ST细胞库的建立及其生物学特性鉴定

从美国典型培养物保藏中心(ATCC)引进ST细胞系,扩大培养后液氮冻存建立了种子细胞库和工作细胞库;细胞复苏后对细胞的活力、形态、生长曲线、微生物污染、核型及荧光蛋白质粒转染表达等特性进行了研究。结果表明,ST细胞系呈成纤维型,生长状态良好;生长曲线呈S型;细菌、病毒、支原体检测均为阴性;染色体为19对;外源质粒在该细胞中能进行复制和表达。建立的细胞库,可为开展ST细胞及猪瘟疫苗的研究提供基础理论依据和细胞资源。     

BHK-21细胞库的建立及其生物学特性鉴定

从美国典型培养物保藏中心(ATCC)和中国兽医药品监察所(中监所)引进2株BHK-21细胞系,传代扩增培养后液氮冻存,建立相应的细胞库;复苏细胞后对活力、形态、生长曲线、微生物污染、染色体核型及荧光蛋白质粒转染表达等特性进行研究。结果显示,2个来源的BHK-21细胞系均呈成纤维型,生长状态良好;生长曲线呈S型;细菌、真菌、支原体及外源病毒检查均为阴性;染色体为21对,二倍体均占主体;外源质粒在该2株细胞系中能进行复制和表达。这可为开展BHK-21细胞系及口蹄疫疫苗的研究与开发提供基础理论依据和细胞资源。     

Hi5-SF悬浮细胞系的建立及鉴定

从中国典型培养物保藏中心(CCTCC)引进High Five细胞并对其进行驯化,获得无血清悬浮驯化株(Hi5-SF)。依据《中华人民共和国药典(2010年版三部)》对其进行细胞生物学特性研究、微生物污染、病毒外源因子及细胞致瘤性检测。结果发现,Hi5-SF细胞复苏活力76.3%,生长曲线呈"S"型,最大增殖密度可达3.50×10~6 mL~(-1),群体倍增时间为26.2h;细菌、真菌及支原体检查均为阴性;血细胞吸附试验、致细胞病变试验和特异性病毒荧光抗体结合物检查结果均为阴性;致瘤性检查结果也为阴性。说明该细胞株符合兽用疫苗生产用细胞质量要求,可以在疫苗研究与生产中应用。     

静置培养MDCK细胞的生长及代谢动力学研究

为研究MDCK细胞的生长及代谢动力学特征,试验采用低密度静置培养,每天测定细胞生长密度和活力,并利用多参数生化分析仪测定培养液中Gluc、Lac、Gln和NH4+的含量,计算细胞指数生长期的比代谢速率。结果表明:MDCK细胞生长曲线呈"S"型,最大增殖密度为53.8×104 upf·mL-1,倍增时间为24.2h;对数生长期Gluc和Gln的比代谢(消耗速率为-2.20 mg·(106cells·d)-1和-3.85μmol·(106cells·d)-1,Lac和NH4+的比生成)速率为2.25mg·(106cells·d)-1和2.22μmol·(106cells·d)-1。     

奶牛脂肪来源间充质干细胞的分离培养及其生物学特性研究

[目的]建立奶牛脂肪间充质干细胞(AD-MSCs)体外分离培养体系,并进行增殖能力检测、分化能力鉴定及生物学特性研究,为推广AD-MSCs在畜牧生产及其疾病治疗等方面的应用提供理论依据.[方法]通过I型胶原酶消化法分离奶牛AD-MSCs并进行代传培养,绘制P3、P6和P9代奶牛AD-MSCs的生长曲线及测定其群体倍增时间,分别采用流式细胞术及RT-PCR检测细胞表面标志物;使用干细胞成骨/成脂诱导分化完全培养基进行奶牛AD-MSCs成骨成脂诱导分化,经茜素红和油红O染色后鉴定其成骨成脂分化能力;并检测冻存奶牛AD-MSCs复苏后的存活率.[结果]分离及传代培养获得的奶牛AD-MSCs在体外培养条件下呈长梭形,折光性强,生长状态良好,其生长曲线呈典型的S形,符合Logistic生长规律.奶牛AD-MSCs高表达MSCs表面标志物CD44、CD73、CD90和CD105,但不表达白细胞表面标志物CD45;分别使用干细胞成骨/成脂诱导分化完全培养基诱导的奶牛AD-MSCs经茜素红和油红O染色后,显微镜下可观察到大量的钙结节和红色脂肪滴.冻存6个月后进行细胞复苏,奶牛AD-MSCs的存活率高达96%;复苏奶牛AD-MSCs培养4 h内贴壁,呈典型的长梭形,生长状态良好,培养72 h细胞融合达80%~90%.[结论]通过I型胶原酶消化法从奶牛腹部脂肪组织分离获得的奶牛AD-MSCs具有良好的体外增殖能力及多向分化潜能,为畜牧生产研究及奶牛疾病治疗提供了一种良好的种子细胞来源.     

