建立海南黎族永生细胞库

采用EB病毒转化外周血B淋巴细胞加环胞霉素A法,建立了含54株海南黎族永生细胞库(男性56%,女性44%),有亲缘关系者占13%。供血者身体健康,三代无与其他民族或支系通婚史。Abstract:　An immortalized cell bank of the Li Nationality in Hainan Province was successfully established using of cyclosporin A in establishing Epstein-Barr virus transformed human lymphoblastoid cell lines(LCL). This cell bank contains 54 continuous LCL of the RUN LI. All the blood donors are healthy, without intermarriage with other nationalities or different nationality branches for three generations. Moreover male and female make up 56% and 44% of the total donors respectively. 13% of the donors have blood relation.     

树鼩视网膜微血管内皮细胞永生化细胞株的建立及鉴定

目的 建立树鼩视网膜来源的微血管内皮细胞的体外分离培养技术以及永生化细胞株,为体外利用树鼩视网膜微血管内皮细胞开展相关研究提供新的实验材料。方法 利用Ⅱ型胶原酶、分散酶和DNaseⅠ酶消化法分离培养出原代视网膜微血管内皮细胞,利用差速消化法纯化内皮细胞,然后利用携带SV40T基因的慢病毒转染细胞,再挑取单克隆后进行传代培养。对传至50代以上的细胞进行形态学观察、免疫荧光鉴定以及核型鉴定。结果 采用混合酶消化法能够分离获得微血管内皮细胞,纯化后的细胞呈不规则多角形和梭形。经慢病毒转染后的细胞传代培养后细胞形态一致,到第50代时细胞形态结构仍较好。细胞免疫荧光结果显示标志性蛋白VWF、CD34、Claudin1、ZO-1和永生化SV40T表达阳性。生长曲线结果显示：细胞生长旺盛,第2～4天时处于对数生长期,第4天进入平台期。细胞核型结果显示：染色体数与树鼩染色体相同,即所获取的细胞为永生化的树鼩视网膜微血管内皮细胞。结论 成功建立的树鼩视网膜微血管内皮细胞永生化细胞株具有较好的形态结构与功能,为视网膜病变及眼科相关疾病的研究提供了新的实验材料。     

中华民族永生细胞库在生命科学研究中的支撑作用

人类样品是生物医学研究必需的物质基础。B淋巴母细胞系(LCL)是利用Epstein-Barr(EB)病毒转化人的B细胞获得,制备简便,可以无限繁殖,是非常便捷的保存人类样品的形式。中华民族永生细胞库保藏有中国各个民族群体的LCL。目前,已经有详实的LCL的性质研究以及关于LCL的全基因组数据,因而, LCL已经广泛应用于遗传学、免疫学、药学基因组学、再生医学、癌症发生与免疫治疗、筛选制备全人单克隆中和抗体及EB病毒致病机理等研究领域。本文对LCL的特性以及LCL在上述研究领域中的应用进行了综述,最后对中华民族永生细胞库的保藏内容做了简单介绍,以促进广大科研人员进一步了解该细胞库的科研价值,充分发挥该库保藏资源在基础科学、生物医学研究中的科技支撑作用。     

长江江豚脐带永生化成纤维细胞系建立及细胞生长特性研究

取长江江豚(Neophocaena phocaenoids asiaeorientalis)产后胎盘脐静脉,经组织块培养,差异贴壁法纯化,构建长江江豚的原代细胞系;经外源癌基因SV40 T antigens(猿猴病毒T抗原)转染构建稳定脐带细胞系,并对长江江豚的永生化后的成纤维细胞的细胞形态、转染效率、生长曲线和活率等...     

47例染色体异常患者永生淋巴细胞株的建立

采用4种不同的血细胞/EB病毒（Epstein-Barr Virus,EBV）比率系列转染冻存血方法建立永生淋巴细胞株,建株成功率达97.87%,并成功地对47例染色体异常患者建立了永生淋巴细胞株,为今后不断进行的深入研究奠定了基础。     

海南黎族聚居区山栏稻的起源演化研究

以14份海南黎族聚居区的山栏稻为研究材料、以原产于中国的69份亚洲栽培稻和110份普通野生稻为对照组,分别对核中SSⅡ基因、ITS基因和Ehd1基因、叶绿体中ndhC-trnV基因以及线粒体中cox3基因等5段序列进行测序,分析基因序列多样性和单倍型,并揭示海南黎族聚居区山栏稻的起源地和驯化过程。结果表明,黎族聚居区山栏稻的基因多样性低于亚洲栽培稻,而亚洲栽培稻的基因多样性低于普通野生稻;85%左右的山栏稻为偏粳型;山栏稻与广东和湖南的普通野生稻亲缘关系较近,而与海南的普通野生稻的亲缘关系较远,推测黎族的山栏稻可能起源于广东和湖南的普通野生稻。     

