核染质

Use constituent piece to stick mural way depart to foster Niu Er to become fiber cell above all, after classics generation is rarefied, fat simple way will be contained double the skin of the 4th acting ox that outside conveying carrier to turn into body, the particularity of person bacteriolysis enzymatic mammary gland that chooses number fosters becomes fiber cell, g/mLG418 chooses 500 μ 2 weeks, g418 halve continues to develop 3 generation of 2 ～, catch the cell after choosing to undertake PCR detects to turning next reach nucleus analysis.

首先采用组织块贴壁法分离培养牛耳成纤维细胞，经传代纯化后，脂质法将含有双重筛选标记的人溶菌酶乳腺特异性表达载体转入体外培养的第4代牛皮肤成纤维细胞，500μg/mLG418筛选2周，G418减半继续培养2～3代，然后对转染筛选后的细胞进行PCR检测及核型分析。

Ambs S,Dennis S,Fairman J,Stillman IE,Lombardo M.Inhi-bition of tumor growth correlates with the expression level of a human angiostatin transgene in transfected B16F10melanoma cells ［J］.

本实验将大鼠血清白蛋白分泌信号正确连接在endostatin基因的碳末端，成功构建了真核表达载体pcDNA3-SE，并利用Lipofectamine介导转染C6胶质瘤细胞。

RESULTS A eukaryotic expression system for high expression humanmutantCD59 were successfully set up : The recombinant PALTER-MAX plasmid containing human mutantCD59 cDNA and PCDNA plasmid were co-transfected into CHO cell by cation lipoid mediating method ;and the cells were grown in F12 medium containing 400ug/ml G418 for 14 days, positive clones were grown in RPMI1640 medium to get stable expressing cell lines . Highly expressing clones were selected by flow cytometry ,and were named PALTER-CD59-CHO1PALTER-CD59-CHO2 . Flow cytometry indicated that expression rates of PALTER-CD59-CHO1 and PALTER-CD59-CHO2 were 53.7%and 54.5%. Further more, Stable highly expressing CHO cell lines were more detected by immunocytochemistry and immunofluorescence technology . PALTER-CD59 -CHO1 and PALTER-CD59-CHO2 were grown in RPMI1640 to get a large of cells . CD59 protein were obtained by spalling PALTER- CD59- CHO1 and PALTER - CD59 - CHO2 cells . Stable highly expressing cells were further validated by SDS-PAGE, immunoblot analysis and solid enzyme immunoassay . PALTER - CD59 - CHO1 and PALTER - CD59 - CHO2 were glycated in RPMI1640 of 50mM ribose for 72 hours .BCECF releasing test indicted that the releasing rate of PALTER-CD59-CHO1 or PALTER-CD59-CHO2 was less high than PALTER-CHO ,and the releasing rate of glycated PALTER-CD59 -CHOI or PALTER-CD59-CHO2 was higher than unglycated ones . PALTER -CD59-CHO1 and PALTER -CD59 -CHO2 were glycated in RPMI1640 of 50mM ribose for 72 hours .BCECF releasing test indicted that the releasing rate of PALTER - CD59 - CHOI or PALTER - CD59 - CHO2 was less high than PALTER-CHO ,and the releasing rate of glycated PALTER-CD59-CHO1 or PALTER - CD5 9-CHO2 was higher than unglycated ones .

结果 成功构建突变人CD59的真核细胞表达系统：运用阳离子脂质体介导法将含有突变人CD59的PALTER—MAX重组质粒与PCDNA共转染入CHO细胞：用含有400ug／mlG418的F12培养基培养14天，筛选出稳定阳性表达克隆，RPMI1640培养基扩增获得稳定表达细胞株，并用流式细胞术进一步筛选出高效表达细胞株分别命名为PALTER—CD59—CH01、PALTER—CD59—CH02，表达率分别为53.7％、54.5％；应用免疫组化方法、免疫荧光技术进一步鉴定阳性细胞株；RPMI1640培养基大量扩增PALTER—CD59—CH01、PALTER—CD59—CH02细胞株，裂解细胞得到CD59蛋白质；通过SDS—PAGE凝胶电泳技术、免疫印迹技术、固相酶联免疫吸附试验验证了这两中文摘要个阳性细胞株CO59蛋白的高效表达；50mM核糖培养72小时，获得突变人CD59糖化细胞株，BCECF染料释放试验结果显示，PALTER一CD59一CHOI、pALTER一CD59一CHOZ细胞较PALTER一CHO细胞染料释放率低，未糖化PALTER一CD59一CHOI、PALTER一CD59一CHOZ细胞比较糖化后细胞染料释放率低。

Electron microscope was used to observe substantia nigra,caudate nucleus and raphe nucleus stained by methylene blue.

