【作者】 查龙应；

【导师】 许梓荣；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2007， 博士

【摘要】 本课题组前期试验表明纳米级硅酸盐微粒吸附铬(CrNano)能够显著改善肥育猪胴体品质并大幅度提高铬在心、肝、肾、背最长肌中的沉积,预示CrNano可能具有特异的吸收机制和较高的生物可利用率。因此,本试验研究了CrNano、吡啶羧酸铬(CrPic)和三氯化铬(CrCl3)在Caco-2细胞体外吸收模型中的转运和摄取过程,并以Sprague-Dawley (SD)大鼠为试验对象,研究了CrNano、CrPic和CrCl3对SD大鼠生长性能、胴体组成、血清相关指标以及组织铬沉积的影响,旨在比较研究和探讨CrNano、CrPic和CrCl3的吸收机理及其体内生物学效应,为CrNano改善胴体品质的作用效果提供科学依据,同时,促进纳米技术在动物营养与饲料科学领域的研究和应用。将Caco-2细胞接种于Snapwell细胞转运培养槽的聚碳酯微孔滤膜上,在37℃和5%CO2浓度培养条件下培养21 d,细胞跨膜电阻值高于620Ω·cm2；甘露醇透过率低于0.3%·h-1·cm-2；肠腔侧(AP)碱性磷酸酶活性显著高于基底侧(BL)酶活性。表明在本实验室构建的Caco-2细胞模型在形态上与小肠上皮细胞相似,细胞已产生极性,且细胞间已形成紧密连接,可作为小肠吸收的体外模型。CrNano、CrPic和CrCl3在Caco-2细胞模型中的转运和摄取试验分别从两个方向进行：肠腔侧到基底侧(AP-BL)和基底侧到肠腔侧(BL-AP),考察了浓度(0.2-20μmol/L)、时间(120 min)和温度(37℃和4℃)对转运和摄取的影响。结果表明：CrNano、CrPic和CrCl3从AP-BL和BL-AP两个方向跨细胞单层转运都呈浓度和时间依赖型,且转运不受温度影响。CrNano的表观通透系数(Papp)为5.89-7.92×10-6 cm/s,CrPic的Papp为3.52-5.31×10-6cm/s, CrCl3的Papp为0.97-1.37×10-6cm/s。Caco-2细胞从AP-BL和BL-AP两个方向对CrNano、CrPic和CrCl3的摄取都呈浓度和时间依赖型。当将孵育温度从37℃降低到4℃时,Caco-2细胞单层AP侧对CrNano的摄取量显著降低(P<0.05),而对CrPic和CrCl3的摄取量没有影响。37℃孵育条件下,CrNano、CrPic和CrCl3的转运率分别为15.83%±0.76%,9.08%±0.25%和2.11%±0.53%；摄取率分别为10.08%±0.76%,4.73%±0.60%和0.88%±0.08%。试验结果表明CrNano在Caco-2细胞中可能主要是经过跨细胞途径被动吸收,而CrPic和CrCl3可能主要是经过细胞间途径被动吸收,CrNano在Caco-2细胞中的吸收率高于CrPic和CrCl3。将5μmol/L不同影响因素(维生素C、CuSO4、FeCl2、ZnCl2、蔗糖、EDTA、草酸钠和柠檬酸钠)与4μmol/L铬(以CrNano、CrPic和CrCl3形式)共同孵育进行转运试验。结果表明：维生素C、CuSO4、ZnCl2、EDTA、草酸盐、柠檬酸盐和蔗糖对CrNano和CrPic的转运没有显著影响(P>0.05),但FeCl3显著降低了它们在Caco-2细胞单层中的转运量(P<0.05)。维生素C和草酸盐显著增加了CrCl3的转运量(P<0.05)；FeCl3和蔗糖则显著降低了CrCl3转运量(P<0.05)；而CuSO4、ZnCl2、EDTA和柠檬酸盐不影响CrCl3转运量(P>0.05)。以上结果说明不同来源的铬在吸收时所受的影响存在差异,无机CrCl3相对于CrNano和CrPic来说较易受到各种因素的干扰。将40只雄性SD大鼠随机分成四组,第1组饲喂基础日粮,第2、3、4组在基础日粮中分别以CrCl3、CrPic和CrNano的形式添加300μg/kg的铬,试验期间称量体重并记录饲料消耗,试验6周结束,测定体组成、血清指标和组织铬含量。结果表明：CrNano处理使日增重和饲料转化效率分别提高了12.68%(P<0.05)和25.59%(P<0.05)；CrPic和CrCl3对生长性能没有显著影响(P>0.05)。CrNano使大鼠瘦体重增加了15.57%(P<0.05),体脂含量下降了24.42%(P<0.05)；CrPic使大鼠瘦体重提高了14.82%(P<0.05),对体脂含量没有显著影响(P>0.05)；CrCl3对体组成没有显著影响(P>0.05)。CrNano使血清中胰岛素样生长因子Ⅰ、总蛋白和高密度脂蛋白含量分别提高了78.57%(P<0.05)、25.05%(P<0.05)和23.07%(P<0.05),使血清中胰岛素、葡萄糖、甘油三酯和尿素氮含量分别降低了45.38%(P<0.05)、31.84%(P<0.05)、46.67%(P<0.05)和48.75%(P<0.05)。CrPic使血清中胰岛素样生长因子Ⅰ和高密度脂蛋白含量分别升高了38.46%(P<0.05)和26.15%(P<0.05),使血清葡萄糖、甘油三酯和尿素氮含量分别降低了15.05%(P<0.05)、36.00%(P<0.05)和57.62%(P<0.05)。CrCl3对血清相关指标没有显著影响(P>0.05)。同时,CrNano处理组大鼠全血、肝脏、肾脏、空肠和后腿肌肉中的铬含量显著高于对照组、CrCl3组和CrPic组大鼠对应器官中铬含量。这些结果表明,CrNano相对于CrPic和CrCl3来说在大鼠体内具有较高的吸收率；CrNano生物利用率显著高于CrCl3,也有高于CrPic的趋势。将70只雄性SD大鼠随机分为7个日粮处理组,第1组饲喂基础日粮,第2-7组分别在基础日粮中以CrNano形式添加75、150、300、450、600和1200μg/kg铬。试验期6周,试验期间称量体重并记录日粮消耗,试验期末用双能X光测定仪测定大鼠体组成,并收集组织样品,测定组织铬含量。结果如下：以CrNano形式添加75、150、300μg/kg铬显著提高了大鼠的平均日增重(P<0.05)；75、150、300、450μg/kg铬显著提高了饲料转化效率(P<0.05)。300和450μg/kg铬显著提高了大鼠瘦体重(P<0.05),而150、300、450、600μg/kg铬则显著降低了大鼠体脂含量(P<0.05)。75-1200μg/kg铬显著升高了全血中的铬浓度(P<0.05);150～1200μg/kg铬都显著升高了肝脏和肾脏中的铬浓度(P<0.05)；同时300、450和600μg/kg铬则显著升高了大鼠后腿肌肉中铬含量(P<0.05)。这些结果表明日粮中补充CrNano能促进大鼠生长,改善体组成,增加铬在肝、肾、肌肉等组织中的沉积,但这些作用并没有呈现严格的剂量效应。更多还原

