牦牛乳酪蛋白降血压肽对高血压大鼠体内降压效果研究

目的：观察牦牛乳酪蛋白降血压肽对应激性高血压大鼠（stress induced hypertensive rats,SIHR）和正常Wistar大鼠的降压效果,并与降压药物厄贝沙坦（irbesaran）和卡托普利（Captopril）的作用效果进行比较。方法：采用木瓜蛋白酶、中性蛋白酶水解制备牦牛乳酪蛋白降血压肽,分别以20 mg/kg、40 mg/kg和60 mg/kg剂量的牦牛乳酪蛋白降血压肽一次灌胃,每隔2 h测量SIHR收缩压（SBP）,连续测量8 h;长期实验持续6周,每天60 mg/kg一次灌胃,每隔1周测量SBP一次。结果：SIHR给予牦牛乳酪蛋白降血压肽后,血压均显著下降（P〈0.05）,并均在灌胃后3-6 h达最低,血压下降最大幅度为（27±5.13）mmHg,呈现一定的量效关系;而正常大鼠给予牦牛乳酪蛋白降血压肽后血压无显著变化,服用厄贝沙坦和卡托普利后血压则保持显著下降的趋势。长期灌胃牦牛乳酪蛋白降血压肽可使SIHR血压明显下降（P〈0.05）,并且降压效果稳定。结论：牛乳酪蛋白降血压肽对SIHR有明显的体内降压效果,长期服用可稳定血压,对正常大鼠血压无明显影响。     

The development of angiotensin I-converting enzyme inhibitor derived from chicken bone protein

In order to produce angiotensin I‐converting enzyme (ACE) inhibitor for application in functional food, chicken bones were gathered from a meat processing factory and then hydrolyzed with Alcalase, pepsin and trypsin for 12 h. The hydrolysates were lyophilized, stored at ?80°C and tested experimentally every 2 h for pH value, peptide content, degree of hydrolysis (DH), electrophoresis and activity of ACE inhibitor. The hydrolysates of Alcalase had the highest peptide content and DH. The components of more than 66 kDa had disappeared in hydrolysates of Alcalase and trypsin after 2 h of hydrolysis. The hydrolysates of Alcalase were more active in inhibiting ACE, especially when hydrolyzed at 4 and 8 h, and also had low IC50 values of 1.960 and 0.945 mg/mL. According to the results of DH and electrophoresis, the higher activity of ACE inhibitor is assumed to be derived from the low molecular peptides in hydrolysates of Alcalase. Chicken leg bone has a high potential to be utilized to develop ACE inhibitory peptides as a potential ingredient of functional food intended to alleviate hypertension.     

Determination of angiotensin-I converting enzyme inhibitory peptides in chicken leg bone protein hydrolysate with alcalase

This study aims to identify peptides with angiotensin-I converting enzyme (ACE) inhibitory activity in hydrolysate from chicken leg bone protein hydrolyzed with alcalase for 4 h (A4H). The hydrolysate has demonstrated potent in vitro ACE inhibitory activity, and has been shown to attenuate the development of hypertension and cardiovascular hypertrophy in spontaneously hypertensive rats (SHR). A4H is competitive for ACE and was separated using high-performance liquid chromatography (HPLC) with a gel filtration column (Superdex Peptide HR 10/30). The results show that A4H is a mixed non-competitive inhibitor. Eighteen fractions were detected after separation of A4H, and most of them showed ACE inhibitory activity. Five fractions with strong ACE inhibitory activities (above 50%) were labeled from A to E. In addition, there were 10 peptides, consisting of 5–10 amino acid residues that were identified from fraction D that exhibited the strongest ACE inhibitory activity. Three of the identified peptides corresponded to peptides derived from collagen type I and chicken muscular protein. It is revealed that A4H has several peptides that possess ACE inhibitory activities.     

