原细胞

Objective: To study osteogenic capability of rabbit bone marrow stromal cells and biological characteristical identification in vitro in order to explore the appropriate methods of osteoblast culture in vitro and to select an idea source of seed cells for bone tissue engineer.

目的：研究兔骨髓基质细胞在体外培养条件下的成骨能力及生物学特性，探讨简便易行的成骨细胞体外培养方法，为组织工程选择理想的种子细胞来源。方法：取新西兰白兔骨髓液经离心后得骨髓单个核细胞，以1× 106／ml的细胞浓度进行培养，经传代培养，选择条件培养液培养(1640完全培养液中加入地塞米松10-8Smol／l，β-甘油磷酸钠10mmol／l，VitC 50μg／ml)作为骨髓基质细胞增殖和成骨的调节因子，通过倒置显微镜、HE染色、扫描电镜、碱磷酸酶(alkaline phosphatase，ALP)测定、Ⅰ型胶原免疫组化染色和四环素荧光钙染色等手段对获得的细胞进行生物学特性研究。

ResultsMacroscopic examination showed no excrescence,thrombus formation,arm fractures and corrosion.The devices were covered with collagen fibrosis and discrete endocardial cells,apparent inflammatory infiltration in the devices and around the devices 1 month after implantation.The implants were nearly endothelialized,while the inflammatory reaction relieved gradually,with myocardial cells ingrowth at the edges of the device 3 months after implantation.The devices were completely covered with endocardium and fibrous tissue.Moreover,endothelial cells could be found on the smooth microscrew adaptor.The inflammatory reaction diminished with a few chronic inflammatory cells existing.Neovascularization and lymphatic vessels ingrowth could be observed 6 months after implantation.

结果所有封堵装置表面均没有发现赘生物、血栓形成、支架发生断裂及被腐蚀；术后1个月，封堵装置表面被胶原纤维和散在内皮细胞所覆盖，大量炎症细胞浸润，封堵装置边缘有小灶性炎症细胞浸润；术后3个月，封堵装置表面几乎被内皮细胞所覆盖，炎症细胞较1个月时明显减少，封堵装置内见纤维化，封堵装置边缘心肌细胞浸入；术后6个月，封堵装置表面完全被心内膜和纤维组织所覆盖，伞尖表面光滑并有内皮细胞上爬，炎症反应明显消散，但仍有少量慢性炎症细胞存在，装置内有新生的血管、淋巴管长入。

DMBQ, ferulic acid and resorcin were effective chemical signal substances in the process of seed germination and haustorium formation of Cistanche deserticola. 4.The embryo is consist of 12 cells, the endosperm cell degraded so as to provide nutrition for the embryo development .As a result, germ tube differentiated from the embryo and came out through micropylar end, the small cell at the tip of the germ tube held the capacity of cleavage and the other cells grew big ,so the germ tube elongated .The anterior extremity of germ tube swelled to form haustorium under chemical substances treatment, the haustorium cell stained ,especially its portrait because of the lignification of the cells.

肉苁蓉种子的胚是由12个细胞紧密排列形成的圆球状胚，球形原胚分裂增殖的同时，胚乳细胞分解，为胚细胞的增殖提供营养，最终胚上分化出的芽管由珠孔端冒出，芽管前端细胞较小，具有旺盛的分裂能力，后部细胞体积增大，芽管逐渐伸长，在化学物质的诱导下，芽管由中部至前端细胞膨大分化出初生吸器，初生吸器呈帽子状，而这些细胞比其周围的细胞着色深，吸器纵向局部区域的番红染色加深，细胞高度木质化。

We introduced improved primary mixed glial culture and different-attachment method to isolate and purify the OPCs, the cells were proliferated in serum-free medium, flow cytometry and immunohischemistry methods were employed to estimate the purity of cultured OPCs. Their abilities of differentiation and expression of trophic factors were identified by RT-PCR and immunostaining. Several methods including TUNEL and MTT were adopted to estimate the protective effects of conditioned culture medium from oligodendrocyte lineage cells on the primary cultured cerebellar granule neurons. Intravitreal transplant of OPCs, combined with retrograde fluorescent labeling the superior colliculus and intraorbital optic nerve transection, were used to investigate the protective effects of OPCs on the axotomized RGCs in vivo. Intravitreal transplantof OPCs or NSCs on the newborn rats, and retinal transplant of OPCs on the young rats were performed, to observe the myelin formation in the retina at different stages after cellular transplantation. Optic nerve transection was carried out on some rats with myelinated retinae, to study the influence of myelination on the injuried RGCs.

