催乳素调控肝脏细胞脂代谢的信号机制

催乳素(prolactin,PRL)是启动和维持泌乳所必须的激素,在泌乳期间能够优先将机体营养物质和能量流向乳腺,用于乳脂和乳蛋白的合成。本试验采用体外分离培养奶牛肝脏细胞,分别添加PRL和磷脂酰肌醇3-激酶(phosphoinositide 3-kinase,PI3K)抑制剂LY294002、PRL和酪氨酸蛋白激酶2(janus kinase 2,JAK2)抑制剂AG490,通过甘油三脂(triglyceride,TG)测定试剂盒测定细胞内TG含量,以确定PRL的脂质合成通路,进而探讨其对脂合成相关基因的作用。结果显示,PRL通过PI3K-Akt信号通路促进奶牛肝脏细胞TG的合成,此时关键转录因子固醇调节元件结合蛋白-1c(SREBP-1c)的表达活性和转录活性及下游控制脂合成的脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶1(ACC1)基因表达均显著升高。而与之相反,添加LY294002之后,SREBP-1c基因的表达活性和转录活性及下游基因FAS、ACC1的表达则显著降低。结果表明,PRL通过PI3K-Akt通路调节SREBP-1c、FAS和ACC1的表达来调控肝脏细胞的TG合成。     

大鼠脂肪细胞分化过程中脂代谢相关基因转录表达时序的研究

对大鼠附睾和肾周脂肪组织分离的前体脂肪细胞原代培养，采用RT—PCR法分析脂代谢重要酶和转录因子基因在不同分化阶段转录表达的时序变化。结果显示，在前体脂肪细胞阶段，固醇调控元件结合蛋白（SREBP）-1c、碳水化合物反应元件结合蛋白（ChREBP）以及脂肪酸合成酶（FAS）、乙酰辅酶A羧化酶α（ACC1）、硬酯酰辅酶A去饱和酶（SCD）和激素敏感脂酶（HSL）基因均不转录表达；HSL mRNA在分化初期表达，中期表达水平最高，分化末期降低；SCD mRNA在分化中期表达，末期表达水平显著提高；分化初期FAS mRNA水平明显高于中期和末期；ACC1 mRNA仅在末期表达；分化初期SREBP-1c mRNA水平较高，中期和末期显著下降；ChREBP mRNA表达水平在分化末期稍高于中期，但差异不显著。表明，SREBP-1c mRNA的表达与脂肪细胞早期分化调控有关，分化成熟脂肪细胞的ChREBP mRNA表达可能与生脂基因的转录激活有关。     

不同饲养环境合作猪脂肪组织生脂基因 mRNA表达水平的比较研究

合作猪是生长在高寒草原以放牧为主的珍稀高原型猪种,为了探讨其品种特性的分子机制,分析测定了原产地合作猪及长期在不同环境和饲养条件下异地饲养合作猪脂肪组织脂肪生成的关键酶脂肪合成酶(FAS)、乙酰辅酶A羧化酶(ACC)1和转录因子固醇调控元件结合蛋白(SREBP)-1c、碳水化合物调控元件结合蛋白(ChREBP)mRNA表达水平,并与杜×大×长三元杂交猪进行了对比。试验结果发现,合作猪脂肪组织FAS、ACC1及ChREBP mRNA水平显著低于三元杂交猪(P<0.05),在长期异地饲养后,这些基因mRNA表达水平提高(P<0.05),而SREBP-1c mRNA表达水平在3个群体中没有明显变化(P>0.05)。结果提示,合作猪由于对低能量、低碳水化合物粗饲的长期适应,表现出脂肪组织生脂基因表达水平较低的生理学特性。     

