核染质

METHODS: After AIF△1-120 gene was successfully cloned, the shorter truncated human AIF gene (AIF△1-400) was constructed by deleting the Nterminal mitochondrial localization sequence, the coding sequence of flavine adenine dinucleotide binding domain and nicotinamide adenine dinucleotide binding domain of AIF protein.The truncated human AIF gene (AIF△1-400) was inserted into the pIRES2EGFP and pcDNA3 eukaryotic expression vectors, and the coexpression vectors were then transfected into HeLa cells with LipofectAMINE.

在AIF△1-120基因克隆成功的基础上进一步改造，构建截去AIF基因线粒体定位信号，FAD结合结构域和NADH结合结构域（1-400位氨基酸）编码序列的AIF△1-400基因，将其克隆入pcDNA3和pIRES2EGFP真核表达载体，用脂质体法转染HeLa细胞，通过荧光显微镜观察，MTT检测等方法检测该基因在转染细胞中的表达及其对肿瘤细胞的促凋亡活性。

METHODS: After AIF△1-120 gene was successfully cloned, the shorter truncated human AIF gene (AIF△1-400) was constructed by deleting the Nterminal mitochondrial localization sequence, the coding sequence of flavine adenine dinucleotide binding domain and nicotinamide adenine dinucleotide binding domain of AIF protein.

在AIF△1-120基因克隆成功的基础上进一步改造，构建截去AIF基因线粒体定位信号，FAD结合结构域和NADH结合结构域（1-400位氨基酸）编码序列的AIF△1-400基因，将其克隆入pcDNA3和pIRES2EGFP真核表达载体，用脂质体法转染HeLa细胞，通过荧光显微镜观察，MTT检测等方法检测该基因在转染细胞中的表达及其对肿瘤细胞的促凋亡活性。

Results In the medium and high dose F0 groups, it was observed that the atrophy and incrassation of seminiferous tubule, decrease of spermatogenesis, hyperplasia of interstitial tissue, especially in high dose groups spermatozoon abnormality and nucleolus concentration in the rats testis after DU ingestion for 14 months. The changes became more severe with the prolongation of DU ingestion. Such changes occurred in filial rats (F1) after DC ingestion for 5 months. In the medium and high dose F0 groups, it was observed that a little atrophy of kidney glomerulus, hyperplasia of interstitial tissue after DC ingestion for 14 months, and kidney glomerulus fibrosis happened after DC ingestion for 20 months, such changes occurred in filial rats (F1) after DC ingestion for 5 months In the medium and high dose F0 groups, splenic germinal center and periarterial lymphatic sheaths were hyperplasia , companies with lymphopoiesis after DC ingestion for 7 months, splenic white pulp became more small and sparse after DC ingestion for 20 months.

结果 F0代的中、高剂量组大鼠摄入贫铀14个月后可见雄性的精曲小管萎缩，管壁增厚呈空虚网状，生精细胞层次减少，间质细胞增生，但仍见有精子生成；高剂量组可见到精子呈异型性改变，细胞核浓缩深染，且随着摄入时间延长改变愈趋明显；F1代大鼠摄入贫铀5个月后就有上述改变且更为严重。F0代中、高剂量组大鼠摄入贫铀14个月后肾小球轻度萎缩，间质增生明显，20个月时肾小球萎缩纤维化；F1代大鼠摄入贫铀5个月后就有上述改变。F0代中、高剂量组摄入贫铀7个月时脾脏生发中心和淋巴鞘增生，淋巴母细胞增生活跃，20个月时脾小体减少，生发中心稀疏；F1代大鼠摄入贫铀早期和晚期有类似改变。F0和F1代高剂量组摄入贫铀早期肝脏有炎症细胞浸润，晚期骨髓有核细胞减少，脂肪细胞增加。

Cell lines was established in vitro cell culture system. Only one of it grows well during gene targeting operation. The sex of these cell lines was identified by Sry-PCR.Using lipotransfection method to transfer pZJ into pig fetal fibroblast cell. After 7 days G418 selection, 30 neo-positive cell clones were obtained, among them, 12 of which is non-green ones.

用Sry-PCR法和染色体核型分析法鉴定细胞系的性别，采用优化的脂质体转染法将pZJ转染猪胎儿成纤维细胞，经7天G418(600μg／ml)药物筛选，共获得30个neo阳性细胞克隆，其中12个为非绿色细胞克隆，仅有7个可以扩大培养。

Figure5 HE 10×10 The tumor cells are large and have rich cytolymph, red coloration. The nucleolus is round or elliptical shape.

图5 HE10×10 肿瘤细胞较大，胞浆丰富，红染，核圆或椭圆形，染色质细，核仁及核膜清楚，背景可见较多成熟淋巴细胞、嗜酸粒细胞，散在巨噬细胞。

Methods Westernblot was employed to detect the intracellular expressions of caspase-9, hTERT, Bcl-2 and Bax, and mitochondrial and cytoplastic cytochrome C in HepG2 cells transfected by pSliencer 3.1-H1 neo-shTERT.

