Abstract:目的体外培养和鉴定心脏神经嵴细胞,探讨其生物学特性。方法取8·5d小鼠胚胎枕中部至第3体节神经管,组织块法无血清条件培养获心脏神经嵴细胞,采用转录激活因子2α(AP-2α)作为其生物学标记物,观察其迁移、分化等生物学特性。结果从胎鼠神经管中分离培养的细胞AP-2α表达阳性,具有迁移特性,传代后以含血清培养基培养后能自然分化成神经元和神经胶质细胞。结论体外培养可成功获得心脏神经嵴细胞,且具有迁移特性和多向潜能分化能力。

Abstract:目的观察不同浓度外源性视黄酸对斑马鱼早期胚胎和心血管系统发育的影响,为进一步研究视黄酸影响斑马鱼心脏前后轴(A-P轴)发育的分子机制提供形态学依据。方法选择斑马鱼胚胎孵育的3,6,9·5,12h四个时间点,用不同浓度视黄酸(1×10-6,1×10-7,4×10-8,1×10-8mol/L)处理斑马鱼胚胎,在解剖显微镜下实时观察斑马鱼胚胎心脏发育的全过程和视黄酸对斑马鱼心脏发育的影响。并采用胚胎整体原位杂交技术观察flk-1mRNA在斑马鱼胚胎的表达。结果1×10-6mol/L视黄酸可导致斑马鱼胚胎表现出多系统的严重畸形,胚胎很快死亡。在胚胎孵育的9·5、12h给与10-7~10-8mol/L浓度的视黄酸,胚胎只表现出心血管系统的畸形,其他系统无明显异常。胚胎整体原位杂交显示视黄酸对flk-1mRNA在斑马鱼胚胎血管的表达没有影响。结论视黄酸影响斑马鱼胚胎心脏发育有剂量依赖性和严格的时间窗,视黄酸影响心脏前后轴发育的关键时间是原肠胚晚期。视黄酸处理组胚胎的循环缺陷主要为心脏发育异常所致。10-7~10-8mol/L浓度视黄酸在9·5、12h处理斑马鱼胚胎可以作为研究心脏发育调控机制的动物模型。

Abstract:Objective To study the differentiation and neoplastic transformation by retrovirus infection of 12 d visceral yolk sac (VYS) of rats in vitro and in vivo. Methods Under various cultrue conditions and at different transplant sites in vivo, the differentiation of rat VYS was investigated. The VYS was transfected by retroviral vector carrying green fluorescent protein (EGFP) gene. The positive clones selected by G418 in vitro were re-implanted into syngenic rats or immunodeficiency mice in vivo. Results The VYS showed totipotency to differentiate into ectoderm, mesoderm and endoderm, but did not become committed to a particular cell lineage under whatever culture conditions or the implantion sites in the present experiment. Green fluorescent protein was successfully labelled into cloned VYS cells. When a large amount of labelled cells were innoculated s.c. into nude mice, mesenchymal neoplasm developed with GFP positive indicating VYS origin.

Abstract:目的口服氯化汞(HgCl2)造成大鼠的肾间质纤维化模型并探讨相关机制。方法用不同剂量HgCl2[(A组为5mg/(kg·bw)、B组为10mg/(kg·bw)、C组为20mg/(kg·bw)]给大鼠灌胃1周,观察大鼠一般状况、肾功能和肾组织病理变化,免疫组化法观察肾组织纤维连接蛋白(FN)和α-平滑肌肌动蛋白(α-SMA)表达。结果模型大鼠体重下降,肾体比增加,肾功能损害和肾组织Hyp含量呈剂量依赖性升高,肾间质炎性细胞浸润,肾间质胶原沉积增加,肾间质FN和α-SMA表达增强,以C组病变最重。结论20mg/(kg·bw)剂量HgCl2灌胃1周可造成大鼠的肾间质纤维化病变,其部分机制在于HgCl2促使肾间质肌成纤维细胞活化和细胞外基质的生成沉积。

