靶向抑制MAP3K1对小鼠乳腺癌细胞生物学行为的影响

为了在细胞学水平研究靶向抑制MAP3K1(mitogen-activated protein kinase kinase kinase 1)基因对小鼠乳腺癌4T1细胞黏附、运动及侵袭能力等生物学行为的影响,试验用体外构建靶向"沉默"MAP3K1基因的amiRNA(artificial microRNA)干扰载体,以X-treme GENE HP转染4T1细胞(干扰组转染amiRNA干扰载体,对照组转染空载体,空白组未转染质粒)对MAP3K1基因mRNA及蛋白表达水平进行检测,以测定干扰效率;分别以黏附试验检测小鼠乳腺癌4T1细胞的黏附能力,用划痕-愈合试验与Transwell侵袭试验测定小鼠乳腺癌4T1细胞的运动与侵袭能力。结果表明:小鼠乳腺癌4T1细胞转染MAP3K1 amiRNA-3干扰载体与空载体后,干扰4T1细胞MAP3K1 mRNA水平及蛋白水平均被有效抑制,干扰效率高达70%(P0.01)。试验组4T1细胞黏附能力极显著低于对照组(P0.01);干扰组4T1细胞的运动与侵袭能力均显著低于对照组(P0.05)。说明构建的amiRNA干扰载体能够有效抑制靶基因MAP3K1的表达,并能够显著抑制小鼠乳腺癌4T1细胞的黏附、运动与侵袭能力;靶向抑制MAP3K1基因能够广泛地影响小鼠乳腺癌4T1细胞的生物学行为。     

低温诱发肉鸡肺血管重塑过程中肺动脉平滑肌细胞c-Myc蛋白表达的变化

试验旨在研究环境低温诱发肉鸡肺血管重塑过程中肺小动脉血管平滑肌细胞c-Myc蛋白的表达变化,初步探讨肉鸡肺血管重塑的发生机制.120只雄性AA商品代肉鸡15日龄时随机分为对照组((22±1.5)℃)条件下饲养)和低温组((11±2)℃条件下饲养).15～50日龄,每周每组随机取6只,取肺组织做石蜡切片,Weigert-间苯二酚复红染色,观察并测定m管重塑情况;采用免疫组织化学方法榆测肺动脉血管平滑肌细胞c-Myc蛋白表达,并对其进行半定量化.结果显示:(1)低温组肉鸡肺小动脉结构从36日龄开始较对照组发生了明显的重塑(P<0.01或P<0.05);(2)低温组肉鸡肺小动脉血管平滑肌细胞c-Myc蛋白表达从29日龄开始较对照组明显增加(P<0.01).结果表明:低温叫显诱发了肉鸡肺小动脉平滑肌细胞c-Myc蛋白的表达,且参与了肉鸡肺血管重塑的发生发展.     

PD-L1+ HeLa细胞对人外周血活化T细胞的免疫调节作用

目的 探讨人宫颈癌细胞表达PD-L1对人外周血T细胞的免疫调节作用.方法 采用PD-L1质粒转染人宫颈癌HeLa细胞株,将PD-L1+ HeLa细胞与PHA刺激活化的人外周血T淋巴细胞混合培养,MTT法检测T细胞的增殖水平,流式细胞术分析T细胞凋亡率和CD8+/CD4+T细胞比例;ELISA检测PD-L1+ HeLa细胞与PHA刺激活化的人外周血T淋巴细胞混合培养上清液IFN-γ的含量.结果 MTT检测显示与PD-L1+ HeLa混合培养组的T细胞增殖光密度值为(0.31±0.02),明显低于与PD-L1 - HeLa混合培养组的T细胞和对照组T细胞,分别为[(0.62±0.02)和(0.59±0.03),P＜0.01];T细胞凋亡率明显高于与PD-L1 - HeLa混合培养组和对照组T细胞,分别为(32.7%、17.9%和18.3%);CD8 +/CD4+T细胞比值稍低于PD-L1 - HeLa混合培养组和对照组T细胞(分别为0.86、0.91和0.89);与PD-L1+ HeLa混合的T细胞培养上清液IFN-γ的含量低于与PD-L1 - HeLa混合培养组和对照组T细胞,分别为[(801.1±20.5) pg/ml、(1 006.2±30.2 )pg/ml和(1 070.2±113.3) pg/ml,P＜0.01].结论 PD-L1明显抑制人外周血活化T细胞增殖促进其凋亡并下调其免疫功能.     

