multiparous than in primiparous cows. As lactation progressed, proportion of casein fractions were not altered. Only κ‐casein fraction was affected by BCS at −30 days as PL showed a higher concentration than PH. The de novo (4:0–15:1) and mixed‐origin fatty acids (16:0–16:1) in milk fat increased, whereas preformed fatty acids (≥17:0) decreased from WOL 2 to 8. Saturated (SAT) fatty acids tended to be greater and monounsaturated fatty acids (MUFA) were less in multiparous than in primiparous cows. High‐BCS cows had greater concentrations of polyunsaturated (PUFA), conjugated linoleic acid (CLA) as well as n‐6 and n‐3 fatty acids in milk fat than low‐BCS cows. The results indicate that casein and fatty acid fractions in milk were affected by parity and may be modified by a differential nutritional management during the pre‐calving period (BCS at −30 days) in cows under grazing conditions.     

Effects of induced energy deficiency on lactoferrin concentration in milk and the lactoferrin reaction of primary bovine mammary epithelial cells in vitro

A dietary energy restriction to 49% of total energy requirements was conducted with Red Holstein cows for three weeks in mid‐lactation. At the last day of the restriction phase, primary bovine mammary epithelial cells (pbMEC) of eight restriction (RF) and seven control‐fed (CF) cows were extracted out of one litre of milk and cultured. In their third passage, an immune challenge with the most prevalent, heat‐inactivated mastitis pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was conducted. Lactoferrin (LF) was determined on gene expression and protein level. An enzyme‐linked immunosorbent assay (ELISA) was developed to determine LF in milk samples taken twice weekly throughout the animal trial, beginning on day 20 pp (post‐partum) until day 150 pp, in cell culture total protein and in cell culture supernatant. Milk LF increased throughout the lactation and decreased significantly during the induced energy deficiency in the RF group. At the beginning of realimentation, LF concentration increased immediately in the RF group and reached higher levels than before the induced deficit following the upward trend seen in the CF group. Cell culture data revealed higher levels (up to sevenfold up‐regulation in gene expression) and significant higher LF protein concentration in the RF compared to the CF group cells. A further emphasized effect was found in E. coli compared to S. aureus exposed cells. The general elevated LF levels in the RF pbMEC group and the further increase owing to the immune challenge indicate an unexpected memory ability of milk‐extracted mammary cells that were transposed into in vitro conditions and even displayed in the third passage of cultivation. The study confirms the suitability of the non‐invasive milk‐extracted pbMEC culture model to monitor the influence of feeding experiments on immunological situations in vivo.     

Further Development of an Equine Cell Line that can be Propagated over 100 Times

Cell lines originating from horses are necessary for isolation and propagation of equine herpesviruses (EHV). Although we established an equine-derived cell line, FHK-Tcl3, propagation ceased after fewer than 40 passages. In this study, FHK-Tcl3 cell propagation continued beyond 40 passages, achieving over 100 passages. FHK-Tcl3 cells were then cloned by limiting dilution at the 100th passage. Cloned cells were termed FHK-Tcl3.1. FHK-Tcl3.1 cells grew well and were propagated every 3 to 4 days by splitting 1:5. In addition, EHV-1, -2 and -4 showed a clear cytopathic effect (CPE) in FHK-Tcl3.1 cells, and this CPE was very similar to those seen in parental FHK-Tcl3 and primary fetal horse kidney cells. FHK-Tcl3.1 cells continue to propagate and the current passage record is over 100 times after cloning. Therefore, this cell appears to have been immortalized. FHK-Tcl3.1 cells have potential for growth and diagnosis of various equine viruses, including equine herpesviruses.     

牛乳腺上皮细胞体外分离培养模式、方法及其应用进展

乳腺由具有泌乳功能的腺泡组成,乳腺上皮细胞(MEC)以单层方式排列在腺泡外围,是乳腺对外界病原进行免疫保护的重要组分,负责将血液中的营养物质通过一系列复杂生化过程转化为乳汁.牛乳腺上皮细胞(BMECs)的体外分离培养在很大程度上解决了活体试验条件不可控、操作困难、成本高及个体差异大等诸多问题,还可以为体外研究乳腺组织生...     

Expression and Characterization of Constitutive Heat Shock Protein 70.1 (HSPA‐1A) Gene in In Vitro Produced and In Vivo‐Derived Buffalo (Bubalus bubalis) Embryos

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA‐1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA‐1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo‐derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA‐1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA‐1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8–16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA‐1A revealed that it shares 91–98% identity with other mammalian sequences. It can be concluded that higher level of HSPA‐1A mRNA in IVP embryos in comparison with in vivo‐derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA‐1A gene could be used as a stress biomarker during pre‐implantation development.     

