講稿文獻(xiàn)學(xué)位

1、分類號(hào)密級(jí) UDC 幾種重要海水養(yǎng)殖魚類細(xì)胞系的建立、鑒定及應(yīng)用 任 國 誠 陳 松 林 研究員 指導(dǎo)教師申請(qǐng)學(xué)位級(jí)別博士專業(yè)名稱海洋生物答辯日期2007 年 6 月 學(xué)位授予日期 中 國 海 洋 大 學(xué)幾種重要海水養(yǎng)殖魚類細(xì)胞系的建立、鑒定及應(yīng)用完成日期： 指導(dǎo)教師簽字： 答辯委員會(huì)成員簽字： 摘要魚類細(xì)胞培養(yǎng)的研究始于 20 世紀(jì)界上第一個(gè)魚類細(xì)胞系，即虹鱒魚60 年代，由 Wolf 和 Quimby 建立了世細(xì)胞系 RTG22，此后，魚類的細(xì)胞培養(yǎng)研究進(jìn)展十分迅速。目前，魚類細(xì)胞培養(yǎng)技術(shù)已成為一種重要的研究，廣泛應(yīng)用于學(xué)、環(huán)境毒理學(xué)、細(xì)胞生物學(xué)、腫瘤學(xué)、組學(xué)、遺傳學(xué)以及資源保護(hù)等方面。魚

2、類培養(yǎng)細(xì)胞作為實(shí)驗(yàn)對(duì)象，有著魚無法比擬的優(yōu)點(diǎn)：(1) 材料容易獲得，成本低；(2)重復(fù)性好，實(shí)驗(yàn)條件可以精確控制。因此，對(duì)魚類細(xì)胞系進(jìn)行深入研究，無論在理論研究方面, 還是在實(shí)際應(yīng)用方面，都具有深遠(yuǎn)意義。牙鲆（Paralichthys olivaceus ）、大菱鲆（Scophthalmus本研究探索了us）、漠斑牙鲆（Pleuronectiformes）胚胎細(xì)胞分離、培養(yǎng)條件，建立了魚類胚胎細(xì)胞培養(yǎng)技術(shù)。建立了牙鲆胚胎細(xì)胞系（FEC）、大菱鲆胚胎細(xì)胞系（TEC） 和漠斑牙鲆胚胎細(xì)胞系（SFEC），并用酶消化法建立了半滑舌鰨（Cynoglossussemilaevis）肝臟細(xì)胞系（HTLC）。

3、牙鲆胚胎細(xì)胞系已傳至100多代，大菱鲆胚胎細(xì)胞系傳至100多代，漠斑牙鲆胚胎細(xì)胞系傳至80多代，半滑舌鰨肝臟細(xì)胞系傳至30多代。胚胎細(xì)胞系完全培養(yǎng)基采用添加抗生素、胎牛血清（FBS）、花鱸血清（SPS）、成纖維生長因子（bFGF）的DMEM，半滑舌鰨肝臟細(xì)胞系完全培養(yǎng)基添加了抗生素、胎牛血清（FBS）、花鱸血清（SPS）、成纖維生長因子（bFGF）的MEM。胚胎細(xì)胞形態(tài)小而呈多邊形、多角形、圓形，半滑舌鰨肝臟細(xì)胞系呈纖維狀。本實(shí)驗(yàn)檢測(cè)了FBS、bFGF、溫度對(duì)細(xì)胞生長的影響。在一定范圍內(nèi)，細(xì)胞生長速度隨FBS濃度的增高而加快，F(xiàn)BS濃度過高或過低都抑制細(xì)胞的增殖。bFGF對(duì)FEC、TEC、SF