应用透射电镜观察经美兰块染的中脑黑质、脑桥中缝部及尾状核部。

Results (1) The objective compound was synthesized, and its structure was confirmed by mass spectrography, infrared spectrography and nuclear magnetic resonancespectroscopy.

4用重组真核表达载体pLXSN-hBD2通过新型阳离子脂质体对大鼠气道上皮细胞进行转染，用PCR、Western blot检测hBD-2基因的整合与表达。

N perifocal tissue intracerebral hemorrhage there were rarefaction neuron,cell spaces augmentation,cell diminution,distinct demarcation of cell membrane and surrounding,and we discovered a lot of degeneration and necrosis nerve cells,cell body collapsed,pycnosis anachromasis,nucleoli disappeared. In EPO group we discovered that center area of hemorrhage shinked,nerve cells of degeneration and necrosis decreased in perifocal tissue,majority cells morphous were normal and pathological changes were light.

CH对照组在术后皮层出血中心无神经元，仅见少量胶质细胞，细胞间质空泡样改变；出血边缘区神经元稀疏，细胞间隙增大，细胞缩小，胞膜与周围分界清楚，并可见大量变性及坏死的神经细胞，表现为胞体皱缩，核固缩深染，核仁消失。rhEPO治疗组出血中心区变小，边缘区神经细胞变性坏死减少，多数存活细胞形态相对正常，病变较轻。

Methods The NGF, BDNF, and NT3 genes of rats were cloned, the eukaryote expression vectors were established, the three kind of recombinant vectors were used to transfect astrocytes, the positive cloned cells were cultured dilatedly after G418 sifting; using supernates of culture liquid of astrocytes modified by gene to culture PC12 or TrkB-PC12, the expression and its level of gene target cells aimed genes were measured by Western blotting or immunohistochemical method.

克隆大鼠NGF、BDNF和NT3基因，构建真核表达载体；3种重组载体分别转染星形胶质细胞，G418筛选后获得阳性克隆细胞进行扩大培养；取基因修饰星形胶质细胞培养液上清培养PC12细胞或TrkB-PC12细胞；用Western blotting杂交或免疫组织化学方法检测基因靶细胞目的基因的表达及其水平。

Primary cultured chondrocytes are poly-angle, cytoplasm-rich, and their nuclei are either round or oval with clear necleole. Metachromatic and alcian blue positive staining in primary cultured chondrocytes was observed. Intercellular matrix was anti-collagen type Ⅱ staining but not anti-collagen type Ⅰ staining by IHC assay.

原代培养软骨细胞呈多角型，胞质丰富，胞核成圆形或椭圆型，核仁清楚，甲苯胺蓝呈异染性，阿尔新蓝8Gx 染色阳性，细胞外基质Ⅱ型胶原免疫组化染色阳性，Ⅰ型胶原染色阴性。

AIM: To construct the eukaryotic expressing vector of human cbl and test its expression in African green monkey kidney cell line COS 7. METHODS: Human cbl gene tagged with flag was amplified from pEFHAcbl plasmid by PCR. The product was cloned into pGEM T easy, sequenced and then subcloned into eukaryotic expressing vector pcDNA3.1.

以含有人全长cbl cDNA的质粒pEFHAcbl 为模板，采用PCR方法扩增cbl基因，并在其N 末端带上含24 bp的flag标签，克隆到pGEM T easy载体并测序，再亚克隆至真核表达载体pcDNA3 1，酶切鉴定正确后采用脂质体法瞬时转染COS 7细胞，Western blot检测flag cbl在细胞中的表达。

METHODS: Human cbl gene tagged with flag was amplified from pEFHAcbl plasmid by PCR. The product was cAIM: To construct the eukaryotic expressing vector of human cbl and test its expression in African green monkey kidney cell line COS7. METHODS: Human cbl gene tagged with flag was amplified from pEFHAcbl plasmid by PCR. The product was cloned into pGEMT easy, sequenced and then subcloned into eukaryotic expressing vector pcDNA3.1.

以含有人全长cbl cDNA的质粒pEFHAcbl 为模板，采用PCR方法扩增cbl基因，并在其N末端带上含24 bp的flag标签，克隆到pGEMT easy载体并测序，再亚克隆至真核表达载体pcDNA31，酶切鉴定正确后采用脂质体法瞬时转染COS7细胞，Western blot检测flagcbl在细胞中的表达。

Do you know, i need you to come back

你知道吗，我需要你回来

Yang yinshu、Wang xiangsheng、Li decang,The first discovery of haemaphysalis conicinna.

1〕 杨银书，王祥生，李德昌。安徽省首次发现嗜群血蜱。