后成质的

Part one Isolating, culturing, chondroblast induction and evaluation of human MSC in vitroObjective:To examine the property and proliferation ability of primary culturing MSC ,mvestgate the effect of lightly centrifugation and chemical definition medium to induce MSC differentiate into chondroblast.

结果：原代骨髓基质干细胞易于体外培养，增殖旺盛；经轻度离心和化学限定培养基诱导后细胞呈高密度生长，仍保持较旺盛的增殖能力，细胞转化成圆形肥大细胞，甲苯胺蓝染色显示软骨细胞外基质 v的特异性异染，11胶原蛋白免疫细胞化学染色呈强阳性，BT干CR鉴定显示＊胶原mRNA丰富的表达。

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。

PartⅡThe mechanism elucidation about effects ofα-(2,3)/(2,6)sialic acid on the Cx43gap-junction functions.(1) Westernblotting experiment showed that the decrease of sialic acid didn\'t changethe Cx43 expression and its phospholation level.(2) Westernblotting experiment showed sialidase didn,t change the ZO-1 expression,IP and confocal experiment showed sialidase improved the interaction of Cx43 andZO-1.(3) Westernblotting experiment showed sialidase didn\'t change N-cadherin expression,IP and confocal experiment showed sialidase promoted the complex formation ofCx43 and N-Cadherin.(4)Sialidase could increase the ERK1/2 phospholation level,and enchanedintercellular homotypic adhesion,Immunofluorometric assay showed sialidase couldpromote the N-cadherin cluster on the membrance.

第二部分：α—(2，3)／(2，6)唾液酸对肿瘤细胞CX43间隙连接功能影响的机制研究1、Westernblotting结果表明降低细胞膜表面唾液酸并不改变Cx43的表达及其磷酸化水平：但Cx43连接斑形成增多；2、Westernblotting结果表明唾液酸酶作用后细胞ZO—1表达没有改变，IP及免疫荧光共定位结果表明唾液酸酶作用后促进了Cx43与ZO-1的结合；3、Westernblotting结果表明唾液酸酶作用后细胞N-cadherin表达没有改变，IP及免疫荧光共定位结果表明唾液酸酶作用后促进了Cx43与N-Cadherin复合物的形成；4、唾液酸酶作用后细胞内ERK1／2磷酸化水平明显增加，细胞间同质粘附增加，以及免疫荧光表明唾液酸降低后可促进N-cadherin的膜成簇。

Methods:The subcutaneous adipose tissue was obtained from the fold inguen of four SD rats, then digested with collagenase type Ⅰ and cultured in endothelial medium for seven days.

获取4只雄性SD大鼠鼠蹊部皮下脂肪，通过机械分割和组织消化法获取脂肪基质细胞，在内皮细胞培养液中培养7d后，经免疫细胞化学及透射电镜鉴定证实95%以上细胞已具内皮细胞表型特征，再将这些细胞进行成脂(DMEM/F-12培养基内加入0.5mmol/L1-甲基-3-异丁基-黄嘌呤，1μmol/L地塞米松，10μmol/L胰岛素，200μmol/L吲哚美星)定向诱导7d，形态学观察和油红O特殊染色鉴定诱导后的细胞。

RESULTS: hADSCs presented fibroblast-like morphological feature with a flocked array. The 3rd passage of hADSCs had unique immunophenotypes and they were positive for CD73, CD44, CD166, CD105 and CD29, but negative for CD31, CD34, CD45 and HLA-DR. Cell cycle result showed that they had the typical growth characteristics of stem cells, namely, 83.81% cells stayed at G0/G1 stage, only 16.19% cells were stayed at S+G2/M stage; The latent phase of the primary culture cells was 2 days prior to and after incubation, followed by 3-6 days of logarithmical proliferation, reached a peak at day 6, and entering the growth platform phase with lower growth speed.

结果：分离培养的人脂肪间充质干细胞为成纤维细胞样，呈漩涡状排列。P3代人脂肪间充质干细胞呈CD73，CD105，CD166，CD44及CD29阳性，而CD31，CD34，CD45及HLA-DR阴性；具有典型的干细胞增殖特点，83.81%细胞处于G0/G1期，仅16.19%细胞处于S+G2/M期；细胞在接种后前2 d处于生长潜伏期，第3~6天处于对数生长期，第6天达峰值，以后细胞生长速度减慢，进入生长平台期，平均五六天传代1次。

The human cartilages are composed of chondrocyte and extracellular matrix , the form of chondrocytes are hypertrophy and the quantity are less; the ECM of cartilage are compised of type Ⅱ collagen and proteoglycan. Articular cartilages are all hyaline with little fibers. Trauma and arthritis are the main cause of cartilage injury, the ommilayer injury ofcartilage can be recovered by marrow, but because of without stimulation mechanism, the new tissues are merely fibrocartilages, they can not be coincide with hyaline cartilage in menchanics; the purely damage of articular cartilage can not stimulate chondrocyte to regenerate because of without blood circulation, thus, the plerosis of articular catilage can not depend on the proliferation of local chondrocyte. Ever since, people tried their best to find a way to reconstruct articular cartilage.