收集第20代hTERT基因RNAi真核表达载体论定转染细胞、对照细胞及未转染HepG2细饱，采用Western blot检测线粒体，胞质细胞色素C的表达及细胞内caspase-9、Bcl-2、Bax和hTERT的表达。

MSCs were cultured in a conditional medium in subculture including: dexamethasone, vitamin C, and β-glycerophosphate. At the same time, we constructed the eukaryotic expression vector containing VEGF165gene. VEGF165 gene was transfected into MSCs by means of LipofectAMINE?2000. The stably gene expressive cells were screened with G-418 for 14 days.

在脂质体介导下将构建成功的真核表达载体pcDNA3.1- VEGF165转染到诱导培养的MSCs，G-418筛选阳性细胞克隆，将阳性克隆细胞和未转染的MSCs共同接种于β-磷酸三钙上，体外培养7天后，植入原大鼠肌肉内。

Fig.1 SHEE cultured on coverslide, the living cells were growing in single layer with rich cytoplasm, the nuclei were uniform in size with a nucleolus ph ×400 Fig.2 SHEE had a nucleus with ellipse shape, large nucleolus and the cytoplasm contained mitochondria and tonofibrilEM ×10 000 Fig.3 SHEE was spherical in shape, with pseudopods attached on petri dish and abundant villi on cell surface SEM ×5 000 Fig.4 Same as in Fig.3, cell attached on petri dish, appeared stellate or polygonal in shape, with abundant pseudopods and cytoplasmic processes. Protrusive nuclear region in central part of the cell had more micro-villi SEM ×5 000 Fig.5 Chromosomes of SHEE cells belonged to diploidy type Giemsa ×1 000 Fig.6 The SHEE cells of stained in dark brown by Ki67 immunohistochemistry were the proliferative cells Immunohistochemistry ×400 Fig.7 In SHEE cell culture, the nucleus stained red or pink by PI was dead cell, the green nucleus was living cell Fluorescent ×400 Fig.8 The cell labeled by TdT was apoptotic cell in which the chromatin of nucleus condensed in block, a pyknotic nucleus in the upper right conner was seen TdT labeled ×400

图1 SHEE培养在盖坡片上，活细胞单层生长，胞浆较丰富，细胞核大小一致，有核仁×400 图2 SHEE培养细胞细胞核椭圆形，核仁较大，胞浆有较丰富的线粒体和张力原纤维EM ×10 000 图3 SHEE细胞呈球状，有伪足贴壁，表面有密集微绒毛SEM ×5 000 图4 同上细胞贴壁，呈星状或多角形，有丰富伪足和胞浆突，核区隆起有较多微绒毛SEM ×5 000 图5 SHEE细胞染色体仍属二倍体Giemsa染色×1 000 图6 SHEE细胞Ki67免疫组织化学染棕黄色为增殖细胞×400 图7 SHEE培养细胞出现死细胞，胞核和胞浆PI染色呈红色或淡红色，蓝色细胞核为活细胞荧光显微镜×400 图8 细胞TdT标记阳性为凋亡细胞，染色质凝集呈块状，右上角有一固缩细胞核TdT标记×400

Eukaryon expression vectors containing humanbcl-2 and bax cDNA were respectively transfected into HCC-9204 cells,HCC cellsthat express Bax stably and highly were established,and an HCC cell strain thatexpress Bcl-2 at a 100% positive rate was obtained by cloning.

用脂质体介导的基因转染法分别将含有人bcl-2和bax cDNA的真核表达载体转染到HCC-9204细胞中，建立稳定高表达Bax的肝癌细胞，并用克隆化的方法建立100％稳定表达Bcl-2的肝癌细胞株。

Results The eukaryon expression system pcDNA3.1/myc-HisC-bFGF could express the target protein bFGF in vitro. The recombinant plasmids, pcDNA3.1/myc-HisC-bFGF and pCD2-VEGF121 were transferred into muscles flap in vivo successfully. The active proteins bFGF and VEGF121were expressed at high levels.

结果 构建的pcDNA3.1/myc-HisC-bFGF真核表达体系成功转染体外培养HeLa细胞，目的基因在mRNA水平和蛋白水平均有表达。pcDNA3.1/myc-HisC-bFGF和pCD2-VEGF121重组质粒分别转染在体肌瓣，获得外源基因高水平表达。

It has been put forward that there exists single Ball point and double Ball points on the symmetrical connecting-rod curves of equilateral mechanisms.

从鲍尔点的形成原理出发，分析对称连杆曲线上鲍尔点的产生条件，提出等边机构的对称连杆曲线上有单鲍尔点和双鲍尔点。

The factory affiliated to the Group primarily manufactures multiple-purpose pincers, baking kits, knives, scissors, kitchenware, gardening tools and beauty care kits as well as other hardware tools, the annual production value of which reaches US$ 30 million dollars.

集团所属工厂主要生产多用钳、烤具、刀具、剪刀、厨具、花园工具、美容套等五金产品，年生产总值3000万美元，产品价廉物美、选料上乘、质量保证，深受国内外客户的青睐