原细胞

Methods: A 5ml bone marrow was extracted from the lilac of human volunteers. By Percoll fluid and density gradient centrifugation, the MSC was obtained; after the cells filled the bottom of vessel, subcultured them, when they subculture in third generation, redigested them, 500 R/min centrifugate, alter the completed medium to chemical definition medium, examined the form change and prolifration of cells by invert microscope, toluidine blue stain、immunocytochemical stain and RT-PCR to test the type Ⅱ collagen mRNA and proteoglycan.

取健康成人髂后上棘处骨髓5ml，经percoll液分离后密度梯度离心，〓／ml密度接种培养，观察原代细胞的贴壁、增殖状况，细胞长满瓶底后进行传代培养；传至第三代细胞，重新消化后以500转／分钟轻度离心5分钟，〓／ml接种，改用化学限定培养基代替完全培养基培养，倒置显微镜观察细胞生长情况及形态变化，甲苯胺蓝染色观察诱导细胞合成细胞外基质中的蛋白多糖，免疫细胞化学染色检测ECM中Ⅱ胶原的蛋白合成，RT-PCR鉴定诱导细胞Ⅱ胶原mRNA的表达。

The aim are:lTo examine the proliferation ability and potential chondroblast differentiation ;2To find an ideal condition stimulated BMSC differentiate into chondroblast;3To examine the chondroblast proliferation in porous scaffolds and to explore the interaction between cells and materials;4To examine the release of cytokine in vitro.

取健康成人略后上棘处骨髓 5ml，经 percoll液分离后密度梯度离心，10'砌l密度接种培养，观察原代细胞的贴壁、增殖状况，细胞长满瓶底后进行传代培养；传至第三代细胞，重新消化后以500转／分钟轻度离心 5分钟，10V加 l接种，改用化学限定培养基代替完全培养基培养，倒置显微镜观察细胞生长情况及形态变化，甲苯胺蓝染色观察诱导细胞合成细胞外基质QCM）中的蛋白多糖，免疫细胞化学染色检测ECM中＊胶原的蛋白合成，RT干CR鉴定诱导细胞＊胶原mRNA的表达。

Results :①The MVs were formed by osteoblast of the secret type、dentinoblast and process;② There was a mature process of the MVs in the extracellular matrix.The appearance of the hydroxyapatite was a major mature feature;③ HAP formed initially in the MVs entered into collage matrix and eventually mineralized;④ The shifting sign of HAP from MVs to the surface of collage fibril was observed.

结果：①MVs是由分泌型成骨细胞、成牙本质细胞及其突起芽生进入胶原基质；②MVs在细胞外胶原基质中有一个逐渐成熟的过程，并以羟基磷灰石（hydroxyapatite，HAP）晶体的出现为其主要特征；③HAP在MVs内形成、增多，最终进入胶原基质并进一步沉积、钙化；④部分胶原原纤维表面沉积的HAP 晶体与MVs分泌的晶体有明显的移形迹象。

In the normal uterus, Cytokeratins immunolabelling were detected in glandular cell, luminal epithelial cell, Vimentin immunolabelling were detected in stromal cell and endoblastic cell; CK7 immunolabelling were not detected in any tissue of the yak utenus.

研究结果显示：未妊娠时，泛角蛋白在子宫内膜腺上皮细胞、腔上皮细胞内表达，波形蛋白在子宫内膜基质细胞内表达，平滑肌肌动蛋白在子宫平滑肌和血管平滑肌内表达，牦牛子宫任何部位均不表达角蛋白7；妊娠30天左右时，泛角蛋白在子宫内膜腺上皮细胞、子宫内膜腔上皮细胞、滋养层细胞、内胚层细胞和尿囊细胞内表达，波形蛋白在子宫内膜基质细胞和内胚层细胞内表达，平滑肌肌动蛋白在子宫平滑肌和血管平滑肌内表达，角蛋白7在尿囊细胞内表达，偶尔在腔上皮细胞的细胞核边缘表达；消化法进行原代培养时，组织经胶原酶消化并通过100目和400目筛网组合可以有效地分离原代子宫内膜基质细胞和子宫内膜腺上皮细胞；分离得到的子宫内膜基质细胞活率达90%以上，并可在体外传代7次以上；分离得到的子宫内膜腺上皮细胞活率可达85%以上，并可在体外传代5次以上；RPMI1640培养基最适合子宫内膜基质细胞和子宫内膜腺上皮细胞的生长，维持子宫内膜基质细胞正常生长的FBS添加量为20%，维持子宫内膜腺上皮细胞正常生长的胎牛血清添加量为30%。