猪SMYD3基因的克隆、序列分析及其对猪成纤维细胞增殖的影响

试验旨在克隆猪SMYD3(SET and MYND domain-containing protein 3)基因并对其进行序列分析,研究其对猪成纤维细胞增殖的影响。首先克隆猪SMYD3基因,根据其他物种SMYD3基因siRNA和shRNA序列,经同源性比对分析,获得两条猪SMYD3基因shRNA序列,分别构建pSicoR-GFP-SMYD3 shRNA1/shRNA2表达载体,转染HEK293T细胞,利用实时荧光定量PCR分析干扰效率,筛选出抑制效率较好的shRNA,并构建pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体,同时分析SMYD3基因对猪成纤维细胞的增殖作用,检测细胞Nanog、DNMT1及DNMT3a基因表达情况。结果显示,试验克隆得到1 404 bp的猪SMYD3基因编码区序列,生物信息学分析发现,德保猪SMYD3基因与野猪、山羊和野耗牛相应氨基酸序列的同源性分别为99.5%、93.8%和92.9%。shRNA1/shRNA2均能显著抑制SMYD3基因表达(P<0.05),抑制效果分别是34%和54%,选择pSicoR-GFP-SMYD3 shRNA2进行后续研究。通过脂质体转染法将构建的pLVX-IRES-ZsGreen1-SMYD3及pSicoR-GFP-SMYD3 shRNA真核表达载体导入HEK293T细胞,均可观察到清晰的绿色荧光。慢病毒感染细胞及实时荧光定量PCR结果显示,与空白对照组及阴性对照组相比,过表达SMYD3基因促进猪成纤维细胞增殖,Nanog和DNMT1基因表达显著升高(P<0.05);抑制SMYD3基因表达,细胞增殖受到抑制,Nanog、DNMT1、DNMT3a基因表达显著降低((P<0.05),说明SMYD3基因的表达与猪成纤维细胞的增殖显著相关。     

布鲁氏茵Ⅳ型分泌系统效应子VceC功能的初步研究

为研究布鲁氏茵(Brucella)Ⅳ型分泌系统效应子VceC的功能,本研究从羊种布鲁氏菌16M株基因组中克隆vceC基因片段,插入pEGFP-N1中,构建真核表达重组pEGFP-vceC.经脂质体转染HPT-8细胞,荧光显微镜观察、western blot鉴定目的蛋白在HPT-8细胞中表达情况,检测细胞凋亡、NO释放量及LDH活力.Western blot检测显示VceC在细胞中表达,转染pEGFP-vceC后的细胞凋亡、细胞上清液中NO释放量及LDH活力比对照组显著增加(p＜0.05).本研究初步表明VceC蛋白具有细胞毒性作用,为进一步研究VceC的功能奠定了基础.     

小鼠IGFBP-6基因shRNA的设计与筛选

胰岛素样生长因子结合蛋白6(IGFBP-6)是胰岛素样生长因子结合蛋白家族(IGFBPs)的一员,IGFBP 6不同于IGFBP1 5,其与IGF-2的亲和力显著的优先于IGF-1,受到研究者的广泛关注.本研究在小鼠IGFBP-6基因mRNA的806、760和908 bp处找到潜在靶位点,设计3条shRNA干扰载体,并合成DNA Oligo,体外退火成双链DNA,插入pGU6/GFP/Neo载体,获得3条针对目的基因不同靶序列的干扰载体,瞬时转染NIH-3T3小鼠胚胎成纤维细胞48 h后,利用流式细胞技术分选收集转染重组载体的细胞,提取细胞总RNA和蛋白质,实时荧光定量PCR和酶联免疫吸附法分别检测目的基因mRNA水平,蛋白水平对IGFBP-6的表达.结果表明,IGFBP-6(IGFBP-6,806-826)表现出了最高的干扰效率,转染效率为(79.2±2.3)％时,mRNA水平的沉默效率达(68.9±12.2)％,(P＜0.05);蛋白质水平的沉默效率达(46.7±11.3)％,(P＜0.05).本试验为研究IGFBP-6基因对细胞增殖的作用和体内生物学功能奠定了基础.     

不同化学激活方法和电激活参数对猪体外成熟卵母细胞早期孤雌发育的影响

利用屠宰场猪卵巢卵母细胞,在体外成熟培养44～48 h后,对核成熟卵母细胞进行激活.试验1为不同化学激活方法10% 乙醇 10 mg/L 放线菌酮组、2.5 mmol/L 氯化锶 10 mg/L放线菌酮组、5 μmol/L离子霉素 2.5 mmol/L 6-二甲氨基嘌呤(6-DMAP)组、200 μmol/L 硫柳汞 8 mmol/L二硫苏糖醇组和对照组(不用任何激活剂).试验2为用不同电场强度和脉冲时程进行电激活之后放入胚胎培养液中进行体外培养7 d.结果显示,(1)4种不同化学激活方法处理卵子的1原核形成率、2原核形成率和原核形成率都显著比对照组高(P＜0.05),其中离子霉素 6-DMAP组的2原核形成率和总原核形成率最高,分别为(23.1±3.5)%和(65.2±3.5)%,显著高于其他处理组(P＜0.05);(2)4种不同化学激活方法处理组的囊胚率都比对照组高(P＜0.01),其中离子霉素 6-DMAP组的卵裂率及囊胚率最高,分别为(46.6±18.5)% 和(5.6±4.2)%;(3)本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响;(4)电场强度为1.7 kV/cm、脉冲时程为50、70 μs时猪体外成熟卵母细胞激活效果最好,卵裂率、囊胚率、囊胚细胞数分别为(77.4±9.7)%、(12.4±3.7)%、(17.6±5.9)%和(75.1±10.6)%、(12.3±2.6)%、(19.1±8.1).以上结果说明本试验所用的4种化学激活方法均能有效激活猪体外成熟卵母细胞,其中离子霉素 6-DMAP的激活效果最理想;在本实验室条件下,采用1.7 kV/cm的电场强度、50～70 μs的脉冲时程均能有效地激活猪体外成熟卵母细胞;本试验所用的4种不同化学激活方法对猪4-细胞孤雌胚SDS-PAGE电泳的蛋白质表达图谱没有显著影响.     