A study of in vivo antihypertensive properties of enzymatic hydrolysate from chicken leg bone protein

Numerous attempts have been made to develop angiotensin I converting enzyme (ACE) inhibitors from various sources of food protein. Generally chicken leg bones are discarded after industrial chicken meat processing without any substantial benefit. In previous studies, chicken leg bone proteins were hydrolyzed by various enzymes and the results demonstrated that Alcalase hydrolysates have considerable ACE inhibiting activities. In this study, the best ACE inhibitory hydrolysate (A4) (which was derived from chicken leg bone protein by Alcalase after 4 h incubation) was orally administrated (50 mg/kg bw) in spontaneously hypertensive rats (SHRs) to investigate its antihypertensive effects. After oral administration of A4, a maximal reduction activity of about 26 mmHg was found at 4 h and maintained to 8 h. Moreover, SHRs treated with A4 (50 mg/kg bw/day) for eight weeks exhibited a reduction in systolic blood pressure, which is as significant as the effects of Captopril ( P  < 0.05). These results suggested that chicken leg bones have a high potential for utilization to develop ACE inhibitors as potential food ingredients intended to alleviate hypertension.     

镉对体外培养鸡骨髓基质细胞成骨分化的毒性作用

骨是镉毒性作用的主要靶器官之一,但其对鸡骨髓基质细胞(bone marrow stromal cells,BMSCs)增殖和成骨分化的毒性作用仍不清楚.本研究利用差速贴壁纯化法获得鸡BMSCs,加入不同浓度镉处理不同时间,采用CCK-8法检测细胞增殖,碱性磷酸酶(alkaline phosphatase,ALP)和茜素...     

HD11来源NDV Ex对鸡骨髓源树突状细胞活化和细胞因子水平的影响

利用重组鸡粒细胞-巨噬细胞集落刺激因子(chGM-CSF)和重组鸡白细胞介素4(chIL-4)体外诱导鸡骨髓细胞分化为鸡骨髓源树突状细胞(chicken bone marrow-derived dendritic cells,chBMDCs),对诱导条件进行优化,利用鸡巨噬细胞系HD11来源的NDV Ex刺激未成熟ch...     

鸡骨髓源树突状细胞的诱导分化及鉴定

为建立鸡骨髓源树突状细胞(Dcs)的体外培养和鉴定方法,本研究无菌抽取10日龄健康SPF雏鸡的骨髓,体外分离纯化骨髓细胞,将其培养于含有重组的人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素4 (rhIL4)的RPMI 1640营养液中,诱导培养鸡骨髓源DCs.采用显微镜观察其体外培养过程中的形态特征及流...     

复方中草药添加剂对大骨鸡生长性能的影响

研究复方中草药添加剂对大骨鸡生长性能的影响,选用1日龄大骨鸡200只,随机分为4组,每组50只,Ⅰ组为空白对照组,Ⅱ、Ⅲ、Ⅳ组基础日粮中分别添加0.5%、1%、2%复方中草药添加剂,试验期42 d。在不同试验阶段称量体重和耗料,计算平均日增重、耗料量及料重比。结果表明,在日粮中添加不同比例的复方中草药添加剂对其生长性能有提高趋势,添加量以1%为最佳。     

养鸡场供水线真菌的分离鉴定及毒性试验

为调查养鸡场供水线真菌污染状况,对哈尔滨市周边15家养鸡场供水线可疑采样进行真菌的分离、纯化,采用病原形态学法对其进行鉴定,同时测定所分离真菌的毒性。结果显示,从30份可疑采样中共分离到6株真菌,分别鉴定为黑曲霉、菌核曲霉、淡紫青霉、鲜绿青霉、扩展青霉和圆弧青霉;毒性试验提示6株霉菌均有弱毒性。     

Fine-mapping of quantitative trait loci for body weight and bone traits and positional cloning of the RB1 gene in chicken

