昆虫细胞系的建立及其生物学特性与应用研究
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摘要
昆虫细胞培养技术作为细胞工程基础之一,是现代实验生物学上极有价值的手段之一,广泛应用于生物学、医学及农业的各个领域。本文主要从昆虫细胞培养、细胞系建立、昆虫细胞系建立过程的生物学特性及昆虫细胞系利用等方面进行研究,通过研究为昆虫细胞系的建立提供有价值的技术手段,了解昆虫细胞在建系过程中发生的生物学变化规律,为昆虫细胞的研究与利用提供重要的理论依据。主要研究结果如下:
1.不同目昆虫细胞培养技术。通过对来源于直翅目的短翅鸣螽(Gampsocleis gratiosa Brunner von Wattenwyl)、东亚飞蝗(Locusta migratoria manilensis Meyen);鳞翅目的柑橘凤蝶(Papilio xuthus Linnaeus)、枯叶蛱蝶(Kallima inachus Doubleday)、金斑蝶(Danaus chrysippus Linnaeus);鞘翅目的喙尾琵琶甲(Blaps rhynchoptera Fairmaire)、黄粉虫(Tenebrio molitor Linnaeus)的多种组织原代培养条件研究,得出各种组织原代培养时的适宜培养条件,研究结果显示不同目昆虫细胞培养所适宜的条件不同,(1)直翅目昆虫细胞培养的消毒时间为3min,来源于5d胚胎组织的细胞较易取得传代,适宜于改良Grace及Schneider培养基及10%Hyclone+10%民海血清的搭配中培养,培养基的更换时间在30d左右。(2)鳞翅目昆虫细胞培养宜的消毒时间为3min左右,来源于3-4d胚胎、新孵幼虫、蛹血淋巴、蛹脂肪体组织的细胞较易取得传代,适宜于在改良Grace及Grace培养基及10%Hyclone+10%民海血清的搭配中培养,培养基的更换时间在15-20d之间。(3)鞘翅目昆虫培养的消毒时间为5-8min,来源于新孵幼虫、精巢、脂肪体组织的细胞较易取得传代,适宜于改良Grace及Schneider培养基及10%Hyclone+10%民海血清的搭配中培养,培养基的更换时间在25-30d之间。
2.昆虫细胞系的建立。在上述培养技术和条件研究基础上对多种昆虫组织进行培养,8种昆虫细胞发生增殖而进行传代培养,但大部分细胞在传至一定代数后停止增殖,使传代不能正常的进行下去。其中1个柑橘凤蝶新孵幼虫细胞及3个短翅鸣螽胚胎细胞度过了传代的危机,并能稳定增殖,传代超过50代,成为无限细胞系,分别命名为RIRI-PX1、RIRI-GG1、RIRI-GG2、RIRI-GG3。
3.建系过程中的生物学特性研究。RIRI-GG1细胞培养初期细胞形态多样,随着细胞生长的逐渐稳定,细胞形态主要为多边形及梭形细胞,并以多边形细胞为主。增殖动力学特征表现出稳定性,50代及60代细胞的生长曲线的走向变化不大,其群体倍增时间分别为105.3h及104.6h。不同代数的RIRI-GG1细胞染色体制片的条件无明显差异,都只有在0.005M秋水仙素处理4.5h,KCl处理浓度0.50%,处理时间为15min时的效果最好。RIRI-GG1的染色体特征随着传代次数的增加而逐渐变化,在传代的前短翅鸣螽原核型特征为2n=31♂,32♀,细胞染色体平均数在传代过程中有增加的趋势,1-50代细胞染色体平均数分布与31-42之间,各代次的细胞仍是二倍体细胞占多数,各代数细胞染色体形态差异不大,两条染色单体明显,为近端部或端部着丝粒染色体。通过ITS序列及COI基因序列比对,证实RIRI-GG1确实来源于短翅鸣螽胚胎细胞。
RIRI-PX1细胞形态随传代次数增加而逐渐变化,培养初期细胞形态多样,随着细胞生长的逐渐稳定,细胞形态以梭形细胞为主;增殖动力学特征表现较高的稳定性,在50代及60代时的生长曲线的走向变化不大,其群体倍增时间分别为42.5h及42.1h。不同代数的RIRI-PX1细胞染色体制片的条件无差异,最优的条件均为0.005M秋水仙素处理5h,0.65%的KCl低渗溶液处理15min。柑橘凤蝶的原染色体难以计数,随后代数细胞染色体变异度较大,主要表现为为染色体数目众多,且数目分布广泛,染色体异倍化及多倍化严重,且染色体数目的变化不具备规律性,7-50代RIRI-PX1细胞染色体数目平均数在103-199之间,染色体形态多为弥散型着丝粒的短杆状、颗粒状、点状或圆球状。通过COI基因比对,证实RIRI-PX1确实来源于柑橘凤蝶新孵幼虫。
4.昆虫细胞系的利用。采用来源于3个目的14种昆虫细胞系对苜蓿银纹夜蛾核型多角体病毒AcNPV (Autographa californica nuclear polyhedrosis virus)和家蚕核型多角体病毒BmNPV (Bombyx mori nuclear polyhedrosis virus)进行敏感性测定。试验结果表明,各昆虫细胞系对AcNPV及BmNPV的敏感性不同,来源于双翅目及直翅目的细胞系均不能被这两种病毒感染,AcNPV可侵染来源于鳞翅目的Sf21、Sf9、HighFive、RIRI-PX1及HZ,而BmNPV仅能侵染BmN细胞。其中AcNPV对Sf9细胞的感染率最高,接种病毒10 d后感染率平均达84.0%,而RIRI-PX1的AcNPV病毒产量最高,形成的多角体数目为43.9PIBs/感染细胞。接种BmNPV10 d后BmN的感染率为72.2%,形成的多角体数目为23.1.PIBs/感染细胞。
测定了氯氟氰菊酯等4种常用农药及二甲苯在不同浓度水平及时间上对7种昆虫细胞系的毒力。结果表明:4种农药对离体培养的7种昆虫细胞的生长均产生了影响,其中印楝素的毒力效果最为显著,其次是高氯?灭乳油、马拉硫磷及氯氟氰菊酯,二甲苯在设置的浓度下对7种细胞的影响不明显。在同一药剂同一浓度处理下,不同细胞系的反应不同,其中Sf9细胞系最为敏感,其次是NIAS-MaBr-85、SL2、NIH-SaPe-4、C6-36、RIRI-GG1及RIRI-PX1。研究结果表明昆虫细胞对农药的敏感性与农药的作用机理一致,昆虫细胞系用于农药的毒力测定及研究是可行的。