中国鄂温克、鄂伦春、达斡尔族永生细胞系的建立与保存

本采用ＥＢＶ（Ｅｐｓｔｅｉｎ－Ｂａｒｒ Ｖｉｒｕｓ）上清液转化Ｂ淋巴细胞,并加入环胞霉素Ａ（Ｃｙｃｌｏｓｐｏｒｉｎｅ Ａ）抑制Ｔ淋巴细胞,成功地对中国东北地区鄂温克族、鄂伦春族及达斡尔族的部分个体建立了永生细胞系,其中鄂温克族４９株,鄂伦春族４０株,达斡尔族５１例,总计１４０株。永久保存我国特有民族的基因组,为分析其遗传学差异奠定了基础。     

海南润方言黎族药用民族植物学研究

运用关键人物访谈法和参与式观察法,对海南润方言黎族进行了药用民族植物的调查研究.结果表明润方言黎族草医常用的药用植物有282种,隶属于89科222属,以茜草科(13属16种)、大戟科(12属14种)、菊科(11属10种),蝶形花科(9属8种)属数和种数最多;治疗风湿性关节炎、肠胃病、淤血肿痛和外伤出血等常见病、易发病的种类较多,分别为78种(27.7%)、45种(16.0%)、44种(15.6%)、38种(13.5%);治疗方法有内服、外敷(分冷敷和热敷)、蒸熏、酒疗法和食物疗法等,其中内服、冷敷和热敷疗法用得最多,各占125种(44.3%)、108种(38.3%)和95种(33.7%).叶的使用频次最高,为202次(48.7%),其次是根(18.1%)、全草(15.2%)、皮(11.8%)、茎(4.1%)和果(2.2%).与中医用药比较,131种(46.5%)相似,109种(38.7%)不同,42种(14.9%)为黎医特有.药用民族植物学知识在交通便利、汉化程度深的村寨流失严重,需要加强整理与挖掘.     

中国10个民族永生细胞系的建立与保存

永久保存我国各少数民族的遗传信息是中国人类基因组计划的重要内容,为此,采用EB病毒(epstein-barr Svirus EBV)上清液及Hepes转化外周血B淋巴细胞,并加入环孢霉素A(cyclosporine A)抑制T淋巴细胞,成功地对中国哈萨克族、满族、朝鲜族、赫哲族、蒙古族、锡伯族、回族、布依族、四川汉族和福建汉族的部分个体建立了永生细胞系。其中哈萨克族64株,朝鲜族58株,赫哲族18株,锡伯族43株,回族63株,布依族67株,满族65株,蒙古族62株,四川汉族51株,福建汉族58株。总计549株。为保存我国各民族遗传资源、分析各少数民族间的遗传学差异及其起源,奠定了材料基础。Establishment and Preservation of Immortal Lymphoblastoid Cell Lines of the 10 Ethnic Groups in ChinaHUANG Xiao-yi,LIU An,YU Yang,MA Lin-lin,SHI Rong-qian,Lü Fu-qu,JIANG Yan,SUN Wen-jing,XUE Ya-li,FU Song-bin,LI PuDepartment of Medical Genetics,Harbin Medical University,Harbin 150086,ChinaAbstract:The immortal lymphoblastoid cell lines were established by EBV transformation of B cells and addition of cyclosporin A to inhibit the activity of T cells.In the present study,549 immortal cell lines of different ethnic groups of Hazak,Manchu,Korea,Hozhe,Mongolia,Sibe,Hui,Puyi,Han in Fujian and Han in Sichuan were established.Through our research,we found it is harmful for B lymphocytes to transform if excessful leucocytes are inoculated.And it is crisis that cyclosporine A shoud be added the last.Our work is an important part of the research of human genome diversity for the exploration of the origin and evolution of different ethnic groups,and it also provides enough research materials for further studies.Moreover,we have sent 50 cell lines of Hozhe,Mongolia,Sibe,Daur,Oroqen to CEPH.Thus it is possible for us to utilize the genetic resources of CEPH freely.Key words：immortal lymphoblastoid cell line;EBV;ethnic groups;reserved in liquid-nitrogen     

