细胞

To investigate whether the expression of cdc2 and cyclin B1 in spermatogenic cells during spermatogenesis is actually a temperature dependent event, in situ hybridization, Western blotting and immunohistochemistry analysis were used to study the expression of cdc2 and cyclin B1 in normal and cryptorchid testis. Results showed that heat would differentially hurt male germ cells in different developmental stages during spermatogenesis, especially the pachytene primary spermatocytes. Most of spermatogonia in contralateral cryptorchid testis were not harmed fatally by heat as yet, indicating that spermatogonia could resist to beat to a certain extent. In this case spermatogonia could develop to pachytene/diplotene primary spermatocytes, but they could not acquire the ability to complete the transition from mitosis to meiosis, and then appeared to go through apoptosis. Therefore, we could not find the descendants of meiosis: secondary spermatocytes and round spermatids, elongated spermatids and spermatozoon. The abdominal temperature had no significant influence on the transcription of cdc2 and cyclin B1 in the spermatogonia and pachytene/diplotene primary spermatocytes. In normal rabbit testis, cyclin B1 increased in the pachytene/diplotene primary spermatocytes before meiosis and reached its peak in the spermatids.

为了解精子正常发生过程中cdc2和cyclin B1表达的低温依赖性，我们利用原位杂交和免疫组化等方法，研究了正常和隐睾精子发生过程中cdc2和cyclin B1的转录和翻译调控活动，结果表明：（1）热对各阶段的雄性生殖细胞都有损害，粗线期的初级精母细胞尤为敏感，实验性隐睾内的精原细胞尚未完全受到"致命"影响，说明精原细胞对热有一定的耐受性，但即使成为粗线期／双线期初级精母细胞，却未能获得由有丝分裂过渡到减数分裂的能力，呈现不同程度的凋亡，所以在整个切片中找不到源自减数分裂的产物----次级精母细胞、圆形精子细胞，更谈不上长形精子细胞和精子的形成；（2）腹腔高温未明显地影响隐睾精原细胞和粗线期/双线期初级精母细胞中cyclinB1和cdc2的转录，说明高温并不是通过影响cyclin B1和cdc2的转录活动而导致生精过程阻断的；（3）正常兔睾丸组织中，〓在精原细胞和粗线期／双线期精母细胞中均有表达：cyclin B1蛋白在减数分裂前期的粗线期／双线期初级精母细胞中的表达量增加，于变态末期的精子细胞中达高峰。

The results of character evolution history reconstruction shows the SC in PT and AD clade might be a homoplasy character; also, the SC in PT clade might have multiple origins. SC in Pteridaceae has some special characteristics:(1) the cell long axes usually run parallel to the lateral veins;(2) Most of Pteris and Adianum species have VSC only in lower epidermis;(3) Species have ISC also have VSC.(4) Presence of spicular cells is highly correlated with the long veinal epidermal cells. Species have type III silica deposition type usually have longer and narrower veinal epidermal cell compared with ordinary epidermal cells; Species have type IV silica deposition type also have long epidermal cells, but not significantly different from ordinary epidermal cells.

本实验中亦发现凤尾蕨科之矽异形细胞具有几项特色：(1)矽异形细胞之长轴多与叶片侧脉方向平行(2)发现多数铁线蕨属以及凤尾蕨属植物仅下表皮具有脉上矽异形细胞；(3)具有脉间矽异形细胞的种类会同时具有脉上矽异形细胞；(4)长条形的侧脉上表皮细胞与矽异形细胞具有相关性，仅具脉上矽异形细胞的分类群之脉上表皮细胞均较一般表皮细胞细长；具有脉间矽异形细胞之物种之脉上表皮细胞亦为长条形但形态与一般表皮细胞常无明显形态差异。

Decidual tissue is a very complex and highly concordant tiny environment between mother and embryo during pregnancy, which is constituted of the variety cells such as extravi11ous trophoblast,endometrium cells, bone marrow cells, decidua l microphage,T cells and a few B cells and so on.