4、EC、HTLC細(xì)胞生長均具有刺激作用，細(xì)胞的速度在含bFGF的培養(yǎng)基中比在bFGF的培養(yǎng)基中要快的多。在2430之間細(xì)胞生長良好，當(dāng)溫度高于30或低于12時(shí)，細(xì)胞生長速度降低。FEC的二倍體核型為2n=48t，TEC的二倍體核型為2n=4m+12st+28t，SFEC的二倍體核型為2n=6st+42t，HTLC的二倍體核型為2n=42t，半滑舌鰨雌魚中期相具（ZW），雄魚中期（ZZ）。將GFP報(bào)告基有異型相具有同型因通過脂（genejammer）介導(dǎo)的方法轉(zhuǎn)入SFEC和HTLC中并成功地獲得了表達(dá)，轉(zhuǎn)化率為20%左右。用牙鲆淋巴囊腫（LCV）和大菱鲆虹彩TRBV）顆粒。用FEC，觀察到了細(xì)胞病

5、變效應(yīng)（CPE），在細(xì)胞內(nèi)發(fā)現(xiàn)了大量鰻弧菌處理TEC，檢測(cè)了不同時(shí)間抗菌肽（Hepcidin）表達(dá)的變化。用HTLC，通過熒光原位雜交（Fluorescence in situ hybridization，F(xiàn)ISH）技術(shù)了半滑舌鰨特異片段，在雌魚W上獲得了特異雜交信號(hào)?？傊疚慕⒘唆~類胚胎細(xì)胞分離培養(yǎng)技術(shù)，在世界上首次建立了牙鲆胚胎細(xì)胞系，大菱鲆胚胎細(xì)胞系和漠斑牙鲆胚胎細(xì)胞系，同時(shí)，用傳統(tǒng)方法建立了半滑舌鰨肝臟細(xì)胞系。在細(xì)胞系應(yīng)用方面做了一些探索，為進(jìn)一步利用細(xì)胞系進(jìn)行基礎(chǔ)理論和實(shí)驗(yàn)研究奠定了基礎(chǔ)。：胚胎細(xì)胞系 核型 熒光原位雜交 GFP 報(bào)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目錄1. 綜 述 ·························&#

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

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22、9101011111112141415161920232427271.2 魚類細(xì)胞培養(yǎng)研究進(jìn)展及細(xì)胞系的應(yīng)用 ································1.2.1 魚類細(xì)胞培養(yǎng)研究進(jìn)展······&

23、#183;·························1.2.2 魚類細(xì)胞系在各領(lǐng)域的應(yīng)用·····················&

24、#183;··········學(xué)發(fā)展概況和魚類細(xì)胞系的應(yīng)用 ····································

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30、3;······························毒理學(xué) ···················

31、·············魚類腫瘤 ································生理學(xué) ···

32、83;····························免疫學(xué) ·····················

33、;···········遺傳學(xué) ································內(nèi)學(xué) ·····

34、3;··························保護(hù) ·······················&

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39、#183;································抗差減 cDNA 文庫的構(gòu)建 ··············

40、3;·················顯微注射 ································

41、;打靶受體細(xì)胞 ································環(huán)境質(zhì)量監(jiān)測(cè) ···············

42、83;················功能的篩選 ································細(xì)胞轉(zhuǎn)

43、化 ································細(xì)胞遺傳圖 ················

44、3;···············細(xì)胞融合技術(shù)································RNA 干擾技術(shù) &

45、#183;·······························FISH 技術(shù) ·················

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54、;························22.1牙鲆胚胎細(xì)胞系的建立、鑒定及應(yīng)用······················

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

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

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60、83;··2828282.2.1 實(shí)驗(yàn)材料 ································2.2.2 主要儀器和試劑··········

61、83;··························2.2.3牙鲆胚胎細(xì)胞的原代培養(yǎng)和繼代培養(yǎng) ····················

62、;············2.2.4 不同溫度對(duì) FEC 生長的影響··································&#

63、183;········2829292930313131333335······················2.2.52.2.62.2.72.2.8不同濃度 FBS 和 bFGF 對(duì)細(xì)胞生長的影響········&#

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

65、3;··········實(shí)驗(yàn)································透射電鏡觀察······&#

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70、183;···············2.3 結(jié)果 ·································&

71、#183;····················2.3.12.3.22.3.3胚胎細(xì)胞系的建立·························&#