【Abstract】 Our previous studies showed that dietary supplementation of chromium (Cr) in the form of chromium-silicate nanocomposite (CrNano) could positively alter carcass characteristics in finishing pigs and greatly increase Cr deposit in heart, liver, kidney and longissimus muscle, which indicated that CrNano might have unique absorption mechanism in vivo and high bioavailability. Therefore, the present studies were conducted to investigate the transport and uptake of CrNano, Cr picolinate (CrPic) and Cr chloride (CrCl3) by using Caco-2 cell monolayer model. At the same time, the effects of CrNano, CrPic and CrCl3 on growth performance, carcass traits, serum parameters and tissue Cr deposit in Sprague-Dawley (SD) rats were also evaluated. Our goal was to investigate, compare and probe into the absorption mechanisms of CrNano, CrPic and CrCl3 as well as their in vivo bioeffects. The potential results will provide scientific explanation for positive effects of CrNano on carcass traits and will promote the research and application of nanotechnology in animal nutrition and feed science.The Caco-2 cells were seeded onto the polycarbonate microporal membranes in Snapwell bichambers and cultured on 37℃and in an atmosphere of 5% carbon dioxide. After 21 days of culture, the transepithelial electrical resistance was higher than 620Ω·cm2, the transport rate of mannitol across Caco-2 cell monolayers was less than 0.3%·h-1·cm-2, and the activity of alkaline phosphatase in apical (AP) side was greatly higher than that in basolateral (BL) side. These results indicated that the Caco-2 cells cultured in our laboratory had similarity to intestinal epithelial cells in morphology, produced polarity, formed well tight junctions between cells, and could be used as an in vitro model to investigate absorption mechanisms of substances.The transport and uptake of CrNano, CrPic and CrCl3 in Caco-2 cell monolayers were conducted from AP to BL and BL to AP, respectively. The effects of concentration (0.2-20μmol/L of Cr), time (120 min of incubation) and temperature (37℃and 4℃on transport and uptake of CrNano, CrPic and CrCl3 were investigated. The results were as follows. Transports of CrNano, CrPic and CrCl3 across Caco-2 monolayers both from AP to BL and BL to AP direction were concentration-and time-dependent. Lowering the incubation temperature from 37℃to 4℃had no effect on transport amounts of CrNano, CrPic and CrCl3 both from AP to BL and BL to AP direction. The apparent permeability coefficient (Papp) of CrNano was between 5.89×10℃cm/s and 7.92×10-6 cm/s. Papp of CrPic was between 3.52×10-6 cm/s and 5.31×10-6 cm/s, and that of CrCl3 was