细胞化学

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。

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。

Thunberg Fritillary Bulb is one of the most popular Traditional Chinese herbs. Some trials in vivo and animals indicated that Peimine and Peiminine, extracted from Thunbery Fritillary Bulb, have the function of reversing MDR of two different kinds of mechanisms. They can improve the cytotoxic of ADR to cancer cells by 5～10 folds under concentration of 10% cancer cell growth inhibitory rate. Peimine and Peiminine not only feather typical character of modifier of MDR, but also differ from other moldifiers such as Calcium channel blocker, Cyclosporines in chemical structure, they belong to cevine groups alkaloid, a subgroup of C-Nor-D-homosteroid alkaloid.

浙贝母是临床常用中药，体外及动物试验证明，浙贝母中的有效活性成分贝母甲素和贝母乙素具有逆转肿瘤细胞MDR的作用，而且能逆转两种不同机制的多药耐药肿瘤细胞的耐药性，在无明显细胞毒剂量下，可使阿霉素对耐药肿瘤细胞的杀伤活性提高5-10倍，不但具备明确的多药耐药逆转剂的特征，而且在化学结构上属异甾类生物碱中的瑟文类生物碱，完全不同于钙离子拮抗剂、免疫抑制剂和其它现有的耐药逆转剂，具有作用靶点宽、化学结构独特的特点。

Horny layer is the outmost layer of epidermis, its thickness is decided by different parts of human body, it's about 0.02mm at the thinnest parts, and 0.5mm at the thickest. It is made up of 5-10 layers of enucleated cornified cells that contains keratin and horny fat, which has the capability of resisting weak acids and weak bases , they array closely and become a natural barrier to moisture content and some chemical substances, so as to prevent the water logging of bodily fluids and endosmose of chemical substances.

角质层是表皮的最外层，其厚度依身体部位的不同而定，最薄处约0.02mm，最厚处约0.5mm由5-10层含有角蛋白和角质脂肪的无核角化细胞组成，角质层细胞有抵抗弱酸弱碱的能力，角质层细胞排列紧密，对水分及一些化学物质有天然屏障作用，因而可以阻止体内液体的水渗和化学物质的内渗。

Results are as followed:1 Exposure of HELF cells to BP caused c-Jun activation,and increased the activity of MAPK,PI-3K,p53 and cyclin D1 pathway.2 BP-induced c-Jun activation was inhibited by dominant negative mutants of extracellular signal-regulated protein kinase or c-Jun NH_2-terminal kinase,but not by p38,impling that JNK and ERK pathways medicate c-Jun activation induced by BP.3 Overexpression of dominant-negative mutants PI-3K and Akt potently blocked phosphorylations of c-Jun and ERK,but not JNK in response to BP,suggesting that PI-3K/Akt pathway positively regulates BP-induced c-Jun activation through ERK.4 Inhibition of p53 by its chemical or molecular inhibitor markedly increased the phosphorylation levels of c-Jun,Akt and ERK upon BP stimulation,indicating that p53 negatively medicates BP-induced c-Jun activation through PI-3K/Akt/ERK pathway.5 The cell lines expressed TAM67 exhibits no significant affecting normal cell growth properties.6 TAM67 was able to significantly block G_1-S transition and subsequent cell proliferation,suggesting that c-Jun is essential for cell cycle alternations elicited by BP.7 Overexpression of TAM67 impaired BP-induced cyclin D1 activation,decreasing expression of E2F1 and pRb,indicating that c-Jun participates in the modulation of BP-induced activation of cyclin D1/pRb/E2F1 pathway.8 Stably expression of TAM67 led to the increases in the expression levels of p53 and p21,elevating phosphorylation level of p53,clearly indicating that c-Jun regulates p53/p21 pathway activation induced by BRCollectively,PI3K/Akt/ERK pathway mediated BP-induced c-Jun activation through p53-dependent mechanism.

结果显示：1BP刺激细胞可促进c-Jun活化，并伴随着MAPK、PI-3K、p53和cyclinD1通路各组成成分的活性增强。2利用MAPK通路的显性失活突变体分别阻断细胞外信号调节激酶和c-Jun氨基末端激酶活性，均可明显抑制BP诱导的c-Jun活化，但阻断p38活性对BP引起的c-Jun活化无明显影响，提示JNK和ERK通路参与调控BP诱导的c-Jun活化。3过表达PI-3K和Akt的显性失活突变体也可显著抑制BP诱导的c-Jun活化，并降低磷酸化ERK的表达水平，但对磷酸化JNK的表达水平无明显影响，说明PI-3K/Akt通路通过ERK正性调控了BP诱导的c-Jun活化。4p53的化学/分子抑制剂能使BP作用的细胞内c-Jun活性明显增加，并同时诱导Akt和ERK的磷酸化水平的升高，表明p53可通过PI-3K/Akt/ERK通路对BP诱导的c-Jun活化进行负性调控。5随后观察转染细胞的生长情况，发现TAM67对细胞正常生长和形态无明显影响。6稳定表达TAM67可有效抑制BP诱导的S期细胞数的增加，提示c-Jun在BP致细胞周期改变的过程中发挥了重要作用。7TAM67过表达能够抑制BP诱导的cyclin D1活化，降低磷酸化Rb以及E2F1蛋白表达水平，表明c-Jun参与调控BP诱导的cyclin D1/Rb/E2F1通路的活化。8过表达TAM67可使BP刺激的细胞中p53、p21总蛋白以及p53磷酸化的表达水平明显升高，可见c-Jun也参与调控BP诱导的p53/p21通路活化。