正常蜕膜组织是母-胎界面间一个非常复杂而又高度协调的微环境，它由多种细胞如侵袭至蜕膜的绒毛外滋养细胞(extravi11ous trophoblast，EVT)、子宫内膜源性细胞(如上皮细胞和血管内皮细胞)、骨髓源性细胞(如大颗粒淋巴细胞、蜕膜巨噬细胞、T细胞和少许B细胞)构成，这些细胞以及由这些细胞、母体和胎儿所分泌到蜕膜的细胞因子共同形成了母-胎间信息交流的平台，并对胚胎滋养细胞"有控性"侵袭、子宫内膜的免疫容受性、蜕膜化和胎盘的形成进行精细的调控。

The neurohypophysis is only found in the dorsal region between the RPD and the PPD. The growth hormone cells do not differentiate in the PPD of the adenohypophysis, occupying 2/3 of the PPD until the larva is 5 days old. The other hormone-producing cells in the RPD and in the PT do not differetiate. In the 10-day old larva, the hormone-producing cells except the GH cells in the adenohypophysis do not differentiate. In 15-day old larva, the adrenocorticotropic hormone cells differentiate in the RPD and the other hormone-producing cells in the adenohypophysis do not differentiate. In the 30-day old larva, the all kinds of hormone-producing cells are formed in the adenlhypophysis.

神经垂体只见于RPD和PPD交界的背上方。5日龄仔鱼腺垂体内GH细胞已分化，占PPD的2/3，其他激素分泌细胞未见分化。10日龄仔鱼脑垂体内除GH细胞外，未见其他种类激素分泌细胞分化。15日龄仔鱼脑垂体RPD内ACTH细胞已分化，PPD和PI内除GH细胞外，其他种类激素分泌细胞均未分化。30日龄仔鱼RPD、PPD和PI内各种激素分泌细胞都已分化，共有6种激素分泌细胞，分别是促肾上腺皮质激素分泌细胞，催乳激素分泌细胞，生长激素分泌细胞、促甲状腺激素分泌细胞、促性腺激素分泌细胞和促黑激素分泌细胞。

To investigate the effect of p38MAPK signal conducting pathway on livercancinoma cell's malignant phenotype induced by VEGF,we take theexperiment with cell growth test,scanning microscope and laser scanningconfocal microscope so that observe effect of p38MAPK signal conductingpathway on liver cancinoma cell growth,pseudopodium formation andframework of cytoskeleton induced by VEGF.results indicate that the cellbecame ellipse and there were more and thick pseudopodium in the cell'ssurface after being treated by VEGF,and destroyed framework ofcytoskeleton,which can be blocked by pretreated with a special inhibitor ofp38MAPK SB203580 so that VEGF promote metastasis by enhancing livercell migration and movement via p38MAPK signal conducting pathway,butVEGF promote cell growth without p38MAPK signal conducting pathway.

为进一步探讨VEGF通过p38信号传导通路诱导肝癌细胞转移时，对肝癌细胞恶性表型的影响，采用细胞增殖实验、扫描电子显微镜、激光扫描共聚焦显微镜观察VEGF对肝癌细胞增殖作用、细胞伪足形成、细胞骨架微丝结构的影响以及p38MAPK信号通路调控作用。结果显示，VEGF能够不通过p38信号通路促进肝癌细胞增殖；VEGF诱导肝癌细胞丝状伪足增多、增长，使细胞骨架微丝结构破坏、甚至消失。阻断p38MAPK信号通路，可以抑制VEGF诱导的肝癌细胞伪足形成，细胞骨架丝状结构呈束状，排列较规整。上述结果表明，VEGF可以通过p38信号传导通路诱导肝癌细胞伪足增多、增长，促使细胞骨架微丝结构破坏，使肝癌细胞迁移、运动能力增加，促进肿瘤转移。VEGF并不通过p38信号通路诱导肝癌细胞增殖作用。