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。

Special staining methods, such as Masson and the Van Gieson staining were used to study the distribution of collogen fibers and elastic fibers. ResultsBy HE staining, the subepithelial connective tissues and vessels in the pterygium were more prominent than normal conjunctival tissues. An amorphous subepithelial superficial hyalinized zone and coarse eosinophilic granular materials were observed in the pterygia, but they were not found in normal conjunctival specimens. Coarse fibers were visible only in the deeper subepithelial connective tissues of pterygial samples. With Masson′s staining, the dense staining of collagen fibers was also more prominent in the pterygium than in the subepithelial connective tissues of normal conjunctiva. Abnormal collagen fibers were visible in the deeper sub-epithelial connective tissues of pterygial samples. With Van Gieson staining, abnormal collagen fibers were visible in the deeper subepithelial connective tissues. Dark coarse elastic fibers were found in the abnormal fibers only in the subepithelial deep connective tissues of pinguecula in the pterygia but not in the conjunctiva. With immunohistochemistry staining, MMP-3 was strong in the pterygial epithelium, moderate in fibroblast and absent from pterygial vascular walls. LN was strongly expressed in the blood vessel wall, moderately in the epithelial basement membrane and absent from the entire stroma.

结果HE染色：翼状胬肉组织上皮下基质中存在结缔组织的增生和血管形成；基质浅层存在一无定形物质透明区及粗糙的颗粒样嗜酸性物质，在翼状胬肉体部深层基质中存在粗糙的纤维组织；正常球结膜组织细胞排列整齐；基质为疏松结缔组织，胶原纤维平行排列，其间可见成纤维细胞，散在少量中性粒细胞、毛细血管；Masson染色：翼状胬肉浅层基质中存在致密的胶原纤维染色，深层基质中的胶原纤维存在变性样改变；VG染色：翼状胬肉组织深层基质中存在大量变性的胶原纤维，其间夹杂黑色的弹性纤维；免疫组化染色法：MMP-3在翼状胬肉上皮细胞中呈强表达，成纤维细胞中呈中等强度表达，血管内皮细胞中未见表达；LN在血管壁中呈强表达，在上皮细胞基底膜中呈中等强度表达，在整个基质中未见明显表达；col Ⅲ在整个翼状胬肉基质中呈强表达。

Methods Constructed the model of fetal scarless wound healing and investigated the expression of collagen in fetal and adult rat wounds by immunohistochemistry and in situ hybridization.

染色，Ⅰ、Ⅲ型胶原免疫组化，Ⅰ、Ⅲ型胶原原位杂交。结果胎鼠伤口愈合快，组织结构再生完全，没有瘢痕形成，胶原呈网状正常排列，Ⅰ、Ⅲ型胶原蛋白及表达均增多，同时伴细胞数增多，Ⅲ型胶原首先形成网状排列；成年伤口胶原纤维沉积慢，平行排列的胶原逐渐呈束状填塞伤口以致瘢痕形成，伤口内Ⅰ、Ⅲ型前胶原mRNA表达均增多，细胞数目增多不明显。

Objective: To explore the mechanisms resulting in the recurrence of urethral scar which make urethral strictures difficult to be cured, a series experiments were conducted to find potential effective factors involved in urethral scar formation and degradation, including the studies of extracellular matrix component of urethral stricture scar, the characteristics of urethral scar fibroblast, and the effects of urine on urethral fibroblast in vitro, as well as the studies to compare the difference of collagenase activity, type Ⅰ collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the tissues and cultured fibroblasts from normal urethra and strictured urethra respectively, and the studies to investigate the effect of antisense TIMP-1 oligodeoxyonucleotide on cell proliferation and collagenase activity of urethral scar fibroblast.