中文题名人骨髓基质干细胞成软骨诱导及多孔复合材料作为细胞载体的体外实验研究副题名外文题名 Cartilage induction of human mesenchymal stem cells and experiment on compound porous materials as cells' scaffold in vitro 论文作者刘晓岚导师周江南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中南大学学位授予日期2003 论文页码总数68页关键词骨组织工程软骨细胞骨髓基质干细胞壳聚糖高分子外消旋聚乳酸馆藏号BSLW /2003 /R68 /10 造成人体关节软骨损伤的原因主要为创伤和关节炎，关节软骨全层损伤可由于骨髓中间充质干细胞的高速增殖修复，但这种修复由于缺乏相应的刺激机制，只能形成纤维软骨，而不能形成符合关节生理、力学要求的透明软骨；单纯软骨部分损伤软骨组织内无血管，软骨细胞迁移迟缓，无法使损伤区域软骨细胞再生，因此，关节炎及关节创伤后的软骨修复不能依赖于软骨细胞的增殖和迁移。

In order to enhance the miscibility between the inorganic and the organic phases,the silanol coupling agent is made from the slane VTMS and the colloid silica. Then the silanol coupling agent and nano-particles of ZnSb2O6 as the conductive filler are successfully bonded and the nano-particles of ZnSb2O6 will become more organic-like. The colloid silane-SiO2-ZnSb2O6 precursor solution is subsequently mixed with a homogeneous lsopropylalcohol solution of SR-399 acrylic monomers (Dipentaerythritol pentaacrylate) and the active free radical agent709. Under nitrogen purging, this reaction solution prepolymerize at 60℃. Then, the solution is spin-coated with the spin coator at room temperature. At last, the coated thin film is cured and crosslinked by the UV curing reactor to form the highly transparent thin film with good optoelectronic properties from hybrid organic-inorganic nano-composite.

首先以矽氧烷VTMS和colloid silica制备矽氧偶合剂，再以此偶合剂对分散於异丙醇溶液之奈米级的colloid ZnSb2O6做有机化改质，以增进其与有机高分子基材的键结相容性，我们将此colloid silane-SiO2 - ZnSb2O6前驱物与SR-399有机基材单体(Dipentaerythritol pentaacrylate)的异丙醇溶液与活性自由基光起始剂709均匀掺混后，在充满氮气反应系统下，加热至60℃进行预聚合反应，接著在室温下以旋转涂布机旋转涂布成厚度均匀薄膜，最后将此薄膜直接高温硬化或以UV硬化机网状键结硬化成透明与具部份导电性等光电特性之无机/有机奈米混成薄膜。

Methods:Human CD80 cDNA was transfected into B16 melanoma cells by lipofectin-mediated gene transfer before the expansion of tumor cells were tested by MTT method in vitro;C57BL/6 mice were inculated subcutaneously either mock-transfected B16 cells (B16-neo)or CD80-transfected B16 cells (B16-CD80),then the latency,survival times and tumor mass growth were investigated.The lymphocytes were examined for both proliferation indices and specific cytotoxic activity by MTT method and improved LDH-releasing method,after syngenic Mixed Lymphocyte tumor cultures.

通过脂质体介导将CD80基因导入小鼠黑色素瘤B16细胞后，利用SABC法检测CD80的表达；MTT法测定肿瘤细胞体外增殖能力；将肿瘤细胞接种至同源小鼠皮下后观察成瘤期、荷瘤小鼠存活期及肿瘤结节生长速度；在同源淋巴细胞肿瘤细胞混合培养后，通过测定淋巴细胞增殖指数和CTL杀伤活性检测肿瘤细胞的免疫原性。

Damage hair is qualitative must first daub of the ability after attenuant emollient , also can allocate with 3 agents, scale chooses according to the damage level of the hair, if discover after bate judgement error brings about bate excessive should pour water instantly, develop the 3rd agent, if the hair is sere only OK redo, if the hair has resembled cotton shrink into oneself is washed-up.

受损发质一定要先稀释软化剂后才涂抹，也可以用三剂调配，比例则根据头发的受损程度来选择，如在软化后发现判断失误导致软化过度则应立即冲水，冲水后涂抹上第三剂，如头发只是干枯则可以再做，如头发已经像棉花一样缩成一团就不行了。

The crushed pine bark raw materials through ferment,thoroughly decomposed,can be made into planting media using in container seedling,slight plant in a pot for viewing,colored leaf bush and arbor container seedling,etc All these planting media Call be widely used in production,cuttage,planting,media,they can also be combil'ed with other mediums such as:Peat,pearlier,vermiculite,rice husk,cinder,etc.

经过粉碎的松树皮原料，通过充分发酵堆泄完全腐熟后，可制作有机生态型栽培基质，使用于容器苗、观赏小盆载、彩叶花灌木和乔木容器苗等，使用于生产、扦插、栽培、基质，也可以与其他介质使用如：泥炭、珍珠岩、蛭石、稻壳、煤渣等混合使用，配制成复合介质。

What would he tell Judith and the children?

他将怎样告诉朱迪丝和孩子们呢？

I this is at that time, the opinion with peacockish true girl is full of in a heart.

这就是当时的我，一个心中布满虚荣的女孩子真实的想法。