血清剥夺对原代培养大鼠脂肪细胞脂代谢相关基因转录表达的影响

利用血清剥夺／血清培养实验模型，采用油红O染色提取法、甘油试剂盒和RT-PCR，诱导和测定了大鼠附睾脂肪垫分离和培养的脂肪细胞生脂和脂解及其相关酶和转录因子mRNA水平的变化，探讨脂肪细胞脂代谢通路中关键酶和转录因子基因转录表达的规律。结果显示，依血清剥夺时间延长，脂肪细胞激素敏感脂酶（HSL）mRNA表达水平显著提高（P〈0．05），恢复血清培养后，表达水平显著降低（P〈0．05），与脂解活性的变化一致；脂肪生成及生脂相关基因脂肪酸合酶（FAS）、乙酰辅酶A羧化酶（ACC1）和碳水化合物反应元件结合蛋白（ChREBP）mRNA表达水平依血清剥夺持续时间明显下降（P〈0．05），恢复血清培养，随培养时间延长显著提高（P〈0．05）；血清剥夺显著降低固醇调控元件结合蛋白（SREBP）-1cmRNA水平，但血清剥夺72h与36h无明显差异（P〉0．05）；血清剥夺72h后恢复血清培养36h和72h，SREBP-1cmRNA的表达水平没有显著性变化（P〉0．05）。结果表明，生脂及FAS和ACC1表达与ChREBP mRNA水平一致，但与SREBP-1c不一致，提示ChREBP与成熟脂肪细胞生脂基因转录激活可能有更密切的关系，但尚待在蛋白水平进一步证实。     

胰岛素、胰高血糖素对体外培养犊牛肝细胞CPT-Ⅰ和CPT-ⅡmRNA表达的影响

体外原代培养犊牛肝细胞,添加不同浓度的胰岛素（insulin,INS）（0、1、10、100、1 000nmol/L）和胰高血糖素（glucagon,GLN）（0、1、10、100、1 000nmol/L）,每个处理做3个重复,分别培养1h后,提取总RNA。实时荧光定量PCR（qRT-PCR）的方法,检测肉碱棕榈酰转移酶Ⅰ（carnitine palmitoyl transferase,CPT-Ⅰ）和肉碱棕榈酸转移酶Ⅱ（carnitine palmitoyl transferase,CPT-Ⅱ）mRNA表达的变化。结果显示,随着培养液中INS含量的升高,肝细胞中CPT-ⅠmRNA和CPT-ⅡmRNA的表达量逐渐降低,呈现一定的剂量依赖性。随着培养液中GLN含量的升高,肝细胞中CPT-ⅠmRNA和CPT-ⅡmRNA的表达量均升高,呈现一定的剂量依赖性,且当GLN的添加浓度高于100nmol/L时,CPT-ⅠmRNA和CPT-ⅡmRNA的表达量显著升高（P〈0.01）。结果表明,INS可在一定程度上抑制脂氧化关键酶的表达,GLN可显著促进脂氧化关键酶的表达。     

CLA对奶牛乳腺上皮细胞脂肪酸合成相关酶的影响

为了研究体外添加c9,t11共轭亚油酸(CLA)和t10,c12 CLA对奶牛乳腺细胞(BMECs)中脂肪酸(FA)合成关键酶基因mRNA转录和SCD酶指数的影响,试验采用组织块分离培养BMECs,两种CLA浓度分别为0,50,100,150μmol/L,作用细胞24 h后采用荧光定量PCR(QRT-PCR)对目的基因进行相对定量。结果表明:与对照组相比,50μmol/L c9,t11 CLA显著抑制BMECs中的ACC、FAS、D5和D6基因mRNA,但150μmol/L却显著促进;50μmol/L t10,c12 CLA显著上调了ACC、SCD5、D5和D6基因的mRNA转录水平,对FAS转录无影响(P>0.05),但150μmol/L显著受到抑制;CLA均抑制SCD1的转录,促进SCD5转录。C9,t11 CLA使SCD酶指数显著增加,t10,c12 CLA则相反。     