鸽骨骼肌卫星细胞的分离、培养及成肌特性

【目的】探讨白羽王鸽Columba livia骨骼肌卫星细胞的分离、培养和鉴定的方法,建立完整的家鸽骨骼肌卫星细胞的培养体系。【方法】选择孵化16 d的鸽胚作为试验材料,采用组织块贴壁法和胶原酶消化法分离胸肌的骨骼肌卫星细胞并绘制其生长曲线。待卫星细胞分化出肌管后,采用免疫荧光法检测肌球蛋白重链(MyHC)的表达;在细胞分化出肌管前、后分别提取总RNA,采用RT-qPCR方法检测Desmin、Pax7、MyoG和MyoD1基因在细胞分化出肌管前、后的相对表达量。【结果】组织块贴壁法和胶原酶法均能成功地分离出骨骼肌卫星细胞,其生长曲线呈"S"型;该细胞经含体积分数为20%FBS的DMEM高糖培养液培养7 d后视野中出现大量明显可见的肌管,成肌特异性标志MyHC表达呈阳性。RT-qPCR结果表明,Desmin和MyoG基因分化后的相对表达量分别是分化前的5.68和10.38倍,而Pax7和MyoD1基因分化前的相对表达量分别是分化后的7.01和5.51倍。【结论】建立了鸽骨骼肌卫星细胞的培养体系,为今后进行家鸽肌肉发育的研究提供细胞模型。     

‘马身猪’皮下前体脂肪细胞的分离培养及成脂诱导分化研究

旨在建立‘马身猪’皮下前体脂肪细胞的体外培养体系,探讨其分化过程中脂质合成及代谢相关基因的表达。对8日龄仔猪的颈部皮下脂肪组织进行采样,用Ⅰ型胶原酶进行消化,分离出原代前体脂肪细胞,进行传代和诱导分化;在此过程中观察细胞的形态、绘制细胞的生长曲线,对诱导后的细胞进行油红O染色,并采用qRTPCR法检测脂肪沉积相关基因PPARγ、LPL、FABP4、FABP3和脂肪代谢相关基因ATGL与HSL的mRNA表达变化规律。结果显示,分离出的细胞形态呈不规则梭形,生长曲线呈S型,诱导15d后在细胞内有大脂滴融合,经油红O染色呈红色;PPARγ、LPL、FABP4、FABP3和HSL基因在诱导2d时表达量达到最高,极显著高于其他时间段(P 0.01),随后降低,到诱导10d时表达量与0d时基本无差异(P0.05)。ATGL基因表达量在诱导2d时与对照组0d相比无差异(P0.05),诱导4d后极显著降低(P 0.01),诱导8d时表达量最低。     

延黄牛前体脂肪细胞的培养及生物学特性研究

近几年来延黄牛因肉质优良、口味独特、有典型的大理石花纹等特点入选为我国五大良种牛之一,越来越多地受到消费者的青睐。为建立延黄牛前体脂肪细胞的体外培养体系,从细胞和分子水平探索延黄牛脂肪细胞的生物学特性及潜在的分化机制,采用胶原酶消化法对延黄牛皮下前体脂肪细胞进行分离,对其进行形态学观察并绘制生长曲线,油红O染色法检测成脂诱导分化过程中的脂质积累,利用甘油三酯酶法测定细胞内甘油三酯,采用实时荧光定量RT-PCR的方法检测脂肪细胞成脂标志基因过氧化物酶体增殖体激活受体γ(PPARγ)、CCAAT/增强子结合蛋白α(C/EBPα)的mRNA在延黄牛前体脂肪细胞分化过程中的表达。结果表明:经过分离的延黄牛原代脂肪细胞可贴壁生长,传代后成分均一贴壁良好,呈带角的梭型、形似成纤维细胞;第2天开始细胞进入对数生长期,第4天以后为细胞生长的平台期,增殖速度明显变慢,细胞生长曲线近视"S"形,符合正常的细胞生长规律;经诱导分化后的细胞可被油红O染色,呈现典型的圆形脂滴,具有成熟脂肪细胞的典型特征;甘油三酯含量在第0~4天变化缓慢,在第4天以后显著升高;成脂标志基因PPARγ、C/EBPα随着分化过程的进行,在分化早期显著上升,随着分化进程的结束略有下降。综上所述,本研究成功建立了延黄牛前体脂肪细胞的分离培养方法,为进一步研究延黄牛脂肪沉积作用机制以及改善牛肉品质奠定了基础。     

