| Abstract |
Insect cell lines are very valuable tools for biological research. This
study was focused on how to establish cell lines of insect fat bodies
through primary culture and to explore the mechanism of proliferation of
insect fat body cells in vitro, in order to provide reliable methods to
establish more insect cell lines, in which the methods of explanted
culture of animal tissues was referred. The cells of primary culture were
obtained successfully from larval fat bodies of healthy Anoplophora
glabripennis (Coleoptera: Cerambycidae). We tried to find a method to
establish new cell lines that adapted to our lab through comparing the age
of the larvae, the size and integrality of the fat body tissue, the
surface of the tissue culture, the medium and its pH, the scale of the
accretion (phenylthiourea, PTU) and the adherence of primary culture
tissues to culture surface. We also researched the methods, culture
conditions and occasions of subculture. As a result, the integrated fat
body tissues of the pre-pupae larvae were used as the best material in
establishing insect cell lines. The new plastic culture flask is the best
choice. The optimum medium that was used as the primary medium was TNM-FH
(80%) supplemented with FBS(10%) and PTU(10%). Its pH ranged from 6.2 to 6.8.
The two key steps from primary culture to first subculture included
making the primary culture tissue attach to the surface of the culture
flask tightly and transferring the culture to a new flask carefully
without disturbing the primary culture tissues when proliferating cells
full of the bottom of the flask. Six new cell lines were initiated from the
larval fat bodies of Spodoptera exigua (Lepidoptera: Noctuidae), which
were designated as IOZCAS-Spex-II, IOZCAS-Spex-III, IOZCAS-Spex-IV,
IOZCAS-Spex-V, IOZCAS-Spex-VI, IOZCAS-Spex-VII, respectively. One new cell
line (IOZCAS-Ha-I) was established from the larval fat bodies of Helicoverpa
armigera (Lepidoptera: Noctuidae). The spherical cells were predominant
among the various cell types. The population doubling times for
IOZCAS-Spex-II and IOZCAS-Ha-I were 108.8h and 65h, respectively.
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