一种新的大鼠隐睾模型的建立

目的改善大鼠隐睾模型的制作方法,提高隐睾模型的质量,并对新模型的稳定性进行研究。方法28只大鼠随机分为对照组（ctrl）和模型组（modl）采用模拟失重大鼠模型,对大鼠进行3周尾部悬吊进行造模,随后模型组大鼠解悬吊恢复8周观察该模型的稳定性。结果经过3周的尾部悬吊,模型组所有大鼠睾丸均滑入腹腔,同时和对照组相比,睾丸和附睾的重量出现极显著的降低（P〈0.01）。HE染色发现对照组大鼠睾丸的生精小管结构排列紊乱,精原细胞消失,附睾尾中成熟精子消失。经过8周的恢复,大鼠的睾丸及附睾仍未恢复到正常水平（P〈0.01）,HE染色显示其生精小管结构和精原细胞数量并未出现明显的改善。结论尾吊法所建立的大鼠隐睾模型效果稳定,可以有效模拟大鼠隐睾时的睾丸温度变化情况,同时对大鼠的伤害较小,操作比较简单。     

HFRSV内影像型抗独特型人单抗重链可变区基因克隆

利用反转录－ＰＣＲ方法,从分泌肾综合症出血热病毒（ＨＦＲＳＶ）内影像型抗独特型人单抗杂交瘤细胞系（Ｃ８）中,成功地克隆了抗ＨＦＲＳＶ１ｄ人单抗重链可变区基因,并将此基因重组入Ｍ１３噬菌体ＤＮＡ中,测定了此重链可变区基因全序列。经计算机分析,基因全长共３５１ｂｐ、编码１１７个氨基酸,氨基酸序列同源性分析发现所推得的该单抗ＣＤＲ２区与ＨＦＲＳＶＧ２蛋白Ｃ端有同源区,此区可能具有较强的抗原性。     

Rab23 在鳞癌细胞系中的表达及其对鳞癌细胞侵袭的影响*

目的:观察Rab23在鳞癌细胞系中的表达及其对鳞癌细胞侵袭的影响。方法:western blot检测Rab23在四个头颈部鳞癌细胞系中的表达,对鳞癌细胞系HSQ-89用脂质体转染小干扰RNA,对Sa3用脂质体转染Rab23质粒,分别干涉或过表达Rab23,用铺有基质胶的transwell小室检测细胞侵袭能力,细胞划痕试验检测迁移能力。结果:Rab23在四个鳞癌细胞系中均有表达,在Hacat中不表达,HSQ-89细胞经Rab23干涉后细胞侵袭能力减弱,而Sa-3细胞过表达Rab23后细胞侵袭能力增强。干涉或过表达Rab23后,鳞癌细胞的迁移能力无差异。结论:Rab23可促进鳞癌细胞侵袭。     

人Era不同结构域在酵母双杂交系统中的表达及对报告基因激活作用的检测

ｅｒａ基因是 1986年在测定大肠杆菌基因组序列时发现的一个开放读框 .由于其编码的氨基酸序列与人及酵母的ＲＡＳ蛋白有部分相似之处 ,命名为Ｅ .ｃｏｌｉＲＡＳ ｌｉｋｅ(ｅｒａ)基因[1] .ｅｒａ基因广泛存在于原核生物中 ,与细胞周期和细胞分裂有关 ,是细菌生存繁殖所必需的重要基因[2 ] .在真核生物如蠕虫、小鼠和人的组织中都发现有与细菌ｅｒａ高度同源基因存在[3 ,4 ] .人ｅｒａ基因 (ｈｅｒａ)的全长ｃＤＮＡ于 1998年被克隆 ,与原核ｅｒａ基因具有很高的同源性[5] .人Ｅｒａ蛋白与大肠杆菌Ｅｒａ蛋白的氨基酸序列有 30 %相同 ,两者…     

Tyrosine phosphorylation of caveolin 1 by oxidative stress is reversible and dependent on the c-src tyrosine kinase but not mitogen-activated protein kinase pathways in placental artery endothelial cells

Acute H(2)O(2) exposure to placental artery endothelial cells induced an array of tyrosine-phosphorylated proteins, including caveolin 1 (CAV1) rapid and transient tyr(14) phosphorylated in a time- and concentration-dependent manner. Basal tyr(14) phosphorylated CAV1 was primarily located at the edges of cells and associated with actin filaments. Phosphorylated CAV1 was markedly increased and diffused with the disorganization of actin filaments at 20 min, disappeared at 120 min treatment with 0.2 mM H(2)O(2). Treatment with H(2)O(2) also disorganized actin filaments and changed cell shape in a time-dependent manner. Pretreatment with antioxidants catalase completely, whereas the other tested superoxide dismutase, N-acetyl-l-cysteine and sodium formate partially attenuated H(2)O(2)-induced CAV1 phosphorylation in a concentration-dependent manner. Acute treatment with H(2)O(2) activated multiple signaling pathways, including the mitogen-activated protein kinases (MAPK) members (MAPK3/1-ERK2/1, MAPK8/9-JNK1/2, and MAPK11-p38(mapk)) and the c-src tyrosine kinase (CSK). Pharmacological studies demonstrated that, among these pathways, only the blockade of CSK activation abolished H(2)O(2)-induced CAV1 phosphorylation. Additionally, H(2)O(2)-induced CAV1 phosphorylation was reversible rapidly (<10 min) upon H(2)O(2) withdrawal. Because maternal and fetal endothelia must make dynamic adaptations to oxidative stress resulting from enhanced pregnancy-specific oxygen metabolism favoring prooxidant production, which is emerging as one of the leading causes of the dysfunctional activated endothelium during pregnancy, these unique features of CAV1 phosphorylation on oxidative stress observed implicate an important role of CAV1 in placental endothelial cell biology during pregnancy.     