Abstract:Objective To investigate the effects of serum containing substances of a Chinese traditional drug, Zuogui Pill, on expression of IL-1, IL-6 and COX-2 in osteoblasts, and to study the mechanism of action of Zuogui Pill treatment for osteoporosis. Methods Osteoblasts were obtained from newborn rats calvaria by collagenase-trypsin digestion. Three groups were included in the experiment: normal serum group, serum of ovariectomized (OVX) model group and serum of Zuogui pill-treated OVX model group. Immunohistochemical techniques were used to detect the expression of IL-1, IL-6 and COX-2. Results Expression of IL-1, IL-6 and COX-2 in osteoblasts in the serum of OVX model group was increased compared with that in the normal serum group, and their expression in the serum of Zuogui pill-treated OVX model group was significantly decreased as compared with those in serum of OVX model group, but did not show significant difference from those of normal serum group. Conclusion The serum containing Zuogui pill substances can inhibit the secretion of IL-1, IL-6 and PGE_2, which may be one of the mechanisms of action of Zuogui Pill treatment for osteoporosis.

Abstract:Objective To investigate the effects of extracts obtained from different parts of Portulaca olerace on blood glucose and lipid metabolism in insulin resistant type 2 diabetic rats. Methods The experimental type 2 diabetic rat model was established by injection of streptozotocin (25 mg/kg) and fed on high-calory laboratory chow. The model rats were treated separately with extracts from different parts of Portulaca oleracea by gastrogavage for twelve weeks, and randomly divided into the model group, Portulace oleracea group, Portulace oleracea MH group and Portulace oleracea MS group, and normal control group and polyene contralcon control group as well. The differences of values of parameters between each experimental group and model group were observed and analyzed: oral glucose tolerance (OGTT), serum insulin, insulin sensitivity index (ISI), total cholesterol (TC), triglycerides (TG), high density lipoproteins-cholesterol, free fatty acid and the ratio of apolipoprotein A I (ApoA I) and apolipoprotein B (ApoB). Results There was a significant difference in improving the blood glucose metabolism between Portulace oleracea group and the model group, and between Portulace oleracea group and polyene contralcon control group. Portulace olerace MH exerted an approximately significant effect in comparison with the model group. Portulace oleracea MS showed almost no effects. As regards the lipid metabolism, compared with the model group, Portulace olerace group increased HDL-c most significantly, simultaneously reduced TC, TG and FFA. Compared with the model group, the Portulace oleracea MH lowered TG most significantly, meanwhile lowered TC and FFA, but did not increased HDL-C distinctly. In comparison with the model group, Portulace oleracea MS lowered FFA most significantly, approximately significantly lowering TG, but did not show a significant effect on TC and HDL-c metabolism. Each treatment group of Portulace oleracea had no obvious effects on the ratio of apolipoproteinA I (ApoA I) and apolipoprotein B (ApoB). Conclusion Chinese medicinal herb Portulaca oleraceaa treatment may improve the blood glucose metabolism in type 2 diabetic rats. Portulace oleracea MH has a tendency to improve the blood glucose metabolism, but the difference is not significant. All Portulace oleracea, Portulace oleracea MH and Portulace oleracea MS may significantly improve the lipid metabolism, but affecting different aspects, which might related to different mechanisms or of action or different steps of the metabolism.

Abstract:Objective To establish a murine model with local skin infection caused by Fusarium solani. Methods Fusarium solani suspension in three different concentrations were inoculated on the intact or abrated skin of normal or immunocompromised ICR mice. Histopathologic examination and fungal culture were performed on 7th d, 14th d, 21st d and 28th d after inoculation, respectively. Results The lesions were developed on abrated skin of all normal and immunocompromised mice, and were more severe and persisted longer in immunocompromised mice than those in normal mice. Moreover, hyphae appeared mostly in histopathologic examination and fungal culture. No lesion was found on undamaged skin. No disseminated infection of Fusarium solani was revealed in all tested mice. Conclusion Inoculation of Fusarium solani cell suspension in concentration of 10~9 or 10~ 10 CFU/ml on the abrated skin of immunocompromised ICR mice can effectively generate a mouse cutaneous Fusarium solani infection model, and to serve experimental research on dermatological infection of this fungus.

