犬五联病毒活疫苗制备用犬肾细胞系的建库筛选与检定

 以人类子宫颈癌细胞系Hela为阳性对照 ,以连传 3代的犬、猫肾原代细胞 (CKC、FKC)作为阴性对照 ,对来自国内外不同单位收集的另外 4株MDCK传代细胞系培养 2 5～ 6 7代的完整活细胞进行裸鼠致癌 /致瘤实验观察 ,筛选出致癌性极低、符合细胞遗传学要求、无传染因子污染的 2株MDCK细胞系用于制苗 ,并建立了相应的细胞种子库和工作库 ,供科研和生产使用 ,4年运转很好。不同代次MDCK细胞系染色体众数所占比率的相差率一般不超过 5 %～ 15 % ,结构畸变率一般为 0～ 3%。研究表明 ,MDCK细胞染色体遗传特征决定致瘤性质 ,并具有种属特异性 ,MD CK细胞不论核型如何 ,始终具有致癌性 ,但其致癌 /致瘤性差 (2 3/ 5 9) ,且一般致上皮源性恶性肿瘤 ,多为高中分化腺癌。降低制苗毒液中细胞系基因含量 ,完全可以将MDCK细胞系 (WB和H株 )用于犬五联苗生产。MDCK细胞超二倍体YB株和亚二倍体与超二倍体KA株不能用作病毒活疫苗培养基质。找出了MDCK细胞系的染色体变异率、软琼脂中克隆形成率、对植物凝集素的凝集性和在裸鼠体内形成癌肿的潜力之间的可能相关性 ,发现细胞系染色体数目增加、克隆形成率增高、凝集性增强 ,则致癌 /致瘤性相应提高     

Sf9昆虫细胞系的致瘤性研究

为确定Sf9昆虫细胞系作为疫苗生产细胞株的安全性,检测了Sf9细胞对裸鼠的致瘤性。将3周龄SPF级雌性裸鼠随机分为5组,分别为Sf9基础细胞库细胞组、Sf9最高限制代次细胞组、阳性对照Hep-2细胞组、阴性对照CEF细胞组和空白对照组,以各自细胞悬液皮下接种裸鼠。在接种后21 d和84 d观察接种部位肿瘤形成情况,并进行病理组织学检查。结果显示,接种后21 d,Hep-2细胞组裸鼠注射部位形成米粒大小结节,经病理组织学检查为鳞状细胞癌,而CEF细胞组和Sf9细胞组注射部位均无结节。接种后84 d,经病理组织学检查,CEF细胞组和Sf9细胞组均无肿瘤形成。本研究表明基础代次和最高限制代次Sf9细胞均不具有致瘤性,Sf9细胞可用于疫苗生产。     

花生四个类型的染色体组型分析

花生（Arachis hypogaea L.）四类型的染色体组型分析表明：⑴四类型的染色体数,2n = 4x = AABB = 40,20对的同源染色体中,有1对具随体的B染色体的B染色体组和1对最小的A染色体的A染色体组。⑵四类型的染色体长度范围分别为5.7-15.1μ（西班牙型）,4.9-14.4μ（秘鲁型）,5.30-14.7μ（巴伦西亚型）和4.5-12.1μ（弗吉尼亚型）。四类型的染色体组型都不同,西班牙型2n = 4x = 17m + 1sm（SAT）+ 2 st,秘鲁型2n = 4x = 17m + 2sm（1SAT）+1st,巴伦西亚型2n = 4x = 12m + 7sm（1SAT）+1st和弗吉尼亚型2n = 4x = 16m + 3sm（1SAT）+1st。     

天然二倍体和四倍体泥鳅鳍细胞系染色体组构成研究

对传代20次的天然二倍体和天然四倍体泥鳅鳍细胞系进行染色体标本制备,并对其染色体核型、Ag-NORs及CMA3/DA/DAPI三重荧光染色等进行研究。结果显示,1二倍体泥鳅鳍组织培养细胞的染色体数目为2n=50,核型公式为:2n=10m+4sm+36t,NF=64;四倍体泥鳅鳍组织培养细胞的染色体数目为4n=100,核型公式为:4n=20m+8sm+72t,NF=128。2天然二倍体泥鳅鳍培养的细胞中期染色体分裂相有2个Ag-NORs,天然四倍体泥鳅鳍培养的细胞中期染色体分裂相有4个Ag-NORs,均位于第1对中部着丝粒染色体(M1)的末端。3天然二倍体泥鳅鳍培养的细胞染色体中期分裂相中CMA3阳性位点为2个,天然四倍体泥鳅鳍培养的细胞染色体中期分裂相中CMA3阳性位点为4个;均位于核仁组织区,即第1对中部着丝粒染色体(M1)的末端。结果表明,天然二倍体和四倍体泥鳅鳍培养细胞制备的染色体数目、核型和带型与体细胞一致。     

