Evaluation of endothelial cell migration with a novel in vitro assay system

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349–354). An aliquot of 15–20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor (TNF). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm² and 5.3 mm² for TNF-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater – 6.4 mm² and 6.5 mm² with and without TNF treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30–40% on vWF/collagen, and 60–80% on FN/collagen, with increased proliferation observed with TNF- treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.     

5992例孕产妇梅毒筛查与分析

目的 了解妊娠期梅毒的流行情况及对围产结局的影响。方法 对5992例孕产妇进行甲苯胺红不加热血清试验（TRUST）筛查，对TRUST阳性病例再进行梅毒螺旋体血清颗粒凝集试验（TPPA）确诊。并对围产结局进行观察分析。结果 5992例孕产妇，检出梅毒患者43例（0．72％），其中30例经青霉素治疗的患者其新产儿经半年以上随访无1例感染梅毒，在未治疗的13例患者中有10例分娩的新生儿TPPA滴度均≥1：40，其中2例为先天性梅毒。1例为死亡梅毒婴儿。结论 开展妊娠期梅毒血清学的产前筛查，加强对妊娠期梅毒的治疗，对减少先天性梅毒的发生尤为重要。     

肽抗生素hPAB-β在毕赤酵母中的表达与活性鉴定

目的 构建表达人源肽抗生素hPAB-β的重组毕赤酵母,为大量制备hPAB-β奠定基础.方法 用PCR及引物延伸法得到天然及优化的hPAB-β序列,克隆入pPIC9K质粒,转化DH5α大肠埃希菌,用酶切和核苷酸测序鉴定.重组质粒经SalⅠ酶线性化,整合入毕赤酵母GS115中,在DNA,mRNA及蛋白水平鉴定阳性重组子.结果 构建的含天然及优化序列的重组质粒酶切均可得到相应大小的插入片段,与预期相符,测序结果表明序列和阅读框均正确.线性化的重组质粒转入GS115后,得到7个不同的高拷贝阳性转化子,均能表达重组肽并具有良好的抑菌活性.结论 本研究成功构建含天然及优化hPAB-β序列的重组毕赤酵母工程菌,能够用于肽抗生素hPAB-β的制备.     

精液分析中精子密度室内质量控制方法研究

目的建立一种可行的精液密度测定的质量控制方法.方法采用血细胞计数板和计算机辅助精液分析(CASA)两种方法,对所选取的与精子头部直径相近的质控珠进行高、中、低三种密度测定,分别于日内及日间测试12次.结果采用血细胞计数板方法测试高、中、低三种密度结果的日内变异系数分别为12.09%、8.86%、4.72%,日间变异系数分别为16.71%、10.65%、10.22%.CASA法日内变异系数分别为6.65%、5.93%、3.80%,日间变异系数分别为7.90%、6.84%、6.14%.结论质控珠可用于以上两种测试精子密度方法的室内质量控制,从而达到保证实验室测试精子密度质量的目的.     

内皮素性大鼠门静脉高压模型的建立

目的： 建立内皮素-1(ET-1)引起门静脉压力升高的动物模型。方法: 正常雄性SD大鼠48只，随机分为生理盐水组、ET-1低剂量组(0.3 μg/kg)，ET-1中剂量组(1.0 μg/kg)和ET-1高剂量组(3.0 μg/kg)。生理盐水组大鼠经股静脉注入生理盐水，其余各组大鼠按相应剂量经股静脉注入ET-1溶液，观察门静脉压力和颈动脉压力的变化情况,并筛选出能使门静脉压力升高最适宜的剂量；另取15只大鼠随机分为对照组、ETAR阻断剂(BQ-123)组和ETBR阻断剂(BQ-788)组，实验开始前30 min经各组大鼠股静脉分别注入生理盐水、ETR阻断剂BQ-123(给药剂量为12.5 μg/kg)和BQ-788(给药剂量为15 μg/kg)，然后以选定的适宜剂量匀速注入ET-1溶液，观察各组大鼠门静脉压力变化。结果: 不同剂量的ET-1均能使门静脉压力升高，尤以高剂量组最为明显；而提前注入ETR阻断剂之后，再注入ET-1溶液门静脉压力虽然升高，但升高的幅度较小。结论: 成功创建了内皮素性大鼠门静脉高压模型，此模型可用于研究ET-1在PHT发病机制中的作用和药物对PHT时血中ET-1的影响。     

