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     

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。     

中国鄂温克、鄂伦春、达斡尔族永生细胞系的建立与保存

本文采用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株,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。Abstract：The immortal lymphoblastoid cell lines were established by EBV transformation of B cells and addition of cyclosporin A to inhabit the activity of T cells.In the present study ,140 immortal cell lines of the Ewenki,the Oroqen and the Daur ethnic groups in the Northeast China were established .This is an important part of the research of human genome diversity for the exploration of the origin and progression of different ethnic groups ,and also provide enough research materials for further studies.     

9例罕见异常染色体新核型

我科于1985年10月一1987年10月期间共发现9例世界首报新核型;其中从流产、早产病人中检出斗例;闭经病人中检出3例;从阵发性睡眠性血红蛋白尿（简称PNH）和无精症的病人中各检出1例。现分别报告如下:     

人的外周血淋巴细胞培养及染色体制作技术

从肘静脉采血于含有肝素的一次性注射器中,采用RPMI1640全培养基进行体外淋巴细胞培养。通过对采血后细胞培养时间、秋水仙素浓度及加入时间、低渗时间及培养温度等条件的模索．建立了较成熟的淋巴细胞体外培养方法及染色体制作技术。     

流行性出血热病毒对健康人外周淋巴细胞和大白鼠骨髓细胞染色体影响的研究

用流行性出血热病毒(EHFV)A9株,滴度为TCID_(50)10~(-5)/0.1ml,加入10名健康人外周血,作淋巴细胞姊妹染色单体互换(SCE)和染色体畸变的检测。每份血分对照组(不加病毒悬液)和A、B、C实验组(根据加不同病毒量而分)。其结果:一、SCE频率,实验组A(8.9±0.19)、实验组B(9.9±0.2)、实验组C(11.6±0.22)与对照组(6.57±0.15)比较,A、B、C、实验组均分别高于对照组,P<0.01,差异有高度显著性,A、B、C三个实验组比较,P<0.01,差异有高度显著性。二、染色体畸变,A、B、C三个实验组分别与对照组比较,P>0.05,差异均无显著性。用EHFV HA 108株,ID_(50)10~(-6)/0.02ml接种2—5日龄大白鼠脑内,15天后颈动脉放血处死,取骨髓细胞培养,另取幼大白鼠骨髓细胞培养作对照,检测SCE和染色体畸变。结果:一、SCE频率,实验组(9.8±0.35)高于对照组(5.4±0.19),P<0.01,差异有高度显著性。二、染色体畸变,实验组与对照组比较,P>0.05,差异无显著性。以上两个实验结果表明,EHFV作用于细胞,无论是在机体或试管内,都引起SCE频率增高,即EHFV促使DNA产生初级损伤,但不致染色体畸变。     

RFLPs研究三例X染色体结构异常的起源和形成机理

本文对三例X染色体结构异常46,X,dup(X)(p21);46,X,del(X)(p11);46,X,i(Xq)患者及其父母,用X染色体短臂或长臂上的限制性片段长度多态性(RFLPs)作为遗传标记,研究了异常X染色体的起源和形成机理。结果表明,dup(X)(p21)和del(X)(p11)起源于父方,而i(Xq)起源于母方。dup(X)(p21)是由X染色体姊妹染色单体不均等的互换所引起的,del(X)(p11)是由于X染色体断裂后丢失所致,i(Xq)的发生是由于卵母细胞X染色体着丝粒错分裂。     

Y染色体异常29例分析

本文从1992例遗传咨询病例中收集29例Y染色体异常的病例,其中Y染色体数目异常(47,XYY)2例;Y染色体结构异常8例:Y/Y易位1例、Yp+3例、de l(Y)3例、嵌合体dic(Y)1例;Y染色体长度变异19例。对Y染色体这几种异常类型的遗传效应进行分析。Abstract:Twenty nine cases of Y chromosome abnormalities were found in 1992 patients asking genetic counseling.Different kinds of Y chromosome abnormalitics were detected by G and banding techniques.These were 47,XYY(2 cascs);46,X,del(Y)(3 cascs);46,X,Yp+(3 cases);46,X,t(Y;Y)(1 case);45,X/46,X,dic(Y)(1 case) and length changes of Y chromosome(19 cases).The genetic effects of Y chromosome abnormalities have been analyzed in this report.     

