多染细胞

In HeLa cells trasnfected with GFP-BGLF4 expressing plasmids, cells were sorted into two groups with various levels of BGLF4 expression by FACS. We found that these two populations of cells display dramatically different cell cycle profile.

若利用HeLa细胞将GFP-BGLF4转染送入细胞后，对BGLF4表现量多或表现量少的细胞群各别分析其DNA含量，则发现BGLF4表现量高时S-phase细胞明显增加，而且这些细胞的细胞周期将无法离开S-phase往G2/M-phase过渡转换时期推进。

Then we transfected transitorily the recombinant of green fluorescent protein gene and middle molecular weight neurofilament cDNA into wide type N2a (N2a/wt) and N2a/tau40 to observe the effect of tau accumulation on GFP-NFM fusion protein transport in cellular processes in living cells. At last we used an apoptotic inducer, camptothecin (an inhibitor of topoisomerase-1) to treat N2a/wt and N2a/tau40 cell lines, and compared their apoptotic response.

主要结果如下：一、tau蛋白过度表达和聚积对细胞形态的影响：倒置显微镜下观察两种细胞的形态，发现N2a/wt细胞的突起多而长，而N2a/tau40细胞胞体变圆，突起明显缩短；免疫印迹结果显示转染了tau40的细胞内tau的免疫反应约增加14倍，免疫荧光结果显示N2a/tau40细胞胞体内呈现出较强的红色荧光，tau主要分布在核周和突起起始部分的胞质内，而N2a/wt细胞内的荧光很弱。

The stable clones are further identified by RT-PCR and Western blot; 6 MTT assay is used to investigate the effect of ZNRD1 on the cell growth of cells (AGS, SGC7901, MKN28, NIH3T3, GES-1); 7 Soft agar assay is used to investigate the effect of ZNRD1 on the clonality of cells (AGS, MKN28); 8 Nude mice assay is used to investigate the effect of ZNRD1 on the cell growth of gastric cancer cells (AGS, MKN28); 9 Flow cytometry is used to investigate the effect of ZNRD1 on the cell cycle distribution of cells (AGS, MKN28, NIH3T3, GES-1); 10 Flow cytometry is used to investigate the effect of ZNRD1 on the cell apoptosis of cells (AGS, MKN28, NIH3T3); 11 MTT assay is used to investigate the effect of ZNRD1 on the drug sensitivity of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR) in vitro; 12 SRCA is used to investigate the effect of ZNRD1 on the drug sensitivity of gastric cancer cells (SGC7901, SGC7901/VCR) in vivo; 13 Flow cytometry is used to investigate the effect of ZNRD1 on adriamycin accumulation of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR); 14 Transmission electron microscope is used to investigate the effect of ZNRD1 on the sensitivity of SGC7901 cells towards drug-induced apoptosis; 15 Flow cytometry and DNA ladder assay are used to investigate the effect of ZNRD1 on the sensitivity of cells (SGC7901, SGC7901/VCR, HL-60/VCR) towards drug-induced apoptosis; 16 Microarray is used to investigate the profiling of ZNRD1-responsive genes in gastric cancer cells (AGS, MKN28, SGC7901, SGC7901/VCR); 17 RT-PCR and Western blot are used to identify the results of microarray; 18 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of cyclin D1; 19 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of MDR1; 20 Kinase assay is used to investigate the effect of ZNRD1 on the activity of cyclin E-CDK2 kinase; 21 The antisensenucleic acids of p21 is used to inhibit the expression of p21, and flow cytometry is used to investigate the effect of p21 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 22 The antisensenucleic acids of p27 is used to inhibit the expression of p27, and flow cytometry is used to investigate the effect of p27 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 23 Liposome is used to up-regulate the expression of Skp2, and flow cytometry is used to investigate the effect of Skp2 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 24 Western blot is used to investigate the effect of ZNRD1 on the stability of Skp2 and p27 in gastric cancer cells; 25 MVD assay is used to investigate the effect of ZNRD1 on the angiopoietic activity of gastric cancer cells; 26 ELISA is used to investigate the effect of ZNRD1 on the expression of VEGF165 in gastric cancer cells; 27 The roles of DARPP-32 in MDR of gastric cancer cells are investigated using gene transfection, MTT assay, SRCA, flow cytometry and DNA ladder assay.