紫苏叶注射液对犬血管搭桥术后血管内膜增生的影响

为了探讨紫苏叶注射液对犬动脉搭桥术后血管内膜增生的影响。将20只试验犬随机分为高、中、低剂量组(1 434mg/kg,478 mg/kg,159 mg/kg)及对照组,采用同侧自体颈外静脉重建颈总动脉,术后头静脉滴注紫苏叶注射液,持续给药5周,术后3个月牺牲动物,制作病理切片,测定搭桥血管远心端吻合口内膜厚度,探讨紫苏叶注射液抑制血管搭桥术后血管内膜增生的作用机理。结果显示,不同剂量的紫苏液注射液对血管搭桥术后血管内膜增生的抑制程度不同。模型对照组血管内膜厚度为103.40±18.91μm,低、中、高剂量紫苏叶注射液组血管内膜厚度分别为66.05±10.27μm、49.31±5.61μm、27.80.±4.47μm,与模型对照组组相比,低、中、高剂量紫苏叶注射液血管组能极显著降低血管内膜增生(P<0.001),内膜增生的程度随给药剂量的增加而减轻,呈现一定的量效关系。表明紫苏叶注射液能明显抑制犬搭桥术后的血管内膜增生。     

猪miR-199a-3p对mTOR基因表达的调控

通过生物信息学预测miR-199a-3p与mTOR-3′UTR的结合位点,人工合成猪mTOR-3′UTR及突变型片段克隆至双荧光素酶载体上,构建野生型(psiCHECK-2-W-mTOR-3′-UTR)和突变型(psiCHECK-2-M-mTOR-3′-UTR)双荧光素酶报告质粒。给PK-15细胞分别转染miR-199a-3p mimics、negative control和mTOR-3′-UTR野生型/突变型双荧光素酶报告载体,用双荧光素酶试剂盒检测荧光素酶活性,分别采用RT-qPCR和Western blot检测mTOR基因的mRNA和蛋白表达水平。结果表明,将含mTOR-3′-UTR的野生型和突变型双荧光素酶报告质粒与miR-199a-3p mimic共转染PK-15细胞,野生型报告质粒表达的荧光素酶活性显著低于其阴性对照组(P0.05);转染miR-199a-3p mimics能显著下调mTOR基因mRNA和蛋白的表达(P0.05),而转染miR-199a-3p Inhibitor组和Inhibitor-NC组相比,mTOR基因蛋白表达差异不显著(P0.05)。成功构建了猪野生型及突变型mTOR-3′-UTR双荧光素酶报告质粒,通过双荧光素酶试验、RT-qPCR及Western blot证实miR-199a-3p与猪mTOR-3′-UTR存在靶向调控关系。     

miR-145-5p靶向IGF1R介导AKT通路抑制猪骨骼肌卫星细胞增殖和分化

旨在探讨miR-145-5p对猪骨骼肌卫星细胞增殖和分化的影响。本研究采用qRT-PCR检测miR-145-5p的组织表达谱和发育性表达规律;选用1 d晋汾白猪趾长伸肌进行骨骼肌卫星细胞的分离与培养,分别转染miR-145-5p模拟物(mimics)及其对照组(mimics NC),miR-145-5p抑制剂(inhibitor)及其对照组(inhibitor NC),每个处理3个重复,利用qRT-PCR、EdU和CCK-8方法检测增殖相关基因表达量、EdU阳性细胞数和细胞增殖活性;待猪骨骼肌卫星细胞分化后利用qRT-PCR、Western blot和免疫荧光染色方法探究分化相关基因mRNA和蛋白水平及肌管形成情况;采用双荧光素酶试验、qRT-PCR和Western blot探究miR-145-5p对下游IGF1R和AKT通路的影响。结果显示,miR-145-5p基因在肝和肺中表达量最高,心和皮下脂肪中次之(P<0.05);随着日龄的增加,miR-145-5p在猪背最长肌中的表达量持续升高(P<0.05)。在猪骨骼肌卫星细胞体外培养过程中,转染miR-145-5p mimi...     

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 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.

     

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 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 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.