癌症患者淋巴细胞核仁形成区活性变异的研究

应用银染-G带复合显示方法研究了肺癌、胃癌、肠癌、乳腺癌患者的外周淋巴细胞核仁形成区(NOR)活性。与正常对照相比,肺癌第15号染色体的Ag-NOR频率及Ag-NOR总频率增加,乳腺癌第14号染色体的Ag-NOR频率减少,胃癌第14号染色体Ag-NOR频率减少而第22号染色体的Ag-NOR频率增加。肠癌未见明显变异。结果提示不同部位的肿瘤具有不同的优势银染型,rRNA基因的表达可能存在肿瘤部位的特异性。     

荧光原位杂交技术检测骨髓增生异常综合征染色体异常的临床意义

目的:探讨荧光原位杂交(FISH)技术检测骨髓增生异常综合征(MDS)染色体异常的敏感性,特异性及临床意义。方法:采用细胞遗传学分析(CCA)和组合探针CSF1R/D5S23,D5S721(5q33),EGR1/D5S23,D5S721(5q31),D7S486/CSP7(7q31),D7S522/CSP7(7q31),D20S108/CSP8(20q12/CSP8)检测45例MDS患者骨髓细胞的染色体异常,并比较检测结果。结果:两种方法共检出染色体异常26例(58%),染色体数目异常9例,占34.6%;染色体结构异常13例,占50%;复杂核型4例。CCA检出+8和20q-各3例,7q-2例;FISH检出7号染色体异常8例占17.8%(8/45),两组间比较差异有统计学意义(P=0.0441713)。FISH检出+8和20q-各5例,5q-异常4例。7号染色体异常和复杂核型组与核型正常组比较转白率高。结论:组合探针检出MDS中5q-,-7/7q-,+8,20q-核型异常高于CCA,CCA结合FISH技术能提高MDS染色体异常的检出率,对于疾病诊断,判断预后具有重要价值。     

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis¹. LCL have been used to present antigens in a variety of immunologic assays^{2, 3}. In addition, LCL can be used to generate human monoclonal antibodies^{4, 5} and provide a potentially unlimited source when access to primary biologic materials is limited^{6, 7}.A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide⁸, and pokeweed mitogen⁹ to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells^{7, 10-12}.The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23^hiCD58⁺ cells observed as early as three days post-infection indicates a successful outcome.     

Comparison of Established Cell Lines at Different Passages by Karyotype and Comparative Genomic Hybridization

Two established cancer cell lines, MCF-7 and Ishikawa, were both obtained directly from a cell repository and through another laboratory. The karyotypes from the two MCF-7 cell lines had up to 83 chromosomes and similarities for chromosomal gain and structural abnormalities. The two Ishikawa cell lines had up to 60 chromosomes with only a missing X as the common chromosome abnormality. CGH studies were performed by co-hybridizing the two Ishikawa or MCF-7 cell lines to normal metaphases. The differences seen between the two MCF-7 cell cultures reflect changes due to passage number and culture conditions. For Ishikawa, DNA polymorphic data and mutation studies suggest that the two cell lines are not derived from the same established tumor cell line. Our study shows the utilization of CGH in comparing cell lines originating from the same specimen. Our study also demonstrates the necessity for periodically evaluating cell lines to confirm their origin.     

东方蓼的染色体核型分析

目的:对药用植物东方蓼的染色体数目、核型、体积等进行了研究。方法:采用常规制片方法,结合显微摄影对染色体的数目进行统计分析。结果:东方蓼体细胞染色体数目2n=22,相对长度组成2n=22=10M2+12M1;核型公式为K(2n)=2X=22=20m+2M;全体染色体总长19.16μm,长臂总长10.93μm,核型不对称系数(AS.K%)为57.00%,属于"1A"型,全体染色体总体积为20.30μm3。结论:东方蓼体细胞染色体的数目、核型、体积等清晰准确。     

从异常核型人胚胎干细胞中筛选不同X染色体失活状态的亚系

目的:从异常核型人胚胎干细胞系中分离两种不同X染色体失活(XCI)状态的细胞,建立亚系,并进行对其XCI状态特征和多能性标记进行鉴定。方法:G显带鉴定人胚胎干细胞系ch HESC-3早晚期代数细胞的核型,H3K27me3免疫荧光染色鉴定早晚期ch HESC-3表观遗传差异,RT-PCR检测早晚期ch HESC-3中XIST基因的表达。利用单细胞克隆的培养分选亚系,H3K27me3、RNA polymeraseⅡ以及DAPI三种标记的共染后每种表观标记各选两株进行RT-PCR,检测两种亚系中XIST基因的表达。并对这四株细胞进行干细胞标记鉴定。结果:G显带结果证明早期ch HESC-3为正常核型,晚期代数核型为异常核型,牵涉到8条染色体的复杂结构变异。H3K27me3免疫荧光染色证明异常核型ch HESC-3中有部分细胞出现了H3K27me3凝集点,而正常核型细胞中未发现。正常核型细胞(ch HESC-3N)没有XIST基因表达,异常核型细胞(ch HESC-3C)中有表达。在RNA polymeraseⅡ着色缺口中发现H3K27me3凝集点的细胞亚株XIST基因表达阳性,polymeraseⅡ着色缺口中未发现H3K27me3凝集点的细胞亚株XIST基因表达阴性,XIST阳性和阴性细胞各选两株进行多能性标记免疫荧光染色均为阳性。结论:成功从异常核型人胚胎干细胞系中分离两种不同XCI状态的细胞并建立亚系,两种表观类型的亚系均保持多能性标记并能在长期培养中保持各自特性。     