应用杂交瘤技术制备ZNRD1的首个单克隆抗体；2）利用RT-PCR、Western blot和免疫组化检测ZNRD1在胃癌组织、胃炎组织、正常胃上皮组织、胃癌细胞和正常胃组织上皮细胞中的表达；3）构建ZNRD1的小干扰RNA载体，并测序鉴定；4）利用脂质体将ZNRD1的真核表达载体及其空载体转染胃癌细胞（AGS、SGC7901、MKN28）和小鼠成纤维细胞（NIH3T3），G418筛选后进行鉴定；5）利用脂质体将ZNRD1的小干扰RNA载体及其空载体转染药敏胃癌细胞（SGC7901）、正常胃组织上皮细胞（GES-1）、对长春新碱耐药的胃癌细胞（SGC7901/VCR）、药敏白血病细胞（HL-60）、对长春新碱耐药的白血病细胞（HL-60/VCR），G418筛选后进行鉴定；6）利用MTT实验检测ZNRD1高/低表达对细胞（AGS、SGC7901、MKN28、NIH3T3、GES-1）生长的影响；7）通过软琼脂克隆形成实验检测上调ZNRD1对AGS、MKN28细胞克隆形成能力的影响；8）通过裸鼠成瘤实验检测上调ZNRD1对AGS、MKN28细胞体内成瘤性的影响；9）通过流式细胞仪分析ZNRD1高/低表达对细胞（AGS、MKN28、NIH3T3、GES-1）的细胞周期的影响；10）通过流式细胞仪分析上调ZNRD1对细胞（AGS、MKN28、NIH3T3）的凋亡的影响；11）通过MTT实验检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60、HL-60/VCR）体外药物敏感性的影响；12）通过肾包膜下移植法检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR）体内药物敏感性的影响；13）通过流式细胞仪分析ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60、HL-60/VCR）内阿霉素蓄积和泵出的影响；14）通过透射电镜检测上调ZNRD1对SGC7901细胞凋亡敏感性的影响；15）通过流式细胞仪和DNA梯度试验检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60）凋亡敏感性的影响；16）通过基因芯片检测ZNRD1高/低表达对胃癌细胞内基因表达谱的影响；17）利用RT-PCR、Western blot对基因芯片的结果进行鉴定；18）利用报告基因实验检测ZNRD1对cyclin D1的启动子活性的调节作用；19）利用报告基因实验检测ZNRD1高/低表达对MDR1的启动子活性的调节作用；20）利用激酶试验检测ZNRD1对cyclin E-CDK2 激酶活力的影响；21）利用反义核酸技术抑制p21的表达；通过流式细胞仪检测抑制p21对ZNRD1介导的细胞周期阻滞的影响；22）利用反义核酸技术抑制p27的表达；通过流式细胞仪检测抑制p27对ZNRD1介导的细胞周期阻滞的影响；23）利用脂质体转染法上调Skp2的表达；通过流式细胞仪检测上调Skp2对ZNRD1介导的细胞周期阻滞的影响；24）利用Western blot检测ZNRD1对p27和Skp2的蛋白稳定性的影响；25）利用微血管密度实验检测ZNRD1对AGS、MKN28细胞裸鼠移植瘤微血管形成的影响；26）利用ELISA检测ZNRD1对AGS、MKN28细胞培养上清和移植瘤匀浆中VEGF165含量的影响；27）利用脂质体转染法、MTT实验、肾包膜下移植法、流式细胞仪和DNA梯度试验检测新耐药相关分子DARPP-32对细胞（SGC7901、SGC7901/VCR、对阿霉素耐药的胃癌细胞SGC7901/ADR）多药耐药表型的影响；利用脂质体转染法和MTT实验检测下调ZNRD1对DARPP-32介导的胃癌多药耐药的调控作用。

293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein.

转染了ACE2的293T细胞能与表达S蛋白的细胞形成多核体，而同样的293T细胞转染了人免疫缺陷病毒-1则不行。

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

The efficiency of selection was monitored by comparing the number of phage recovered from the acid elution and cell lysate in each round,and by testing EGFR binding specificity of polyclonal phagescFv on CHOEGFRGFP1 and CHOK1 cell with cell ELISA. Bacterial PCR was used to select clones containing a 1 kb insert. Cell ELISA was used to determine EGFR binding specificity of monoclonal pscFv on EGFR positive and negative cell. The number of individual EGFRbinding clones was determined with nucleotide sequencing. Results 500fold enrichments were observed by tittering phages in the cell lysate after five rounds of selection.

