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Fibroblasts have the ability to secret many kinds of extracellular matrix and cytokines relevant to hepatogenic differentiation. So, we investigate whether coculture of hADSCs with fibroblasts without cell-cell contact play a role in hepatic differentiation of hADSCs by RT-PCR analysis of hepatic specific gene expression and real-time PCR analysis of Alb expression.

成纤维细胞能够分泌很多利于干细胞向肝细胞分化的细胞外基质和细胞因子，我们采用非接触共培养的方式对成纤维细胞与人脂肪干细胞共培养能否引发其向肝细胞的分化进行了研究。

Positive rate was relatively high, and the foreign gene could also maintain in testicular tissue for a long time, it could still be detected in the tissue in 7 months after being injected. However. Deferent results could be gained when the detection were made with different primers on different sites of the recombinant plasmid. This could be explained by transgene lost when the foreign gene recombinated and integrated in heterogenetic cell, or transgene degradated by cell nucleases.

然而，我们也看到，在后代检测中，利用不同的引物扩增重组质粒不同部位时，检测结果存在差异，转基因阳性率不同，这种差异说明当外源基因进入到异源细胞内，在重组整合时发生了外源基因丢失的现象，并由此导致外源基因的整合率下降，也可能是受细胞内核酸酶的作用，使得外源基因遭到降解。

The PSCA_3 fragment was selected for its superior expression level in eukaryotic cells.Then the sig-PSCA_3-Fc-GPI genetic fragment was cloned into pVAX1-neo-IRES-GM/B7 vector to construct the final immunological inhanced DNA vaccine pVAX1-PSCA_3-FcGB. Immunofluorescence and flow cytometry were used to confirm the expression of PSCA_3 fragment by transfected into Cos7 cell.Finally,the anti-tumor effect of pVAX1-PSCA_3-FcGB was tested in murine prostate cancer model generated by RM-1 cell line.The animal was immunized with pVAX1-PSCA_3-FcGB DNA vaccine by intramuscular injection plus electroporation,pVAX1 and pVAX1-PSCA_1-FcGB plasmid were used as control.The inhibitory effect of tumor was investigated by observion of forming time,volume and inhibition ratio of tumor.Results:DNA sequencing conformed that the heterological PSCA fusion antigen fragment which was synchronized by overlapping-extending-PCR,was consistent to design.Enzyme digestion analysis showed that the 1 to 4 copies heterological PSCA fusion antigen fragments were constructed successfully.

方法(1)检索GenBank，选择包含人主要T细胞抗原表位序列的人PSCA基因片段，应用异种化抗原设计技术，保留人T细胞抗原表位，设计异种化PSCA融合抗原片段；(2)根据核酸序列按中心模板法设计引物，应用重叠延伸PCR技术拼接合成异种化PSCA融合抗原片段基因，以PCR、限制性酶切和DNA序列测定法进行鉴定：(3)利用DNA限制性内切酶BssHⅡ和MluⅠ酶切后粘端互补的特点，采用同尾酶法构建1—4拷贝异种化PSCA融合抗原片段(PNCA_1-PSCA_4)，并将上述片段分别插入真核表达载体pCI-neo-Fc-GPI中，转染293T细胞，借助免疫荧光+流式细胞术考察插入片段表达效率，最终选定PSCA_3片段进行下一步研究；(4)将sig-PSCA_3-Fc-GPI基因片段自pCI-PSCA_3-Fc-GPI质粒上切下，插入pVAX1-neo-IRES—GM/B7载体中，构建免疫增效DNA疫苗pVAX1-PSCA_3-FcGB，并应用转染Cos7细胞+免疫荧光/流式细胞术方法鉴定其在真核细胞中的表达情况；(5)给8周龄雄性C57BL/6小鼠皮下种植RM-1细胞，制备小鼠前列腺癌模型，并采用股四头肌肌肉注射+电脉冲法(Electroporation，EP)接种DNA疫苗质粒pVAX1-PSCA_3-FcGB，同时接种pVAX1空载体质粒和pVAX1-PSCA_1-FcGB质粒作为对照，通过观察计算免疫动物的成瘤时间、肿瘤体积和抑瘤率，来评价该DNA疫苗在小鼠体内的抑瘤效果。

A strain of breast tumor cell line ZHL2006 was established and biologically identified. The ZHL2006 cells grew fast in vitro and the population doubling time was 43.6h. Electronmicroscopical observation showed that there were microvilli on the surface of the cells, nucleus irregularity, karyoplasmic ratio increased, nucleolus distinctness, et al.. Chromosome number and structure were abnormal. Immunohistochemistry showed that PCK were expressed. The ZHL2006 cell line could form onto clone in soft agar and had oncogenicity in athymic mice.