催乳素对奶牛乳腺上皮细胞乳脂和乳蛋白合成相关基因表达的影响

本试验旨在探讨催乳素对奶牛乳腺上皮细胞(BMECs)乳脂和乳蛋白合成相关基因表达的影响。选取中国荷斯坦奶牛BMECs为试验材料,经纯化培养后,培养基中添加不同浓度催乳素[0(对照)、100、300、500和1 000 ng/m L],继续培养24 h。通过四甲基偶氮唑盐(MTT)比色法检测细胞活力;利用试剂盒检测胞内甘油三酯的含量;采用实时定量PCR法检测乳脂和乳蛋白合成相关基因的表达。结果表明:1)催乳素浓度为100、300 ng/m L时,BMECs相对增殖率显著高于对照组与其他试验组(P0.05)。2)与对照组相比,300 ng/m L催乳素能够显著提高BM ECs乙酰辅酶A羧化酶(ACC)、二酰甘油酰基转移酶(DG AT)、脂肪酸结合蛋白3(FABP3)基因表达量及甘油三酯的含量(P0.05),硬脂酰辅酶A去饱和酶(SCD)、过氧化物酶体增殖物激活受体γ(PPARγ)基因表达量有增加的趋势。3)与对照组相比,100、300 ng/m L催乳素能够显著提高哺乳动物雷帕霉素靶蛋白(m TOR)、催乳素受体(PRLR)基因表达量(P0.05);300 ng/m L催乳素能显著提高αS1酪蛋白(CSN1 S1)基因表达量(P0.05)。综上所述,100~300 ng/m L的催乳素对BM ECs乳脂和乳蛋白合成有较好的促进效果。     

Assessment of Meiotic Spindle Configuration and Post‐Warming Bovine Oocyte Viability Using Polarized Light Microscopy

The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule‐polymerized protein in in vitro‐matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro‐matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule‐polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p < 0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n = 651) were exposed or not (controls) to PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(?) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule‐polymerized protein in in vitro‐matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post‐warming viability in vitrified bovine oocytes.     

Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT‐MECs)

Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post‐parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT‐MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF‐7 cells. hT‐MECs on passage 42 grew vigorously and ‘S’ sigmoid curves of growth were observed. Moreover, hT‐MECs maintained a normal chromosome modal number of 2n = 60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta‐casein protein was synthesized and secreted. Beta‐casein expression was enhanced by prolactin (P < 0.05). Lipid droplets were found in hT‐MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT‐MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT‐MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time.     

Degradation of two soluble proteins – casein and egg protein by a macro in vitro method

Degradation of casein and egg protein was studied with whole rumen contents (RC) in a macro in vitro system to elucidate previous findings of initial rapid disappearance of soluble proteins in vitro. Five to 7.5 kg of RC from a dry and/or a lactating cow were incubated with buffer and casein or egg protein for 180 min with frequent sampling. Degradation was measured as loss of trichloroacetic acid precipitable N (TCA‐N) from the inocula. Normal (39 °C) and low (2 °C) temperature incubations were examined in Exp. 1, using 1 g of TCA‐N from casein. Four levels of casein (0–12 g TCA‐N) in Exp. 2 and four levels of egg albumin (0–24 g TCA‐N) in Exp. 3 were fermented at 39 °C. Initial recovery of casein TCA‐N was 106% at 2 °C and 56% at 39 °C (Exp. 1). Casein (TCA‐N) recovered initially increased in Exp. 2 from 21% at 3 g to 86% at 12 g TCA‐N, while absolute loss remained relatively constant at 358 mg TCA‐N/kg RC (SD = 47). Fractional degradation rate was highest (0.03/min) at the intermediate dosage level. In the absence of rumen fluid (Exp. 4), no casein was lost. Initial egg protein recovery was on average 103% (Exp. 3). Recovery seemed unaffected by dosage level, and absolute degradation rate was relatively constant over time and increased with dosage level (p < 0.001) from 1.48 to 2.95 mg TCA‐N/(kg RC × min). Maximum degradation rate [mg TCA‐N/(kg RC × min)] and affinity constant (mg TCA‐N/kg RC) were estimated at 261 and 1650, respectively. It is concluded that a surprisingly constant amount of casein disappears immediately from warm rumen fluid and that this does not occur either with chilled RC, in the absence of rumen fluid, or when replaced with egg protein. The mechanisms for this disappearance are yet to be discovered.     

