组织培养

As the growth rate of callus is slow in the solid medium in the process of culture, callus is not fit for industrialization of secondary metabolite. The cell suspension culture line was established using the callus in the solid medium.

由于在固体培养基上进行培养的愈伤组织生长速率较慢，不适于培养物中目标次生代谢产物的工业化生产，实验中又进一步以固体培养基上的愈伤组织无性系为材料建立了银杏细胞悬浮培养系。

To establish novel cell lines from brown-marbled grouper, Epinephelus fuscoguttatus, primary cultures of fin, heart and swim bladder tissues were initiated in this study.

为建立褐点石斑鱼鳍、心脏和鳔组织细胞系，本文利用不同培养液和培养温度对褐点石斑鱼鳍、心脏和鳔组织细胞进行了原代培养和传代培养。

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。

METHODS: The model of tissue damaged submandibular gland in 10 rats was made by deligation. One week later, the gland tissue was obtained to harvest submandibular gland stem/progenitor cells by enzyme digestion in vitro. Following 10-14 days of primary culture, small round cells were collected, purified and subcultured for monoclonal culture.

10只大鼠采用结扎下颌下腺主导管、抑制腺体分泌来建立组织损伤模型，1周后切取腺体组织，酶消化法体外分离下颌下腺干/祖细胞，原代培养10~14 d后，挑取培养皿中形成的小的类圆形、类上皮细胞集落予以纯化，传代后进行单克隆培养。

Our study demonstrates that cardiac cells possess the ability to reestablish 3-D tissue architecture in vitro;Simulated microgravity by HARV bioreactor facilitates the metabolism and function of the 3-D cardiac cell cultures.

体外培养的乳鼠心肌细胞具有形成具有同步自律收缩的三维组织结构的能力；模拟微重力的培养环境有利于改善心肌细胞三维组织样培养物的代谢和功能。

Methods: The fibroblasts companying human hepatoma were primarily cultured with explant culture method, first passaged after they spread to the bottom of the culture bottle, and pured by enzyme digestion and repeated strapping the cells were identificated through observing the morphologic change using invert microscope and detecting expression of vimentin, keratin by iMmunohistochemical method.

应用组织块培养法进行人肝癌伴生成纤维细胞的原代培养，细胞铺满培养瓶底后首次传代，通过胰酶消化法和反复贴壁法进行成纤维细胞的纯化；通过倒置显微镜观察细胞形态、免疫组织化学方法检测细胞膜波形蛋白、角蛋白表达情况，对细胞进行鉴定。

①Rat MSC and VSMC were cultured and identified, respectively. MSC were labeled with DAPI firstly, and then co-cultivated with VSMC. The changes of morphology and ultrastructure of co-cultured cells were observed. Immunfluorescence analysis was performed by using monoclonal antibodies against specific antigen.②We established the regulatable system in two steps: a stable MSC line expressing rtTA has been constructed and characterized firstly by transfected with pUHD 17-1hyg and then selected by hygromycin B; in a second step, this line was used for trandfer the AT2R gene to MSC to get the well establishing double stable MSC lines;③The expression of AT2R regulated by doxycycline was evaluated by western blot;④The MSCs were transduced into rat carotid arteries with regulatable AT2R gene after the establishment of rat carotid balloon injury restenosis model. The intimal/medial area ratio were measured by digital analysis system.

研究方法：(1)密度梯度离心法及胶原酶消化法分别培养原代大鼠MSC及VSMC，细胞共培养并行免疫荧光化学染色和透射电镜观察超微结构；(2)组成受Dox调控的哺乳动物表达系统的四种成分的转化、扩增及提纯并酶切鉴定；(3)采用常规分子生物学方法连续两个回合转染体外培养的MSC，并分别采用发光计检测不同细胞克隆萤光素酶活性改变以及RT-PCR方法检测AT2R目的基因mRNA表达情况，根据各个细胞克隆受Dox调控表达的程度，选择低背景、高诱导表达AT2R的细胞系，作为双重稳定MSC细胞系；蛋白免疫印迹法观察该细胞系在Dox调控下AT2R表达的时相性、持续性及在不同浓度Dox调控下的表达情况；(4)建立大鼠颈动脉球囊损伤动物模型，将双重稳定MSC在术中导入血管，分别于14 d、28 d进行病理切片，检测可调控AT2R对新生内膜增生的影响；采用RT-PCR免疫组织化学免疫荧光等技术观察AT2R基因在新生内膜中的表达以及细胞外基质成分表达的改变，TUNEL法检测血管组织中细胞凋亡的变化情况。

Fungus cultured: yeast like cells were found in the culture at 37℃ or in tissues.

真菌培养：在37℃培养或组织中马尔尼菲青霉菌呈酵母型，组织细胞外真菌长，弯曲，有横膈的腊肠状；在25℃培养中呈菌丝型，并产生红色色素扩散入培养基中。

By suspending cell culture from the callus of P. thugbergii and by fluorescent observation, a new method, which is easy and sensitive to observe cell activity and to test the phytotoxicity of various bacteria, was used. This method can be utilized to study the bacterial toxins. Cultured filtrates of some strains of the nematode-carrying bacteria were bioassayed for their toxicity to suspending cultured cells from P. thugbergii callus. Strong toxic effects were observed by the filtrates of Pseudomonas spp., P. fluorescence, and Sphingomonas spp., these bacteria species are coincide with those in the early stage of pine wilt disease.

本试验使用了黑松愈伤组织的细胞悬浮培养方法，用荧光方法观察各种细菌对植物细胞的毒性测定，这种方法具对细胞的活性观察容易、灵敏度高等特点，证明可用于细菌的毒素研究；对松材线虫携带的细菌的部分菌株，用其培养滤液对黑松愈伤组织的悬浮培养细胞的生物测定初步证明，松材线虫携带的细菌中以假单胞菌、荧光假单胞菌和少动鞘氨醇单胞菌的滤液对黑松细胞具有较强的毒性，这与松树发病早期出现的细菌种类相一致。

The optimized conditions for isolation and culture of endophytic bacteria isolated from grasses in alpine area in this study. The different tissues of grasses were immersed in 0.1% of SDS for 15 min, 3% of NaClO for 3 min, 0.1% of mercuric chloride for 10 min, 75% of alcohol for 1 to 2 min. The endophytic bacteria were isolated after aseptic grinding and diluted to 10^(-3) and using TSA culture medium for 5 to 7 days under 28℃.

通过对高寒牧草内生细菌的分离培养方法研究，得出了优化的分离培养条件，即牧草不同组织器官用0.1%SDS浸泡15min、3%NaClO浸泡3min0.1%升汞浸泡10min、75%酒精1~2min处理后（各步间均用无菌水冲洗3~4次），研磨并稀释至10^(-3)，涂布于TSA培养基上，置28℃恒温条件下培养5-7d，可从高寒牧草组织中分离获得数量、种类较多的内生细菌。

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 。