Previously, a quantitative trait locus (QTL) that affects body weight (BW) at 4-12 weeks of age and carcass weight at 12 weeks of age had been mapped on chicken chromosome 1. After including more markers and individuals, the confidence interval was narrowed down to approximately 5.5 Mbps and located this QTL near a microsatellite marker (ADL328). This QTL is the same as the QTL for 12 bone traits, including metatarsus length and metatarsus circumference at 4, 6, 8, 10 and 12 weeks of age and keel length and metatarsus claw weight at 12 weeks of age, that was identified using the same population. In the current study, 1010 individuals from the Northeast Agricultural University F(2) resource population were used and 14 single-nucleotide polymorphism (SNPs) around ADL328 were developed to construct haplotypes, and an association analysis was performed to fine-map the QTL. The haplotypes were constructed on the basis of a sliding 'window', with three SNP markers included in each 'window'. The association analysis results indicated that the haplotypes in 'windows' 6-12 were significantly associated with BW and bone traits and suggested that the QTL for BW and bone traits was located between SNP8 and SNP14 or was in linkage disequilibrium with this region. The interval from SNP8 to SNP14 was approximately 400 kbps. This region contained five RefSeq genes (RB1, P2RY5, FNDC3A, MLNR and CAB39L) on the University of California Santa Cruz website. The RB1 gene was selected as a candidate gene and five SNPs were identified in the gene. The association results indicated that the RB1 gene was a major gene for BW and bone traits. The SNPs g.39692 G>A and g.77260 A>G in RB1 gene might be two quantitative trait nucleotides for BW and bone traits.     

鸡和猪脾转移因子活性组分的提取及免疫活性研究

采用Sephadex G-25柱层析法分离提取鸡、猪脾转移因子(Transfer Factor,TF)各组分。利用环磷酰胺建立小鼠免疫抑制模型,通过MTT法比较鸡脾TF各组分及TF原液对免疫抑制小鼠和正常小鼠的淋巴细胞转化的作用,同时比较了不同来源的鸡脾和猪脾TF各组分及原液对正常小鼠淋巴细胞转化的作用。结果表明鸡脾和猪脾TF均由三大组分构成,其中鸡脾TF三大组分及原液均对免疫抑制小鼠的外周血淋巴细胞转化具有明显的免疫增强作用,与对照组相比均表现差异显著(P<0.01),其中组分Ⅲ的淋巴细胞转化最佳,组分Ⅰ与组分Ⅱ接近,但均优于原液。而对正常小鼠的外周血淋巴细胞转化却表现不同的结果,其中组分Ⅰ和原液具有显著的免疫增强作用(P<0.05),组分Ⅱ作用不显著,组分Ⅲ则起到极显著的抑制作用(P<0.01)。而不同来源的猪脾和鸡脾TF各组分及原液对正常小鼠的淋巴细胞均表现出一致的免疫活性。     

鸡源SUMO标签蛋白的鉴定及在IBDV VP2原核表达中的促溶功能

VP2蛋白是传染性法氏囊病病毒(infectious bursal disease virus,IBDV)的保护性抗原。原核表达中能否获得具有天然构象的可溶性VP2蛋白是研发IBD亚单位疫苗的关键问题。为了解是否存在鸡自体促溶标签蛋白以及外源促溶标签能否在一定程度上促进蛋白的正确折叠,试验对不同物种来源的类泛素蛋白修饰分子(small ubiquitin-like modifier,SUMO)进行比对研究。结果显示,获取到的3种鸡源SUMO蛋白与酵母源SUMO蛋白的氨基酸同源性均高于40%,其中SUMO-1与SUMO-3两种蛋白可以在大肠杆菌中可溶性表达;通过SUMO-1、SUMO-3与VP2蛋白的融合表达,证明了鸡源SUMO蛋白对VP2具有促溶效果,得到的重组菌株P-28A-s1-VP2和P-28A-Os3-VP2琼脂扩散效价均达到1∶16;经过纯化后,融合蛋白SUMO-1-VP2的琼脂扩散效价可达到1∶256;使用SUMO蛋白酶酶切融合蛋白,SDS-PAGE和Western blot结果表明鸡源SUMO蛋白可以被成功切割,说明试验发现的鸡源SUMO蛋白可以作为促溶标签,在亚单位疫苗关键抗原表达中具有广阔的应用前景。     

Osteogenic potential of mesenchymal cells derived from canine umbilical cord matrix co-cultured with platelet-rich plasma and demineralized bone matrix

Canine mesenchymal cells (MSCs) derived from Wharton''s jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.     