The technique of insect cell culture, as one of the bases of cell engineering, is a valuable method of modern experimental biology. It is widely applied in various fields of biology, medicine and agriculture. The insect cell culture, establishment of insect cell lines, characterization of insect cell lines in the establishing process, and the utilization of insect cell lines were studied in this paper. This research provided reliable methods to establish more insect cell lines, understanding of biological characteristics change rule of insect cell lines during the establishing process, and expect to find useful insect cell lines. The main results of the study are as follows:
1.The cell culture of insects from different Orders. In this research, different tissues of Gampsocleis gratiosa, Locusta migratoria manilensis, Papilio xuthus, Kallima inachu, Danaus chrysippus, Blaps rhynchoptera, and Tenebrio molito were cultured in different culture conditions, and the suitable culture conditions of each tissue have been found out. The results showed that the suitable culture conditions of the insects from different Orders are different, (1) Cell culture for the insects from Orthoptera: Sterilized the insecst for 3min could achieved the best sterilized effects, and the 5days old embryos was easier to obtain subculture than other tissue of Orthopteran insects. The modified Grace’s medium and Schneider medium with 10% Hyclone FBS plus 10% Minghai FBS were suitable for the cell culture of Orthopteran insects, and the suitable interval of mediun change was 30d. (2) Cell culture for the insects from Lepidoptera: Sterilized the insects for 3min could achieved the best sterilized effects, and the 3-4days old embryos, neonate larvae, hemocytes of pupa, fat body of pupa were easier to obtain subculture than other tissue of Lepidopteran insects. The Grace’s medium and modified Grace’s medium with 10% Hyclone FBS plus 10% Minghai FBS were suitable for the cell culture of Lepidopteran insects, and the suitable interval of mediun change was 15-20d. (3) Cell culture for the insects from Coleoptera: Sterilized the insecst for 5-8min could achieved the best sterilized effects, and the neonate larvae, testes, fat body were easier to obtain subculture than other tissue of Coleopteran insects. The modified Grace’s medium and Schneider medium with 10% Hyclone FBS plus 10% Minghai FBS were suitable for the cell culture of Coleopteran insects, and the suitable interval of mediun change was 25-30d.
