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Culture of Trichinella Spiralis Muscle Larvae in Vitro

Author: WangLi
Tutor: CuiJing
School: Zhengzhou University
Course: Pathogen Biology
Keywords: Trichinella Muscle larvae Molt Semi-solid medium Intestinal epithelial cells Invasion
CLC: R383
Type: Master's thesis
Year: 2009
Downloads: 27
Quote: 0
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Abstract


meat and other animal infections in the acute phase of clinical persistent high fever, eyelid and facial edema, generalized muscle aches, allergic rash, increased eosinophils in the blood and other allergic manifestations, critically ill patients may be due to concurrent disease and death. Trichinosis Commission (International Commission on Trichinellosis, ICT) reported more than 10,000 cases of patients between 1995-1997, is now included in the re-ravaging disease (re-emerging disease). Trichinella not in vitro culture hinder its intrusive host molecular mechanisms row of insects infected host intestinal reaction molecular mechanism of anti-Trichinella drug screening and Trichinella vaccine development research, therefore, Trichinella spiralis Muscle vitro study is especially important. This study will Trichinella spiralis Muscle in different liquid medium for culture, observed larvae the molting larvae proportion molting time and after various incubation times, and not molting muscle larvae lengths were measured: to observe the liquid medium (complete medium of T84 cells ), semi-solid medium (complete medium, agarose, complete medium agarose T84 cells) on the larval molt; agarose Caco-2 cells in semi-solid medium (complete medium) observed in larvae treated with different activator After its molting treatment, and compare the two kinds of intestinal epithelial cells of the larval molting. Materials and methods the Trichinella worm species, laboratory animals and cell lines in this study used the Trichinella Trichinella Henan strains (Trichinella spiralis, T1), Kunming mice passaged species preservation, artificial digestion method and the Bayesian method to collect pure T. spiralis muscle larvae; experimental animals healthy male w age Kunming mice and Sprague Dawley (SD) rats; cells are human colon carcinoma cell line (human colonic carcinoma cell line) T84 and Caco-2 cells. 2. Preparation of rat intestinal contents and mouse bile ① fasted for 8 h rats were sacrificed, the small intestine with 5 ml of normal saline containing antibiotics, centrifuged at 1 200 g for 10 min to remove large debris, supernatant of the intestinal contents, 1:2 diluted with saline at -20 ℃ before normal saline 1:10 dilution; (2) will be fasting to 8 h Xing Benedict macrophages baking? remove the complete gallbladder collect bile After at -20 ℃ diluted with saline before 1:20. Spiralis muscle larvae cultured in culture medium ① fresh muscle larvae in saline, PBS, RPMI-1640 culture medium containing 5% fetal bovine serum (fetal bovine serum, FBS) RPMI-1640 culture medium cultured in liquid medium; ② from the culture of 12 h, every 2 h under an inverted microscope to observe and record the muscle larvae in the culture medium molting; ③ every 24 h culture bottles removed from the incubator, mixed even after the lessons muscle larvae microscope and counting the larvae molt and photographed; measuring length select about 15 at both ends no significant molting larvae camera. Trichinella spiralis muscle larvae cultured in three different medium fresh muscle larvae after incubation in rat intestinal contents were in complete medium (containing 10? S DMEM/F12 medium) T84 cells, semi-solid medium (1.75% agarose complete medium), semi-solid medium T84 cells cultured for 24 h, the microscope and count one (not the slough under complete epidermal larvae) and 4 (the slough under complete epidermis larvae) larvae. 5 different activator cultured larvae molt fresh muscle larvae in saline, RPMI-1640 culture medium, rat intestinal contents or a mouse bile After incubation, suspended to a semi-solid medium (containing 10? SDMEM The medium was 1.75% agarose), and cover to Caco-2 cell layer culture, 24 h after the microscope and count the larvae of a 2 to 4. 6 intestinal epithelial cells in culture the larvae molt impact observed in semi-solid medium in the T84 and Caco-2 cells cultured larvae molt. Results Trichinella muscle larvae begin molting period of observation at 37 ° C for 5% CO 2 conditions, muscle larvae in RPMI-1640 culture medium, PBS containing 5? S RPMI-1640 culture medium and physiological the brine began molting time were 15 h, 19 h, and 23 h and 37 h, molting majority from the larvae trailing. 2 Trichinella the muscle larvae activity and molting observation Trichinella muscle larvae inoculated into the culture medium is very active, stretching and curling movement cultivation in saline and PBS 3 d activity decreased after 5 d basic non- activities, containing 1 to 4 layers? S RPMI-1640 culture medium in the culture to 21 d some parasites still active; larvae molt a layer only in saline and PBS, RPMI-1640 culture medium molting 5378 muscle larvae observed, the number of larvae molt layers 1 to 4 were 386 (62.96%), 377 (25.60%), 71 (1.32%) and 14 (0.26% ); larvae containing 5? molt layer 1 to 2 S RPMI-1640 culture medium, accounting for the observed larvae 66.78% (3699/5539) and 0.45% (25/5539). Not molt muscle larvae length changes observed in the 1 ~ 21 d not molt cultured in RPMI-1640 culture medium

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