Studies On Establishment Of Insect Cell Lines And Its Biological Characteristics And Application

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.