靶基因xylE质粒载体转基因小鼠的建立

目的建立在基因组中整合有ｐＵＣ１１８ＮＸ质粒的转基因小鼠致突变检测模型。方法将含ｘｙｌＥ靶基因的ｐＵＣ１１８ＮＸ质粒ＤＮＡ显微注射至Ｃ５７ＢＬ／６Ｊ小鼠３７６枚受精卵雄性原核中,存活的２２５枚受精卵移入１１只假孕母鼠双侧输卵管内发育成个体后,用ＰＣＲ－Ｓｏｕｔｈｅｒｎｂｌｏｔ、质粒回收转化试验和酶切鉴定方法筛选存活仔鼠。结果假孕母鼠中有７只妊娠,共产仔鼠２９只,存活２５只,其中ＰＣＲ－Ｓｏｕｔｈｅｒｎｂｌｏｔ检测阳性小鼠１８只,阳性率为７２％。最后选择２只在基因组中完整整合ｐＵＣ１１８ＮＸ质粒的健康雄性小鼠作为Ｆｏｕｎｄｅｒ鼠,进行转基因小鼠致突变检测和建系工作。结论成功地建立了在基因组ＤＮＡ中整合有ｐＵＣ１１８ＮＸ质粒的Ｃ５７ＢＬ／６Ｊ转基因小鼠。     

浙江省吸毒人员HIV,HBV,HCV感染的血清学研究

为了解浙江省吸毒人员ＨＩＶ、ＨＢＶ、ＨＣＶ感染情况,收集浙江省戒毒人员血浆１８２份,其中静脉吸毒者（ＩＶＤＵｓ）６４份,非静脉吸毒者（ＮＩＶＤＵｓ）１１８份。用ＥＬＩＳＡ法检测抗ＨＩＶ,用ＥＩＡ法检测ＨＢＶ标志物和抗ＨＣＶ。结果发现,浙江省吸毒戒毒人员抗ＨＩＶ阳性率为０％,ＩＶＤＵｓ组ＨＢＶ总感染率为６７．１８％（４３／６４）,ＨＢｓＡｇ携带率为１０．９３％（７／６４）,ＨＢｅＡｇ阳性７．８１％（５／６４）,抗ＨＣＶ阳性２１．８８％（１４／６４）;ＮＩＶＤＵｓ组ＨＢＶ总感染率为７１．１９％（８４／１１８）,ＨＢｓＡｇ携带率为１５．２５％（１８／１１８）,ＨＢｅＡｇ携带率为１５．２５％（１８／１１８）,ＨＢｅＡｇ阳性５．９３％（７／１１８）,抗ＨＣＶ阳性０．８４％（１／１８）。ＨＢＶ标志物阳性率ＩＶＤＵｓ和ＮＩＶＤＵｓ间无显著性差异（Ｐ＞０．０５）,抗ＨＣＶ阳性率两组间有非常显著性差异（Ｐ＜０．００１）。结果提示,浙江省为ＨＩＶ感染低发区,为ＨＢＶ感染高流行区,静脉吸毒为ＨＣＶ感染的高危因素     

表没食子儿茶素没食子酸酯抗突变的实验研究

目的 研究表没食子儿茶素没食子酸酯（ＥＧＣＧ）抗突变作用。方法 以昆明纯种小鼠的骨髓细胞为实验材料,用公认的诱变剂环磷酰胺（ＣＰ）诱发的微核（ＭＮ）率和姐妹杂色单体互换（ＳＣＥ）频率为指标,研究ＥＧＣＧ对ＣＰ诱导小鼠骨髓细胞ＭＮ和ＳＣＥ的影响。结果 ＥＧＣＧ对ＭＮ和ＳＣＥ均具明显抑制作用。结论 ＥＧＣＧ具有抗突变性和潜在的预防肿瘤的作用。     

