成年牛瘤胃上皮细胞原代培养方法研究

为了更加经济便捷,微观科学地研究成年牛瘤胃生理及营养代谢机制,本试验着手建立成年牛瘤胃上皮细胞(ruminal epithelium cell,REC)的原代培养方法,其主要分为瘤胃微生物去除和细胞分离培养两部分。首先用9种不同种类和剂量的抗生素洗涤液,冲洗瘤胃上皮组织。根据微生物去除结果,确定最佳种类和剂量的抗生素洗涤液;然后采用0.25%胰蛋白酶+0.02%EDTA在不同条件下对瘤胃上皮进行连续消化和分步消化以及先用0.1%胶原酶I消化再用0.25%胰蛋白酶+0.02%EDTA在37℃分步消化,根据所得的细胞特征以及细胞增殖活性来确定适合成年牛瘤胃上皮细胞的分离方法。结果表明,瘤胃上皮需分别经过6倍青链霉素和6倍庆大两性霉素洗涤液的清洗后能有效去除瘤胃上皮表面微生物。而瘤胃上皮先经过0.1%胶原酶I消化30min再经0.25%胰蛋白酶+0.02%EDTA分步消化后可获得较多的上皮细胞,且活性较好,分离效果较理想。本试验为成年牛瘤胃上皮细胞的原代培养提供了参考方法。     

Culture of epithelial cells from bovine ruminal mucosa

A method is reported for the primary in vitro culture of epithelial cells derived from bovine ruminal mucosa. That the cultures of ruminal epithelial cells consisted exclusively of stratum spinosum, stratum basale and stratum granulosum was confirmed by immunoperoxidase and immunofluorescence staining using carbonic anhydrase isoenzyme as a marker.     

The effect of sodium butyrate supplementation on ruminal and fecal pH and serum lipopolysaccharide-binding protein after ruminal acidosis challenge in nonlactating cows

The objective of this study was to evaluate effects of sodium-butyrate supplementation on gastrointestinal function and the inflammatory response to ruminal acidosis (RA) challenge in cows. Four nonlactating cows with a rumen cannula were assigned to two treatments in a crossover design. Treatments were ruminal administration of sodium-butyrate (BUT) or control (CON). Sodium-butyrate was provided as Gustor BP70 and administered at a butyrate dose of 0.04% per kg body weight. The CON premix was made by replacing sodium-butyrate with wheat bran. Experimental periods were 28 days long with 21-day washout period separating the treatments. On Day 25 of each period, corn starch was ruminally administered at 0.7% per kg body weight as RA challenge. After RA challenge, ruminal pH was lower, and endotoxin concentration was higher for cows provided with BUT than those with CON, but the increase in fecal starch and the decrease in fecal pH were attenuated by BUT. The effect of butyrate supplementation on serum lipopolysaccharide-binding protein after RA challenge was not found. From these findings, butyrate supplementation mitigated rectal acidosis by reducing the flux of fermentable carbohydrate into the large intestine. An anti-inflammatory effect of butyrate was not observed, possibly due to lower pH and higher endotoxin concentration in the rumen.     

Technical note: isolation and characterization of sheep ruminal epithelial cells

A system for the isolation and characterization of sheep ruminal epithelial cells has been developed. Ruminal papillae from the ventral cranial sac of 12- to 24-wk-old Dorset ram lambs were subjected to serial tryptic digestions. Initial digestions (two 15-min periods) in the series were used to remove keratinized cells of the stratum corneum, which were able to produce only small amounts of beta-hydroxybutyrate from butyrate (5.37 +/- .72 nmol/120 min/per milligram of dry cell weight). Later digest fractions, containing primarily cells from the stratum basale, exhibited high viabilities (75 to 95%) and proved capable of converting butyrate to beta-hydroxybutyrate at relatively high rates (33.6 +/- 6.7 nmol/120 min per milligram of dry cell weight). Neither acetate nor propionate underwent significant conversion to beta-hydroxybutyrate. However, addition of acetate inhibited (77.4% of control) and addition of propionate stimulated (200% of control) beta-hydroxybutyrate production. Acetate addition reduced the propionate-induced stimulation of beta-hydroxybutyrate production from butyrate (136% of control). These results are similar to those obtained from in vitro incubations of ruminal papillae and suggest that an isolated cell system may prove useful in the further study of ruminal epithelial metabolism.     