戴胜(Upupa epops)的染色体组型研究

 本文报道了戴胜(Upupa epops)的染色体组型研究结果。其染色体二倍体数2n=128。这是迄今已研究过的鸟类染色体二倍体数目最多的一种。在常染色体中,1—10号为双臂染色体(中、近中或近端着丝粒染色体);11—63号全为端部着丝粒染色体。雄性性染色体ZZ是整个染色体组中最长的一对近端着丝粒染色体;雌性的W染色体也为近端着丝粒染色体,其长度仅次于第3号常染色体。本研究结果为鸟类细胞分类学和研究鸟类的核型进化提供了新的细胞遗传学资料。     

内蒙古不同类型白绒山羊染色体组型分析研究

本文通过对二狼山、阿尔巴斯、辽宁和大青山白绒山羊染色体组型的分析研究,首次证实了内蒙古白绒山羊类型不同,各染色体之间存在着共同的特点即染色体形态均为端着丝点,染色体数目均为２ｎ＝６０;但各染色体之间的相对长度存在着极显著差异,即绒山羊品种不同,染色体相对长度不同。这一研究结果为今后不同类型绒山羊互相辨别提供了依据,为今后内蒙古绒山羊选育提供了理论依据。     

六个油菜物种的染色体组型分析

对油菜的三个基本种和三个复合种体细胞染色体的核型研究表明:六个种特别是三个基本种的染色体形态是大致相似的;但随体的形态和数目存在明显的多样性和杂合性;同时,三个复合种中随体数目是其基本种亲本之和。     

高粱染色体组型及G-带的研究

本文首次用EBHQ法研究了高粱不同地理生态类型的22个品种的核型及部分品种的G—带带型。结果表明,高梁不同地理生态类型的核型较为一致,其基本核型为:2n=20=16m+2Sm+2st(2SAT),只有一对染色体具有随体,且属于“大随体”类型。用该法不仅在晚前期、早中期和中期染色体上诱导出了丰富的G-带,且首次在早后期染色体及随体上获得了G-带。     

梭鱼Mugil so-ing Basiewsky染色体组型的研究

鱼类染色体组型的研究是鱼类细胞生物学、细胞遗传学不可缺少的一部分工作。它对鱼类的分类、进化、遗传、变异以及杂交育种等研究都具有重要意义。然而，鱼类的染色体一般是数目较多而且个体极小，颇难进行核型分析。     

泰国斗鱼的染色体组型分析

对泰国斗鱼Betta splendens的染色体组型进行了分析。采用植物血球凝集素和秋水仙素活体注射法,取泰国斗鱼的肾和鳃分别制备雌、雄染色体滴片,分析其染色体组型。结果表明:泰国斗鱼的染色体数目为2n=42,其中有2条为亚中部着丝粒染色体(sm),8条为亚端部着丝粒染色体(st),其余32条为端部着丝粒染色体(t),染色体组型公式为2sm+8st+32t,臂数NF=44,雌、雄染色体组型无差异,也未发现异型的性染色体。     

Experimental Researches on Carcinogenesis or Tumorigenicity of MDCK Canine Kidney Cell(CKC) Lines and Analysis of Their Chromosome Karyotypes