钙调素拮抗剂三氟拉嗪对犬肾上皮细胞增殖的影响

为探讨钙调素拮抗剂三氟拉嗪(TFP)对犬肾上皮细胞(MDCK)增殖的抑制作用及对细胞各周期时相的影响,采用细胞计数法、流式细胞术、[γ-32P]ATP掺入外源性底物的液体闪烁法和免疫组化技术,分别测定5、10和15μmol/L浓度的TFP对MDCK细胞增殖的抑制作用、细胞各周期时相的百分率、胸苷激酶(TK)的活性以及增殖细胞核抗原(PCNA)表达的阳性率。结果表明:给药3 d后,各浓度TFP对MDCK细胞生长均具有明显抑制作用;G0/G1期的细胞比率均显著增加(P<0.05),而S期、G2/M期细胞的比率则有所下降,其中高浓度TFP组显著下降(P<0.05);细胞内TK的活性和PCNA的表达显著降低,呈明显的时间剂量效应关系。说明TFP可通过阻断钙调素信号通路,影响DNA合成和细胞周期而抑制MDCK细胞的增殖。     

稳定表达鸡氨肽酶N的MDCK细胞系构建及其对传染性支气管炎病毒感染的影响

为研究鸡氨肽酶N(Chicken aminopeptidase N,c APN)在鸡传染性支气管炎病毒(IBV)感染细胞过程中作用,构建稳定表达c APN的MDCK细胞系。将重组质粒pc DNA3.1-c APN通过脂质体转染到MDCK细胞中并利用G418筛选,克隆纯化获得稳定表达c APN的MDCK细胞系。通过RT-PCR和间接免疫荧光检测表明c APN在MDCK细胞中持续稳定表达。IBV可以感染稳定表达c APN的MDCK细胞并连续传代,该细胞系接种IBV阳性样品24 h即可产生典型细胞融合病变,而对照MDCK细胞接种后没有细胞病变出现。结果表明c APN在介导IBV感染宿主细胞过程中发挥重要作用。     

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.     

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.     

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。     

河曲马睾丸组织成纤维细胞系的建立及生物学特性研究

采集河曲马睾丸组织,用热消化法制备原代细胞,通过差速消化和差速贴壁法纯化细胞,扩大培养至第3代用液氮保存,细胞复苏后进行活力、形态、生长曲线、微生物污染、核型、乳酸脱氢同工酶谱及荧光蛋白质粒转染表达等特性研究。结果显示,河曲马睾丸组织原代和传代细胞呈成纤维型,生长良好,最大增殖数量为4.32×105 mL-1,倍增时间为34.58h;细菌、真菌、病毒、支原体检测呈阴性;染色体2 N=64,二倍体为85%,占主体;乳酸脱氢酶同工酶电泳图谱有明显特征,共有4条谱带,其中LDH3浓度较高,活性较强;外源质粒在该细胞中能进行复制和表达。表明已成功建立河曲马睾丸组织成纤维细胞系,使河曲马这一重要种质资源在细胞水平上得以保存。     

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.     