高山榕染色体制片优化及核型分析

以高山榕种子的根尖为材料进行染色体常规压片,比较不同预处理方法和解离方法对高山榕染色体制片的影响,以选择最优的压片方法制片并进行核型分析。结果显示,预处理24 h的总体效果优于12 h;3种预处理液的总体作用效果为0.05%秋水仙素>混合液>0.002 mol.L-18-羟基喹啉,综合比较后认为混合液低温4℃处理24 h为最佳预处理方法。解离方法选择1 mol.L-1HCl中60℃水浴2~3 min较为适宜。首次报道了高山榕核型公式为2n=2x=30=22m(2SAT)+6sm+2T,核型不对称系数为61.54%,核型属于Stebbins核型分类中的2C类型。     

陕北野生山丹丹染色体数目与核型分析研究

首次采用酸解去壁低渗法,以陕北地区野生山丹丹根尖为材料,观察染色体并对野生山丹丹的染色体数目与核型进行研究分析。结果表明:陕北野生山丹丹的染色体数目为2n=24,核型公式为2n=2x=8m+2sm+12st+2t,相对长度变化范围为5.84%～12.75%,核型不对称系数为72.16%,属"3B"型。     

稳定表达人Fhit突变体细胞系的建立

目的:构建人脆性组氨酸三联体(Fhit)突变体真核表达载体并建立稳定表达人Fhit突变体的细胞株,以便进一步研究Fhit与复制蛋白A(RPA)在体内的相互作用。方法:将3种人Fhit突变体cDNA克隆至带有HA标签的真核表达载体pREP10上,构建人Fhit突变体真核表达载体,转染HeLa细胞,经潮霉素B加压筛选阳性克隆,用Western印迹鉴定稳定表达Fhit突变体蛋白FhitA、FhitD和FhitF的阳性细胞株。结果:经PCR鉴定及序列分析,Fhit突变体基因真核表达载体pREP10/FhitA/D/F-HA构建正确,转染人HeLa细胞,筛选出Fhit突变体表达较高的细胞株。结论:建立了3株稳定表达Fhit突变体的细胞株HeLa-FhitA/D/F,为研究Fhit与RPA的相互作用在DNA损伤应答中发挥的作用奠定了基础。     

中华蚊母树染色体制片及核型分析

以中华蚊母树根尖为材料,采用常规压片法制片,比较材料的不同采集时间、预处理方法、固定剂、解离方法、解离时间及染色方法对中华蚊母树根尖染色体制片的影响.结果表明最佳的制片技术为:取材时间为上午9:00~11:00或下午13:00~15:00,以饱和对二氯苯预处理3 h,用1 mol?L-1盐酸60℃下解离8 min,卡诺固定剂固定,以改良石碳酸品红染色10 min.以该最佳制片方案对中华蚊母树进行体细胞染色体核型分析,首次揭示了中国特有植物———中华蚊母树体细胞染色体数目为2n=2x=24,染色体基数x=12,染色体核型公式为2n=2x=24=12m(2SAT)+10sm+2st,主要由中部和近中部着丝点染色体组成;染色体相对长度组成为2n=24=2L+8M2+12M1+2S,核型不对称指数为64.29%,属于2A型.结果显示中华蚊母树核型对称程度较高,在进化中属于比较原始的类型.     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidopteran Cell Lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33℃ and 35℃ was carried out by shifting the culture ...     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidop teran Cell Lines

Lepidopteran heat-tolerant(ht)cell lines have been obtained with sf-9,sf-21 and several Bombyx cells.They have a distinct karyotype,membrane lipid composition,morphology and growth kinetics from the parental cell lines.In this paper,we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility.Adaptation of cell lines sf-9,BTI-TN-5131-4(High5)and BTI-TN-MG1(MG 1)to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature.The process of adaption to a higher culture temperature was accomplished over a period of 2 months.The cell lines with the temperature adaption were designated as sf9-ht33,sf9-ht35,High5-ht33,High5-ht35,MG1-ht33,MG1-ht35.These cell lines have been subcultured over 70 passages.Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines.The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines.Cell shapes did not show obvious change,however,the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption.When the cell lines were infected with Autographa californica nuclear polyhedrosis virus(AcMNPV)at 28℃,33℃,35℃ and 37℃,production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.