以稳定转染的CHOEGFRGFP1细胞和未转染的CHOK1细胞分别作为EGFR阳性和阴性细胞，采用负筛选的方法进行筛选；通过比较每轮投入及洗脱出噬菌体的效价比以及细胞ELISA检测多克隆pscFv与阳性、阴性细胞结合情况对筛选过程进行监测；采用菌落PCR挑选含有全长scFv片断的菌落，进一步用细胞ELISA检测单克隆pscFv与EGFR阳性及阴性细胞结合特异性；挑选EGFR特异性单克隆pscFv采用DNA测序法确定克隆多样性。

Cell ELISA was used to determine EGFR binding specificity of monoclonal pscFv on EGFR positive and negative cell. The number of individual EGFRbinding clones was determined with nucleotide sequencing. Results 500fold enrichments were observed by tittering phages in the cell lysate after five rounds of selection.

以稳定转染的CHOEGFRGFP1细胞和未转染的CHOK1细胞分别作为EGFR阳性和阴性细胞，采用负筛选的方法进行筛选；通过比较每轮投入及洗脱出噬菌体的效价比以及细胞ELISA检测多克隆pscFv与阳性、阴性细胞结合情况对筛选过程进行监测；采用菌落PCR挑选含有全长scFv片断的菌落，进一步用细胞ELISA检测单克隆pscFv与EGFR阳性及阴性细胞结合特异性；挑选EGFR特异性单克隆pscFv采用DNA测序法确定克隆多样性。

Estrogen levels are high, there are more smear Diversification cells, nuclear deep into dense; Low levels of estrogen, Smear a small round or oval, blue-stained nuclear osteoporosis bottom of the cell.

雌激素水平高时，涂片中有较多角化细胞，核致密深染；雌激素水平低时，涂片中出现小而圆或卵圆形，核疏松蓝染的底层细胞。

CGRP content in myocardium gradually enhanced from normal group to controls group, and model group's was the highest. We found immunopositive neurons and nerve fibers of nNOS and CGRP by immunochemistry in normal myocardium. They were mainly round-shaped and oval-shaped, also tadpole-shaped、 shuttle-shaped、triangle-shaped、ring-shaped and irregular-shaped. Nv of neurons and nerve fibers in atria were more than in ventricle in some degree; Those in right part of heart were more than in left. Morphology of CGRP neurons changed in model group, the number of ring-shaped neurons increasing, while the round-shaped and oval-shaped decreasing, and Nv and Sv of model group were markedly less than normal. Expression of nNOS mRNA was overly higher than normal group, but CGRP was opposite.

心肌组织CGRP的含量由正常组、实验对照组至CHF组逐渐升高，差异显著；免疫组化研究表明心肌组织中存在nNOS和CGRP免疫阳性神经细胞及纤维，细胞形态多样，正常多以圆形和椭圆形为主，还有梭形、蝌蚪形、三角形、指环形及不规则形，细胞密度心房高于心室，右心高于左心；CHF组CGRP神经细胞形态有一定改变，淡染的指环形细胞增多，而深染圆形或椭圆形细胞减少，其面、数密度均明显低于正常组，差异显著；用RT-PCR测得CHF组nNOS mRNA极度表达，显著高于正常组，而CGRP表达明显低于正常组。

The transient transfection efficiency was measured by flow cytometry. The stable transfected cells were screened by G418. Results Electrotransfection had the highest transient transfection efficiency (56.8%), with the most masc clones of stably transfection and the least time taken to form big masc clones (20 days). Cationic polymer mediated transient transfection had the lowest efficiency (1.7%), with the least masc clones and the longest time taken to form the big masc clones (30 days). Cationic liposome transfection had a transient transfection efficiency of 39.9% and took 25 days to form the big masc clones.

结果 电转染有最高的瞬时转染效率(56.8%)，稳定转染得到的阳性克隆最多，且从细胞筛选到扩增至得到阳性大克隆所用的时间也最少（约20d）；用阳离子聚合物瞬时转染率低(1.7%)，稳定筛选同样可以得到阳性大克隆，但是数量很少且从细胞筛选到扩增至阳性大克隆所用的时间也最多（约30d）；阳离子脂质体转染效果居中，瞬时转染率为39.9%，从细胞筛选到扩增至得到阳性大克隆所用的时间约25d。

My first article for Screen Power Magazine, which is to this day is the official magazine of Jackie Chan, was a DVD review of the Hong Kong actioner Gen-X Cops, starring Nicholas Tse and Daniel Wu.

我的第一篇文章为屏幕力量杂志，在对这天是Jackie Chan 正式杂志，是香港actioner Gen X 警察的DVD 回顾，担任主角Nicholas Tse 和丹尼尔吴。

His lips grazed hirs, Now what are you going to do?

他的唇使吃草了 hirs，现在你将要做什麼？