Effect of increasing ruminal butyrate on portal and hepatic nutrient flux in steers.

Six Holstein steers (mean +/- SE BW = 344 +/- 10 kg) fitted with hepatic, portal, and mesenteric vein and mesenteric artery catheters and a ruminal cannula were used in a 6 x 6 Latin square design to evaluate the effects of increasing ruminal butyrate on net portal-drained visceral and hepatic nutrient flux. Steers were fed a 40% brome hay, 60% concentrate diet in 12 portions daily at 1.25 x NEm. Water (control) or butyrate at 50, 100, 150, 200, or 250 mmol/h was supplied continuously via the ruminal cannula. Simultaneous arterial, portal, and hepatic blood samples were taken at hourly intervals from 15 to 20 h of ruminal infusion. Portal and hepatic blood flow was determined by continuous infusion of P-aminohippurate, and net nutrient flux was calculated as the difference between venous and arterial concentrations times blood flow. Ruminal and arterial concentrations and total splanchnic flux of butyrate increased (P less than .01) with increased butyrate infusion. Arterial concentrations of acetate (P less than .10), alpha-amino-N (P less than .05), and glucose (P less than .01) decreased with increased butyrate, whereas arterial beta-hydroxybutyrate (P less than .01) and acetoacetate (P less than .05) increased. Increased butyrate produced an increased portal-drained visceral flux of acetoacetate and an increased net hepatic flux of beta-hydroxybutyrate. Urea N and glucose net portal and hepatic fluxes were not affected by ruminal butyrate. Alpha-amino-N uptake by the liver decreased with increased butyrate (P less than .10). Simple linear regression (r2 = .985) indicated that 25.8% of ruminally infused butyrate appeared in portal blood as butyrate. Only 14% could be accounted for as net portal-drained visceral flux of acetoacetate plus beta-hydroxybutyrate.     

The effects of beta agonists on muscle cells in culture

Increases in protein synthesis of 12% were found with two myogenic cell lines (L6 and G8-1) on treatment for 6 hr with the beta-adrenergic agonist cimaterol. In L6 cells, propranolol blocked the effect. Protein breakdown measured over 18–24 hr was unchanged. The K_d for cimaterol binding to the L6 beta-receptor was 26 nM which was compatible with its EC₅₀ for the stimulation of protein synthesis (approx 5 nM). Evidence provided with muscle cell lines indicatesa direct effect of cimaterol on protein synthesis, which may contribute to muscle accretion in cimaterol-fed animals.     

牛乳腺上皮细胞的分离培养

为了对牛乳腺上皮细胞(MECs)进行分离、培养和鉴定,并研究细胞分泌功能,试验通过胶原酶消化法分离得到了牛乳腺上皮细胞,采用传代法对细胞进行纯化,对细胞标志蛋白进行免疫荧光染色鉴定,通过体外诱导和RT-PCR分析鉴定细胞的分泌功能。结果表明:分离到的牛乳腺上皮细胞具有典型乳腺上皮细胞的形态特征,表达广谱角蛋白,经诱导后可分泌β-酪蛋白。     

荷斯坦奶牛乳腺上皮细胞的分离培养

目的：建立荷斯坦奶牛乳腺上皮细胞的分离培养方法。方法：采用组织块种植法培养奶牛乳腺上皮细胞,利用胰蛋白酶差时消化法分离、纯化上皮细胞。结果：成功培养出奶牛乳腺上皮细胞,显微镜下观察,纯化的乳腺上皮细胞呈典型上皮细胞形态,细胞之间排列紧密,呈鹅卵石铺路样,形态均一,多角形的单层聚集。通过荧光免疫细胞染色方法对细胞骨架蛋白-角蛋白18进行鉴定,呈现阳性反应。乳腺上皮细胞增殖旺盛,经25次以上传代后长势仍然良好。结论：采用组织块种植法结合胰酶差时消化法成功获得纯化的奶牛乳腺上皮细胞。     

Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro

The present study evaluates the effects of embryonic age and proteolytic enzymes on the isolation and primary culture of chicken enterocyte and to establish an effective technique for chicken intestinal epithelial cell (IEC) cultivation. Fourteen‐day‐old, 16‐day‐old and 18‐day‐old embryos (average weight: 52.23 ± 0.76 g, 50.86 ± 0.99 g, 48.98 ± 1.03 g) were the source for preparation of enterocyte culture, and trypsin‐ethylene diamine tetraacetic acid, collagenase, thermolysin and combination of collagenase and thermolysin were used for digestion medium. Optimal culture protocols were determined by qualitative assays of proliferation. Cells isolated by using 14‐day‐old embryo and collagenase obtain the best attachment and growth in culture, and the production of continuously growing IEC cultures. Thus, we conclude that the use of collagenase as a dissociating enzyme and 14‐day‐old embryo as a source can be advantageously applied to the isolation of chicken IEC and this method may be useful for various applications and basic studies of the intestinal tract concerning such objects as physiology, immunology and toxicology.     

牦牛支气管上皮细胞的分离培养与鉴定

为了分离培养牦牛支气管黏膜上皮细胞并传代培养,利用支气管结扎灌注酶冷消化法和支气管组织贴壁法分离培养牦牛支气管黏膜上皮细胞并传代培养,第二代细胞制作细胞爬片通过H.E.染色,扫描电镜和免疫荧光分别对培养的上皮细胞的形态结构进行鉴定,结果两种方法都获得了牦牛支气管黏膜上皮细胞,细胞活性好,纯度高;H.E.染色可见细胞形态呈梭形、圆形、多角形等,细胞核深染,细胞质淡红色;扫描电子显微镜检测中,明显可见细胞的纤毛;免疫荧光鉴定细胞核清晰可见,细胞核呈亮绿色,表明分离培养的细胞为上皮细胞,可进一步为牦牛支气管黏膜上皮细胞粘附模型的建立提供材料和奠定技术基础。     

Isolation and monolayer culture of bovine oviduct epithelial cells

Oviduct epithelia obtained from 32 cows were cultured. The oviducts were classified into follicular and luteal phases and divided into ampulla and isthmus regions. The epithelial cells were dissociated by enzyme digestion and cultured in plastic dishes with Dulbecco's modified Eagle's medium/Ham's F12 (1:1) containing 10% calf serum. After enzyme treatment, the epithelial suspension showed free ciliated and non-ciliated cells, and cell mass. The non-ciliated cells contained secretory granules in the cytoplasm. The cell mass was composed of ciliated and secretory cells. The cell mass adhered to the dish within 12-24 hr, while the free ciliated cells attached on Day 2 of the culture. The cells grew into confluent monolayers on Day 4. The cell monolayers contained ciliated and non-ciliated cells. The monolayered non-ciliated cells showed a few secretory granules. When the cells were further cultured without subculturing, ciliary activity diminished on Day 5 and was rarely detected on Day 9. When the cells were subcultured on Day 3, ciliary movement was detected on the monolayers for only 2 days. Cell mass that did not adhere to the dish and remained floating in the medium formed ball-like structures on Day 2. Active ciliary beating was observed on the cells that were cultured in the medium supplemented with 10(-5) and 10(-9) M estradiol-17 beta, however, the ciliary activity diminished on Day 5. No difference in the cell growth was observed between the follicular and luteal phases or between the ampulla and isthmus regions.     