海南岛黎族体质特征之研究

黎族的体征明显属黄种人。与其他少数民族相比,在体征上与壮族、布依族、彝族和高山族较相似;与汉族相比,则与广西、广东、福建和湖南的汉族相似。因此,作者认为黎族的体征属华南类型。黎族四个组群间,在容貌上较难区分,但测量的尺寸略有不同,其中(亻考)黎与岐黎较接近,本地黎与美孚黎较接近。     

Multicentric investigation of ionising radiation-induced cell death as a predictive parameter of individual radiosensitivity

In the present study, the predictive value of ionising radiation (IR)-induced cell death was tested in peripheral blood lymphocytes (PBLs) and their corresponding Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) in an interlaboratory comparison. PBLs and their corresponding LCLs were derived from 15 tumour patients, that were considered clinically radiosensitive based on acute side-effects, and matched controls. Upon coding of the samples, radiosensitivity of the matched pairs was analysed in parallel in three different laboratories by assessing radiation-induced apoptotic and necrotic cell death using annexin V. All participating laboratories detected a dose-dependent increase of apoptosis and necrosis in the individual samples, to a very similar extent. However, comparing the mean values of apoptotic and necrotic levels derived from PBLs of the radiosensitive cohort with the mean values of the control cohort did not reveal a significant difference. Furthermore, within 15 matched pairs, no sample was unambiguously and independently identified by all three participating laboratories to demonstrate in vitro hypersensitivity that matched the clinical hypersensitivity. As has been reported previously, apoptotic and necrotic cell death is barely detectable in immortalised LCL derivatives using low doses of IR. Concomitantly, the differences in apoptosis or necrosis levels found in primary cells of different individuals were not observed in the corresponding LCL derivatives. All participating laboratories concordantly reasoned that, with the methods applied here, IR-induced cell death in PBLs is unsuitable to unequivocally predict the individual clinical radiosensitivity of cancer patients. Furthermore, LCLs do not reflect the physiological properties of the corresponding primary blood lymphocytes with regard to IR-induced cell death. Their value to predict clinical radiosensitivity is thus highly questionable. The authors B. Greve, K. Dreffke and A. Rickinger are treated as first authors.     

Correlation between the expression of CD4 and the level of CD4 mRNA in human B-cell lines

Lines of Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cells (B-LCLs) differ in the expression of surface CD4 glycoproteins. The aim of the present study was to correlate the expression of CD4 molecules on B-LCL cells with the synthesis of CD4 mRNA. RT-PCR assays were performed with oligonucleotide primers designed to detect mRNA corresponding to intracellular, transmembrane, or extracellular portions of the CD4 molecule. RT-PCR assays with all sets of primers were positive in T-cell populations, but were negative in various B-cell lymphoma lines. The majority of the LCLs established by EBV transfection of non-selected B-cells yielded positive results with at least some of the primer sets used for detection of CD4 mRNA. A significant positive correlation was found between the proportion of CD4+ cells in various B-LCLs and the concentration of CD4 mRNA. LCLs established from B-cells which synthesized various antibodies did not express CD4 molecules and either failed to synthesize CD4 mRNA or produced very low concentrations. These findings indicate that the expression of CD4 on B-LCLs is directly correlated with the concentration of CD4 mRNA synthesized and with the differentiation stage in which B-cells were immortalized by EBV infection.     

Theileria parva: Early events in the development of bovine lymphoblastoid cell lines persistently infected with macroschizonts

The cellular origin and development of bovine lymphoblastoid cell lines persistently infected with macroschizonts of Theileria parva was studied. Cultures of lymphoblastoid cells isolated from cattle with patent East Coast fever were compared with those obtained by infecting normal lymphocytes in vitro with sporozoites. The young lines were contrasted with a continuous line which had been isolated earlier. The mononuclear cells were separated from the blood and the inoculum enriched for lymphoblastoid cells and/or lymphocytes by removing the monocytes. The lines arose directly from lymphoblastoid cells transplanted into culture or from lymphocytes infected by sporozoites. In primary cultures of lymphoblastoid cells from the peripheral blood, there was an increase in the proportion of infected cells without the eclipse of the parasite, the macroschizonts were larger than those observed in the inoculum or the continuous line, and there was concurrent microschizont differentiation. In lymphocyte cultures challenged with sporozoites, small mononucleated trophozoites were observed after 2 days which differentiated into typical macroschizonts but microschizonts were rare. In all cultures, the infected cells had mitotic indices of 4 to 5%. As the young lines were passaged, the parasites came to resemble those of the continuous line. The macroschizont size in the continuous line was stable and most had six to eight nuclei but when cultured at high cell concentrations the number of parasite nuclei increased. Minicultures of lymphocytes were used to quantitate the infectivity of sporozoites obtained from organ cultures of Rhipicephalus appendiculatus savliary glands. Sporozoites from ticks fed on rabbits for 5 days were approximately six times more infective than those from glands of ticks fed for 2 days and then cultured at 32 °C for 3 days. Glands from unfed ticks cultured for 5 days failed to yield infective sporozoites.     