2.The establishment of insect cell lines. There were 8 insect cell lines proliferated and got subculture, but most of the cells stop proliferated after several passages, so that the subculture could not to be continued. Just one cell line from the neonate larvae of Papilio xuthus and three cell lines from embryos of Gampsocleis gratiosa have lived through the crisis of subculture, and the cells exhibited stable growth and proliferation. Therefore, they were considered to be a cell line after the fiftieth passage and named RIRI-PX1, RIRI-GG1, RIRI-GG2, and RIRI-GG3.
3.Characterization of insect cell lines in the establishing process. The morphology of RIRI-PX1 have been gradually changed with the times of subculture. At the early stages of culture, cells exhibited variable morphologies, the morphologies of the cells gradually changed from diversiform to spindle with the gradual stable growth of the cells. The proliferation characteristics of RIRI-PX1 showed high stability, the growth curve of passage50 and passage60 were similar, and the population doubling time of the two passages were approximately 42.5h and 42.1h. There was no difference for the karyotype conditions of the different passages of RIRI-PX1, The suitable karyotype conditions for RIRI-PX1 were 0.005M colchicines solution treated with 5h and 0.65% hypotonic KCl solution treated with 15min. The original chromosome numbers of Papilio xuthus was difficult to count, then the chromosomes of RIRI-PX1 exhibited deeply mutation, these cells had a great variation in chromosome number, and had deeply heteroploid. The change of the chromosome number did not have the regularity, the chromosome range of passage7 to passage 50 was from 103 to 199. Chromosomes without apparent centeromere were short pole-like and pellet-like. The mitochondrial cytochrome c oxidase subunit I gene sequence analysis indicated that the RIRI-PX1 cell line was derived from Papilio xuthus.
4.The utilization of insect cell lines. The morphology of RIRI-GG1 have been gradually changed with the times of subculture. At the early stages of culture, cells exhibited variable morphologies, the morphologies of the cells gradually changed from diversiform to polygonal and spindle with the gradual stable growth of the cells,and polygonal cells were predominant.
The proliferation characteristics of RIRI-GG1 showed high stability, the growth curve of passage50 and passage60 were similar, and the population doubling time of the two passages were approximately 105.3h and 104.6h. There was no difference for the karyotype conditions of the different passages of RIRI-GG1. The suitable karyotype conditions for RIRI-GG1 were 0.005M colchicines solution treated with 4.5h and 0.50% hypotonic KCl solution treated with 15min. The chromosome numbers of RIRI-GG1 cells changed with increasing passages of subculture. The original chromosome numbers of Gampsocleis gratiosa were 2n=31 for males and 2n=32 for females, the average number of chromosomes was increased with increasing passages of subculture, ranging from 31 to 42 at the passage1 to passage 50, but most of the cells remained as diploid cells at every passage. The chromosome profile exhibited no obvious changes at every passage, the two chromatids were obvious, all chromosomes were acrocentric. The rDNA internal transcribed spacer and the mitochondrial cytochrome c oxidase subunit I gene sequence analysis indicated that the RIRI-GG1 cell line was derived from Gampsocleis gratiosa.