ZHL2006细胞在体外生长迅速，群体倍增时间为43.6h；倒置相差显微镜及Giemsa染色镜下观察细胞为多边形及梭形；透射电镜观察可见细胞表面有微绒毛，核高度不规则，核浆比例增大，核仁明显等；染色体数目和结构异常；广谱细胞角蛋白染色阳性，说明其自上皮而非来自基质；同时ZHL2006细胞系在软琼脂中可形成克隆，具裸鼠致瘤性。

Results ⑴ The cell proliferation was inhibited by TET. It showed thatdivision index and cell number during G0-G1 were decreased, while S and G2-M phases were lengthened with dosage and time dependency in dosages of TET from 2mg/L to 8mg/L; besides, oval-shap cells, karyopyknosis and fragmentation were observed under light microscope. The inhibitory effect in T2 group was statistically similar to that in triamcinolone.

结果⑴ TET在2mg/L~8mg/L之间呈剂量及时间依赖性地抑制瘢痕成纤维细胞的增殖，使生长曲线不同程度的压低、分裂指数降低、G0-G1期细胞数减少、G2-M期增多、S期与G2-M期延长，光镜下见细胞突起变短，细胞由梭形变成椭圆形，核固缩、碎裂。P.C的改变近似于T2组(4mg/L TET)。

A time sex usage the 滤 divided by the leuco - cell blood transfusion the 器 to make use of the gravity blood or blood component the approval filter divided by the leuco - cell with the 滤, and combine to infuse into the human body or for nothing bag amid.

一次性使用滤除白细胞输血器是利用重力将血液或血液成分通过过滤器以滤除白细胞，并注入人体或空袋中。

The experimental study is to explore the effect about rhEPO on neuromotor function, secondary pathology change and expression of NTF and the apoptosis of neural cell, which administered at different time after traumatic spinal cord injury. The experimental study is devided into three parts: The first part The effect of rhEPO on NT-3 and NGF expression after traumatic spinalcord injury Objective Nerve growth factor and neurotrophic factor–3(NT-3) are chief members in neurotrophic factors family, which can promote nerve cell survival、growth and differentiation.

本研究拟在脊髓损伤后不同时间应用促红细胞生成素干预，探讨其对神经运动功能和继发性病理改变的保护作用及对神经营养因子表达和神经细胞凋亡的影响，共分为以下三个部分：第一部分脊髓损伤后促红细胞生成素对NGF、NT-3表达的影响目的：神经生长因子和神经营养因子-3(NT-3)是神经营养因子家族的主要成员，是机体产生的能够促进神经细胞存活、生长、分化的一类多肽或蛋白质因子。

The experimental group corneas were preserved by organ culture for 4 weeks, the corneal thickness was measured with ultrasonic corneal pachymeter. Then every corneas were divided into half -chip, there are 48 half-chip total. It was divided into 4 groups, there are 12 half-chip in every groups. The corneal endothelial cell density of 12 half-chip were counted through Alizarin Red-Trypan blue staining; 12 half-clip corneas were fixed with 4% neutral formalin solution, HE staining was performed, the expression of AQP-1 in corneal stroma and corneal endothelial cell were detected through immunohistochemical staining; Na~+-K~+-ATPase activities in 12 half-clip corneas were examined with Na~+-K~+-ATPase kit; the expression of AQP-1 mRNA were detected through real-time fluorescent quantitation PCR.