Abstract:Objective To investigate the protective effect of heat stress pretreatment on the hepatic ischemia resperfusion (I/R) injury and its mechanism of action. Methods To establish the rat model of hepatic ischemia resperfusion injury using Pringle's maneuver with or without heat stress pretreatment. The rats were randomly divided into pretreatment group (HP+IR) and non-pretreatment group (IR). The activity of NOS in the liver, the activity of serum enzyme LDH and the pathological changes of the liver were assessed at 0, 4, 8, 12 and 24 h after I/R. Results At each time point set after I/R, the activity of NOS in the liver of HP+IR group was lower than that of IR group. Maximal HSP70 expression was observed at 12 h after heat pretreatment. While in the HP+IR group, the level of liver enzyme was significantly reduced and the pathological changes were improved in comparison with those in IR group. Conclusion HSP70 induced by heat pretreatment protects the liver against I/R injury may be through inhibiting the NOS activity and therefore reducing the insults of oxygen free radicals on the liver.

Abstract:Objective To illucidate the variation of anatomical patterns of brain anterior and posterior communication arteries in adult Mongolian gerbils. Methods 128 adult gerbils were included in this study. The animals died from cerebral ischemia or were sacrificed by anesthesia and were autopsied. The arteries on the ventral surface of the brain were studied under a dissection microscope. Results Seven different patterns of anterior communication arteries (ACA) and five patterns of posterior communication arteries (PCoA) were found in the gerbils. Absence of left or right ACA was observed in 46.1% of this group and 85.2% of the gerbils was lack of PCoA. Conclusion The brain anterior and posterior communication arteries in adult gerbils have several anatomical patterns, especially some gerbils have complete posterior communication arteries.

Abstract:Objective To develop a protocol of isolation and culture of human keratinocytes in order to serve as seeding cells in tissue engneering of skin and the treatment of severe burns and other traumatic skin defects or lesions. MethodsForeskin obtained from 3 to 9 year-old boys with circumcision were treated with dispase II. The epidermis was treated with trypsin to make single epidermal cells suspension. Keratinocytess were cultured in medium DMEM supplemented with serum or medium K-SFM. The rate of colony formation was calculated and the growth conditions were evaluated. ResultsThe cells grew well in both DMEM and K-SFM culture media. However, the time needed for colony formation in K-SFM medium was significantly shorter and the efficiency of colony formation was significantly higer than that in DMEM medium. ConclusionsIt is an simple and efficient method to isolate keratinocytes by dispase II and trypsin and to culture them in K-SFM medium.

Abstract:Objective To find out whether porcine circovirus type 2 (PCV_2) total genome could be expressed by E.coli and whether the recombinant proteins still be biologically active. MethodsThe N-end fragment of PCV2 ORF2 gene of 737-421nt was PCR amplified, cloned and fusion expressed with the expression vector pGEX-6p-1 in E. coli, resulting in the recombinant clone of pGEX-PCV737-421. Both the C-end fragment of the cap gene and the rep gene of PCV2 virus were expressed by E. coli previously. Then SDS-PAGE assay of the three recombinant proteins was performed and Western-blotting against PCV2-specific antiserum was carried out in turn. Finally both the recombinant proteins of the Cap gene were inoculated into SPF BALB/c mice to prepare anti-mouse polyserum in order to find out if the serum could be used to identify the virus antigens propagated in PK15 cell line through indirect fluorescence assay (IFA) experiment. ResultBoth Western-blotting and IFA assays could identify the expressed protein bands and virus antigens specifically. ConclusionThe immunogen assay demonstrated that E.coli expressed recombiant proteins of PCV2 total genome have immunoreactivity.