The fourteen inscet cell lines which from three different orders were used to be infected with AcNPV and BmNPV. The result showed that the susceptibility for two viruses were different among different insect cell lines. The cell lines which come from Diptera and Orthoptera could not be infected with AcNPV and BmNPV. The five cell lines, Sf21、Sf9、HighFive、RIRI-PX1 and HZ which come from Lepidoptera original, could be infected with AcNPV. The BmN cell line could be infected only with BmNPV. The infection rate of AcNPV to Sf9 was the highest, reached 84.0% after 10 days infection. RIRI-PX1 infected with AcNPV could produce the highest yield of polyhedral inclusion bodies which reached 43.9 PIBs/cell at 10 day post infection. The infection rate of BmNPV to BmN was 72.2% after 10 days infection, and the yield of polyhedral inclusion bodies was 23.1PIBs/cell.
The toxicity of lambda-cyhalothrin、beta-cypermethrin plus emulsion, E,C、alathion、azadirachtin and xylene at the different concentration and treat time was studied in seven insect cell lines. The results showed that the four types of pesticides have toxic effect on the seven cell lines, while the xylene at the three concentrations has no obvious toxic effect on the cell lines. The tests indicated that azadirachtin was significantly more toxic than other pesticides, then in turn are beta-cypermethrin plus emulsion,E,C, alathion, and lambda-cyhalothrin. Respectively, the extent of cytotoxic effect among different cell lines at the same test concentration of same pesticide was different, the sensitivity different of seven cell lines was obvious. sf9 was more sensitive than other cell lines, which in turn are NIAS-MaBr-85,SL2,NIH-SaPe-4,C6-36,RIRI-GG1 and RIRI-PX1。The results indicated that the sensitivity of insect cell lines to the pesticides is accord to the mechanism of pesticides, and the insect cell lines used for the determination of pesticide cytotoxicity is feasible.
1.不同目昆虫细胞培养技术。通过对来源于直翅目的短翅鸣螽(Gampsocleis gratiosa Brunner von Wattenwyl)、东亚飞蝗(Locusta migratoria manilensis Meyen);鳞翅目的柑橘凤蝶(Papilio xuthus Linnaeus)、枯叶蛱蝶(Kallima inachus Doubleday)、金斑蝶(Danaus chrysippus Linnaeus);鞘翅目的喙尾琵琶甲(Blaps rhynchoptera Fairmaire)、黄粉虫(Tenebrio molitor Linnaeus)的多种组织原代培养条件研究,得出各种组织原代培养时的适宜培养条件,研究结果显示不同目昆虫细胞培养所适宜的条件不同,(1)直翅目昆虫细胞培养的消毒时间为3min,来源于5d胚胎组织的细胞较易取得传代,适宜于改良Grace及Schneider培养基及10%Hyclone+10%民海血清的搭配中培养,培养基的更换时间在30d左右。(2)鳞翅目昆虫细胞培养宜的消毒时间为3min左右,来源于3-4d胚胎、新孵幼虫、蛹血淋巴、蛹脂肪体组织的细胞较易取得传代,适宜于在改良Grace及Grace培养基及10%Hyclone+10%民海血清的搭配中培养,培养基的更换时间在15-20d之间。(3)鞘翅目昆虫培养的消毒时间为5-8min,来源于新孵幼虫、精巢、脂肪体组织的细胞较易取得传代,适宜于改良Grace及Schneider培养基及10%Hyclone+10%民海血清的搭配中培养,培养基的更换时间在25-30d之间。