The chromosomal number variations & structural aberrations of the MDCK cell line, primary feline or canine kidney cell(FKC or CKC) and Hela cell line were investigated and their karyotypes of conventional chromosome bands were analyzed. The carcinogenesis or tumorigenicity testing of these cell lines in about 232 nude mice and for colony formation in soft agarose and for haemagglutination under different concentration of plant lectins of these cells were carried out. Under the prerequisite that the incidence of cancer or tumor in negative-control nude mice inoculated subcutaneously with primary feline or canine kidney cell cultures purified in vitro at passage 3 was 0 (0/22) and 0 (0/10), respectively. The incidence of the progressively-growing malignant tumor(MT) in positive-control nude mice inoculated subcutaneously with Hela cell cultures of KB, X, or NM20/X strain was 10/10, 25/25 and 5/51, respectively. The results showed that the incidence of tumor in nude mice with tetrapioid YA strain of MDCK cell during 20 - 45 passages, with hypodiploid JB strain of MDCK cell on passage 25, with di-and hypoploid JC strain of MDCK cell during 2 - 15passages or with hypoploid M strain of MDCK cell during 9 - 27 passages was 28/58, 1/5, 4/18 and 0/31,respectively. The chromosomal analysis results showed that the ratio of difference in the rate of modal chromosome number between high (mcs + n) and lowest (mcs)passages was not more than 5 % - 15 % and the structure aberrations was generally 0 - 3%. These results proved that the genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species-specific. MDCK line has tumorigenicity no matter what its chromosome karyotype is, at least it has very low tumorigenicity even when its modal chromosome number is hypoploid. The repeatedly frozen, thawed and split controls of tumorigenicity-positive cell lines(X strain of Hela, M strain of BHK-21, JA strain of Vero, YA strain of MDCK) have much lower tumorigenicity or are even non-carcinogenesis, and the repeatedly frozen, thawed and split controls of very low tumorigenicity cell lines (M or JC strain of MDCK) are certainly non-carcinogenic and never have increased tumorigenicity.It is thus evident that MDCK cell of M, JB or JC strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YA strain can not be approved as substrate for the preparation of comattenuated viral vaccines. In summary, all strains of MDCK cell line have tunorigenicity, at least have low tumorigencity, never have non-cancinogenic MDCK, but very low tumorigenicity MDCK cell strains can certainly be used for the approval production of canine viral vaccines if the DNA content in viral cell cultures was remarkably decreased through conventional means in manufacturing process. Therefore, the master cell stock and working cell bank of MDCK line used for vaccine manufacture were established in China, which are free of infectious agents, and described with respect to cytogenetic characteristics and tumorigenicity. Tests showed that there were correlations among cell line chromosome number variations, anchorage independence in soft agarose, haemagglutination under plant lectins, and tumor-forming ability in nude mice, thus all the in vitro tests are economic, simple and reliable means for monitoring the tumor-forming ability of MDCK line in nude mice.     

Further Report on Carcinogenesis or Tumorigenicity of MDCK Canine Kidney Cell(CKC) Lines and Analysis of Their Chromosome Karyotypes

Using Hela cell cultures as positive control and primary canine kidney cell (CKC) or feline kidney cell (FKC) cultures purified in vitro on passage 3 as negative control, the tumorigenicity of Madin-Darby canine kidney (MDCK) cells was tested in ＞273 nude mice, and colony formation in soft agarose and haemagglutination under different concentration of plant lectins of these cells were carried out at the same time. Subsequently, very low tumorigenicity strains of MDCK line were successfully selected; these were evaluated for the production of canine or feline combination viral vaccines, free of infectious agents, and of known cytogenetic and tumorigenic. It is thus evident that MDCK cell of M, JB, JC, WB or H strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YA, YB and KA strains can not be approved as substrate for the preparation of attenuated viral vaccines. The heritable character of these cell sub-lines is comparatively stable, and shows little significant difference between passages.     

应用套式PCR在MDCK细胞系中发现犬细小病毒

本研究自１９９７年至１９９９年从国内部分大学实验室、卫生防疫站和动检局陆续收集１１个犬肾（ＭＤＣＫ）细胞系样品,选取犬细小病毒基因组的ＶＰ２基因上４段核苷酸序列作引物,对样品ＤＮＡ进行套式ＰＣＲ（Ｎｅｓｔｅｄ ＰＣＲ）检测;ＰＣＲ扩增产物经０．０１ｇ／ｍＬ琼脂糖凝胶电泳和克隆到ｐＧＥＭ＾－Ｔ载体并进行核苷酸序列测序鉴定后,发现我国ＭＤＣＫ细胞系中存在犬细小病毒的传代病毒株。该病毒基因组部分序列测定结果显示与犬细小病毒ＣＰＶ－Ｎ株有９１％同源性。     

Establishment of Master Cell Stock and Working Cell Bank of MDCK Lines and Selection and Evaluation of the Lines as Candidate Viral Substrates for Approval Production of Combinational Canine Attenuated-live Virus Vaccines

Under the prerequisite that the incidence of cancer or tumor in negative-control nude mice inoculated subcutaneously with primary feline or canine kidney cell cultures purified in vitro at passage 3 was 0(0/22) and 0 (0/10), respectively. The incidence of the progressively-growing malignant tumor(MT) in positive-control nude mice inoculated subcutaneously with Hela cell cultures of KB, X, or NM20/X strain was 10/10, 25/25 and 5/51, respectively. The results showed that the incidence of tumor in nude mice with di-and hyperploid YB strain of MDCK cell during 17 - 23 passages, with hyper- and hypoploid KA strain of MDCK cell during 6 - 8 passages, with hypoploid WB strain of MDCK cell on passage 6, with hyper-and hypopioid H strain of MDCK cell during 8 - 24 passages was 2/24, 6/10, 5/10 and 10/15, respectively. The chromosomal analysis results showed that the ratio of difference in the rate of modal chromosome number between high(mcs + n) and lowest (mcs)passages was not more than 5- 15% and the structure aberrations was generally 0-3%. These results proved that the genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species-specific. MDCK line has tumorigenicity no matter what its chromosome karyotype is, at least it has very low tumorigenicity even when its modal chromosome number is hypoploid. It is thus evident that MDCK cell of WB or H strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YB or KA strain can not be approved as substrate for the preparation of attenuated viral vaccines.     