抗生素对莱氏衣原体膜流动性和Mg~(2+)-ATPase活性的影响

本文以莱氏衣原体AIH089为材料,用DPH荧光偏振等技术研究红霉素和土霉素对莱氏衣原体膜流动性和Mg~(2+)-ATPase活性的影响,并用聚丙烯酰胺梯度凝胶电泳技术进一步分析膜蛋白的组成,发现红霉素和土霉素能使莱氏衣原体膜的流动性显著增加,使Mg~(2+)-ATPase活性显著降低。红霉素和土霉素对莱氏衣原体膜流动性和膜上Mg~(2+)-ATPase活性的影响与它们的抑菌能力有很好的相关性。     

大肠杆菌中高表达重组Era可溶性蛋白的纯化及其生化特性

质粒pCE31含有在P_L起动子控制下的重组era基因,在大肠杆菌中、42℃诱导高表达出Era蛋白,经溶菌酶处理裂菌、沉淀离心洗涤后所得的纯Era是不溶的,生物活性也不高。改在40℃诱导2h,在Era底物GDP的存在下裂菌,60％以上的Era处于可溶状态。用蛋白酶抑制剂防止Era蛋白降解,经盐析、Q-SepharoseFF柱层析分离,获得可溶性纯Era蛋白。该蛋白能特异地与鸟苷酸结合、并具有GTP酶活性,表明Era是一种G蛋白。动力学定量分析结果:在4℃时,每分子Era肽链能结合一分子GTP或GDP,表明所纯化的Era蛋白几乎都具有活性;4℃下,Era蛋白与GTP或GDP结合的解离常数,分别为5.49和1.01μmol/L;37℃时,Era蛋白GTP酶的Km值为9.0μmol/L,催化GTP水解的最大速度为9.8mmolCTP/mol Era/min。     

Global protein expression profiling underlines reciprocal regulation of caveolin 1 and endothelial nitric oxide synthase expression in ovariectomized sheep uterine artery by estrogen/progesterone replacement therapy

Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17beta (E2, 5 microg/kg bolus + 6 microg kg(-1) day(-1)), or P4 + E2 for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 microg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a approximately 133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E2, P4, and their combination, whereas a approximately 22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E2 and E2/P4, but not P4 treatments. Western blot analysis revealed that E2, P4, and their combination all increased NOS3 protein, whereas E2 and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E2, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E2. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E2, but not P4, causes uterine vasodilatation and that E2 and P4 increase NOS3 expression, but only E2 decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.     

花背蟾蜍视网膜感光细胞分化的研究

经电镜及间接荧光抗体对花背蟾蜍视网膜感光细胞分化的研究,结果表明:视网膜感光细胞在视网膜中央首先分化,同一切片中位于中央的细胞分化程度最高,离它越远、分化越低。以中央细胞为准,胚胎发育到21期,感光细胞开始分化,22期外节出现,外节盘膜是直接以外凸折叠形成的。同时在视网膜杆锥层出现视蛋白特异性荧光,说明光敏蛋白的合成,感光细胞轴突与双极细胞建立突触联系。至此(22期末),感光细胞行使功能的结构基本形成。到25期,外节盘数达300多层,突触结构发育成熟。     

大肠杆菌中rnc与era基因的共表达

 rnc,era基因是大肠杆菌两个相邻的基因。用S_1核酸酶图谱法测得转录从rnc基因伸延到era基因。当rnc基因突变使所产生的RNaseⅢ丧失活性时,大肠杆菌中RNaseⅢ和Era蛋白的合成速度同对上升,且上升幅度相同,合成量相等。将此二基因分别或一起克隆入质粒并置于P_L起动子下游时,rnc能表达产生RNaseⅢ;rnc-era能表达产生RNaseⅢ和Era两种蛋白质,单独era则虽能转录却不产生Era蛋白。实验证明:era基因的表达依赖于rnc基因的表达,在体内两者共表达体现在转录和翻译水平上,两个基因同属于一个操纵子,共同受RNaseⅢ活性的反馈调节。单独转录的era-mRNA因缺乏有效的翻译起始序列而不能被翻译。