2.昆虫细胞系的建立。在上述培养技术和条件研究基础上对多种昆虫组织进行培养,8种昆虫细胞发生增殖而进行传代培养,但大部分细胞在传至一定代数后停止增殖,使传代不能正常的进行下去。其中1个柑橘凤蝶新孵幼虫细胞及3个短翅鸣螽胚胎细胞度过了传代的危机,并能稳定增殖,传代超过50代,成为无限细胞系,分别命名为RIRI-PX1、RIRI-GG1、RIRI-GG2、RIRI-GG3。
3.建系过程中的生物学特性研究。RIRI-GG1细胞培养初期细胞形态多样,随着细胞生长的逐渐稳定,细胞形态主要为多边形及梭形细胞,并以多边形细胞为主。增殖动力学特征表现出稳定性,50代及60代细胞的生长曲线的走向变化不大,其群体倍增时间分别为105.3h及104.6h。不同代数的RIRI-GG1细胞染色体制片的条件无明显差异,都只有在0.005M秋水仙素处理4.5h,KCl处理浓度0.50%,处理时间为15min时的效果最好。RIRI-GG1的染色体特征随着传代次数的增加而逐渐变化,在传代的前短翅鸣螽原核型特征为2n=31♂,32♀,细胞染色体平均数在传代过程中有增加的趋势,1-50代细胞染色体平均数分布与31-42之间,各代次的细胞仍是二倍体细胞占多数,各代数细胞染色体形态差异不大,两条染色单体明显,为近端部或端部着丝粒染色体。通过ITS序列及COI基因序列比对,证实RIRI-GG1确实来源于短翅鸣螽胚胎细胞。
RIRI-PX1细胞形态随传代次数增加而逐渐变化,培养初期细胞形态多样,随着细胞生长的逐渐稳定,细胞形态以梭形细胞为主;增殖动力学特征表现较高的稳定性,在50代及60代时的生长曲线的走向变化不大,其群体倍增时间分别为42.5h及42.1h。不同代数的RIRI-PX1细胞染色体制片的条件无差异,最优的条件均为0.005M秋水仙素处理5h,0.65%的KCl低渗溶液处理15min。柑橘凤蝶的原染色体难以计数,随后代数细胞染色体变异度较大,主要表现为为染色体数目众多,且数目分布广泛,染色体异倍化及多倍化严重,且染色体数目的变化不具备规律性,7-50代RIRI-PX1细胞染色体数目平均数在103-199之间,染色体形态多为弥散型着丝粒的短杆状、颗粒状、点状或圆球状。通过COI基因比对,证实RIRI-PX1确实来源于柑橘凤蝶新孵幼虫。
4.昆虫细胞系的利用。采用来源于3个目的14种昆虫细胞系对苜蓿银纹夜蛾核型多角体病毒AcNPV (Autographa californica nuclear polyhedrosis virus)和家蚕核型多角体病毒BmNPV (Bombyx mori nuclear polyhedrosis virus)进行敏感性测定。试验结果表明,各昆虫细胞系对AcNPV及BmNPV的敏感性不同,来源于双翅目及直翅目的细胞系均不能被这两种病毒感染,AcNPV可侵染来源于鳞翅目的Sf21、Sf9、HighFive、RIRI-PX1及HZ,而BmNPV仅能侵染BmN细胞。其中AcNPV对Sf9细胞的感染率最高,接种病毒10 d后感染率平均达84.0%,而RIRI-PX1的AcNPV病毒产量最高,形成的多角体数目为43.9PIBs/感染细胞。接种BmNPV10 d后BmN的感染率为72.2%,形成的多角体数目为23.1.PIBs/感染细胞。
测定了氯氟氰菊酯等4种常用农药及二甲苯在不同浓度水平及时间上对7种昆虫细胞系的毒力。结果表明:4种农药对离体培养的7种昆虫细胞的生长均产生了影响,其中印楝素的毒力效果最为显著,其次是高氯?灭乳油、马拉硫磷及氯氟氰菊酯,二甲苯在设置的浓度下对7种细胞的影响不明显。在同一药剂同一浓度处理下,不同细胞系的反应不同,其中Sf9细胞系最为敏感,其次是NIAS-MaBr-85、SL2、NIH-SaPe-4、C6-36、RIRI-GG1及RIRI-PX1。研究结果表明昆虫细胞对农药的敏感性与农药的作用机理一致,昆虫细胞系用于农药的毒力测定及研究是可行的。
The technique of insect cell culture, as one of the bases of cell engineering, is a valuable method of modern experimental biology. It is widely applied in various fields of biology, medicine and agriculture. The insect cell culture, establishment of insect cell lines, characterization of insect cell lines in the establishing process, and the utilization of insect cell lines were studied in this paper. This research provided reliable methods to establish more insect cell lines, understanding of biological characteristics change rule of insect cell lines during the establishing process, and expect to find useful insect cell lines. The main results of the study are as follows:
