染色质的

3Chondrogenesis was assessed using Alcian blue staining, Toluidine bluestaining, Safranin O/Fast Green staining at 7 and 14 days, still collagenⅡimmunohistochemistry and aggrecan immunofluorescence at 4, 7 and14 days after initial chondrogenic induction of adipose -derivedmesenchymal stem cells.

3观察脂肪间充质干细胞在诱导后的生长增殖情况、形态变化，于诱导7、14天后应用阿尔新兰染色、甲苯胺兰染色、藩红0／固绿染色，以及4、7、14天后应用Ⅱ型胶原蛋白免疫细胞化学、aggrecan免疫荧光评价其成软骨能力。

The extracellular matrix wasstained positively for Alcian blue, Toluidine blue and Safranin O / FastGreen at 7 and 14 days after initial chondrogenic induction of adipose -derived mesenchymal stem cells, and indicated collagenⅡand aggrecanwere expressed positively.

2脂肪间充质干细胞在高密度的"微团"培养条件下，于诱导7、14天后阿尔新兰染色、甲苯胺兰染色、藩红0／固绿染色阳性，诱导4、7、14天后Ⅱ型胶原蛋白免疫细胞化学、aggrecan免疫荧光阳性表达。

Intimal areas were measured using morphometric analysis of perfusion-fixed vein graft specimens, and intimal thickness was calculated using circumferential measurements. The SMC proliferation was studied by the immunohistochemical detection of proliferating cell nuclear antigen. Expression of MMP-2、MMP-9 mRNA in vein grafts and unoperated control Vein grafts was detected by reverse transcription polymerase chain reaction. Substrate gel zymography was used to determine the proteolytic activity.

分别行HE染色、Verhoeff弹性纤维染色观察组织病理变化，计算机病理图象分析系统测量新生内膜厚度及面积，免疫组织化学方法检测静脉壁增殖细胞核抗原表达以观察细胞增殖情况，半定量逆转录-聚合酶链反应检测静脉壁基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)mRNA的表达，明胶酶谱法检测MMP-2、MMP-9活性，比较各组之间的差别。

The study showed the recombinant wt/mCREG protein depressed the VSMC proliferation depending on dose and the optimal concentration was 400nM;2biologic function of CREG protein and the membrance receptor mechanism:①effect on VSMC migration: the wound healing experiment showed the OB2 cells migration was slower significantly after added wt/mCREG(400Nm) in supernatant. The HITASY cells migration were very slowly and no remarkable change. The gelatinase digestion and Western blot analysis showed the matrix metalloproteinase was decreased and TIMPs was increased;②effect on differentiation: after added wt/mCREG(400nM), the expression of myocardin, SMα-actin, MHC and caldesmin were increased and that of LM-1 and FN were decreased in OB2 cells. These effects were more significant when adding wtCREG.;③effect on VSMC proliferation: Cell cycle assay and BrDU stain showed: after added the wtCREG and mCREG protein, the ratio of cell in G0/G1 phase increased to 0.5773 and 0.5572 from 0.5308 respectively in OB2 group, which increased to 0.7369 and 0.7034 respectively from 0.6297 in HITASY group;3Role of M6P/IGF2R in CREG biologic function:①ELISA and co-immunoprecipitation showed the wt/mCREG binding to M6P/IGF2R directly.②antibody blocking test: when the anti-IGF2R was added to medium at the same time with wt/mCREG at different concentration(2μg/mL、4μg/mL、8μg/mL),the effects of CREG protein which depressing proliferation, migration, secretion and promoting differentiation were blocked, which had the positive correlation to the concentration of added anti body. The studies showed two combinant CREG promoted VSMC switch to differentiation phaenotype, at the same time, depress VSMC proliferation, migration and secreting extracellular matrix.

上述实验结果证实：两种重组CREG蛋白对VSMC增殖均有剂量依赖性的抑制作用，并且相同浓度的糖基化的CREG蛋白对细胞增殖的抑制效应更为显著，最佳效应浓度为400nM；2两种重组CREG蛋白添加后对HITASY和OB2细胞生物学行为的影响：①CREG蛋白对VSMC迁移的影响：刮伤实验发现，加入最佳效应浓度的wtCREG和mCREG蛋白24h后，OB2组迁移能力下降，HITASY组无明显变化；细胞外基质金属蛋白酶-2，9(Matrix metallo-proteinase 2，9，MMP2 ，9)明胶酶电泳检测和Western blot检测结果证实，两种CREG蛋白均可以使OB2细胞合成细胞外基质MMP2，9减少，而组织金属蛋白酶抑制物(Tissue Inhibitors of Metalloproteinases，TIMPs)增加；②CREG蛋白对VSMC分化的影响：加入400nM的wtCREG和mCREG蛋白12h后，OB2细胞myocardin、SMα-actin、MHC、caldesmin表达增加，LM-1、FN表达减少；③流式细胞仪分析细胞周期和BrDU染色分析证实，加入400nM的wtCREG和mCREG蛋白后，OB2组G0/G1期细胞由0.5308分别增加至0.5773和0.5572，HITASY组G0/G1期细胞由0.6297分别增加至0.7369和0.7034；3M6P/IGF2R在重组CREG蛋白的生物学功能中的调控作用：①免疫共沉淀和免疫荧光双染色分析结果显示，CREG蛋白与M6P/IGF2R存在直接结合；②应用抗体阻断实验：将不同浓度的anti- M6P/IGF2R(2、4、8μg /mL)与两种CREG蛋白同时加入培养液中，CREG蛋白抑制VSMC增值、迁移和合成细胞外基质、促进分化的效应减弱，而且与加入anti- M6P/IGF2R浓度正相关。

In the present study, the expression of bFGF and TGF β1 in normal and hyperplasic prostate were determined with immunohistochemistry and in situ hybridization.

本研究采用免疫组化及原位杂交方法检测bFGF和TGF-β1在正常前列腺及BPH中的表达情况，结果显示，正常及增生前列腺组织中上皮及间质细胞中均见bFGF、bFGFmRNA及TGF-β_1、TGF-β_1mRNA阳性染色，间质细胞染色强于上皮细胞。

After osteoplastic induction, peripheral blood MSCs had strongly positive reactions for alkaline phosphatase staining, total collagen staining, alizarin red staining.

外周血间充质干细胞成骨诱导后，碱性磷酸酶染色、总胶原染色及茜素红染色均呈强阳性；成脂诱导后油红染色呈阳性，有一定数量的脂滴形成。

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。

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。

GFAP overexpessed but NOS neuron decreased, distorted and pale. The glia in SM-DB complex of ONR hypertrophied, but NOS neuron no degeneration. The increased GFAP immunoreactivity and the morphometric and cytological changes in SM-DB complex astrocytes may reflect a sustained upregulation of cellular activity with the insults of neurons, resulting in hypertrophy of glial perikarya and cell processes.

老年减退组星形胶质细胞的细胞体密度较老年正常组和青年组明显增加（P 在GFAP免疫细胞化学和NOS组织化学双重染色的切片上，老年减退大鼠SM-DB星形胶质细胞明显肥大，突起粗壮弯曲，GFAP超量表达，而NOS阳性神经元数量减少，胞体变形，突起减少，染色深度下降。

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