实验组经器官培养保存4周后以角膜测厚仪测量角膜厚度，然后每个角膜被分成两半，共48个半片角膜，再分成4组，每组12个半片。12个半片用茜素红-台盼蓝染色染色行角膜内皮细胞计数；12个半片角膜用4%中性福尔马林溶液固定行HE染色、应用免疫组化染色检测AQP-1在角膜基质和内皮细胞表达的改变；12个半片角膜用Na~+-K~+-ATP酶试剂盒测量角膜内皮细胞Na~+-K~+-ATP酶活性；12个半片角膜用实时荧光定量PCR检测AQP-1mRNA表达改变。

The general situations of the eye were observed and the corneal thickness were measured with ultrasonic corneal pachymeter after the animal models was established. After a week, the corneas were removed after the experimental animals are put to death. The corneal endothelial cell density of 12 half-chip were counted through Alizarin Red-Trypan blue staining; 12 half-clip corneas were fixed with 4% neutral formalin solution , HE staining was performed, the expression of AQP-1 in corneal stroma and corneal endothelial cell were detected through immunohistochemical staining; Na~+-K~+-ATPase activities in 12 half-clip corneas were examined with Na~+-K~+-ATPase kit; the expression of AQP-1 mRNA were detected through real-time fluorescent quantitation PCR.

术后观察眼球大体情况、测量角膜厚度。1周后处死实验动物取角膜，用茜素红-台盼蓝染色染色行角膜内皮细胞计数；用4%中性福尔马林溶液固定行HE染色、应用免疫组化染色检测AQP-1在角膜基质和内皮细胞表达的改变；用Na~+-K~+-ATP酶试剂盒测量角膜内皮细胞Na~+-K~+-ATP酶活性；实时荧光定量PCR检测AQP-1mRNA在角膜内皮细胞表达的改变；并于正常对照组角膜比较。

After the cell growth curves was recorded, RPE cells of the 3-5th passages were utilized. 2、Three different siRNA (siRNAl,siRNA2,siRNA3) targeting against human cx43 gene and one negative control siRNA were designed and transfected into cultured human RPE cells via liposome reagent. The most effective siRNA can be determined by semi-quantitative reverse transcription PCRRT-PCR. 3、To the most effective siRNA, after transfected into human RPEs with different concentration, the cellular proliferate activities were messured by MTT colorimetry ; the percentages of RPE in different cell circle phase was assayed by FCM; the changes of phenotypical properities were observed with SCM; the protein expression of cx43 was studied through immunocytochemistry stain and Weston blot; the communication intercellular was calculated with FRAP; and the ability of recovery was assessed by using an in vitro wound healing model.4、The total proteins of siRNA1 and RPE were seperated by two-dimensional gel electrophoresis and visualized by silver staining. Proteins with significant expression alterations were selected and their peptide mass fingerprints (PMFs were obtained by matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS).The PMFs were used to search NCBInr database by Auto MS-Fit software.

实验方法：1、培养原代的人RPE细胞，经过细胞角蛋白、S-100和神经胶质原纤维酸性蛋白免疫细胞化学鉴定后，通过AO/PI染色技术确定培养细胞的存活率，描记其生长曲线，第3-5代用于以下细胞实验2、生物合成针对人cx43基因的三条小干扰RNA和一条阴性RNA通过脂质体转染RPE细胞后，通过RT-PCR的方法确定抑制效率最高的干扰片断3、将该片段以不同浓度通过阳离子脂质体转染培养的人RPE细胞后，采用MTT法观察其对细胞的增殖力的作用；通过流式细胞仪观察其对细胞周期的影响；通过扫描电镜观察其对细胞形态的影响；通过免疫细胞化学和Weston blot观察其对cx43蛋白表达的作用；采用激光共聚焦和荧光淬灭恢复技术观察荧光恢复速率平均百分率，评价其对细胞间通讯功能的影响；通过制作RPE细胞损伤模型，观察其对损伤修复能力的作用4、分离纯化转染siRNA的RPE组和正常对照组RPE细胞的全部蛋白质，应用等电聚焦电泳和SDS-PAGE双向电泳技术，银染显示分离出的蛋白质斑点，经凝胶图像分析软件对两个样本进行胶图分析，寻找差异蛋白点。

But we don't care about Battlegrounds.

但我们并不在乎沙场中的显露。

Ah! don't mention it, the butcher's shop is a horror.

啊！不用提了。提到肉，真是糟透了。