产气荚膜梭菌β-毒素基因的克隆及CPB-ST融合基因的构建

用聚合酶链式反应（ＰＣＲ）技术，从Ｂ型产气荚膜梭菌染色体基因组中扩增了９３０ｂｐ的β－毒素基因。用限制性核酸内切酶ＢａｍＨＩ和ＥｃｏＲＩ对ＰＣＲ产物进行双酶切处理，然后，通过Ｔ４ＤＮＡ连接酶将其定向连接于事先经同样的双酶切处理的载体质粒ｐＥＴ－２８Ｃ（＋）的多克隆位点，转化至受体菌ＢＬ２１（ＤＥ３）中。经ＢａｍＨＩ和ＥｃｏＲＩ双酶切分析和ＰＣＲ扩增检测。证明重组质粒ｐＥＣＢ２中含有产气荚膜梭菌的β－毒素基因。经核苷酸序列分析，明确了克隆的β－毒素基因在重组质粒中的连接向位和阅读框架是正确的。利用已构建的另一重组质粒ｐＸＳＴ１（带有肠毒素性大肠杆菌的耐热性肠毒素ＳＴ基因），通过ＥｃｏＲＩ和ＳａｌＩ双酶切处理，将切下的ＳＴ基因片段定向连接到ｐＥＣＢ２重组质粒中ＣＰＢ基因的下游。经特定酶切分析鉴定，得到了理想重组子质粒ｐＥＣＢＳＴ１，ＣＰＢ－ＳＴ融合基因在重组质粒ｐＥＣＴ－ＳＴ１中的连接向位是正确的     

乙基亚硝基脲诱导转基因小鼠基因突变的研究

[目的 ]用乙基亚硝基脲 (ENU)进一步验证携带xylE基因的转基因小鼠在体内基因突变研究中的应用价值。 [方法 ]用ENU对转基因小鼠进行诱变处理 (i.p .5 0mg/(kg·d) ,连续 5d)后 ,从脾脏组织DNA中回收带有靶基因xylE的pESnx质粒 ,通过电穿孔法转化大肠杆菌 ,筛选突变体 ,检测突变率 ,并对突变靶基因进行测序分析以确定突变类型。 [结果 ] (1)ENU诱发转基因小鼠脾脏组织xylE基因的突变率为 1 2 48× 10 -4 ,显著高于自发突变率 ;(2 )ENU诱发基因突变的类型包括碱基的颠换、转换以及由于碱基的缺失或插入所引起的移码突变。 [结论 ]该转基因小鼠是检测体内基因突变的一种有效的动物模型。     

CPB-ST融合基因的构建及表达研究

用限制性核酸内切酶ＥｃｏＲＩ和ＳａｌⅠ双酶切含有大肠杆菌耐热性肠毒素ＳＴ１前体蛋白基因的质粒ｐＸＳＴ１，回收３２５ｂｐ的ＳＴ基因片段，然后，通过Ｔ４ＤＮＡ连接酶将其定向连接于事先经同样的双酶切处理的带有产气荚膜梭菌β－毒素基因（ＣＰＢ）的重组质粒ｐＥＣＢ２中ＣＰＢ基因的下降，转化受体菌ＢＬ２１（ＤＥ３）中，经ＢａｍＨＩ和ＥｃｏＲＩ酶切反应鉴定重组质粒，得到了理想重组质粒ｐＥＣＢ－ＳＴ１。重组菌株Ｂｌ２１（ＤＥ３）（ｐＥＣＢ－ＳＴ１）经ＩＰＴＧ诱导后，其表达产物经ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎ－ｂｌｏｔｉｎｇ及ＥＬＩＳＡ检测，结果表明重组菌株可以表达ＣＰＢ－ＳＴ融合蛋白，而且该融合蛋白无天然β－毒素和ＳＴ１的生物毒性。     

