疟原虫的培养方法-试验方案

疟原虫的培养方法
点击： 作者： 来源：dxy 日期：2007-12-20 本站论坛

Trager W, Jensen JB. Human malaria parasites in continuous culture[J ] . Science , 1976 , 193 (4254): 673 - 675.

Lambros C, Vanderberg J P. Synchronization of Plasmodium falciparum erythrocytic stages in culture[J ] . Parasitol, 1979, 65 :418 - 420.

疟疾不完全培养基(Uncomplete Malaria-Culture Medium，UMCM)成分包括RPMI1640 10.43 g/L，Hepes 5.92g/L，NaHCO3 3g/L，调至pH 7.3-7.4，不加血清，过滤除菌，–20℃保存。临用前加AB+血清至终浓度为10%即配成疟疾完全培养基 (Complete Malaria-Culture Medium，MCM)，4℃保存，限用两周。人O+ 红细胞(Red Blood Cell，RBC)由本实验室志愿者提供，人AB+血清来自xx医院输血科，使用前56℃灭活60 min。

红内期疟原虫体外连续培养
50% RBC悬液的制备：从储存RBC中取出适量RBC至干净无菌离心管中，加入5mLUMCM，轻缓吹打悬浮，1500 rpm离心5min，先吸除RBC表层白细胞，然后弃去上清。如此洗涤两次后弃去上清，加入与细胞压积等体积的MCM，轻缓混匀。储于4℃备用，两周内使用有效。
疟原虫体外培养按照本教研室的培养方法进行。常规每天MCM换液一次，每逢裂殖体期添加一次50% RBC悬液。

疟原虫同步化处理
疟原虫同步化处理按照本教研室方法进行。选择原虫密度较高且环状体较丰富的生活阶段，将培养原虫吸至刻度离心管中，2000 rpm离心5 min，吸弃上清，于细胞压积中加入5倍体积37℃预热的5% D-山梨醇(即0.274 mol/L)，充分混匀，于37℃作用5-10min，2000 rpm离心5 min后吸弃上清及上层棕色沉淀，加入至少2倍体积MCM，充分混匀，同上离心，弃去上清，加等体积MCM和2-3倍体积50%的RBC悬液，用MCM将培养物稀释为5%的细胞悬液后，完全转入培养瓶，放于CO2孵箱培养。48 h(一个生活史周期)后再进行一次同步化处理。

I. Culturing of erythrocytic asexual stages of Plasmodium falciparum and P. vivax

I:A. The candle-jar technique of Trager–Jensen
by Malin Haeggström1, Martha Schlichtherle1, and Ahmed Bolad2
1Microbiology and Tumor Biology Center (MTC), Karolinska Institutet and Swedish Institute for Infectious Disease Control, Box 280, SE-171 77 Stockholm, Sweden
2Immunology, Stockholm University, SE-106 91 Stockholm, Sweden
e-mail: malin.haeggstrom@mtc.ki.se, ahmed@imun.su.se

Equipment
incubator (37 癈)
glass desiccator (e.g., candle jar)
cell culture flask
candles
centrifuge
sterile pipettes
sterile tubes
glass slides and coverslips
microscope, fluorescence or light

Materials and reagents
purified erythrocytes (or human blood type O+ in CPD-adenine (Terumo) or S.A.G.M. (“Sagman” solution or EDTA)
MCM (see below)
Tris (Sigma)
Albumax II (Gibco)
RPMI 1640 (Gibco)
gentamicin
1 M HEPES (Gibco)
Hanks’ balanced salt solution (Gibco)
acridine orange (10 礸/mL) or Giemsa 5%

optional:
human serum
glucose
hypoxanthine (Sigma)
Tris-buffered Hanks’ (TH)

Preparations
Prepare malaria culture medium (MCM) and Tris-buffered Hanks’ (TH) for washing cells.

Albumax complete medium:
10.43 g RPMI 1640 powder (Gibco)
25 mL 1 M HEPES solution or 6 g HEPES (Gibco)
2 g NaHCO3
0.5 mL gentamicin (from 50 mg/mL stock)
5 g Albumax II
Add distilled water to 1 liter. Filter-sterilize.
Use within 10 days, store at −20 °C.

Comment: For growing parasites from patient blood, use 10 g of Albumax for 1 liter of complete MCM. The vast majority of cultures will survive at least 2 weeks. It is also important to avoid serum in the culture for preparation of crude parasite antigen (see SEROLOGY, section III:. Not all strains can be adopted to Albumax II medium.

Alternative MCM:
10.43 g RPMI 1640 powder (Gibco)
25 mL 1M HEPES or 6 g HEPES
2 g NaHCO3
0.5 mL gentamicin (from 50 mg/mL stock)
Add distilled water to 1 liter. Filter-sterilize and store at −20 °C in 45-mL aliquots.

For complete MCM (cMCM), add 5 mL of human blood type AB+ serum (inactivated at 56 °C for 60 min; then stored at −20 °C) to 45 mL of medium. Complete MCM can be used for up to one week if stored at 4 °C.

MCM can also be made from commercial liquid RPMI with sodium bicarbonate and HEPES buffer (Gibco). Just add 5 mL of 100 L-glutamine (Gibco) and 0.25 mL gentamicin (Gibco) to a 500 mL bottle of the RPMI.

TH (0.15 M Tris-buffered Hanks’) (pH 7.2):
2.11 g Tris–HCl
0.2 g Tris-base
7.88 g NaCl
Dissolve in distilled water and bring volume to 1 liter.
Mix 1 volume of Tris buffer with 1 volume of Hanks’.

In vitro cultures in tissue-culture flasks
• Wash the erythrocytes 3 times in TH or RPMI 1640 to remove CPD, serum, and leukocytes if present. Dilute to 5% hematocrit with cMCM in small flasks of 25 cm2 (0.2 mL of packed cells to 4 mL of cMCM) or in 75-cm2 flasks (1.0 mL to 20 mL).
• Add parasites to an appropriate parasitemia (see below).
• Put the flask in a candle jar and loosen the screw cap. Produce low oxygen by burnt out candle and place the jar at 37 °C.

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