Micronucleus induction and chromosome loss in transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN) is a potent inhibitor of protein synthesis produced by a number of cyanobacterial species, the most common being Cylindrospermopsis raciborskii. CYN contains a uracil moiety attached to a sulphated guanidino moiety, suggesting that it may have carcinogenic activity. This report describes the use of the WIL2-NS lymphoblastoid cell-line in the well-validated cytokinesis-block micronucleus (CBMN) assay to test this hypothesis. Centromeres (CENs) were identified in micronuclei (MNi) of binucleated cells (BNCs) by fluorescent in situ hybridisation of alpha centromeric DNA sequence repeats. The results indicate that CYN induced a significant increase in the frequency of MNi in BNCs exposed to 6 and 10 μg/ml, and a significant increase in CEN-positive MNi at all concentrations of CYN tested (1, 3, 6, and 10 μg/ml). However, despite this apparently greater sensitivity of WIL2-NS cells to induction of CEN-positive MNi at low CYN concentrations, at the higher concentrations the magnitude of the increase in CEN-positive MNi did not account for the greater increase in MNi in BNCs, indicating that both CEN-positive and CEN-negative MNi were induced. This suggests that CYN acts to induce cytogenetic damage via two mechanisms, one at the level of the DNA to induce strand breaks, the other at the level of kinetochore/spindle function to induce loss of whole chromosomes (aneuploidy). C. raciborskii occurs in a number of human drinking water sources worldwide and so these findings may have important public health implications.     

海芒果叶中三萜类成分的研究

采用多种柱色谱等分离纯化手段对海芒果叶中的三萜类成分进行分离,得到7个化合物,通过理化性质和波谱方法鉴定化合物为熊果醇(1),(23Z)-9,19-cycloart-25-ene-3β,24-diol(2),大戟醇(3),乌苏酸(4),2α-羟基乌苏酸(5),乙酰乌苏酸(6),α-香树脂醇(7)。化合物1～6均为首次从该属植物中分离得到。     

Traces of copper ions deplete glutathione in human hepatoma cell cultures with low cysteine content

BACKGROUND: Cell death induced by intracellular glutathione depletion has been reported to be dependent on the presence of trace amounts of extracellular copper ions. Since little is known about the relationship between glutathione depletion and copper homeostasis, we have in the present study further investigated the role of low amounts of copper ions in glutathione depletion. METHODS: Glutathione turnover was investigated in HeLa and hepatoma cell cultures with normal and low cysteine content in the presence of copper ions (1 and 10micromol/L) and two other glutathione-stimulating agents (lipoic acid and mercury ions). RESULTS: Copper ions (10micromol/L) caused relatively small increases in total amount of glutathione (the sum of the intracellular and the extracellular amount of glutathione) in HeLa and hepatoma cell cultures with normal cysteine levels (420nmol/mL) compared to control cell cultures, whereas lipoic acid and mercury ions strongly increased total glutathione in both types of cell cultures. Lower amount of total glutathione was observed in cell cultures with a lower cysteine levels (84nmol/mL), which is similar to that in human plasma. A strongly decreased total amount of glutathione in the presence of copper ions was observed in hepatoma cell cultures with lower cysteine levels, whereas the other agents showed effects similar to those described for cell cultures with normal cysteine levels. CONCLUSION: Glutathione synthesis in hepatoma cell cultures is probably more sensitive to a low cysteine level than HeLa cell cultures, and the presence of copper ions further decreases the availability of cysteine probably by increasing the disulfide binding to cysteine residues in extracellular proteins, which causes a further decrease of total glutathione.     