美国ACUSON ASPEN SN31893彩超特殊故障1例

美国ＡＳＰＥＮＳＮ３１８９３彩超系美国ＡＣＵＳＯＮ公司的最新一代产品 ,为新一代全功能彩超 ,该机型在部队医院拥有量较大 ,据对经销商了解还没有出现过类似的故障。故障现象开机后机器开始自检 ,４ＭＢＤＲＡＭＯＫ。然后是ＡＣＵＳＯＮＡＳＰＥＮ的杉树图象出现 ,机器进入ＰＯＷＥＲＤＩＡＧＮＯＳＴＩＣＳ ,一段时间后出现错误提示 :ＰＯＷＥＲＯＮＤＩＡＧＮＯＳＴＩＣＳＳＵＩＴＥＣＯＭＰＬＥＴＤ ,ＮＵＭＢＥＲＯＦＴＥＳＴＳＦＡＩＬＥＤ :１ＳＣＰＴＥＳＴ０６７７ＦＡＩＬＥＤ屏幕最下方提示按任意键继续 ,按照提示按任一键结…     

转基因细胞CHL－2B6的建立和在遗传毒理试验代谢活化中的应用

应用稳定表达ＣＹＰ２Ｂ６的转基因细胞系ＣＨＬ－２Ｂ６对一组前致突变物／致癌物进行双核微核试验，同时以母细胞系ＣＨＬ为对照，结果表明：５种前致突变物ＮＮＫ、ＤＥＮ、ＮＭ、ＭＢＩ和ＣＰ均能诱导ＣＨＬ－２Ｂ６细胞的徽核率显著增加，其中ＮＮＫ、ＣＰ和ＡＦＢ１具有剂量－反应关系，在最高受试浓度微核率约增加３～８倍。显示转基因细胞系ＣＨＬ－２Ｂ６在化学致癌物的遗传毒理学研究方面具有重要的应用价值．     

VSV在两种小鼠原代贤细胞上的接种效率和最高产量的比较

Ｈ－２Ｕｄ基因是ＢＡＬＢ／Ｃ小鼠针对ＶＳＶ的Ｉ型ＭＨＣ限制性等位基因＾（１）。缺失了Ｈ－２Ｌ＾ｄ基因的ＢＡＬＢ／Ｃ－Ｈ－２＾ｄｍ＾２小鼠，（简称ｄｍ２小鼠）其产生病毒的效率和产量是否会受影响从而给实验带来困难？针对这一问题，利用ＢＡＬＢ／Ｃ和ｄｍ－２两种小鼠的原代肾纤维细胞，进行了ＶＳＶ的种植效率和最高产量的比较。ＢＡＬＢ／Ｃ小鼠细胞的种植效率为１０１．４％，ｄｍ２为９９．８％，前者的最高产量为４     

转基因小鼠（Tg）两种髓鞘蛋白（MBP）的T细胞受体（TCR）之间的差异

实验性脑脊髓炎 (ＥＡＥ)动物模型是用于了解人类脱髓鞘病多发性硬化症 (ＭＳ)发病机理重要手段。ＥＡＥ属于中枢神经系统Ｔ细胞介导的自身免疫性疾病。应用髓鞘成分 ,包括鞘碱性蛋白 (ＭＢＰ)、糖脂、髓鞘少树突糖蛋白或某些多肽类物质可诱发ＥＡＥ。ＣＤ4 + Ｔ细胞介导ＥＡＥ的发生 ,转基因小鼠两种Ｔ细胞受体 (ＴＣＲ) :Ｖα2 3/Ｖβ8 2和Ｖα4 /Ｖβ8 2可分别识别ＭＢＰ的ＮＡｃ 1- 11免疫显性表位。口饲ＭＢＰ可使Ｖα2 3/Ｖβ8 2和Ｖα4 /Ｖβ8 2转基因小鼠 ,免遭ＥＡＥ的侵袭。作者发现这两株Ｔｇ小鼠对ＭＢＰ耐受诱导作用。…     