1.The cell culture of insects from different Orders. In this research, different tissues of Gampsocleis gratiosa, Locusta migratoria manilensis, Papilio xuthus, Kallima inachu, Danaus chrysippus, Blaps rhynchoptera, and Tenebrio molito were cultured in different culture conditions, and the suitable culture conditions of each tissue have been found out. The results showed that the suitable culture conditions of the insects from different Orders are different, (1) Cell culture for the insects from Orthoptera: Sterilized the insecst for 3min could achieved the best sterilized effects, and the 5days old embryos was easier to obtain subculture than other tissue of Orthopteran insects. The modified Grace’s medium and Schneider medium with 10% Hyclone FBS plus 10% Minghai FBS were suitable for the cell culture of Orthopteran insects, and the suitable interval of mediun change was 30d. (2) Cell culture for the insects from Lepidoptera: Sterilized the insects for 3min could achieved the best sterilized effects, and the 3-4days old embryos, neonate larvae, hemocytes of pupa, fat body of pupa were easier to obtain subculture than other tissue of Lepidopteran insects. The Grace’s medium and modified Grace’s medium with 10% Hyclone FBS plus 10% Minghai FBS were suitable for the cell culture of Lepidopteran insects, and the suitable interval of mediun change was 15-20d. (3) Cell culture for the insects from Coleoptera: Sterilized the insecst for 5-8min could achieved the best sterilized effects, and the neonate larvae, testes, fat body were easier to obtain subculture than other tissue of Coleopteran insects. The modified Grace’s medium and Schneider medium with 10% Hyclone FBS plus 10% Minghai FBS were suitable for the cell culture of Coleopteran insects, and the suitable interval of mediun change was 25-30d.
2.The establishment of insect cell lines. There were 8 insect cell lines proliferated and got subculture, but most of the cells stop proliferated after several passages, so that the subculture could not to be continued. Just one cell line from the neonate larvae of Papilio xuthus and three cell lines from embryos of Gampsocleis gratiosa have lived through the crisis of subculture, and the cells exhibited stable growth and proliferation. Therefore, they were considered to be a cell line after the fiftieth passage and named RIRI-PX1, RIRI-GG1, RIRI-GG2, and RIRI-GG3.