The effect of different antioxidants on glutathione turnover in human cell lines and their interaction with hydrogen peroxide

BACKGROUND: Glutathione plays crucial roles in antioxidant defence and glutathione deficiency contributes to oxidative stress and may therefore play a key role in the pathogenesis of many diseases. The objectives of the present study were to evaluate the effects on glutathione turnover of thiol and non-thiol antioxidants in human cell cultures and if any of the antioxidant had a short-term cellular effect against different levels of hydrogen peroxide. METHODS: We have investigated the effect on the total glutathione amount in HeLa and hepatoma cell cultures of thiol antioxidants in comparison with non-thiol antioxidants, such as a copper chelator, Vitamin C, and a flavonoid. Furthermore, we have investigated the short-term (within 24h) interaction of the different antioxidants with hydrogen peroxide. RESULTS AND CONCLUSION: Lipoic acid and quercetin (Quer) were the two antioxidants that showed the highest stimulation of glutathione synthesis in cell cultures as judged by the total glutathione amount. However, no antioxidant protected against hydrogen peroxide present in concentrations that lowered cell protein. This finding may be attributed to the fact that it is necessary to incubate cell cultures with antioxidants or small doses of oxidants for a period before the cultures are exposed to hydrogen peroxide in order to enhance the antioxidant defence. The presence of Quer and Vitamin C lowered cell protein and total glutathione even in cell cultures containing hydrogen peroxide in concentrations that did not lower cell protein. This finding might be attributed to pro-oxidant properties and formation of excess reactive oxygen species in the presence of Quer and Vitamin C.     

Oxidative stress decreases extracellular homocysteine concentration in human hepatoma (HepG2) cell cultures

BACKGROUND: Mild hyperhomocysteinemia is associated with premature vascular disease. The mechanism behind the vascular injuries is, however, still unknown. Homocysteine may be catabolized in the trans-sulfuration pathway to cysteine. Cystathionine beta-synthase, which catalyses the first step in the trans-sulfuration pathway is redox-sensitive. We have therefore investigated total extracellular homocysteine turnover in the presence of oxidative stress in human cell lines. METHODS: The turnover of total extracellular homocysteine in HeLa and hepatoma cell cultures has been investigated in the presence of hydrogen peroxide. Furthermore, the effect of hydrogen peroxide on the removal of high amounts of exogenously added homocysteine was also studied. RESULTS: Total extracellular homocysteine concentration in hepatoma cell cultures decreased in the presence of hydrogen peroxide, whereas the extracellular homocysteine concentration in HeLa cell cultures was not influenced. There was no significant change of intracellular homocysteine in any type of cell cultures. Furthermore, the presence of hydrogen peroxide did not increase the removal of exogenously added homocysteine. CONCLUSION: The presence of hydrogen peroxide probably increases the activity of the trans-sulfuration pathway in hepatoma cell cultures, which increases the intracellular use of homocysteine and lowers its extracellular release. Consequently this mechanism might tend to lower total plasma homocysteine concentration in oxidative stress.     

Characterization of invertebrate cell lines

Summary Cell extracts of five mosquito cell lines and a tick cell line were examined for four cellular isozymes using a cellulose-acetate electrophoretic technique. This method distinguished the cell lines that were derived from the different species. Intraspecies distinctions were not made using the cell lines tested; the significance of this finding is discussed. The usefulness of this technique in identifying a potentially mislabeled cell line was demonstrated. This research was supported by contracts, DADA 17-72C-2170 of the U.S. Army and N00014-78C-0104 of the U.S. Office of Naval Research and grants from the World Health Organization and the Rockefeller Foundation.     

The assessment of anti-tumoral activity of polysaccharide extracted from terrestrial filamentous fungus

Fungal polysaccharides are well-known for the medicinal properties such as antitumor and immunomodulating effects. Hence, this study evaluated antitumor effects of polysaccharide extracted from Fusarium sp. isolated from soil samples of Karaj district, Alborz, Iran along with its taxonomic study. The filamentous fungus strain FK1 was isolated from the soil sample of Karaj, Iran. The strain was identified based on cultural, morphological and 18 S rRNA gene parameters as Fusarium. Further, the strain Fusarium was cultured in fermented broth of modified (PDB) for 10  days at 25 °C. The polysaccharide of strain FK1 was extracted from the mycelium free supernatant by boiling water method and evaluated for antitoxicity effect on two human cancer cell lines: HeLa cell line and Lymphoblastoid cell line (LCL) by MTT method. Findings revealed that water-extracted from mycelia polysaccharide of strain FK1 had the highest cytotoxicity effect against LCL which is the cause of B lymphocyte cancer, at 50  μg/ml concentration dose (114 ± 1.63) followed by 100  μg/ml (105 ± 0.57) and 10  μg/ml (104 ± 0.57), while it did not have a considerable effect on HeLa cell line. Fusarium could be alternative sources as an antitumor component.