为此，本研究采用改良的胶质细胞混合培养与差速贴壁方法获得大鼠OPCs，使用无血清培养基进行扩增、培养，用免疫组织化学和流式细胞技术对培养细胞的纯度进行鉴定，对少突胶质系细胞表达部分营养因子的情况进行检测；采用TUNEL、MTT等方法对少突胶质系细胞条件培养基对原代培养小脑颗粒神经元的保护作用进行检测；将OPCs移植入成年SD大鼠玻璃体内，利用上丘逆行荧光标记技术，观察眼内移植的OPCs对眶内视神经切断时的视网膜神经节细胞的保护作用及其持续时间；将OPCs或NSCs移植入新生和幼年SD大鼠玻璃体或视网膜内，观察不同时期视网膜内髓鞘形成与分布特点，分析髓鞘的超微结构，并观察眼内髓鞘形成对损伤神经节细胞的保护作用。

Result 1 Magnetic nanoparticles, magnetic nanoparticles modified with antisense oligodeoxynucleotide of human telomerase reverse transcriptase induced HL-60 tumor cells to apoptosis, we could see typical morphologic change of apoptosis cells: karyopyknosis, chromation"s condensing and aggregation in nuclear, forming crescent-shaped or annulus structures to lean on edge of cell nucleus"s membrane and posing apoptosis body by Atomic Force Microscope, Fluorescence microscope, transmission electron Microscope 2 There was a significant difference compared with control group(p.01), inhibition ratio had significant positive correlation with medication dosage and time ;during 0.8-8μM dosage amplitude, inhibition ratio accrescenced by dosages increasing. However, the inhibition ratio would decrease when dosage over 8-80μM.

结果 1 磁性纳米粒子、修饰有端粒酶反义寡核苷酸的磁性纳米粒子诱导HL-60细胞发生凋亡，原子力显微镜、光学显微镜、荧光显微镜和透射电镜下均观察到HL-60细胞呈现典型的凋亡细胞的形态变化：细胞核固缩，核内染色质浓缩、凝聚、形成新月形或环状结构紧靠在细胞核膜边缘，并形成凋亡小体。2 磁性纳米粒子、修饰有端粒酶反义寡聚脱氧核苷酸的磁性纳米粒子对HL-60肿瘤细胞的生长和增殖有明显的抑制作用，与对照组相比有显著性差异(p＜0.01)，在剂量为0.8-8μmol／L范围内，抑制率随剂量的增加而增加，当剂量超过8μmol／L时，抑制率反而下降；3 磁性纳米粒子、修饰有端粒酶反义寡聚脱氧核苷酸的磁性纳米粒子可增强p53基因的表达活性，引起DNA降解损伤，反向调节细胞周期活动，促使细胞从G0期进入G1期，抑制肿瘤细胞的生长。4 修饰有端粒酶反义寡聚脱氧核苷酸的量子点能通过内吞作用进入HL-60肿瘤细胞的细胞核，可以在细胞内进行定位和促进HL-60肿瘤细胞的凋亡。

That GER cells which are likely hair cell progenitors could be cultured in vitro and generate stereociliary bundle-like structure when forced to express Hath1 suggests that the misexpression of Hath1 probably can induce the predifferentiation of pure hair cell progenitors into hair cells in vitro.

GER细胞作为毛细胞前体细胞可以实现体外原代培养，在Hath1基因重表达情况下，部分细胞的表面可见不成熟的纤毛样结构，提示Hath1基因也许可以诱导离体培养下的纯的毛细胞前体细胞向毛细胞的初步分化。

Both Fas and NO can lead chondrocyte apoptosis respectively and cause articular cartilage destruction. IGF-Ⅰ, TGF-β, bFGF, BMP and other growth factors are polypeptide agents that can influence cell activity by signal convection. They can accelerate chondrocyte proliferation and proteoglycan synthesis, play the local regulation action on formation and plerosis of bone and cartilage tissue by autocrine or paracrine. They have the ability to induce cartilage formation. Some investigations showed that growth factors can influence chondrocyte metabolism, synthesis of specific Ⅱ type collagen and proteoglycan by co-operation and inhibition. 1. 3 Situation of OA therapeutics The therapeutic methods of OA mainly comprised non-drug treatment, drug treatment, operation treatment, tissue and genetic engineering, et al. Drug treatment is the chief method among them.