Protein-losing enteropathy in dogs is associated with decreased fecal proteolytic activity

Background: Measurement of proteolytic activity in feces is a traditional method for the diagnosis of exocrine pancreatic insufficiency (EPI). A drawback of this method is the occurrence of falsely low results that may lead to a false‐positive diagnosis of EPI. We hypothesized that intestinal loss of serum proteinase inhibitors in protein‐losing enteropathy (PLE) may inhibit fecal proteolytic activity and be a potential source of false low results. Objective: The objective of this study was to determine the effect of PLE on fecal proteolytic activity in dogs. Methods: Fecal proteolytic activity was measured using a radial diffusion casein digestion assay in 12 samples from 4 clinically healthy control dogs and 30 samples from 16 dogs with PLE. Gastrointestinal protein loss was assessed using an ELISA to determine fecal canine α₁‐proteinase inhibitor concentration. The relationship between the concentration of canine α₁‐proteinase inhibitor in the feces and the diameter cleared in the casein digestion assay was determined. The mean clearing diameter was compared between control dogs and dogs with PLE. Results: A significant negative correlation was observed between fecal canine α₁‐proteinase inhibitor concentration and casein clearing diameter (P < .001, Pearson r=—.6317, r 2 =.3999). Mean clearing diameter was significantly lower in dogs with PLE than in control dogs (12.63 vs 16.83 mm, P < .001, two‐tailed Student's t‐test). Conclusion: Increased fecal loss of α₁‐proteinase inhibitor in dogs with PLE is associated with a significant decrease in fecal proteolytic activity and may result in a false positive diagnosis of EPI.     

Effects of Prolonged in vitro Culture and Cryopreservation on Viability,DNA Fragmentation,Chromosome Stability and Ultrastructure of Bovine Cells from Amniotic Fluid and Umbilical Cord

The objective of this work was to study cellular types that did not participated in the gastrulation process, amniotic fluid cells (AFCs) and umbilical cord cells (UCCs), in conditions of long‐term culture and cryopreserved with different solutions. The AFCs and UCCs were used in a comparative study with ear fibroblast cells (EFCs) that were cultured in vitro until 20 cellular passages and cryopreserved in 10% dimethylsulphoxide (DMSO), 5% dimethyl formamide (DMF) and 7% glycerol (Gly) solutions. The cellular viability, ultrastructure, DNA fragmentation and chromosome stability were evaluated to determine the cellular type most resistant. In all cell types, it was possible to evaluate the AFCs until 15 passages and UCCs until 20 passages with different periods of cellular growth to reach the confluence phase. Solutions containing 10% DMSO ensured viability of 90.33 ± 5.58%, 90.56 ± 4.40% and 81.90 ± 3.31%, respectively for EFCs, AFCs and UCCs, being significantly more efficient and with less variation than other cryoprotectant solutions. The AFCs were more sensitive to cryopreservation and presented low viability rate at the passage 20 (17.2 ± 8.87%). There was no change in karyotype and nuclear fragmentation was low in all cellular passages studied. With the scanning electron analysis was possible the characterization of AFCs and UCCs in suspension. The three cellular types of cells presented different shapes and characteristics on the surface. The results demonstrate that bovine AFCs and UCCs can be isolated, cultured in vitro and cryopreserved in 10% DMSO, not causing damage to DNA and chromosomes. The UCCs were more resistant than AFCs in all aspects.     

The preliminary study on the effects of growth hormone and insulin‐like growth factor‐I on κ‐casein synthesis in bovine mammary epithelial cells in vitro

The effects of growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) on protein synthesis and gene expression of κ‐casein in bovine mammary epithelial cell in vitro were studied. The treatments were designed as follows: the growth medium without serum was set as the control group, while the treatments were medium supplemented with GH (100 ng/ml), IGF‐I (100 ng/ml), and GH (100 ng/ml) + IGF‐I (100 ng/ml). The quantity of κ‐casein protein was measured by ELISA, and the κ‐casein gene (CSN3) expression was examined by real‐time quantitative PCR (RT‐qPCR). Compared with the control group, all the experimental groups had greater (p < 0.05) expression of CSN3. The concentration of κ‐casein followed a similar response as CSN3, but the difference between the treatments and the control was not statistically significant (p > 0.05). Furthermore, no synergistic effect of GH and IGF‐I was observed for both the κ‐casein concentration and CSN3 expression. It is therefore concluded that GH or IGF‐I can independently promote the expression of CSN3 in bovine mammary epithelial cells in vitro.