讲稿文献学位

1、分类号密级 UDC 几种重要海水养殖鱼类细胞系的建立、鉴定及应用 任 国 诚 陈 松 林 研究员 指导教师申请学位级别博士专业名称海洋生物答辩日期2007 年 6 月 学位授予日期 中 国 海 洋 大 学几种重要海水养殖鱼类细胞系的建立、鉴定及应用完成日期： 指导教师签字： 答辩委员会成员签字： 摘要鱼类细胞培养的研究始于 20 世纪界上第一个鱼类细胞系，即虹鳟鱼60 年代，由 Wolf 和 Quimby 建立了世细胞系 RTG22，此后，鱼类的细胞培养研究进展十分迅速。目前，鱼类细胞培养技术已成为一种重要的研究，广泛应用于学、环境毒理学、细胞生物学、肿瘤学、组学、遗传学以及资源保护等方面。鱼

2、类培养细胞作为实验对象，有着鱼无法比拟的优点：(1) 材料容易获得，成本低；(2)重复性好，实验条件可以精确控制。因此，对鱼类细胞系进行深入研究，无论在理论研究方面, 还是在实际应用方面，都具有深远意义。牙鲆（Paralichthys olivaceus ）、大菱鲆（Scophthalmus本研究探索了us）、漠斑牙鲆（Pleuronectiformes）胚胎细胞分离、培养条件，建立了鱼类胚胎细胞培养技术。建立了牙鲆胚胎细胞系（FEC）、大菱鲆胚胎细胞系（TEC） 和漠斑牙鲆胚胎细胞系（SFEC），并用酶消化法建立了半滑舌鳎（Cynoglossussemilaevis）肝脏细胞系（HTLC）。

3、牙鲆胚胎细胞系已传至100多代，大菱鲆胚胎细胞系传至100多代，漠斑牙鲆胚胎细胞系传至80多代，半滑舌鳎肝脏细胞系传至30多代。胚胎细胞系完全培养基采用添加抗生素、胎牛血清（FBS）、花鲈血清（SPS）、成纤维生长因子（bFGF）的DMEM，半滑舌鳎肝脏细胞系完全培养基添加了抗生素、胎牛血清（FBS）、花鲈血清（SPS）、成纤维生长因子（bFGF）的MEM。胚胎细胞形态小而呈多边形、多角形、圆形，半滑舌鳎肝脏细胞系呈纤维状。本实验检测了FBS、bFGF、温度对细胞生长的影响。在一定范围内，细胞生长速度随FBS浓度的增高而加快，FBS浓度过高或过低都抑制细胞的增殖。bFGF对FEC、TEC、SF

4、EC、HTLC细胞生长均具有刺激作用，细胞的速度在含bFGF的培养基中比在bFGF的培养基中要快的多。在2430之间细胞生长良好，当温度高于30或低于12时，细胞生长速度降低。FEC的二倍体核型为2n=48t，TEC的二倍体核型为2n=4m+12st+28t，SFEC的二倍体核型为2n=6st+42t，HTLC的二倍体核型为2n=42t，半滑舌鳎雌鱼中期相具（ZW），雄鱼中期（ZZ）。将GFP报告基有异型相具有同型因通过脂（genejammer）介导的方法转入SFEC和HTLC中并成功地获得了表达，转化率为20%左右。用牙鲆淋巴囊肿（LCV）和大菱鲆虹彩TRBV）颗粒。用FEC，观察到了细胞病

5、变效应（CPE），在细胞内发现了大量鳗弧菌处理TEC，检测了不同时间抗菌肽（Hepcidin）表达的变化。用HTLC，通过荧光原位杂交（Fluorescence in situ hybridization，FISH）技术了半滑舌鳎特异片段，在雌鱼W上获得了特异杂交信号。总之，本文建立了鱼类胚胎细胞分离培养技术，在世界上首次建立了牙鲆胚胎细胞系，大菱鲆胚胎细胞系和漠斑牙鲆胚胎细胞系，同时，用传统方法建立了半滑舌鳎肝脏细胞系。在细胞系应用方面做了一些探索，为进一步利用细胞系进行基础理论和实验研究奠定了基础。：胚胎细胞系 核型 荧光原位杂交 GFP 报告The Establishment, Iden

6、tification and Application of Cell Lines from Several Important Marine FishAbstractThe research of fish cell culture has developed rapidly since 1960s when Wolf and Quimby established the first fish cell line- RTG22 in the world. At the presenttime, fish cell culturing hase an essential research tec

7、hnology which has beenused extensively, ranged from virology, environmental toxicology, cytobiology, oncology, genomics, genetics and environmental protection. Cultured fish cells have more advantages than live fish as the experimental material: 1. the materials are cheap and easy to obtain; 2. the

8、experimental condition could be controlled accurately and the experiment could be repeated.In the present study, the culture methods of fish embryonic cell had beenresearched deeply. The results were as follows.1.Three embryonic cell lines had been established: Japanese flounder (Paralichthys olivac

9、eus) embryonic cell line (FEC), turbot embryonic cell line (TEC) and Southern Flounder embryonic cell line (SFEC). At the same time, a liver cell line of half-smooth tongue-sole（Cynoglossus semilaevis) (HTLC) was established. FEC has been cultured for more than 100 passages and the same as the TEC.