Abstract:目的比较正常组、模型组及对照组的图形视觉诱发电位(PVEP)和扫描视觉诱发电位(SVEP)视力,研究各组的视觉电生理表现,探讨单眼睑缝合1周是否可以建立形觉剥夺性弱视模型。方法分析三组PVEP的波形变化、潜时及完成一次诱发反应的时间;比较各组由SVEP得出的客观视力。结果①正常组PVEP由2个波峰组成“M型”(正向波向上),模型组及对照组出现波的分离—波形由多个波组成,而且完成一次反应的时间延长。②缝合侧的N75延迟,SVEP视力较正常组差且差别有统计学意义。③对照组未缝合侧视力恢复到正常水平而缝合侧视力仍差且与正常组有统计学差异。结论短期形觉剥夺的视觉电生理变化是弱视猫的一种表现,单眼睑缝合1周即可建立形觉剥夺性弱视猫的模型。

Abstract:目的建立泰泽氏病原体(Ty)纯化方法,获得纯的菌体供抗原研究,为ELISA诊断抗原的制备提供依据;并尝试建立Ty隐性感染血清学抗原检查方法。方法选择特异性抗体包被磁珠,从感染大鼠肝脏中富集和纯化Ty;用SDS-PAGE和Western blot技术考察纯化Ty的蛋白和抗原图谱;同时用免疫磁珠分离技术(IMS)直接检查隐性感染大鼠肠道上皮细胞内的Ty。结果用辛酸-硫酸铵纯化的Ty单克隆抗体M5以0·5μg/107beads以上浓度包被抗IgG抗体预结合的磁珠4h,可最大效率地富集Ty;分离反应进行1h,敏感性达到103菌体/mL;吖啶黄染色镜检法可以直接、快速地观察到结合于磁珠上的细菌;抗原分析表明,IMS较好地去除了肝组织和真核细胞成分,纯化的Ty RJ株具有3个免疫优势的抗原成分,相对分子质量(Mr)分别为160×103、116×103、55×103;此外,IMS法可直接从隐性感染大鼠盲肠上皮细胞中检测到少量寄生的Ty。结论用IMS技术可有效地富集和纯化Ty,并可以作为Ty隐性感染血清学抗原检查的候选方法。

Abstract:Objective To isolate and identify Sendai virus strain in Heilongjiang province, and to be used as a diagnostic antigen in detecting SV serum antibody of laboratory animals. MethodsThe virus was isolated from conventional mice lungs through chicken embryo passages and identified by haemoglutination test, haemoglutination blocking test and constitution genes sequencing. The virus was purified by sugar density gradient centrifugation and was used as an immunogen to laboratory animals. The titers of immune serum were detected by standard kits. ResultsTwo virus strains with haemoglutination characteristics were isolated from 150 portions of mouse lungs, which were identified as Sendai virus through virus morphological characterization, serum detection and constitution gene sequencing. The nucleocapsid (N)gene sequencing result showed that the nucleotide sequence and deduced amino acid sequence of the isolated virus shares 99.6% and 99.0% homology with Fushimi strain, respectively. ConclusionSendai virus HLJ strain has been isolated and identified, which can be used to develop diagnostic kits for SV antibody detection.

Abstract:目的观察游泳运动对神经毒素(MPTP)致小鼠神经和运动机能损伤的保护作用,探讨可能存在的机制。方法在注射MPTP或生理盐水前1d和注射后1、4、7、10d,测量MPTP游泳组、MPTP非游泳组和生理盐水对照组小鼠的爬杆时间和步伐,第11天用放射自显影法测定纹状体多巴胺转运体密度。结果MPTP两组第1天步伐延长,随后恢复。第4天MPTP非游泳组步伐小于生理盐水组和游泳组,后两组差异无显著性。MPTP两组第1天爬杆时间延长,但与生理盐水组比较差异无显著性。游泳组在随后各时间点爬杆时间依次缩短,并在第7、10天明显短于MPTP非游泳组和生理盐水组。游泳组纹状体多巴胺转运体相对密度较非游泳组和生理盐水组明显下调。后两组无差异。结论游泳运动能增强小鼠的运动机能,减轻MPTP的损伤效应,纹状体多巴胺转运体下调可能是其中机制之一。