Ghrelin对猪小肠上皮细胞增殖的影响

以体外分离培养的猪小肠上皮细胞为试验材料,通过分别添加1×10-5、1×10-6、1×10-7、1×10-8、1×10-9和1×10-10mol/L的Ghrelin液,来测定Ghrelin对猪小肠上皮细胞增殖的影响。试验结果表明,Ghrelin的浓度在1×10-7-1×10-6mol/L时能够显著促进体外培养的猪小肠上皮细胞的增殖(P<0.05),且在刺激后第5天达到增殖高峰。     

The relationship between cell proliferation and phosphatidylinositol metabolism in skeletal muscle cells in culture

Effects of gamma-hexachlorocyclohexane (1 X 10(-4) M) and lithium chloride (10 mM) on the cell proliferation and phosphatidylinositol metabolism of L6 myoblasts were examined. gamma-Hexachlorocyclohexane (inhibitor of phosphatidylinositol synthesis) and LiCl (inhibitor of inositol 1-phosphatase) treatment of these cells resulted in the loss of their proliferative properties. Coordinate with effects on cell proliferation, gamma-hexachlorocyclohexane and LiCl also reduced the levels of phosphatidylinositols and their water-soluble metabolites. However, the protein content of treated cells was higher than control cells. Gamma-hexachlorocyclohexane and LiCl treatment of cells reduced DNA synthesis by about 50% over control values. It was, therefore, concluded that there appears to be a direct relationship between L6 myoblast proliferation and phosphatidylinositol metabolism.     

Both butyrate incubation and hypoxia upregulate genes involved in the ruminal transport of SCFA and their metabolites

Butyrate modulates the differentiation, proliferation and gene expression profiles of various cell types. Ruminal epithelium is exposed to a high intraluminal concentration and inflow of n‐butyrate. We aimed to investigate the influence of n‐butyrate on the mRNA expression of proteins involved in the transmembranal transfer of n‐butyrate metabolites and short‐chain fatty acids in ruminal epithelium. N‐butyrate‐induced changes were compared with the effects of hypoxia because metabolite accumulation after O₂ depletion is at least partly comparable to the accumulation of metabolites after n‐butyrate exposure. Furthermore, in various tissues, O₂ depletion modulates the expression of transport proteins that are also involved in the extrusion of metabolites derived from n‐butyrate breakdown in ruminal epithelium. Sheep ruminal epithelia mounted in Ussing chambers were exposed to 50 mM n‐butyrate or incubated under hypoxic conditions for 6 h. Electrophysiological measurements showed hypoxia‐induced damage in the epithelia. The mRNA expression levels of monocarboxylate transporters (MCT) 1 and 4, anion exchanger (AE) 2, downregulated in adenoma (DRA), putative anion transporter (PAT) 1 and glucose transporter (GLUT) 1 were assessed by RT‐qPCR. We also examined the mRNA expression of nuclear factor (NF) κB, cyclooxygenase (COX) 2, hypoxia‐inducible factor (HIF) 1α and acyl‐CoA oxidase (ACO) to elucidate the possible signalling pathways involved in the modulation of gene expression. The mRNA expression levels of MCT 1, MCT 4, GLUT 1, HIF 1α and COX 2 were upregulated after both n‐butyrate exposure and hypoxia. ACO and PAT 1 were upregulated only after n‐butyrate incubation. Upregulation of both MCT isoforms and NFκB after n‐butyrate incubation could be detected on protein level as well. Our study suggests key roles for MCT 1 and 4 in the adaptation to an increased intracellular load of metabolites, whereas an involvement of PAT 1 in the transport of n‐butyrate also seems possible.     

鸡小肠上皮细胞分离培养及NLRP3在该细胞中的表达

为了探明NLRP3在鸡小肠上皮细胞中表达情况,为鸡的NLRP3基因在小肠细胞中的功能研究奠定基础。采用18胚龄的鸡胚,分离培养鸡的小肠上皮细胞(intestinal epithelial cells,IEC);采用免疫细胞化学(immunocytochemistry,ICC)和反转录PCR(RT-PCR)方法,检测NLRP3基因在鸡小肠上皮细胞中的表达。结果表明:分离培养出活性较强的IEC;ICC检测显示获得的小肠上皮细胞表面NLRP3抗原阳性,RT-PCR法可扩增出520 bp的目的片段。表明成功分离培养出鸡小肠上皮细胞,并证明鸡NLRP3在鸡小肠上皮细胞中表达。     

