后成质

GFP gene eukaryotic express plasmids were transfected into ES cells. The cells were observed under the inverted fluorescent microscope. The clones which expressed the powerful green fluorescence were chosen to be named ES-GFP cells and cultured continuously. The examination of ES cell totipotent including Alkine phosphatase staining, embryonic body formation in vitro and teratomas formation in nude mice was implemented.

1。采用脂质体方法将GFP质粒转染ES-D3细胞株，经筛选后倒置荧光显微镜观察细胞形态及荧光状态，挑取荧光最强的克隆，命名为ES-GFP细胞，进一步扩增培养，观察细胞生长及荧光表达情况，行细胞分化全能性鉴定包括碱性磷酸酶（alkine Phosphtase，AKP）染色、体外胚胎体（embryonic body，EB）形成实验及裸鼠体内成瘤实验。2。

The histopathologic observations of corneas after penetrating keratoplasty in dogs: Thestructure of wholly transparent graft was the same as that of host cornea apart from the upperdonor-host junction where there was minimal irregularity of the corneal lamellae. However, thecorneal lamellae at the lower donor-host junction were neat. The graft with its central part beingtransparent only showed poor coaptation with recipient, in which the graft shifted inward and had marked wrinkles and folds in Descemet"s membrane. On the back of Descemet"s membranethere was hyperplastic stromal fibers that thickened the graft and partially adhered to iris.

5犬穿透性角膜移植术后角膜病理组织学观察完全透明的植片与植床结构一致，仅两者对合处上部纤维紊乱，而下方纤维排列整齐；中央透明的植片与植床对合不良，植片向植床方向嵌入，后弹力层皱缩、折叠，植片下方因有来自植床的基质纤维而增厚，部分对合处与虹膜粘连；浑浊的植片有比较完整的上皮层，植片约2／3深度的基质丧失原来纤维整齐排列结构，代以大量成纤维细胞增生。

A549 cells are more sensitive to CDDP-induced apoptosis(P.01) and less sensitive to THP andβ-Ele-induced apoptosis than A375p(P.01, P.05 respectively). Condensation and crack of nucleus and apoptotic bodies appeared in apoptotic cells of A549 and A375p cell lines in all treated groups and necrocytosis were to be seen in some groups. Fluorescence imaging experiments demonstrated that accumulation of iASPP in cytoplasm but little distribution in nucleus in all groups. untreated groups in two cell lines presented a bright-green colour,followed by CDDP-treated groups,and THP-treated groups is the darkest one in all groups.

免疫荧光显示，未加药物处理的A549和A375p细胞胞质染色均匀呈亮绿色，核区较暗；CDDP及β-Ele处理组细胞为暗绿色，其中可见皱缩的凋亡细胞；THP处理的细胞胞质荧光弱呈灰绿色，由于THP嵌入DNA自发红色荧光与二抗的绿色荧光中和，胞核被染成桔红色，结果提示药物处理后A549和A375p细胞中iASPP的表达有不同程度的下降。

The three types of reconstructed embryos(reconstructed embryo of goat-rabbit, goat-bovine and goat-goat) were produced respectively by nuclear transfer using goat ear firbroblast cells as donors and rabbit, bovine and goat ooctyes as recipients, and the factors of cytoplast environments influencing cloned embryo development in vitro, cytoplast effect in nuclear transfer, the methods of transferring cloned embryo and the effects of pregnancy factor on implantation of cloned embryo were studied in this experiment, in order to supply basis for solving the difficulty of implantation of inter-species cloned embryo in recipient and improving the efficiency of cloning.

本试验以波尔山羊耳成纤维细胞为供体，分别以牛、羊、兔的卵母细胞为受体进行体细胞核移植，构建了山羊-兔、山羊-牛和山羊-山羊三种重构胚。研究了不同的胞质环境对重构胚体外发育的影响与核移植中的胞质效应，并对异种克隆胚胎采用了去透明带移植和配种后再移植的方法，探讨移植方法和妊娠因子对异种克隆胚胎在受体动物体内发育的影响，旨在为解决异种克隆胚移植不易着床发育的难题和为提高动物克隆效率提供理论依据。

Rat glioma cell C6 was infected by the deficiency recombinant retrovirus (named C6TK). The sensitivity of C6TK cells to GCV or ACV increased 450 or 10 times than that of C6 cells respectively. Rat C6 glioma model was successfully built, and SD rats inoculated intracerebrally C6TK cells had normal gliomas. The average survival periods of the rats were about 15 days. However, after treated with ACV, the growth of C6TK tumors obviously regressed, and the survival periods extended to 57.8±8.1 days, especially in rats injected with mixed PA317TK and C6 cells or in situ PA317TK cells, which the tumors nearly disappeared after ACV administration and the survival periods extended to over 120 or 71.4±36.1 days respectively, P＜0.001.