After HRP application to the rostral area (n=10), quite a number of labelled cells were distributed at the ventrolateral part of the nucleus of trapezoid body, the lateroventral periolivary nucleus and the retrotrapezoid nucleus. After HRP application to the caudal area (n=10), HRP labelled cells were mainly located at the lateral reticular nucleus and the ventral part of at the lateral paragigantocellular nucleus LPGi. After HRP application to the intermediate area (n=6), only 8HRP labelled cells were observed at the in 6rats. After HRP application to the control area (n=4), only 6HRP labelled cells were found at the rostral ventrolateral reticular nucleus.

大鼠延髓腹外侧表面的头端化学感受区表面敷贴辣根过氧化物酶后，在延髓斜方体核、外周橄榄腹外侧核和斜方体后核均有HRP标记细胞存在；于尾端化学感受区表面敷贴HRP后，在延髓的巨细胞旁外侧核、外侧网状核和舌下神经核有HRP标记细胞存在；于中间区表面敷贴HRP后，在6只大鼠的延髓内部区域仅观察到8个HRP标记细胞；于对照区敷贴HRP后，在4只大鼠的延髓内部区域仅观察到6个HRP标记细胞，散在地分布在头端腹外侧网状核。

Fig.1 SHEE cultured on coverslide, the living cells were growing in single layer with rich cytoplasm, the nuclei were uniform in size with a nucleolus ph ×400 Fig.2 SHEE had a nucleus with ellipse shape, large nucleolus and the cytoplasm contained mitochondria and tonofibrilEM ×10 000 Fig.3 SHEE was spherical in shape, with pseudopods attached on petri dish and abundant villi on cell surface SEM ×5 000 Fig.4 Same as in Fig.3, cell attached on petri dish, appeared stellate or polygonal in shape, with abundant pseudopods and cytoplasmic processes. Protrusive nuclear region in central part of the cell had more micro-villi SEM ×5 000 Fig.5 Chromosomes of SHEE cells belonged to diploidy type Giemsa ×1 000 Fig.6 The SHEE cells of stained in dark brown by Ki67 immunohistochemistry were the proliferative cells Immunohistochemistry ×400 Fig.7 In SHEE cell culture, the nucleus stained red or pink by PI was dead cell, the green nucleus was living cell Fluorescent ×400 Fig.8 The cell labeled by TdT was apoptotic cell in which the chromatin of nucleus condensed in block, a pyknotic nucleus in the upper right conner was seen TdT labeled ×400

图1 SHEE培养在盖坡片上，活细胞单层生长，胞浆较丰富，细胞核大小一致，有核仁×400 图2 SHEE培养细胞细胞核椭圆形，核仁较大，胞浆有较丰富的线粒体和张力原纤维EM ×10 000 图3 SHEE细胞呈球状，有伪足贴壁，表面有密集微绒毛SEM ×5 000 图4 同上细胞贴壁，呈星状或多角形，有丰富伪足和胞浆突，核区隆起有较多微绒毛SEM ×5 000 图5 SHEE细胞染色体仍属二倍体Giemsa染色×1 000 图6 SHEE细胞Ki67免疫组织化学染棕黄色为增殖细胞×400 图7 SHEE培养细胞出现死细胞，胞核和胞浆PI染色呈红色或淡红色，蓝色细胞核为活细胞荧光显微镜×400 图8 细胞TdT标记阳性为凋亡细胞，染色质凝集呈块状，右上角有一固缩细胞核TdT标记×400

A 46-year-old man presented with a gradually growing nodule on the left dorsal foot, which had been present for over 10 years. In the beginning, the lesion was treated as a keloid. Because of poor response to treatment, biopsy was arranged. Sections showed a dermal tumor composed of numerous foamy cells, hyalinized wiry collagen, spindle cells and entrapped collagen. The foamy cells were positive for KP1(CD68) and negative for S-100 protein and HMB-45. The histological and immunohistochemical results were consistent with the diagnosis of lipidized fibrous histiocytoma.

