荧光镜

The PCR products were examined by agarose gel electrophoresis. The target gene fragments were purified by gel extraction kit and ligated to cloning vector pMD18-T. The recombinant vectors were transformed into host strain E. coli K802 by lithium chloride method, screened and identified with PCR and restrictive enzymatic digestion. Their sequences were confirmed by DNA sequencing.(2) sTWEAK1 gene was subcloned into expression vector pProEx HTb and transformed into E. coli BL21. sTWEAK2 gene was subcloned into expression vector pMAL-C2x and transformed into E. coli TB1. The recombinant vectors were screened and identified with PCR and restrictive enzymatic digestion. The recombinant fusion proteins were induced to express with IPTG, detected by coomassie brilliant blue-stained SDS-polyacrylamide gel electrophoresis , and confirmed by Western blot analysis.(3) The sTWEAK1 fusion protein was purified with Ni-NTA Spin Kit.(4) The biological activity was assayed on transformed and tumor cells by microplate photometer after crystal violet or sulfur rodamine B staining.(5) The contents of IL-8 in the supernatant of 1990 cell cultures were determined by ELISA.(6) The morphological changes of the sensitive cells were observed by light and transmission electron microscopies.(7) The cell cycle and apoptotic rate were assayed by flow cytometry in 1990 and M85 cells.(8) The effect of fusion proteins on induction of NF-κB in 1990 and LOVO cells was detected with Dual-Luciferase Reporter Assay system.(9) The TWEAK gene was subcloned into Adeno-X Viral DNA with pShuttle vector and transfected into HEK293 cells by lipofectamine method.

(1)本研究用RT-PCR方法，从人组织细胞总RNA中扩增可溶性TWEAK胞外区（sTWEAK1和sTWEAK2）的cDNA序列及全长编码序列，用琼脂糖凝胶电泳分析PCR产物，胶回收目的基因片段，连接到pMD18-T克隆载体中，转化大肠杆菌K802，PCR和酶切筛选阳性克隆，全自动DNA测序验证序列；(2)sTWEAK1和sTWEAK2分别亚克隆到pProEx HTb和pMAL-C2x表达载体中，分别转化大肠杆菌BL21和TB1，PCR筛选和酶切鉴定，阳性克隆用IPTG诱导表达，表达产物用SDS-PAGE分析和Western blot验证融合蛋白；(3)用NTA-Ni Spin试剂盒初步分离纯化sTWEAK1融合蛋白；(4)用体外培养的肿瘤细胞和正常对表达产物进行活性检测，贴壁细胞用结晶紫染色法，悬浮细胞用磺酰罗丹明B染色法，酶标仪检测OD值；(5)敏感细胞用ELISA法检测细胞培养上清中IL-8的含量；(6)用光镜和电镜观察敏感细胞死亡和细胞凋亡情况；(7)用流式细胞仪分析表达产物对敏感细胞凋亡率和细胞周期的影响；(8)用双荧光素酶报告基因检测法，测定表达产物对敏感细胞NF-κB的影响；(9)用pShuttle穿梭质粒将TWEAK重组到腺病毒载体上，用脂质体转染法转染HEK293细胞，PCR鉴定重组质粒。

Second is studying the traumatic condition changes in a choosey pressure at different post-PT phases. Moreover, we detected model PLA_2 activity, express of PLA_2 subtype mRNA by fluorescent quantitative RT-PCR technique, protein express and site by Western blot and IHC at different post-PT phases. At last, we studied the PLA_2 inhibited effects and model protection of Variabilin in vivo.

研究在不同压强的外力作用下，模型指定时相点的一般情况和存活率、大体、光镜及电镜下胰腺组织的病理变化和常规酶学变化，得到稳定建模致伤压强；然后，固定致伤压强，研究胰腺创伤后不同时相点的伤情变化(包括一般情况、胰腺组织的病理变化、AMS和LPS活性变化、血常规、血生化、胰腺细胞凋亡以及胰腺组织细胞生长周期变化情况)；再次，采用3H-花生四烯酸标记大肠杆菌膜作为底物的方法检测大鼠胰腺创伤后不同时相点的PLA_2活性，利用荧光定量RT-PCR技术检测其亚型mRNA的表达，利用Western blot和IHC技术检测其蛋白表达量和蛋白组织定位；最后，将Variabilin体内应用于急性胰腺创伤大鼠模型，研究其对模型的保护作用和对PLA_2的抑制效应。

Methods The AGS cellseeded coverslips were prepared, then the two different status of Lactobacillus acidophilus and two different forms of Hp were added in different order. 2 hours later, the bacteria were Garm's stained, and the adherence of Hp to AGS cell were observed by microscope and the adherence index was calculated.

制备人胃上皮腺癌细胞系细胞爬片，将两种状态嗜酸乳杆菌和不同形态Hp菌分组按不同顺序加入，作用2 h，革兰染色后光镜观察分析Hp黏附指数产生的变化，荧光抗体染色法定性评价黏附于AGS细胞的Hp密度的改变。

D. N big mouse model was successfully made by adopting single side kidney cutting with induction, then by 24 week continuos observation on the change of various values of kidney and function, through testing of immunity florescence, electro-scope and optical scope, what we can see from model group is that typical kidney small cell got hardened by knot, and discarded, and dead, also small tube withered, small cell of on base film got thick, cell capsule wall was fibred, and base film got double layered.

实验研究采用单侧肾切除加以链脲佐菌素诱导，成功地复制出糖尿病肾病大鼠模型，对其肾脏结构和功能的改变各项指标，连续进行24周动态观察，并进行免疫荧光、电镜、光镜检测，结果表明：糖尿病肾病模型组可见典型的肾小球节段硬化、废弃、坏死，有大量蛋白管型，肾小球管糖元空泡沉积及脂肪滴，肾小管萎缩，肾小球基底膜明显增厚，球囊壁纤维化，基底膜双层化，有一例结核性肉芽组织及钙化等病理变化。

According to the clear water experiment, aeration performance of the new equipment is good with high total oxygen transfer coefficient and oxygen utilization ratio.

曝气设备的动力效率在叶轮转速为120rpm～150rpm时取得最大值，此时氧利用率和充氧能力也具有较高值。

The environmental stability of that world - including its crushing pressures and icy darkness - means that some of its most famous inhabitants have survived for eons as evolutionary throwbacks, their bodies undergoing little change.

稳定的海底环境─包括能把人压扁的压力和冰冷的黑暗─意谓海底某些最知名的栖居生物已以演化返祖的样态活了万世，形体几无变化。