胰蛋白酶浓度对H9亚型禽流感病毒在MDCK细胞上增殖的影响

[目的]探讨不同胰蛋白酶浓度对低致病力禽流感病毒在MDCK细胞上增殖后的HA滴度。[方法]通过3株禽流感H9亚型病毒分别接种MDCK细胞单层,加入含有不同胰蛋白酶浓度的DMEM维持液,每隔24 h观察细胞病变,并测定上清液中的HA滴度。[结果]当维持液中胰蛋白酶含量为10～20μg/ml时,培养液上清液中的HA滴度最高,达到7 log2（1∶128）。当病毒的接种浓度为10-3和10-4时,MDCK细胞在96 h几乎全部病变,峰值出现在接种后的72～96 h。[结论]当维持液中胰蛋白酶含量为10～20μg/ml时,有利于H9亚型AIV病毒在MDCK细胞上增殖。     

晋北地区不同芸豆品种(系)的适应性评价

为分析不同芸豆品种(系)在晋北地区的适应性,在山西晋北地区,对引进的12个芸豆品种(系)的生育期、农艺性状、产量构成因素、产量等指标进行调查,运用主成分分析法对其适应性进行分析和评价。结果表明,供试的12个品种(系)中除BY2015-1无法在晋北地区成熟,其余11个品种(系)在晋北地区的适应性较好;对11个品种(系)的9个性状进行主成分分析,提取3个主成分因子,即产量与株型因子、生育期与荚长因子和分枝数因子,累积贡献率为86.054%;根据各品种(系)的主成分得分和综合得分,共筛选出4个适宜晋北地区种植的芸豆品种(系),分别为中芸5号、龙12-2614、中芸4号和中芸6号,其中中芸5号的综合得分最高,产量也最高;聚类分析表明,11个芸豆品种(系)可划分为两大类,其中中芸5号、龙12-2614、中芸4号和中芸6号聚为一类。结果为芸豆品种的评价和筛选提供了新的思路和方法。     

8个枇杷品种(系)的RAPD分析

采用RAPD分析,从80个引物中筛选出能稳定扩增的6个引物,对大五星、龙泉1号、川农1号等8个枇杷品种(系)进行了扩增,共扩增出54条带,26条具有多态性,多态性比例占48.15%。利用筛选的6个引物初步建立了大五星、龙泉1号、川农1号等品种(系)的DNA指纹图谱,并找到部分特异谱带。利用特异谱带结合DNA指纹图谱,对参试品种(系)进行了鉴别,并利用NTSYSpc2.1软件进行统计分析,得到品种(系)间的遗传距离及聚类分析结果。     

马铃薯品种(系)的产量及主要农艺性状分析评价

对自育的42个马铃薯品系或高代材料在贵州威宁县进行品种比较鉴定试验,主要对生育期、鲜薯产量及块茎形状、淀粉含量等农艺性状进行了鉴定和分析评价。结果表明:所有参试品种(系)的生育期在72～114d,植株高度在44～126cm,多数品种的芽眼均比较浅。鲜薯产量为1610～3755kg/667m2,其中以9904-2(3755kg/667m2)为最高,比米拉(CK)增产44.98%。商品性最好的品种是9904-2和9904-5。淀粉含量为8.99%～17.96%,最高的品种是C01-34-2。     

应用单体分析法对三个冬小麦品种（系）进行抗白粉基因定位的研究

用单体分析法对3个冬小麦品种（系）进行了基因定位研究。结果表明：小白冬麦和复壮30对白粉病的抗性，分别由1个位于1A染色体、1个位于4D染色体上的隐性基因所控制。由于1A染色体上的两个已知Pm基因Pm3和Pm17的苗期抗性均不如小白冬麦的强，也由于已知Pm基因中没有位于4D染色体上者，故认为小白冬麦和复壮30所含隐性抗白粉基因可能是新的Pm基因。结果还表明，Fr81－8所含抗白粉基因很可能是Pm4b。对于关键组合F2抗感分离比例偏离97∶3标准比例的原因亦作了分析。