靶基因xy1E质粒载体转基因小鼠的建立

OBJECTIVE: To develop a transgenic mouse model carrying with plasmid pUC118NX integrated into its genomic DNA for detecting mutagenesis. METHODS: DNA of plasmid UC118NX in target gene xy1E was injected microscopically into male protonucleus of 376 mouse spermatova, and 225 survival spermatova were transferred into the oviducts in both sides of 11 pseudopregnant female mice to develop their offspring. The genomic DNA in survival young mice were analyzed with polymerase chain reaction (PCR)-Southern blot, plasmid transformation test and endonuclease-digestion. RESULTS: Seven pseudopregnant mice got pregnant, and 29 offspring were delivered and 25 survived of which 18 were identified with positive for PCR-Southern blot (72%). The two stout male mice with intact integration of plasmid pUC118NX in their genome were finally chosen as founders to detect gene mutation in vivo and establish transgenic mouse lineages. CONCLUSION: Transgenic C57BL/6J mice integrated with plasmid pUC118NX into their genomic DNA have been successfully developed.     

C57BL/6J小鼠脂肪细胞抵抗胰岛素抑制脂解的分析

为了从细胞水平探讨Ｃ５７ＢＬ／６Ｊ和Ａ／Ｊ小鼠对胰岛素（Ｉｎｓ）的敏感性,该实验分离了这两种小鼠的脂肪细胞,观察Ｉｎｓ 抑制脂肪细胞在异丙肾上腺素介导下的脂解作用,以甘油的释放量作为脂解的指标。结果表明：Ｃ５７ＢＬ／６Ｊ的脂肪细胞在Ｉｎｓ ４００和８００ ｐｍ ｏｌ时甘油的释放量多于Ａ／Ｊ（Ｐ＜ ０．０１）,即Ｉｎｓ抗脂解能力显著低于Ａ／Ｊ小鼠,提示Ｃ５７ＢＬ／６Ｊ小鼠脂肪细胞抵抗Ｉｎｓ抑制脂解。这与Ｃ５７ＢＬ／６Ｊ为什么易于发生２ 型糖尿病可能存在病理生理方面的联系。     

大剂量γ射线对小鼠造血功能损伤的比较研究

目的探讨大剂量电离辐射对IRM-2小鼠、C57BL/6 J小鼠造血功能损伤及恢复的比较。方法用137Csγ射线分别对IRM-2小鼠、ICR小鼠进行4.0Gy照射,照后9d,检测小鼠造血功能三项(脾指数、CFU-S、DNA)指标的变化;对IRM-2小鼠、C57BL/6 J小鼠进行6.0Gy照射,照后45d,检测小鼠外周血白细胞的变化及绝对值。结果 IRM-2小鼠4.0Gy照射后9 d造血功能三项指标均高于ICR小鼠,CFU-S、DNA差异有统计学意义(P0.001)。6.0Gy照射后45d WBC、RBC及HGB、HCT指标均高于C57BL/6 J小鼠,差异有统计学意义(P0.01)。结论受137Csγ射线大剂量及亚致死剂量照射后,IRM-2小鼠与C57BL/6 J小鼠及ICR小鼠比较,造血系统有较强的恢复功能。     

转基因雄性小鼠不育原因分析

目的研究转基因雄性小鼠不育的主要原因。方法以野生型C57BL／6J作为对照，比较检测健康性成熟雄性转基因小鼠的睾丸、附睾、前列腺、提肛肌的脏器系数和精子数、精子活动度、活精率、精子畸形率；观察睾丸组织病理学变化及睾丸组织葡萄-6-磷酸脱氢酶（G-6-PD）、乳酸脱氢酶（LDH）、乳酸脱氢酶同工酶（LDH-X）及琥珀酸脱氢酶（SDH）的活性。结果与正常非转基因对照鼠相比，转基因鼠睾丸的脏器系数与正常对照鼠相接近，附睾、提肛肌的脏器系数显著降低。精子数、直线运动精子数显著降低（P〈0．01）；转基因鼠睾丸组织曲细精管变薄，生精细胞排列不整齐，层次减少（2层），上皮部分有变性，精原细胞、初级精母细胞分裂异常，精子形成减少；G-6-PD、LDH、LDH-X及SDH的活性均显著低于对照鼠。结论外源基因整合后，引起睾丸组织酶活性下降，影响各细胞的生理、生化功能，进而引起睾丸发育严重障碍，精子减少和活动能力降低而导致不育。     