between 0.97×10-6 cm/s and 1.37×10-6 cm/s. Uptakes of CrNano, CrPic and CrCl3 into Caco-2 cells both from AP to BL and BL to AP direction were concentration-and time-dependent. The uptake amount of CrNano into Caco-2 cells in AP side was significantly decreased (P<0.05) when incubation temperature lowered from 37℃to 4℃, whereas the uptake amounts of CrPic and CrCl3 were unaffected (P>0.05). The transport rate of CrNano, CrPic and CrCl3 was 15.83%±0.76%,9.08%±0.25%, and 2.11%±0.53%, respectively. The uptake rate of CrNano, CrPic and CrCl3 was 10.08%±0.76%, 4.73%±0.60%, and 0.88%±0.08%, respectively. These results indicated that absorption of CrNano in Caco-2 cell monolayers was mainly via transcellular pathway, while absorption of CrPic and CrCl3 were mainly via paracellular pathway. CrNano had higher absorption efficiency than CrPic and CrCl3 in Caco-2 cell monolayers.Transport of 4μmol/L of Cr in the form of CrNano, CrPic and CrCl3 were conducted with 5μmol/L of vitamin C, copper sulfate, ferric chloride, zinc chloride, sucrose, ethylene diaminetetraacetic acid (EDTA), oxalate, or citrate, respectively. Vitamin C, copper, zinc, EDTA, oxalate, citrate and sucrose had no effect on transport of CrNano and CrPic (P>0.05). However, ferric significantly reduced the transport amount of CrNano and CrPic (P<0.05). Vitamin C and oxalate significantly increased the transport amount of CrCl3 (P<0.05). Ferric and sucrose decreased the transport amount of CrCl3 (P<0.05). Copper, zinc, EDTA and citrate did not affect the transport of CrCl3 (P>0.05). These results indicated that dietary factors played different roles in affecting diverse sources of Cr. CrCl3 was easily affected by dietary factors relative to CrNano and CrPic.A total of forty male SD rats were randomly assigned to four dietary treatment groups. Rats in the first group were fed basal diet as a control. Rats in the second, third and fourth group were fed basal diet supplemented with 300μg/kg Cr in the form of CrCl3, CrPic and CrNano, respectively. Feeding trial was conducted for six weeks. During the period of trial, body weight of rats was weighed and feed consumption was recorded. At the end of trial, body composition, serum parameters and tissue Cr content were determined. In comparison to the control, supplemental Cr from CrNano increased average daily gain and feed efficiency by 12.68%(P<0.05) and 25.59%(P<0.05), respectively. Addition of Cr from CrPic and CrCl3 had no effect on growing performance of rats (P>0.05). Supplementation of CrNano increased lean body mass by 15.57%(P<0.05) and decreased percent body fat by 24.42%(P<0.05). Addition of CrPic increased lean body mass by 14.82%(P<0.05) while had no effect on percent body fat (P>0.05). Addition of CrCl3 had no effect on body composition of rats (P>0.05). Supplemental Cr from CrNano increased serum contents of insulin-like growth factor I, total protein and high-density lipoprotein by 78.57%(P<0.05), 25.05%(P<0.05) and 23.07%(P<0.05), respectively, and decreased serum contents of insulin, glucose, triglyceride, and urea nitrogen by 45.38%(P<0.05),31.84%(P<0.05), 46.67%(P<0.05), and 48.75%(P<0.05), respectively. Addition of Cr from CrPic increased serum contents of insulin-like growth factor I and high-density lipoprotein by 38.46%(P<0.05) and 26.15%(P<0.05), respectively, and decreased serum contents of glucose, triglyceride, urea nitrogen by 15.05%(P<0.05),36.00%(P<0.05), and 57.62%(P<0.05), respectively. However, supplementation of Cr from CrCl3 had no effect on serum parameters in the rats. In addition, Cr content in blood, liver, kidney, jejunum and hind leg muscle of rats fed supplemental Cr was higher than that in the corresponding tissues of rats fed basal diet and diet supplemented with CrCl3 and CrPic. These results suggested that CrNano had higher absorption efficiency than CrPic and CrCl3 in SD rats. In addition, CrNano had significantly higher bioavailability than CrCl3 and tended to be more bioavailable than CrPic in rats.A total of seventy male SD rats were randomly allotted to seven dietary treatment groups. Rats in the first group were fed basal diet as control. Rats in the second, third, fourth, fifth, sixth and seventh group were fed basal diet supplemented with 75,150,300,450,600, and 1200μg Cr/kg diet in the form of CrNano, respectively. The feeding trial was conducted for six weeks. The body weight of rats was weighed and feed consumption was recorded during the trial. At the end of the trial, body composition of rats was assessed by dual energy X-ray absorptionmetry. Samples of organs were collected for analysis. Supplementation of 75,150 and 300μg/kg Cr in the form of CrNano significantly increased average daily gain (P<0.05). Feed efficiency was increased by addition of 75,150,300 and 450μg/kg Cr from CrNano (P<0.05). Lean body mass was increased by 300 and 450μg/kg Cr as CrNano (P<0.05). Addition of 150,300,450 and 600μg/kg Cr from CrNano decreased percent body fat of rats significantly (P<0.05). Addition of from 75μg/kg to 1200μg/kg Cr from CrNano significantly increased Cr content in blood (P<0.05). The Cr concentration in liver and kidney was greatly enhanced by supplementation of from 150μg/kg to 1200μg/kg Cr (P<0.05). In addition, Cr content in hind leg muscle of rats was significantly increased by addition of 300,450 and 600μg/kg Cr from CrNano. These results suggested that supplementation of CrNano to the diet of rats could promote growth, positively alter body composition, increase Cr deposit in tissues. However, no restrict dose dependent effect was detected.更多还原