3.Characterization of insect cell lines in the establishing process. The morphology of RIRI-PX1 have been gradually changed with the times of subculture. At the early stages of culture, cells exhibited variable morphologies, the morphologies of the cells gradually changed from diversiform to spindle with the gradual stable growth of the cells. The proliferation characteristics of RIRI-PX1 showed high stability, the growth curve of passage50 and passage60 were similar, and the population doubling time of the two passages were approximately 42.5h and 42.1h. There was no difference for the karyotype conditions of the different passages of RIRI-PX1, The suitable karyotype conditions for RIRI-PX1 were 0.005M colchicines solution treated with 5h and 0.65% hypotonic KCl solution treated with 15min. The original chromosome numbers of Papilio xuthus was difficult to count, then the chromosomes of RIRI-PX1 exhibited deeply mutation, these cells had a great variation in chromosome number, and had deeply heteroploid. The change of the chromosome number did not have the regularity, the chromosome range of passage7 to passage 50 was from 103 to 199. Chromosomes without apparent centeromere were short pole-like and pellet-like. The mitochondrial cytochrome c oxidase subunit I gene sequence analysis indicated that the RIRI-PX1 cell line was derived from Papilio xuthus.
4.The utilization of insect cell lines. The morphology of RIRI-GG1 have been gradually changed with the times of subculture. At the early stages of culture, cells exhibited variable morphologies, the morphologies of the cells gradually changed from diversiform to polygonal and spindle with the gradual stable growth of the cells,and polygonal cells were predominant.
The proliferation characteristics of RIRI-GG1 showed high stability, the growth curve of passage50 and passage60 were similar, and the population doubling time of the two passages were approximately 105.3h and 104.6h. There was no difference for the karyotype conditions of the different passages of RIRI-GG1. The suitable karyotype conditions for RIRI-GG1 were 0.005M colchicines solution treated with 4.5h and 0.50% hypotonic KCl solution treated with 15min. The chromosome numbers of RIRI-GG1 cells changed with increasing passages of subculture. The original chromosome numbers of Gampsocleis gratiosa were 2n=31 for males and 2n=32 for females, the average number of chromosomes was increased with increasing passages of subculture, ranging from 31 to 42 at the passage1 to passage 50, but most of the cells remained as diploid cells at every passage. The chromosome profile exhibited no obvious changes at every passage, the two chromatids were obvious, all chromosomes were acrocentric. The rDNA internal transcribed spacer and the mitochondrial cytochrome c oxidase subunit I gene sequence analysis indicated that the RIRI-GG1 cell line was derived from Gampsocleis gratiosa.
The fourteen inscet cell lines which from three different orders were used to be infected with AcNPV and BmNPV. The result showed that the susceptibility for two viruses were different among different insect cell lines. The cell lines which come from Diptera and Orthoptera could not be infected with AcNPV and BmNPV. The five cell lines, Sf21、Sf9、HighFive、RIRI-PX1 and HZ which come from Lepidoptera original, could be infected with AcNPV. The BmN cell line could be infected only with BmNPV. The infection rate of AcNPV to Sf9 was the highest, reached 84.0% after 10 days infection. RIRI-PX1 infected with AcNPV could produce the highest yield of polyhedral inclusion bodies which reached 43.9 PIBs/cell at 10 day post infection. The infection rate of BmNPV to BmN was 72.2% after 10 days infection, and the yield of polyhedral inclusion bodies was 23.1PIBs/cell.
The toxicity of lambda-cyhalothrin、beta-cypermethrin plus emulsion, E,C、alathion、azadirachtin and xylene at the different concentration and treat time was studied in seven insect cell lines. The results showed that the four types of pesticides have toxic effect on the seven cell lines, while the xylene at the three concentrations has no obvious toxic effect on the cell lines. The tests indicated that azadirachtin was significantly more toxic than other pesticides, then in turn are beta-cypermethrin plus emulsion,E,C, alathion, and lambda-cyhalothrin. Respectively, the extent of cytotoxic effect among different cell lines at the same test concentration of same pesticide was different, the sensitivity different of seven cell lines was obvious. sf9 was more sensitive than other cell lines, which in turn are NIAS-MaBr-85,SL2,NIH-SaPe-4,C6-36,RIRI-GG1 and RIRI-PX1。The results indicated that the sensitivity of insect cell lines to the pesticides is accord to the mechanism of pesticides, and the insect cell lines used for the determination of pesticide cytotoxicity is feasible.
引文
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