胎牛血清和新生牛血清体外培养动物细胞的效果比较

为了比较进口胎牛血清、国产胎牛血清和国产新生牛血清的细胞培养效果,以2批进口胎牛血清、3批国产胎牛血清和2批国产新生牛血清为试验材料,采用细胞贴壁传代培养、最大增殖浓度和倍增时间以及集落形成率3种方法,比较了7批供试血清对VERO、CHO-K1、MDCK和Hela 4种细胞的培养效果。结果表明:连续传代培养时所有牛血清均有较好的促细胞生长的效果;进口胎牛血清、国产胎牛血清和国产新牛血清对4种细胞的平均最大增殖密度为67.8×104、62.1×104和64.8×104 cfu·mL-1,平均倍增时间为22.2、22.5和22.7h;对VERO、CHO-K1和Hela细胞的平均集落形成率为59.2%、57.6%和50.9%。3种牛血清对群体性细胞培养时效果差别不明显,对单细胞克隆培养时胎牛血清优于新生牛血清。     

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。     

转拟蜘蛛牵丝蛋白基因新疆美利奴细毛羊成纤维细胞系的建立及初步鉴定

[目的]建立合有拟蜘蛛牵丝蛋白基因的新疆美利奴细毛羊成纤维细胞系.[方法]采用脂质体转染,将表达拟蜘蛛牵丝基因的质粒pKap-EGFP4S转入新疆美利奴细毛羊成纤维细胞,通过G418毒性筛选获得阳性细胞系,并采用PCR、RT-PCR检测重组细胞中的目的基因表达.[结果]对体外分离培养的成纤维细胞观察表明细胞形态均为长梭形、活性良好（细胞生长曲线呈S型）、染色体数目为2n =54;通过G418筛选出共43株单克隆细胞系,经PCR鉴定拟蜘蛛牵丝蛋白基因随机整合的细胞系15株,其中挑选4个阳性细胞系经过RT-PCR检测目的基因已转录为mRNA.[结论]成功建立了拟蜘蛛牵丝蛋白基因的新疆美利奴细毛羊成纤维细胞系,为进一步通过体细胞核移植技术制备被毛含转拟蜘蛛牵丝蛋白基因克隆羊奠定基础.     

建立猪囊尾蚴细胞系的研究

为建立具有免疫原性的猪囊尾蚴细胞系,试验对猪囊尾蚴细胞进行了体外培养,并将建立的细胞系命名为猪囊尾蚴CC-97免疫细胞系。猪囊尾蚴细胞原代培养30d后形成单层细胞,然后将单层细胞以1：2分种率传代培养,相隔7d传代一次,连传24代。结果表明：光镜下观察发现,细胞系由3种类型细胞组成,以椭圆形细胞占优势,梨形、球形细胞数量接近。放射自显影法测定细胞分裂情况,3H—TdR显示细胞生长指数,结果均表明,细胞对数增殖期长达7d,细胞从12h开始倍增分裂,第四、五天相继达到倍增分裂高峰,细胞增殖率达11倍,增量达6．32X10^（9）／L,后来达到1：37的分种率,细胞数为3．7X10^（10）／L;细胞周期约为32h,细胞系以二倍体细胞为主,染色体有10对2条,细胞蛋白质组分有30个区带,酯酶同工酶谱显示一条区带,分子量约32kDa;细胞系经荧光抗体检测表明,与猪囊尾蚴具有同源性,致肿瘤性与外源污染检验均为阴性。结果提示,建立的猪囊尾蚴细胞系是一个形态均一、生长旺盛、遗传性稳定、免疫原性高、无污染、无致肿瘤性、与猪囊尾蚴同源的生物学新品系。     

奶山羊乳腺上皮细胞系的建立

乳腺上皮细胞系的建立为乳蛋白基因表达调控机制的研究及乳腺表达载体的检测提供有利条件。本研究建立了正常培养的奶山羊乳腺上皮细胞系。利用胶原酶和透明质酸消化法从奶山羊乳腺组织中分离并获得了纯化的乳腺上皮细胞,利用细胞角蛋白18的免疫荧光染色对乳腺上皮细胞进行了鉴定。通过细胞生长曲线、群体倍增时间、细胞接种存活率、细胞活力检测等生物学性状的检测建立并鉴定了奶山羊乳腺上皮细胞系。结果表明,奶山羊乳腺上皮细胞在添加表皮生长因子、胰岛素样生长因子-1、转铁蛋白-硒钠、10%胎牛血清的DF12培养液中生长状态良好,细胞传至30代时仍保持旺盛的增殖活力。通过细胞传代及反复冻存和复苏实现了细胞系的长期保存,建立了乳腺上皮细胞系,获得了大量的乳腺上皮细胞。从而为开展奶山羊、奶牛等产奶动物乳腺生物反应器及泌乳机制的研究提供了便利条件。