乳酸菌Z222产胞外多糖(EPSⅠ)对免疫细胞功能的影响

目的　检测乳酸菌Z2 2 2 产胞外多糖EPSⅠ对体外免疫细胞功能的调节作用。方法　不同浓度的EPSⅠ作用于小鼠的腹腔巨噬细胞和脾细胞 ,分别用中性红染色法检测巨噬细胞的吞噬能力 ,用MTT法检测淋巴细胞在体外的转化能力、NK细胞杀伤肿瘤细胞Yac 1的能力和产细胞因子IL 2的活性。结果　 (1)EPSⅠ单独作用小鼠脾细胞就能促进体外淋巴细胞增殖 ,且表现出剂量依赖关系。EPSⅠ亦可促进ConA诱导的体外小鼠淋巴细胞转化。 (2 )EPSⅠ体外作用于小鼠腹腔巨噬细胞均可提高MΦ的吞噬能力 ,与对照组比较差异都有显著性。 (3)与对照组相比EPSⅠ能增加体外NK细胞的活性 ,杀伤率最大值达 6 2 .4 1%± 2 .5 4 %。 (4 )EPSⅠ使小鼠脾细胞产IL 2的能力与对照组比较 ,差异有显著性。结论　乳酸菌多糖EPSⅠ对小鼠的免疫功能有一定的调节作用。     

Molecular mechanisms underlying IFN-{gamma}-mediated tumor growth inhibition induced during tumor growth inhibition induced during tumor immunotherapy with rIL-12

The present studnt Investigates the molecular by which IFN-produced as a result of in vitroIL-12 addministration exertsits anty-tumor,rIL-12 was administered three or five times intomice bearing CDA1M fibrosarcoma, OV-HM ovarian carcinoma orMCH-1-A1 fibosarcoma. This regimen induced complete regressionof CSA1M and OV-HM tumors but only transient growth inhibitionof MCH-1-A1 tumors. The anty-tumor effects of Il-12 were associatatedwith enhanced induction of IFN-becouse these effects were abrogatedby pretreatment of hosts with anti-IFN- antibody.Exposure inin vitro of the three types of tumor cells to rIFN- resultedin moderate to potent inhibition of tumor cell growth.IFNstimulatedthe expression of mRNAs for an inducible type of NO synthasa(INOS)in CSA1M cells and indoleamine 2,3-dioxygenasa (IDO),an enzyme capable of degrading tryptophan, in OV-HM cells ,but induced only marginal levels of these mRNAs in MCH-I-ALcells. In association withiNOS gene expression, INF--stimulatedCSA1M cells produced a large amount of NO which functioned toinhibit their own growth in vitro. Although OV-HM and MCH-1-A1cells did not produce NO, they also exhibited NO susceptibility.Whereasthe tumor masses from IL-12-treated CSA1M-bearing mice inducedhigher levels of INOS (for CSA1M) or IDO and iNOS (for OV-HM)mRNAs,the MCH-1-A1 tumor mass expressed lower levels of iNOS mRNAalone.Moreover, massive infiltration of CD4⁺and CD8⁺ T cellsand Mac-1⁺ cells was seen only in the CSA1M and OV-HM tumors.Thus, these results indicate that IFN- produced after IL-12treatment induces the expression of various genes with potentialto modulate tumor cells and growth by acting directly on tumorecells or stimulating tumor-infiltrating lymphold cells and thatthe effectiveness of IL12 therapy is assoiated with the operation if these mechanisms.     

羊心左冠状动脉形态学观察

目的　探讨羊心脏左冠状动脉的解剖结构 ,丰富动物实验资料。方法　局解手术学实验后处死山羊 ,取羊心 90例 ,经 10 %甲醛固定 ,手工剥制显示左冠状动脉 ,6例用过氯乙烯 乙酸乙脂填充剂注入冠状动脉中制成动脉血管铸型 ,测量左冠状动脉及其分支。结果　羊冠状动脉分左、右冠状动脉 ,左冠状动脉粗大较浅表 ,前降支行于心肌表面或深达心肌不能明视 ,上多附有心肌桥 ,旋支粗大行程长分支多 ,二者之间多有对角支出现。羊心冠状动脉为左优势性。结论　羊冠状动脉与人相比有较大差异     

人绒毛膜滋养层细胞的分离纯化及鉴定

目的建立一种简便易行、经济有效的纯化培养滋养层细胞的方法.方法:取孕6-8w的正常绒毛组织10例,采用胰蛋白酶消化法分离细胞,将细胞随机分为两组(各5例),一组用淋巴细胞分层液进行纯化,另一组未纯化,最后用含15%胎牛血清的培养基培养细胞.用免疫细胞化学法进行细胞纯度鉴定.结果本纯化的细胞阳性率为(68.6 2±2.69)%,纯化后的细胞阳性率为(91.60±1.55)%,两者之间的差异具极显著性意度( P<0.001).结论该法简便易行,经济有效,可获得纯度较高、合乎试验要求的细胞滋养层细胞.     

THP-1单核细胞氧化低密度脂蛋白过程中基因表达谱的改变

目的检测与巨噬细胞氧化低密度脂蛋白可能相关的信号传导相关蛋白基因。方法利用低密度脂蛋白作用人单核细胞系THP-1,通过基因芯片分析人信号相关蛋白的表达谱,期望获得阐明氧化机制的有价值的线索。结果在被检测的1 651个基因中,13个基因的表达变化超过了2倍以上,其中9个基因表达增加,4个基因表达减少。在表达增加的基因中,有2个基因已在新近的报道中被认为可能与动脉粥样硬化相关。结论我们的发现可能为揭示巨噬细胞氧化低密度脂蛋白的机制提供全新的思路。