Get high purity DCs by Cultured plastic-adherent monocytes isolated from healthy human peripheral blood with GM-CSF and IL-4 for 7 days. To observe the morphology of DCs by inverted phase contrast microscope ,electron microscope and laser confocal microscope. Analyse phenotype of DCs with flow cytometry. Investigate the endocytosis ability of DCs as a group by Horseradish peroxidase endocytosis assay. To appraise allogeneic mixed lymphocytes reaction of DCs by MTT reduction assay. Analyse the levels of IL-12 and TNF in liquids of cultured medium by ELISA and MTT reduction assay respectively. Soluble antigens of HCCs was obtained by 3 freeze-and-thaw cycles. Biological characteristics of HC soluble antigens pulsed DCs were monitored by flow cytometry. According to MTT reduction assay estimated the cell proliferation of self lymphocytes activated by HC antigens pulsed DCs. Get high purity BCG HSP 70 protein by SDS-PAGE electrophoresis and determined its biological activity with ELISA. Analyse phenotype of antigen pulsed DCs primed by BCG HSP70 with flow cytometry. By MTT reduction assay estimated the cell proliferation of self lymphocytes and the MLR of DC based vaccine. Analyse expression of HLA-DR molecule on surface of HCC lines. The IFN-γ mRNA in lymphocytes after actived by DC vaccine and the Fas-L expression on DC and DC vaccine primed lymphocytes were detected by in situ hybridization and flow cytometry respectively. Specific cytotoxity lysis of T lymphocytes and nonspecific inhibition of liquids in culture medium against HCC lines were also tested. Detect expression of hAFP on four HCC lines with Cell-ELISA. Induce apoptosis of HCCs with actinomycin-D. Interaction of DCs and apoptotic cells was observed under transmission electron microscope. Growth inhibition test of DC against HCC lines was also performed. Establish the nude mouse model bearing human HC xenografts and indentify the characteristic of tumour by histochemistry and immunohistochemistry techniques. Prevent and treat transplanted human HC on nude mouse with Freezing and anabiotic HC specific lymphocytes.

用GM-CSF和IL-4从健康人外周血诱导DC；分别用倒置相差显微镜、电子显微镜及激光共聚焦显微镜观察DC形态；流式细胞术检测DC表型；HRP吞噬实验测定DC的群体内吞能力；MTT法检测同种异体混合淋巴细胞反应；ELISA法和MTT法分别测定DC培养上清液中IL-12和TNF水平；冻融法制备肝癌细胞可溶性抗原；流式细胞术检测负载肝癌可溶性抗原后DC的生物学特性；MTT法检测DC负载肝癌抗原后对自身淋巴细胞增殖的影响；SDS-PAGE制备电泳纯化BCG HSP70并鉴定纯度，ELISA测定活性；流式细胞术检测负载抗原DC经BCGHSP 70活化后的表型；MTT法检测肝癌DC疫苗对自身淋巴细胞增殖的影响和混合淋巴细胞反应；流式细胞术检测肝癌细胞表面HLA-DR表达；MTT法检测肝癌DC疫苗对自身淋巴细胞的活化；原位杂交法检测肝癌DC疫苗活化后的淋巴细胞IFN-γmRNA表达；流式细胞术检测DC和肝癌DC疫苗活化后淋巴细胞表面Fas-L；MTT法分别检测肝癌DC疫苗活化的淋巴细胞和其培养上清对肝癌细胞的特异性杀伤和非特异性抑制作用；Cell-ELISA检测人肝癌细胞hAFP表达；MTT法检测负载AFP表位肽和凋亡肝癌细胞DC对自身淋巴细胞增殖的影响；ELISA法和MTT法分别测定活化后淋巴细胞培养上清中TNF和IL-12水平；肝癌细胞凋亡的诱导和检测；DC吞噬凋亡肝癌细胞后的电子显微镜观察；DC对肝癌细胞的生长抑制试验；人肝癌裸鼠皮下移植瘤动物模型的建立及其组织学和免疫组织化学鉴定；DC及肝癌特异性淋巴细胞预防和治疗人肝癌裸鼠皮下移植瘤；冻存和复苏后的肝癌特异性淋巴细胞预防和治疗人肝癌裸鼠皮下移植瘤。

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。

MTT assay FAK signaling pathway inhibitor genistein on human corneal epithelial cell cytotoxicity;RT-PCR detection of human corneal epithelial cells adhesion to fungus at different times,extracellular matrix protein including laminin,fibronectin,FN glass,Ⅳcollagen,transmembrane protein integrinαⅤ,integrinβ1(ITGβ1),as well as the FAK signaling pathway FAK1,FAK2 and Paxillin gene expression;Western blot detection of the signal transduction pathway adhesion-associated protein ITGβ1,FAK and PAX expression and the inhibition of genistein. Immunocytochemical method was used to observe the LN,FN and FAK expression in human corneal epithelial cells during interaction with the fungues;Laser scanning confocal microscope had a cell positioning on FN,FAK and PAX,observed the changing of the human corneal epithelial cytoskeleton after stimulated by fungues;Quantitatived by flow cytometry to detect of human corneal epithelial cells with PAX at ITGβ1 fungal expression after adhesion;Optical microscopy quantitied the fungues and human corneal epithelial cell adhesion and recorded to determination the integral optical density afrer adhesion;Scanning and transmitted electron microscope observed the changing of cell ultrastructure after fungues and human corneal epithelial cell adhesion.