不同品种猪肌内脂肪合成代谢相关基因表达水平的研究

为了研究肌内脂肪与脂类合成代谢相关基因的关系,试验采用荧光定量PCR的方法检测了8头乌金猪和6头长白猪背最长肌脂肪酸合成酶(FAS)、固醇调节元件结合蛋白1c(SREBP-1c)和硬脂酰CoA去饱和酶(SCD)基因mRNA相对表达量,同时采用索氏提取法检测背最长肌肌内脂肪含量,并对两者进行相关性分析。结果表明:乌金猪FAS基因mRNA相对表达量极显著高于长白猪(P0.01);SCD基因mRNA相对表达量显著高于长白猪(P0.05);SREBP-1c基因mRNA相对表达量虽然高于长白猪,但差异不显著(P0.05)。乌金猪背最长肌肌内脂肪含量高于长白猪,且差异显著(P0.05)。乌金猪和长白猪的FAS和SCD基因mRNA相对表达量都与肌内脂肪含量呈极显著正相关(P0.01);乌金猪SREBP-1c基因mRNA相对表达量与肌内脂肪含量呈极显著正相关(P0.01),长白猪SREBP-1c基因mRNA相对表达量与肌内脂肪含量呈显著正相关(P0.05)。说明脂肪酸合成关键基因的mRNA相对表达量在乌金猪和长白猪间存在品种差异,这种差异可能是肌内脂肪含量不同的主要原因之一。     

甘薯渣替代白酒糟对育肥牛肌内脂肪沉积相关基因表达的影响

本试验旨在研究甘薯渣替代白酒糟对育肥牛肌内脂肪(IMF)沉积相关基因表达的影响。选择30头西杂阉公育肥牛(西门塔尔牛×本地黄牛)随机分为3组,每组10头,每个重复1头牛。A组(对照组)饲喂基础饲粮,B组和C组分别用甘薯渣替代基础饲粮中50%和100%的白酒糟,试验期为8周。结果表明:1) C组的平均日增重和IMF含量显著低于A组和B组(P0.05)。2) C组的血清甘油三酯、总胆固醇、高密度脂蛋白和低密度脂蛋白含量显著低于A组(P0.05)。3) C组背最长肌脂肪酸合成酶(FAS)和乙酰辅酶A羧化酶(ACC)活性显著低于A组和B组(P0.05),激素敏感脂肪酶(HSL)和肉碱转移酶-1(CPT-1)活性显著高于A组和B组(P0.05)。B组背最长肌FAS和ACC活性显著低于A组(P0.05),HSL和CPT-1活性显著高于A组(P0.05)。4) C组背最长肌固醇调节元件结合蛋白-1(SREBP-1)、FAS、ACC和过氧化物酶增殖激活受体γ(PPARγ)基因表达量显著低于A组和B组(P0.05),HSL和CPT-1基因表达量显著高于A组和B组(P0.05)。B组背最长肌SREBP-1、FAS、ACC和PPARγ基因表达量显著低于A组(P0.05),HSL和CPT-1基因表达量显著高于A组(P0.05)。由此可见,提高甘薯渣替代白酒糟比例,可下调育肥牛背最长肌脂肪酸合成相关基因(SREBP-1、FAS、ACC和PPARγ)表达以及上调脂肪分解相关基因(HSL和CPT-1)表达,减少背最长肌IM F沉积。     

脂肪酸结合蛋白5对奶牛乳腺上皮细胞乳脂合成的影响

脂肪酸结合蛋白5（fatty acid binding protein 5,FABP5）是一种胞内脂质运载体。本试验应用甘油三酯测定法及BODIPY染色法检测奶牛乳腺上皮细胞（bovine mammary epithelial cell,BMEC）中甘油三酯及脂滴分泌情况,采用实时荧光定量PCR、Western blotting技术检测BMECs中FABP5对固醇调节元件结合蛋白-1c（SREBP-1c）的表达影响。结果显示,试验成功获得高纯度的BMECs,构建了pGCMV-IRES-EGFP-FABP5真核表达载体,且与空白对照组和空载体组相比,FABP5过表达组的甘油三酯和脂滴分泌量极显著增加（P<0.01）,同时SREBP-1c及靶基因FAS、ACC的表达量均极显著增加（P<0.01）;试验成功筛选了FABP5 siRNA1为最佳干扰片段,当FABP5抑制时,抑制组的甘油三酯、脂滴分泌量极显著减少（P<0.01）,SREBP-1c、FAS、ACC的表达量均极显著降低（P<0.01）。表明FABP5可通过上调SREBP-1c的表达从而促进BMEC中乳脂的合成。     