奶牛乳腺上皮细胞和成纤维细胞的体外培养与鉴定

为体外培养纯化出稳定的奶牛乳腺上皮细胞和成纤维细胞,试验通过外科手术的方法取妊娠后期或泌乳期的荷斯坦奶牛乳腺组织,分离乳腺腺泡,用组织块法体外培养奶牛乳腺细胞,应用差时胰酶消化法和差速贴壁法将奶牛乳腺上皮细胞和成纤维细胞分别纯化出来,并用免疫组化的方法对细胞的纯度进行鉴定。结果表明:纯化的奶牛乳腺上皮细胞多为多角形,细胞核呈圆形或椭圆形,核仁清晰可见,多呈鹅卵石样或铺路石样生长,并可分泌乳滴,角蛋白-18反应阳性,波形蛋白反应阴性;纯化的奶牛乳腺成纤维细胞多为长梭形,呈旋涡状或放射状生长,角蛋白-18反应阴性,波形蛋白反应阳性;经纯化后2种细胞的纯度均可达95%以上,可满足后续试验的要求。     

The effect of replacing corn with glycerol on ruminal bacteria in continuous culture fermenters

The effects of substituting corn with glycerol on DNA concentration of selected ruminal bacteria were investigated using continuous fermenters. Four continuous culture fermenters were used in a 4 × 4 Latin Square design with four 10 days consecutive periods. Treatment diets (60:40 forage to concentrate) were fed at 45 g/day dry matter (DM) in three equal portions. Glycerol (0.995 g/g glycerol) was used to replace corn in a grain mix at proportions of 0% (T0; control), 15% (T15), 30% (T30) and 45% (T45). On day 10 of each period, samples were collected from each fermenter 3 h after the morning feeding and analysed for volatile fatty acid and bacterial DNA concentration. Glycerol substitution was related to significantly higher butyrate, valerate and isovalerate concentrations. Compared with the T0 diet, acetate concentration was significantly lower with the T30 and T45 diets whilst propionate concentration was higher only with the T45 diet. The DNA concentrations for Butyrivibrio fibrisolvens and Selenomonas ruminantium decreased with the T30 and T45 diets compared with the T0 diet. No differences in the DNA concentrations for Ruminococcus albus and Succinivibrio dextrinosolvens amongst diets were observed. The findings show that substituting 15% of the dietary corn with glycerol had no substantive effects on fermentation processing or ruminal bacteria. Higher substitution levels, however, may adversely affect ruminal bacteria and negatively impact acetate production.     

马羊膜上皮干细胞的分离培养和鉴定

通过Tryp LE消化马羊膜收集羊膜上皮细胞,经过传代培养后,用免疫荧光染色、RT-PCR及流式细胞技术(FACS)分析其干细胞特征。免疫荧光染色发现马羊膜上皮细胞表达SSEA-1、SSEA-3、SSEA-4、TRA-1-60和TRA-1-81,只有少量的半贴壁细胞表达Oct4;RTPCR进一步证实马羊膜上皮细胞表达Oct4、Nanog和Sox2,而不表达STAT-3;流式细胞技术分析显示马羊膜上皮细胞也表达CD44、CD90、CD105,但不表达CD45。马羊膜上皮细胞培养三代时,平均倍增时间为(1.25±0.17)d。研究结果表明,通过Tryp LE消化马羊膜上皮可以分离获得马羊膜上皮细胞。     

Potential protective effects of thiamine supplementation on the ruminal epithelium damage during subacute ruminal acidosis

In ruminants, the ruminal epithelium not only has the function of absorbing nutrients but also is an important tissue to prevent harmful substances in the rumen from entering the blood circulation. Thus, the normal function of ruminal epithelium is critical for ruminants. However, subacute ruminal acidosis induced by high-concentrate diets often damages the barrier function of ruminal epithelium in ruminants. Recently, many studies have shown that dietary supplementation with thiamine is an effective method to alleviate subacute ruminal acidosis. In order to provide theoretical reference for the in-depth study of subacute ruminal acidosis and the application of thiamine in the future, this review introduces the effects of subacute ruminal acidosis on morphological structure, inflammatory response, and tight junction of ruminal epithelium. In addition, this paper summarizes the role of thiamine in maintaining ruminal epithelial function of ruminants during subacute ruminal acidosis challenge.