第一部分真菌激活人角膜上皮细胞FAK信号转导通路的体外实验研究将三种常见致病真菌(白色念珠菌、烟曲霉菌和茄病镰刀菌)分别与人角膜上皮细胞共孵育，MTT法检测FAK信号通路抑制剂染料木黄酮的对人角膜上皮细胞的细胞毒性作用；RT-PCR检测真菌黏附人角膜上皮细胞后不同时间细胞外基质层连蛋白、纤连蛋白、玻连蛋白、Ⅳ型胶原、跨膜蛋白整合素αV、整合素β1(ITGβ1)，以及FAK信号通路中FAK1、FAK2和桩蛋白基因的表达情况；Western blot的方法检测黏附信号转导途径相关蛋白ITGβ1、FAK和PAX的表达，以及染料木黄酮对真菌刺激人角膜上皮细胞FAK信息通路活化的抑制作用；免疫细胞化学方法观察人角膜上皮细胞与真菌相互作用过程中LN、FN和FAK的表达；激光共聚焦显微镜对FN、FAK和PAX进行了细胞定位，并观察真菌刺激后人角膜上皮细胞骨架的变化；流式细胞仪定量检测人角膜上皮细胞ITGβ1与PAX在真菌黏附后表达的改变；光学显微镜观察真菌与人角膜上皮细胞黏附数量，记录并测定了黏附后积分光密度值OD扫描及投射电镜观察了真菌与人角膜上皮细胞黏附后，细胞超微结构的改变。

The apoptosis of 80 mg/kg ﹒ d group was mainly located in spermatogenous cell and first spermatocyte; the apoptosis of 120 mg/kg ﹒ d group was mainly located in spermatogenous cell , first spermatocyte and spermatocyte of the second order; the apoptosis of 240 mg/kg ﹒ d group was mainly located in spermatogenous cell and first spermatocytes, and seemingly, the apoptosis located in first spermatocyte was dominant; whereas the apoptosis of 480 mg/kg ﹒ d group was located in all levels spermatogenic cells.

结果表明：各试验组小鼠生精细胞凋亡指数随着浓度的升高有增加的趋势，而且与对照组差异显著（ P ＜0.05或 P ＜0.01）。80 mg/kg · d 组凋亡细胞主要定位于精原细胞和初级精母细胞；120 mg/kg · d 组凋亡细胞主要定位于精原细胞、初级精母细胞和次级精母细胞；240 mg/kg · d 组凋亡细胞主要定位于精原细胞、初级精母细胞，以初级精母细胞为最多；而480 mg/kg · d 组凋亡细胞定位于各级生精细胞。

These results indiacated that tranferring macrophages combinedwith cytokines could be a new adoptive immunotherapy protocol foradvanced cancer,and rTNF used in vivo locally could induce andactivate macrophages to kill tumor cells.Monocytes when activatedunderwent a series of phenotypic and functional changes includingthe expression of IL-2R which may provide an important immunoregu-latory pathway.The presence of lectin-like molecules on thesurface of monocytes and tumor cells may bring theeffector/target cells together,thus facilitating the inductionof apoptosis in target cells by triggering the production ofcytolytic factors and the modification of target of targer cell surface anti-gens.(such as HLA-DR).

综合以上结果可以得出以下结论：转输巨噬细胞并结合细胞因子的联合应用是肿瘤继承性免疫中的又一新的、有潜力的方法；抗肿瘤细胞因子rTNF可通过激活巨噬细胞在体内抑制对其不敏感的肿瘤细胞的生长；人单核细胞在一定条件下激活后，可表达IL-2R，进而一方面增强其自身对IL-2的敏感性，另一方面在体内也具有调节T细胞功能的作用；单核细胞和肿瘤细胞上存在的凝集素类受体可促使两种细胞之间在凝集素介导下相互接近，并诱发单核细胞产生细胞毒因子，以及通过调节靶细胞上表面抗原的表达，促进诱导靶细胞的细胞凋亡。

I didn't watch TV last night, because it .

昨晚我没有看电视，因为电视机坏了。

Since this year, in a lot of villages of Beijing, TV of elevator liquid crystal was removed.

今年以来，在北京的很多小区里，电梯液晶电视被撤了下来。