紫苏籽提取物对育肥牛肌内脂肪沉积的影响

本试验旨在研究紫苏籽提取物对育肥牛生长性能、屠宰性能、肉品质和肌内脂肪(IMF)沉积相关基因表达的影响。选用20头西杂阉公育肥牛(西门塔尔牛×本地黄牛),随机分为2组,每组10头。对照组饲喂基础饲粮,试验组在基础饲粮中添加0.03%紫苏籽提取物(干物质基础),即添加0.04‰紫苏籽提取物活性成分(干物质基础)。试验期为120 d。结果表明:1)与对照组相比,饲粮中添加紫苏籽提取物对育肥牛初重、末重、平均日增重、日均干物质采食量、料重比、胴体重、净肉重、屠宰率、胴体产肉率、背最长肌pH和背最长肌系水力均无显著影响(P>0.05)。2)与对照组相比,饲粮中添加紫苏籽提取物显著提高了育肥牛背最长肌IMF含量(P <0.05),显著降低了背最长肌剪切力(P <0.05)。3)与对照组相比,饲粮中添加紫苏籽提取物显著提高了育肥牛背最长肌脂肪酸合成酶(FAS)和乙酰辅酶A羧化酶(ACC)的活力(P<0.05),显著降低了背最长肌激素敏感脂肪酶(HSL)和肉碱转移酶-1(CPT-1)的活力(P<0.05)。4)与对照组相比,饲粮中添加紫苏籽提取物显著提高了育肥牛背最长肌固醇调节元件结合蛋白-1(SREBP-1)、FAS、ACC和过氧化物酶增殖激活受体γ(PPARγ)基因的相对表达量(P<0.05),显著降低了背最长肌HSL和CPT-1基因的相对表达量(P<0.05)。由此可见,饲粮中添加紫苏籽提取物可上调育肥牛背最长肌脂肪酸合成相关基因(SREBP-1、FAS、ACC和PPARγ)的表达,下调脂肪酸分解相关基因(HSL和CPT-1)的表达,提高脂肪酸合成关键酶(FAS和ACC)的活力,并降低脂肪酸分解关键酶(HSL和CPT-1)的活力,从而促进IMF的沉积。     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Heartworm in dogs in Canada in 1991

In late November 1991, 1883 clinics in Canada were sent a questionnaire to assess the status of Dirofilaria immitis in dogs in 1991 and there was a 60.0% response. There were 344,031 dogs tested for heart-worm (HW), 627 were found infected and the prevalence of HW infection was 0.18%. There were 417 dogs with HW in Ontario, 116 in Manitoba, 38 in Quebec, 53 in British Columbia, three in Alberta, and one in Nova Scotia. In British Columbia, all of the infected dogs but one were from the Okanagan valley which, as from 1991, is a new focus of infection in Canada. Most dogs with HW had not been on preventive medication in 1990, and the prevalence among dogs tested and unprotected was 0.59%. That prevalence was considerably higher in endemic areas. Companion dogs, over three years of age and maintained primarily outdoors in rural areas, were most frequently infected. One cat was diagnosed with D. immitis and 33 dogs had Dipetalonema reconditium.     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1983

In late December 1983, 2 800 veterinarians across Canada were sent a questionnaire in order to assess the status of heartworm disease in Canada in 1983 and 26% of them responded. Veterinarians reported that 59 504 dogs were blood-tested to check for microfilariae and 771 dogs (1.30% of those tested) were found with Dirofilaria immitis. Heartworm disease was diagnosed in all provinces except New Brunswick and Newfoundland but most (733) of the cases were in Ontario.

Heartworm disease was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 31% of the cases were observed with clinical signs of heartworm disease and 64% had a history of not having left Canada. Southwestern Ontario continues to be the focus of the infection and most of the dogs there had not left the province previously.