结果：构建了带有HSV-tk基因的反转录病毒载体GINaTK，应用脂质体转移方法将GINaTK导入反转录病毒包装细胞PA317，成为产病毒细胞PA317TK，用带有HSV-tk基因的复制缺陷型反转录病毒感染C6细胞，命名为C6TK细胞，对GCV和ACV的敏感性分别高于亲本450倍和10倍；成功地建立了大鼠脑胶质瘤模型，并证实转染细胞C6TK的成瘤效应未改变，存活期约为15天；而经ACV治疗后，含有C6TK细胞的肿瘤生长明显被抑制，大鼠生存期延长为57.8±8.07天，尤其是采用PA317TK细胞混和治疗组和原位治疗组，肿瘤基本消失，大鼠生存期显著延长，混和治疗组存活120天以上，原位治疗组存活至71.4±36.1天。t检验，P值均小于0.001。

Results: GINaTK retroviral vector containing HSV-tk was constructed and transduced into PA317 retrovirus packaging cell by lipofectin (named PA317TK). Rat glioma cell C6 was infected by the deficiency recombinant retrovirus (named C6TK). The sensitivity of C6TK cells to GCV or ACV increased 450 or 10 times than that of C6 cells respectively. Rat C6 glioma model was successfully built, and SD rats inoculated intracerebrally C6TK cells had normal gliomas. The average survival periods of the rats were about 15 days. However, after treated with ACV, the growth of C6TK tumors obviously regressed, and the survival periods extended to 57.8±8.1 days, especially in rats injected with mixed PA317TK and C6 cells or in situ PA317TK cells, which the tumors nearly disappeared after ACV administration and the survival periods extended to over 120 or 71.4±36.1 days respectively, P ＜0.001.

结果：构建了带有HSV-tk基因的反转录病毒载体GINaTK，应用脂质体转移方法将GINaTK导入反转录病毒包装细胞PA317，成为产病毒细胞PA317TK，用带有HSV-tk基因的复制缺陷型反转录病毒感染C6细胞，命名为C6TK细胞，对GCV和ACV的敏感性分别高于亲本450倍和10倍；成功地建立了大鼠脑胶质瘤模型，并证实转染细胞C6TK的成瘤效应未改变，存活期约为15天；而经ACV治疗后，含有C6TK细胞的肿瘤生长明显被抑制，大鼠生存期延长为57.8±8.07天，尤其是采用PA317TK细胞混和治疗组和原位治疗组，肿瘤基本消失，大鼠生存期显著延长，混和治疗组存活120天以上，原位治疗组存活至71.4±36.1天。t检验，P值均小于0.001。

Results:(1)NSCs form typical neurospheres under adequate concentration in vitro, which are immunoreactive to Vimentin. Typically and terminally differentiated mature neural cells could not be found without the stimulus of mitogen or only under NSCs self-regulation and self-induction;(2)NSCs derived from hippocampus maintain the character of stem cells much longer with better biological behavior; NSCs passed to the 2-3 passage are the best to graft since they have not differentiated;(3)NSCs cultured in vitro could self-regulate and differentiate into neurospheres and progenitors positively immunoreactive to specific antibodies representing neurons, astrocytes, oligodendrocytes and Schwann cells;(4)There are widespread synaptic contacts between various kinds of descendent clones and cells;(5)Neurospheres could be formed without the stimulus of mitogen when NSCs and OECs are cocultured. Many neurospheres and cells immunoreactive to Vimentin, GFAP, MAP2, 02, p75NGFR, GFAP, S-100, Synaptosis, Vimentin, Tau (Tau is only positive in cocultureof HNSCs+HOECs) could be found;(6)The supernatant fluid triturated from adult rat spinal cord stimulates NSCs to differentiate into neurons, but do not terminally differentiate;(7)Fibroblasts and O4 oligodendrocytes are not supported to grow under this culture medium.Part II: Isolation, culture and identification of rat and human olfactory ensheathing cellsOlfactory ensheathing cells/glials are the most powerful cells to enable the regeneration of axons in the central nervous system.

结果表明：①在适宜的浓度体外培养条件下，NSCs能形成典型的神经干细胞克隆球，Vimentin免疫荧光染色阳性，单靠丝裂原刺激或NSCs自我调节和分化诱导，不会产生典型的终末分化的成熟神经细胞；②海马源性的NSCs维持干细胞特性的时间更长，生物学特性更优；③传至第2～3代的NSCs尚未分化时移植最佳；④体外培养的NSCs能自我调控分化为神经元、星形胶质细胞、O2少突胶质细胞、雪旺氏细胞染色阳性克隆球和前体细胞；⑤各种子代克隆球和细胞存在广泛的突触联系；⑥NSCs与OECs联合培养时，不需丝裂原刺激即能形成克隆球，获得大量Vimentin、GFAP、MAP2、O2、p75NGFR、GFAP、S-100、Synaptosis、Vimentin、Tau(Tau只有人HNSCs+HOECs联合培养时出现阳染)染色阳性的克隆球和细胞；⑦脊髓研磨后的上清液刺激神经干细胞向神经元方向分化，但并不出现终末分化；⑧本研究培养条件不利于成纤维细胞、O4生长。

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。

With cellulose as the skeleton, gelatinized cassava starch solution and tungsten carbide were used as porogenic agent and inert densifier, respectively. The matrix prepared showed good sphericity and a logarithmic symmetrical distribution of particle size.

取得的主要结果如下：纤维素基复合层析基质的物理功能化：以再生纤维素为骨架，碳化钨粉末为增重剂，糊化木薯淀粉为制孔剂，采用&反相悬浮热再生法&和成球后去除淀粉方法，成功制备了纤维素/碳化钨复合微球，可用作层析介质的基质。

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 split between the two groups can hardly be papered over.

这两个团体间的分歧难以掩饰。

This approach not only encourages a greater number of responses, but minimizes the likelihood of stale groupthink.

这种做法不仅鼓励了更多的反应，而且减少跟风的可能性。