72、183;········溫度對(duì) FEC 生長的影響·······································&

73、#183;···················不同濃度的 FBS 和生長因子對(duì) FEC 生長的影響 ·························

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

75、;································2.3.5和電鏡觀察················&

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

78、183;··································3 大菱鲆胚胎細(xì)胞系的建立、鑒定與應(yīng)用 ············

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

80、83;······3.2 材料和方法 ································3.2.1 實(shí)驗(yàn)材料 ········

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85、183;·····3.2.23.2.33.2.4主要儀器和試劑········································

86、3;···大菱鲆胚胎細(xì)胞的原代培養(yǎng)和繼代培養(yǎng) ································不同溫度對(duì) TEC 生長的影響·········&

87、#183;···········································3.2.5 不同濃度 FBS 和 bFGF 對(duì)細(xì)胞生長的影響 ·

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89、183;··················3.2.7 利用細(xì)胞系檢測(cè)抗病3.3 結(jié)果 ···························

90、3;····································的表達(dá) ·············

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93、···3.3.13.3.23.3.3胚胎細(xì)胞系的建立································溫度對(duì) TEC 生長的影響·········

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95、··································不同濃度的 FBS 和生長因子對(duì) TEC 生長的影響 ···········

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

97、3;··············································3.3.5 不同濃度鰻弧菌處理后抗菌肽的表達(dá)

98、 ····································513.4 討論 ············&#

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100、183;·······················514 漠斑鲆胚胎細(xì)胞系的建立與鑒定·······················&#

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

102、;······4.2 材料和方法································4.2.1 實(shí)驗(yàn)材料 ········

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107、;·····4.2.24.2.34.2.44.2.54.2.64.2.7主要儀器和試劑·····································原代培養(yǎng)和繼代培養(yǎng)&

108、#183;·······························溫度對(duì)細(xì)胞生長的影響················

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110、83;························不同濃度FBS和bFGF對(duì)細(xì)胞生長的影響······················&

111、#183;················分析································細(xì)胞轉(zhuǎn)化&

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116、#183;4.3 實(shí)驗(yàn)結(jié)果 ································4.3.1 胚胎細(xì)胞系的建立·············&#

117、183;····················4.3.2 溫度對(duì)細(xì)胞生長的影響··························

118、3;·································58596062634.3.3 FBS和各種生長因子濃度對(duì)細(xì)胞生長的影響 ··········

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

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

121、;···4.3.5 GFP在SFEC中的表達(dá) ···········································

122、3;···································4.4 討論 ·············

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

124、83;5 半滑舌鰨肝臟細(xì)胞系的建立、鑒定與應(yīng)用································665.1 引言 ············

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

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

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

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

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130、183;······················主要儀器和試劑··························&

131、#183;··········原代培養(yǎng)和繼代培養(yǎng)································溫度對(duì)細(xì)胞生長的影響····

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

133、·····································不同濃度FBS和bFGF對(duì)細(xì)胞生長的影響·········

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

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

138、3;·······························5.3 實(shí)驗(yàn)結(jié)果 ·················&

139、#183;··············5.3.15.3.25.3.35.3.4半滑舌鰨肝臟細(xì)胞系的建立······························

140、;··溫度對(duì)細(xì)胞生長的影響································不同濃度FBS和bFGF對(duì)細(xì)胞生長的影響···········

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

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

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

144、#183;········5.3.5 GFP在HTLC中的表達(dá) ······································

145、········································5.4 討論 ·········

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

147、;·····6 半滑舌鰨細(xì)胞系在FISH技術(shù)中的應(yīng)用·····································776.1 引言 ···

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

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161、料································主要儀器和試劑················

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

163、3;·································6.2.3.16.2.3.2雄魚··············

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

165、······································雌魚6.2.46.2.56.2.6探針的········

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167、183;·····························6.2.6.1 變性 ··················

168、83;·············6.2.6.2 雜交 ································6.2.6.3 洗脫 ·

169、;·······························6.3 結(jié)果 ·················

170、3;··············6.3.1 探針標(biāo)記效果檢測(cè)································6.3.1 雜交

171、結(jié)果································6.4 討論 ·················