若其活性发生改变，则将导致关节软骨基质成分的丢失和进行性破坏；软骨细胞凋亡与OA的发病密切相关，Fas与NO可各自独立介导软骨细胞凋亡，造成关节软骨破坏；IGF-Ⅰ、TGF-β、bFGF、BMP等生长因子是一组通过细胞间信号传递影响细胞活动的多肽因子，具有促进细胞生长、增殖与合成等作用，可通过自分泌或旁分泌方式对骨和软骨的形成和修复起局部调节作用，可促进软骨细胞增殖、分化与蛋白多糖的合成，具有较强的诱导软骨形成的能力，研究表明多种生长因子相互促进、相互抑制，以协同或拮抗方式影响软骨细胞代谢，影响软骨细胞特异性Ⅱ型胶原和蛋白多糖的合成分泌。

Primary culture of goat mammary gland cells could be derived by outgrowth of migrating cells from fragment of tissue. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Complex cultures treated by 0.25% trypsin in Hanks' for 5~7min at 37℃, the dispersed cells were mainly fibroblasts,then the rest treated by 0.15% trypsin-0.02% EDTA in Hanks'for 5~8min at 37℃, the majority cells harvested were mammary epithelial cells.

用组织块培养法可获得良好的山羊乳腺细胞原代培养物；培养的山羊乳腺成纤维细胞比上皮细胞对胰蛋白酶更敏感，据此可在传代过程中将二者分离纯化，获得纯细胞系；混合培养物先用0.25%胰蛋白酶在37℃消化5~7min 所收集的细胞主要为成纤维细胞；再加0.15%胰蛋白酶-0.02鞹A 消化液在37℃继续消化5~6min 所回收的细胞绝大多数为上皮细胞，经过2~3 代，即可得到纯化的乳腺上皮细胞系。

Primary culture of goat mammary gland cells could be derived by outgrowth of migrating cells from fragment of tissue. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Complex cultures treated by 0.25% trypsin in Hanks' for 5~7min at 37℃, the dispersed cells were mainly fibroblasts,then the rest treated by 0.15% trypsin-0.02% EDTA in Hanks'for 5~8min at 37℃, the majority cells harvested were mammary epithelial cells.

用组织块培养法可获得良好的山羊乳腺细胞原代培养物；培养的山羊乳腺成纤维细胞比上皮细胞对胰蛋白酶更敏感，据此可在传代过程中将二者分离纯化，获得纯细胞系；混合培养物先用0.25%胰蛋白酶在37℃消化5~7min 所收集的细胞主要为成纤维细胞；再加0.15%胰蛋白酶-0.02%EDTA 消化液在37℃继续消化5~6min 所回收的细胞绝大多数为上皮细胞，经过2~3 代，即可得到纯化的乳腺上皮细胞系。

Immunohistochemical identification of cultured conjunctival cellsWhen the second cultured generation of conjunctival cells close to confluence, the glass coverslips were removed in situ fixation by methanol and DAB color immunohistochemical staining by CK13 monoclonal antibody in time.3. growth curve of conjunctival cellsThe 1st,3rd,5th passage cells were selectecd randomly and subcultured at the density of 5×10~4/ml,after culturing for another 1-9 days, were counted and compared the cell number every day.

采用消化后组织块法培养结膜原代细胞，并进行传代。2、培养结膜细胞的免疫组化鉴定把第2代的结膜细胞接种于置在培养皿中的盖玻片上，当细胞接近融合时，及时取出作原位甲醇固定，CK13单抗、DAB显色免疫组化染色。3、结膜细胞生长曲线的测定随机选取第1、3、5传代细胞，以5×10~4／ml密度传代、分别培养1-9天，每天分别细胞计数并进行分析比较，重复3次。

Lugalbanda was a god and shepherd king of Uruk where he was worshipped for over a thousand years.

Lugalbanda 是神和被崇拜了一千年多 Uruk古埃及喜克索王朝国王。

I am coming just now,' and went on perfuming himself with Hunut, then he came and sat.

我来只是现在，'歼灭战perfuming自己与胡努特，那麼，他来到和SAT 。