Intermittent availability of ethanol does not always lead to elevated drinking in mice

Aims: Intermittent access (IA) to an alcohol (ethanol) solution can lead rats to higher ethanol intakes than continuous access, and a recent report showed increased drinking in C57BL/6J mice offered 20% ethanol vs. water 3X/week (Prior studies have offered ethanol during 24?h periods, either continuously or intermittently.). Methods: We tested the high-preference C57BL/6J inbred mice: we also studied High Drinking in the Dark (HDID) mice, a line we have selectively bred to reach intoxicating blood ethanol levels after a short period of access to a single bottle of 20% ethanol. Results: Neither HDID or C57BL/6J male mice offered ethanol every other day during only a 4-h access period showed greater daily intake than mice offered ethanol daily for 4?h. There was a small increase in drinking with 24?h IA in C57BL/6J mice. An experiment with HDID mice and their control heterogeneous stock stock modeled closely after a published study with C57BL/6J mice (Hwa, Chu, Levinson SA et al. Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% ethanol. Alcohol Clin Exp Res 2011;35:1938-1947) showed no significant elevation with 24?h IA exposure in either sex of any genotype. Finally, a near replication of the Hwa et al. study showed modestly greater intake in C57BL/6J mice, confirming the efficacy of 24?h IA. Conclusion: We conclude that 4?h of IA is likely insufficient to elevate drinking in mice. The lack of effect in HDID mice and their controls further suggests that not all genotypes respond to intermittency.     

Depression of thymus-dependent immunity in wasting protein-energy malnutrition does not depend on an altered ratio of helper (CD4+) to suppressor (CD8+) T cells or on a disproportionately large atrophy of the T-cell relative to the B-cell pool.

We report cell numbers within major subsets of lymphocytes in the spleen, mesenteric nodes, and recirculating pool of weanling mice subjected to protein-energy malnutrition (PEM). PEM and thymus (T)-dependent immunodepression were induced in male C57BL/6J mice by a low-protein (LP) diet fed ad libitum. Recirculating lymphocyte numbers were estimated by enumerating labeled and unlabeled cells after equilibration of a known number of fluorescein isothiocyanate-labeled C57BL/6J donor lymphocytes within well-nourished or LP recipients. Involution of the recirculating lymphocyte pool of the LP group was proportionately less than the lymphoid atrophy of the spleen and mesenteric nodes. The LP protocol exerted no influence on the ratio of helper (CD4+) to suppressor (CD8+) T cells and increased the ratio of T cells to B cells in the secondary lymphoid organs and recirculating pool. These results challenge two established concepts: that T-dependent immunodepression in PEM depends on a reduced CD4(+)-CD8+ ratio and that PEM induces greater involution within the T-cell system than within the B-cell system.     

Characterization of the ethanol-deprivation effect in substrains of C57BL/6 mice.

Ethanol craving plays a major role in relapse drinking behavior. Relapse and ethanol craving are an important focus for the treatment of alcoholism. The ethanol-deprivation effect (EDE) is a widely used animal model of alcohol craving. While the EDE is widely studied in rats, the molecular mechanisms underlying EDE are not clearly understood. The C57BL/6 inbred mouse strain is widely used for behavioral and molecular analyses of ethanol drinking but studies on the EDE have not been reported in this strain. In the present study, we characterized a simple behavioral protocol that rapidly and reliably induced EDE in C57BL/6 mice. Briefly, single-housed adult male C57BL/6NCrl and C57BL/6J mice were presented at the beginning of dark phase with two-bottle choice drinking containing either 10% wt/vol ethanol or tap water for 18 h/day, as well as food ad libitum. Following ethanol drinking for 4 days or 14 days, mice were deprived of ethanol for a period of 4 days. To study EDE, mice were reinstated with two bottles containing either ethanol (10% wt/vol) or water. Mice were exposed to single or multiple ethanol-deprivation cycles. Ethanol consumption (g/kg/18 h) and percent ethanol preference (% preference/18 hrs) was recorded for individual mice. C57BL/6NCrl mice consumed moderate amounts (4.78+/-0.63 g/kg) of ethanol but showed robust EDE after ethanol-drinking episodes (4 days or 14 days) as evidenced by increased ethanol consumption and ethanol preference following reinstatement of ethanol. While repeated ethanol deprivation in C57BL/6NCrl mice transiently increased ethanol consumption and ethanol preference, the magnitude of these behaviors was reduced as compared to the first deprivation cycle. In contrast, the C57BL/6J substrain consumed substantially higher levels (9.65+/-0.90 g/kg) of ethanol but did not show a clear EDE after single or multiple ethanol-deprivation cycles. In conclusion, we established a simple and reliable behavioral model to study EDE in C57BL/6NCrl mice. A reliable behavioral model to study EDE in inbred C57BL/6NCrl mice could greatly facilitate further studies on molecular mechanisms of ethanol craving behavior.     