10、SFEC has been cultured for more than 80 passages while HTLC has been cultured for more than 30 passages.The embryonic cells were cultured in DMEM medium supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), basic fibroblast growth factor (bFGF). The HTLC cells were cultured

11、 in MEM medium supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), basic fibroblast growth factor (bFGF).The embryonic cells are small and polygon, rotundity. The liver cells of half-smooth tongue-sole are fibriform. Effect of temperature, fetal bovine serum (FBS) concent

12、ration and bFGF on the growth of cells was examined. To some extent, the growth rate of cellsdepends on FBS concentration, and can be restrained by higher or lower FBS2.concentration. Addition of bFGF to the medium can significantly increase the growth rate of cells. The cells grow well in at the te

13、mperature range of 24-30, but have a reduce growth rate at temperature below 18 or above 30.Chromosome analysis reveals that FEC cells have a normal diploid karyotype with 2n=48t; TEC cells have a normal diploid karyotype with 2n=4m+12st+28t; SFEC cells have a normal diploid karyotype with 2n=6st+42

14、t; HTLC cells have a normal diploid karyotype with 2n=42t. Heterotypic sex chromosome (ZW) was investigated on the metaphase chromosome of half-smooth tongue-sole () and homotypic sex chromosome were investigated on the metaphase chromosome of half-smooth tongue-sole ().GFP reporter gene were transf

15、erred into SFEC and HTLC cells with Genejammer-mediated and successfully expressed in two cell lines. Thetransformation efficiency was about twenty percent. FEC cells were injected by3.4.Flounder lymphocystis diseaseLCV）and Turbot reddish body irido(TRBV).replicated rapidly in FEC and the cytopathic

16、 effect (CPE) wasinvestigated,. The mRNA expression levels of Hepcidin gene in TEC after the challenge of Pichia pastoris were measured by semi-quantitative RT-PCR. Sex-specific bands located on the W sex chromosome of females of half-smooth tongue-sole by fluorescence in situ hybridization (FISH) a

17、nalysis.In summary, the technology of establishing embryonic cell lines is founded. it is the first time that FEC, TEC and SFEC were established in the world while the HTLC was established by traditional method. Some research was explored in the application of cell lines and laid foundation of using

18、 cell lines to study theory and experiment.Key Words: embryonic cell lines, karyotype, fluorescence in situ hybridization,GFP reporter gene目录1. 综 述 ·························&#

19、183;······1.1 前言··········································&#

20、183;··········································1·······&

21、#183;················································1224478

22、9101011111112141415161920232427271.2 鱼类细胞培养研究进展及细胞系的应用 ································1.2.1 鱼类细胞培养研究进展······&

23、#183;·························1.2.2 鱼类细胞系在各领域的应用·····················&

24、#183;··········学发展概况和鱼类细胞系的应用 ····································

25、3;·················································

26、3;·················································

27、3;·················································

28、3;·················································

29、3;·················································

30、3;······························毒理学 ···················

31、·············鱼类肿瘤 ································生理学 ···

32、83;····························免疫学 ·····················

33、;···········遗传学 ································内学 ·····

34、3;··························保护 ·······················&

35、#183;·················································&

36、#183;·················································&

37、#183;·················································&

38、#183;·················································&

39、#183;································抗差减 cDNA 文库的构建 ··············

40、3;·················显微注射 ································

41、;打靶受体细胞 ································环境质量监测 ···············

42、83;················功能的筛选 ································细胞转

43、化 ································细胞遗传图 ················

44、3;···············细胞融合技术································RNA 干扰技术 &

45、#183;·······························FISH 技术 ·················

46、;··················································

47、;··················································

48、;··················································

49、;··················································

50、;··················································

51、;··················································

52、;··················································

53、;··················································

54、;························22.1牙鲆胚胎细胞系的建立、鉴定及应用······················

55、83;·········引言 ········································

56、··············································2.2材料和方法 ··

57、83;·················································

58、83;·················································

59、83;·················································

60、83;··2828282.2.1 实验材料 ································2.2.2 主要仪器和试剂··········

61、83;··························2.2.3牙鲆胚胎细胞的原代培养和继代培养 ····················

62、;············2.2.4 不同温度对 FEC 生长的影响··································&#

63、183;········2829292930313131333335······················2.2.52.2.62.2.72.2.8不同浓度 FBS 和 bFGF 对细胞生长的影响········&#