Abstract:Objective To establish an useful animal model for research on diagnosis, treatment and drug development for endometriosis. MethodsFemale virginal rats with sexual maturation were induced by estrogen prior to operation. During the laparotomy, a part of right uterus was taken and the endometrium was implanted to the left abdominal wall. A cyst was formed and it was taken out at 16 weeks after implantation, examined by histology and histochemistry. ResultsThe ectopic endometrium growing in the abdominal wall formed a cystic mass with a large cavity containing much mucus. It showed an endometrial structure similar to that of normal endometrium. Glycogen and RNA were observed in the ectopic endometrium. ConclusionThe ectopic endometrium established by this method forming a cystic mass, is palpable as early as at one week after implantation. It is a reliable and convenient animal model for research on endometriosis diagnosis and treatment, and pharmacological and pharmaceutical studies.

Abstract:Objective To induce expression of nucleoprotein gene of Sendai virus (SV) in prokaryotic system. MethodsA pair of primers were designed and synthesized according to the SV nucleoprotein gene sequence published by GenBank. Nucleoprotein gene was amplified by RT-PCR. Recombinant pET-SN was constructed after the SN gene was sequenced. BL21(DE_3)pLysS was induced with 1 mmol/L IPTG. ResultsSDS-PAGE showed that the recombinant protein was about 60 Ku which could react with rabbit-anti Sendai virus serum in Western blot assay. ConclusionThe recombinant nucleoprotein as an antigen may provide a basis for diagnosis of Sendai virus.

Abstract:目的观察全网饲养对大、小鼠其生长发育的影响。方法在传统的大小鼠饲养盒底部40mm高处加不锈钢金属网格,网孔大小为10mm×100mm,网格上用于饲养动物。雌雄动物分别分为实验组和对照组,观察体重、行为、供食量和供水量的变化。结果全网养小鼠生长不如传统饲养方式生长快,但没有统计学差异;全网养大鼠生长发育正常,与传统饲养方式相比,未见差异。结论全网养大鼠不需要垫料,值得进一步推广;全网式饲养小鼠必须慎重。

Abstract:Objective The Sprague-Dawley rat strain is one of most popular animal models for studies of folliculogenesis, oocyte maturation and reproductive toxicology. The aim of the present study was to determine the optimal conditions for in vitro fertilization (IVF) and culture system of Sprague-Dawley rat oocytes. Methods Rat oocytes obtained from superovulation or natural ovulation cycle were fertilized and cultured in the HTF and IVF-30 media, modified by supplement with NaCl in different concentrations. Penetration of sperm, pronuclear and blastocyst formation were observed. Results Oocytes fertilized and subsequently cultured in IVF-30 or HTFplus supplemented with 30 mmol/L NaCl showed fertilization rates (67%, 70%) with subsequent blastocyst formation rates, 22% and 32%, respectively, significantly higher than the fertilization rates (15% and 13%, respectively) with no blastocyst formation in original media. More oocytes from natural ovulation cycles (28%) developed into blastocyst than that from superovulation (16%). Conclusion An in vitro fertilization and culture system for SD rat oocytes has been successfully established.

Abstract:Objective Gene knock-out animal is a species of experiment animal which can let people research gene function at individual level. It is based on the gene knock-out technique and embryonic stem cells. Now, it is widely used in many fields of life science research. The gene knock-out technique can not be replaced by RNA interference technique which grew over the last few years. The study and application of gene knock-out animal were reviewed in this article. Comparison between gene knock-out technique and RNA interference technique was also overviewed.

Abstract:Objective This paper discussed the basic theory and practice for establishing of animal models of Heptitis B Virus infection. The advantages and disadvantages of those models were compared, that may provide useful information for researchers to choose suitable animal model.