一位46岁男性从十年前开始於左足背上出现一个逐渐长大之结节，一开始被当作蟹足肿给予治疗，因为对治疗的反应不好，故安排了切片检查，病理显示一个由大量泡沫状细胞，坚硬而透明之胶原纤维，梭状细胞，胶原纤维束所构成的真皮肿瘤，免疫组织化学染色显示KP1(CD68)阳性，S-100 protein与HMB45阴性，综合病理与免疫组织化学染色结果，诊断为脂肪性纤维组织细胞瘤。

objective:to investigate the expression and significance of rb in the occurrence, development and regression of infantile hemangiomas.methods:the expression of rb was examined in the proliferative stage and catagen of human hemangiomas and normal skin tissues by using immunohistochemical technique.immunohistochemical technique for factorⅷ-related antigen was used to prove that the cells which expressed rb were endothelium.image analysis system was applied to measure the expression level of rb at different stages of hemangiomas and in normal skin tissues.results:the expression of rb was significantly lower in proliferating hemangiomas than that in involuting hemangomas(p.05).conclusion:rb might play an important role in the regression of human hemangioma endothelial cells and anti-angiogenesis.

目的：探讨rb蛋白在血管瘤发生、发展及退化过程中的表达状况及其意义。方法：采用免疫组织化学方法检测人皮肤血管瘤增生期、退化期及正常皮肤组织中rb的表达水平，并结合第ⅷ因子相关抗原的免疫组织化学染色证实表达rb的细胞是血管内皮细胞。利用计算机图像分析技术测量不同时期血管瘤组织和正常皮肤组织rb表达的积分光密度和面积。结果：增生期血管瘤内皮细胞rb表达水平低于退化期，差异有显著性（p.05），退化期血管瘤内皮细胞rb表达水平与正常皮肤组织相比，差异有显著性（p.05）。结论：rb通过抑制血管瘤内皮细胞增殖和血管生成而在血管瘤的退化过程中起重要作用。

The results showed that (1) COX-2 mRNA and protein existed in the mature male rat testis. COX-2 was localized in spermatocytes by immunohistochemistry.(2) COX-2 also existed in adult man testis. COX-2 protein was positively stained in spermatocytes and Leydig cells.(3) 2 weeks after administration of rofecoxib, the level of testosterone in the whole testis reached its lower values, being only 50% of control values. However, testosterone level recovered during 4 weeks. After such treatment, histologic examination of these testes showed atrophy of the seminiferous tubules and maturation arrest of spermatogenesis.(4) 2 weeks post-EDS, expression of COX-2 decreased significantly (P.005), in comparison with vehicle-treatment control. 4 weeks after treatment, a new generation of fetal like Leydig cells repopulated in the testicular interstitium resulting in COX-2 expression partially recovered. Although the expression of COX-2 mRNA and protein are enhanced at 8th week after using external testosterone, it wasn't significant higher than control group.

实验研究证明：(1)正常成年雄性大鼠睾丸组织中COX-2在mRNA和蛋白质水平均存在表达，免疫组织化学结果显示COX-2定位于曲细精管内的生精细胞；正常成年男性睾丸组织中同样存在COX-2表达，免疫组织化学结果显示COX-2定位于曲细精管内的生精细胞和Leydig细胞；(2)服用特异性COX-2酶抑制剂rofecoxib 2周后，实验组大鼠睾丸组织内睾酮的含量减少，为正常对照组的50％；持续用药4周后睾丸组织内睾酮浓度逐渐恢复至正常水平；COX-2酶活性降低后病理组织切片显示睾丸内曲细精管萎缩，生精紊乱，持续用药4周时影响最明显；(3)注射特异性Leydig细胞杀灭剂EDS 2周后，实验组大鼠睾丸组织内睾酮浓度降至极低水平时，COX-2的蛋白和基因表达水平也显著低于正常空白对照组（P.005）；使用EDS后第4周，睾丸组织中的睾酮浓度逐渐回升，同样组织中COX-2蛋白表达和mRNA表达水平也相应提高接近正常水平；使用EDS后4周开始给予外源性睾酮，6周和8周时COX-2表达水平的绝对值虽有提高，但与正常大鼠空白对照组比较并无显著性差异，说明外源性雄激素刺激睾丸合成COX-2的作用并不明显。

The concept of equivalent rotationally rigidity is offered and the formula of rotationally rigidity is obtained.

主要做了如下几个方面的工作：对伸臂位于顶部的单层框架—筒体模型进行分析，提出了等效转动约束的概念和转动约束刚度的表达式。

Male cats normally do not need aftercare with the exception of the night after the anesthetic.

男猫通常不需要善后除了晚上的麻醉。