印楝油氯仿提取物杀螨活性成分的分离纯化及其生物学活性

将印楝油氯仿提取物经硅胶柱层析和丙酮重结晶进行生物活性跟踪分离纯化,并运用互补重对数模型(CCL模型)分析活性化合物的离体生物活性,求出半数致死浓度(LC50)和半数致死时间(LT50).结果显示,从印楝油氯仿提取物中分离出一种白色雪花状丙酮结晶物,熔点为60～61℃,经结构鉴定为18-碳酸-3,4-呋喃二酯,其对兔疥螨幼虫的杀螨活性呈时间浓度依赖性,24 h的LC50和LC90分别为0.0818和9.842 4 g/L,7.500 g/L时的LT50和LT90分剐为15.332 4 h和24.678 4 h.这表明印楝油氯仿提取物的杀螨活性成分主要为酯类物质,对兔疥螨具有较强的杀螨活性.     

Purification and characterization of an angiotensin I‐converting enzyme inhibitory peptide derived from porcine troponin C

To search for a novel angiotensin I‐converting enzyme (ACE) inhibitory peptide, porcine skeletal troponin was hydrolyzed with pepsin. This hydrolysate showed ACE inhibitory activity, and was applied to various kinds of chromatography to separate an active peptide. Analysis using a protein sequencer identified this peptide as RMLGQTPTK (9mer). This sequence was estimated to occur at the 44–52 position of troponin C, and its 50% inhibitory protein concentration (IC₅₀) was 34 µM. RMLGQTP (7mer), a partial peptide of 9mer, showed activity with an IC₅₀ of 503 µM. RP‐HPLC analysis of a reaction mixture of 9mer and ACE showed that 9mer was slowly hydrolyzed by ACE. On the other hand, 7mer was rapidly hydrolyzed by ACE. Activity of 9mer was reduced as its hydrolysis by ACE proceeded. To estimate the resistance of 9mer to digestive proteases after oral administration, it was reacted with pepsin, α‐chymotrypsin, or trypsin. In each of these reaction mixtures, a significant amount of 9mer remained as a substrate after digestion. Remaining ACE inhibitory activity was close to that of 9mer. These results suggest that 9mer might not be digested after oral administration, because of its relatively high resistance to digestive proteases. Therefore, 9mer might be expected to work well in vivo as an ACE inhibitor.     

不同代次大鼠骨髓间充质干细胞和脂肪间充质干细胞成骨分化比较

本研究旨在观察不同代次骨髓间充质干细胞(BMSCs)和脂肪间充质干细胞(ADSCs)体外培养的生长特点和体外诱导成骨能力。通过密度梯度离心和贴壁培养法分离培养大鼠骨髓间充质干细胞和脂肪间充质干细胞,用含地塞米松、抗坏血酸、β-甘油磷酸钠的培养液定向诱导传代细胞向成骨细胞分化,并利用茜素红染色、碱性磷酸酶染色及PCR方法检测成骨细胞。结果表明骨髓及脂肪间充质干细胞呈成纤维细胞样生长,增殖能力强,生长迅速。第5、10、15、20代BMSCs及ADSCs经诱导培养后茜素红染色呈阳性并且出现"矿化"、碱性磷酸酶活性强,随着细胞代次的递增,诱导后细胞碱性磷酸酶活性呈递减趋势;诱导后的两类细胞传代后细胞仍能继续分化,并形成正常的"矿化"结节,且碱性磷酸酶染色均弱于初次诱导。结果提示,BMSCs及ADSCs易于分离培养及体外扩增,诱导条件下成骨能力强且成骨细胞传代培养仍具有成骨能力,适合作为再生医学骨组织工程的种子细胞。