Effects of kiwifruit extracts on colonic gene and protein expression levels in IL-10 gene-deficient mice

Inflammatory bowel disease (IBD) is a collective term for conditions characterised by chronic inflammation of the gastrointestinal tract involving an inappropriate immune response to commensal micro-organisms in a genetically susceptible host. Previously, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa) have demonstrated anti-inflammatory activity using in vitro models of IBD. The present study examined whether these kiwifruit extracts (KFE) had immune-modulating effects in vivo against inflammatory processes that are known to be increased in patients with IBD. KFE were used as a dietary intervention in IL-10-gene-deficient (Il10(-/-)) mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 × 2 factorial design. While all Il10(-/-) mice developed significant colonic inflammation compared with C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. These findings are in direct contrast to our previous study where KFE reduced inflammatory signalling in primary cells isolated from Il10(-/-) and C57BL/6J mice. Whole-genome gene and protein expression level profiling indicated that KFE influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, expression levels across gene sets related to adaptive immune pathways were significantly reduced using three of the four KFE in C57BL/6J mice. The present study highlights the importance of investigating food components identified by cell-based assays with appropriate in vivo models before making dietary recommendations, as a food that looks promising in vitro may not be effective in vivo.     

Strain dependent differences in a histological study of CD44 and collagen fibers with an expression analysis of inflammatory response-related genes in irradiated murine lung

Using a mouse model, we investigated the mechanisms of heterogeneity in response to ionizing radiation in this research. C57BL/6J and C3H/HeMs mice were irradiated with gamma rays at 10 and 20 Gy. The animals were sacrificed at times corresponding to the latent period, the pneumonic phase, and the start of the fibrotic phase for histological investigation. Small areas of fibrosis initially appeared in C57BL/6J mice at 4 weeks postirradiation with 20 Gy, whereas small inflammatory lesions appeared at 4 and 8 weeks after 20 and 10 Gy, respectively. The alveoli septa were thickened by an infiltration of inflammatory cells, and alveoli were obliterated in lungs from C57BL/6J mice after 20 Gy irradiation. At 24 hours and from 2 to 4 weeks postirradiation, fourfold more CD44 positive cells had accumulated in the lungs of C3H/HeMs than in C57BL/6J mice. Hyaluronan accumulated 12 hours after irradiation, and the rapid resolution was achieved within 2 weeks in the lungs in both strains of mice. C57BL/6J mice lungs accumulated dense collagen at 8 weeks. Quantitative RT-PCR assay was performed for several genes selected by cDNA microarray analysis. The expression of several genes, such as Cap1, Il18, Mmp12, Per3, Ltf, Ifi202a, and Rad51ap1 showed strain-dependent variances. In conclusion, a histological investigation suggested that C3H/HeMs mice were able to induce a more rapid clearance of matrix after irradiation than C57BL/6J mice. The expression analysis showed that the several genes are potentially involved in interstrain differences in inflammatory response causing radiation-induced lung fibrosis.