中文题名尿道瘢痕基础研究副题名胶原酶活性，TIMP-1的表达及其反义基因治疗外文题名 Experimental study on urethral scar-activity of collagenase，expression of TIMP-1，and antisense TIMP-1 gene transfection of urethral scar fibroblast 论文作者黄翔导师杨宇如教授学科专业外科学研究领域\研究方向学位级别博士学位授予单位四川大学学位授予日期2002 论文页码总数104页关键词尿道手术瘢痕胶原酶成纤维细胞尿道瘢痕馆藏号BSLW /2003 /R699 /12 目的：研究尿道瘢痕的细胞外基质的组成，尿道瘢痕成纤维细胞的生物学特性以及尿液对其生长的影响；比较胶原酶活性，金属蛋白酶组织抑制因子-1（TIMP-1）以及Ⅰ型胶原含量在尿道瘢痕和正常尿道组织及体外培养的成纤维细胞中的差异；研究反义TIMP-1寡核苷酸对尿道瘢痕成纤维细胞增殖以及胶原酶活性的影响。

The hFCECs were cultured by sticking tissues piece method that the cornea were divided into the limbus and central tissue piece and digestive method,respectively.For digestive method,the digestive juice of 5mg/ml DispaseⅡ+0.25%trypsin was chosed.D/F12 with 10%fetal bovine serum and 100 U/ml penicillin and streptomycin were used in this study,the culture condition was 37℃and 5%CO2,and culture medium changed every three days2、Passage of hFCECsAfter more than 80%confluenced,cells from corneal limbus hFCECs were digested for 5-15min with different concentrations of trypsin/EDTA at 37℃,and then passaged at a ratio of 1:2.The cells were subcultured on empty plates,mouse 3T3 fibroblast feeder layer,fetal corneal stromal cell feeder l ayer or HTK feeder layer respectively.And D/F12, mouse 3T3 fibroblast conditioned medium,fetal corneal stromal cells conditioned medium or HTK conditioned medium with 10%FBS and 100 U/ml penicillin and streptomycin were used respectively as the culture medium.And the culture medium were changed every three days as well.

方法一、人胎儿角膜上皮原代和传代培养1、原代培养严格无菌操作获取人胎儿角膜片，采用组织块贴壁法、酶消化法原代培养人胎儿角膜上皮细胞。2、传代培养角膜缘部的细胞生长融合达80%以上，不同稀释浓度的胰酶/EDTA消化液消化传代分别接种于空板、小鼠3T3成纤维细胞饲养层、胎儿角膜基质细胞饲养层及HTK饲养层，培养液分别采用D/F12、小鼠3T3成纤维细胞条件培养液、胎儿角膜基质细胞条件培养液、HTK条件培养液。

Results 1、 Generally, we can see the original blue and white, shiny, no cracks in the articular surface of the cartilage after the stress increases gradually yellow, surface roughness, cracks appear; when the pressure decreases, the yellowing, rough, the color of the fracture restore gradually and become shiny.2、the shiny smooth surface can be seen under a light microscope, formation, cell distribution, tidy, clear the level of cartilage at the articular surface stress increases, the surface roughness changes, defects, disordered cells, uneven dyeing ; when the articular surface of the pressure gradually decreased, the cartilage gradually repair and the surface of cells at the surface appear only disorder.3、immunohistochemical observation can be seen throughout the observation period, cartilage cells are type Ⅱ collagen expression and expression after 3 weeks gradually weakening, when the seventh week begin to strong gradually.4、 electron microscopy shows that when stress increases the articular surface, the cartilage cells became flat, the cytoplasm in the endoplasmic reticulum, Golgi apparatus decreased with collagen disorders; and when stress decreases the articular surface, cartilage cells gradually returned normal, cytoplasm in the endoplasmic reticulum, Golgi body gradually restore quantity; collagen fibers with a gradual rules.

结果：①大体观察可见到原本蓝白色、有光泽、无裂纹的软骨在关节面压力增大后，逐渐呈灰黄色，表面粗糙，出现裂隙；当压力逐渐减小后，变黄、粗糙、有裂隙的软骨颜色逐渐恢复，变得有光泽②光镜下可见表面光滑、平整，细胞分布均匀、整齐，层次清楚的软骨在关节面压力增大后，表面变粗糙、缺损，细胞排列紊乱、染色不均；当关节面压力逐渐减小后，软骨表面逐渐修复，细胞仅在表层排列紊乱③免疫组织化学观察可见整个观察期内软骨细胞胞浆内均有Ⅱ型胶原表达，术后3周内表达逐渐变弱，从第7周时开始逐渐变强。④电镜下可见当关节面压力增大后，软骨细胞逐渐变扁，胞质中内质网膜、高尔基体减少，胶原排列紊乱；当关节面压力减小，软骨细胞形态逐渐恢复正常，胞质中内质网膜、高尔基体数量逐渐恢复；胶原纤维排列逐渐有规则。

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