64、183;···························核型分析·····················

65、3;··········实验································透射电镜观察······&#

66、183;·················································&#

67、183;·················································&#

68、183;·················································&#

69、183;·················································&#

70、183;···············2.3 结果 ·································&

71、#183;····················2.3.12.3.22.3.3胚胎细胞系的建立·························&#

72、183;········温度对 FEC 生长的影响·······································&

73、#183;···················不同浓度的 FBS 和生长因子对 FEC 生长的影响 ·························

74、3;··2.3.4 核型分析 ··············································

75、;································2.3.5和电镜观察················&

76、#183;·················································&

77、#183;··················36392.4 讨论 ·····························&#

78、183;··································3 大菱鲆胚胎细胞系的建立、鉴定与应用 ············

79、·······················423.1 引言 ·························

80、83;······3.2 材料和方法 ································3.2.1 实验材料 ········

81、··················································

82、··················································

83、··················································

84、························4243434343444444454646474849·····················&#

85、183;·····3.2.23.2.33.2.4主要仪器和试剂········································

86、3;···大菱鲆胚胎细胞的原代培养和继代培养 ································不同温度对 TEC 生长的影响·········&

87、#183;···········································3.2.5 不同浓度 FBS 和 bFGF 对细胞生长的影响 ·

88、···································3.2.6 核型分析·············&#

89、183;··················3.2.7 利用细胞系检测抗病3.3 结果 ···························

90、3;····································的表达 ·············

91、··················································

92、··················································

93、···3.3.13.3.23.3.3胚胎细胞系的建立································温度对 TEC 生长的影响·········

94、··················································

95、··································不同浓度的 FBS 和生长因子对 TEC 生长的影响 ···········

96、83;················3.3.4 核型分析 ·······························

97、3;··············································3.3.5 不同浓度鳗弧菌处理后抗菌肽的表达

98、 ····································513.4 讨论 ············&#

99、183;·················································&#

100、183;·······················514 漠斑鲆胚胎细胞系的建立与鉴定·······················&#

101、183;······················544.1 引言 ··························

102、;······4.2 材料和方法································4.2.1 实验材料 ········

103、83;·················································

104、83;·················································

105、83;·················································

106、83;·······················5455555555555656575757·······················

107、;·····4.2.24.2.34.2.44.2.54.2.64.2.7主要仪器和试剂·····································原代培养和继代培养&

108、#183;·······························温度对细胞生长的影响················

109、83;·················································

110、83;························不同浓度FBS和bFGF对细胞生长的影响······················&

111、#183;················分析································细胞转化&

112、#183;·················································&

113、#183;·················································&

114、#183;·················································&

115、#183;·················································&

116、#183;4.3 实验结果 ································4.3.1 胚胎细胞系的建立·············&#

117、183;····················4.3.2 温度对细胞生长的影响··························

118、3;·································58596062634.3.3 FBS和各种生长因子浓度对细胞生长的影响 ··········

119、83;·······················4.3.4 核型分析·························

120、;··················································

121、;···4.3.5 GFP在SFEC中的表达 ···········································

122、3;···································4.4 讨论 ·············

123、83;·················································

124、83;5 半滑舌鳎肝脏细胞系的建立、鉴定与应用································665.1 引言 ············

125、3;···················5.2 材料和方法·····························&

126、#183;·················································&

127、#183;·················································&

128、#183;·················································&

129、#183;··66676767676768686969····························5.2.15.2.25.2.35.2.45.2.55.2.65.2.7实验材料·········&#

130、183;······················主要仪器和试剂··························&

131、#183;··········原代培养和继代培养································温度对细胞生长的影响····

132、··················································

133、·····································不同浓度FBS和bFGF对细胞生长的影响·········

134、3;·····························分析····················&#

135、183;···········细胞转化·····································

136、3;·················································

137、3;·················································

138、3;·······························5.3 实验结果 ·················&

139、#183;··············5.3.15.3.25.3.35.3.4半滑舌鳎肝脏细胞系的建立······························

140、;··温度对细胞生长的影响································不同浓度FBS和bFGF对细胞生长的影响···········

141、;··················································

142、;·····················697070727474·······核型分析···················&

143、#183;·················································&

144、#183;········5.3.5 GFP在HTLC中的表达 ······································

145、········································5.4 讨论 ·········

146、;··················································

147、;·····6 半滑舌鳎细胞系在FISH技术中的应用·····································776.1 引言 ···

148、·····························6.2 材料和方法 ···················&#

149、183;·················································&#

150、183;·················································&#

151、183;·················································&#

152、183;·················································&#

153、183;·················································&#

154、183;·················································&#

155、183;·················································&#

156、183;·················································&#

157、183;·················································&#

158、183;·················································&#

159、183;·················································&#

160、183;····························7778787879797980808181818182828384·············6.2.16.2.26.2.3材

161、料································主要仪器和试剂················

162、3;·················································

163、3;·································6.2.3.16.2.3.2雄鱼··············

164、··················································

165、······································雌鱼6.2.46.2.56.2.6探针的········

166、3;··封闭 DNA 的···································荧光原位杂交··········&#

167、183;·····························6.2.6.1 变性 ··················

168、83;·············6.2.6.2 杂交 ································6.2.6.3 洗脱 ·

169、;·······························6.3 结果 ·················

170、3;··············6.3.1 探针标记效果检测································6.3.1 杂交

171、结果································6.4 讨论 ·················