小鼠肝脏线粒体的制备

Rat liver is an ideal source for functional intact mitochondria for a number of reasons. We use Sprague-Dawley albino male rats for our studies. Female rats can be used, however for serious studies we usually stick to males to avoid complications due to the estrous cycle. Animal tissue is preferable to plant tissue because they do not have cell walls, which make homogenization difficult. The metabolism of endotherms requires that some tissues maintain a high density of mitochondria, so the potential yield is high. The isolation of mitochondria from highly structured animal tissues such as muscle can be technically difficult since a high proportion of the organelles remain trapped in cell and tissue fragments. Liver is a fairly homogeneous soft tissue with relatively little connective tissue, so one can quickly and easily obtain a substantial quantity of mitochondria within less than an hour's preparation time.

For the mitochondria preparation, a good weight range for the animal is 200 - 250 gms, perhaps a bit larger. A liver from this size animal yields up to two ml or so of concentrated mitochondria, enough for dozens of oxygraph experiments. In the teaching lab, two sets of lab partners share one animal, dividing the liver tissue into equal starting amounts by weight. The animal must be killed humanely, according to government guidelines. Since the liver is responsible for detoxification processes including metabolism of anesthetic agents, and because both an anesthetic and its metabolites can affect liver function, it is preferable from an experimental viewpoint to avoid using anesthetic or tranquilizing drugs. Guidelines may require anesthesia; otherwise, simple decapitation using a conventional rat guillotine is quick, humane, and does not compromise the experiment.

A medial incision (vertical, up the middle of the animal - don't be a goober and call it a "medical" incision) from groin to sternum, first separating the skin then the underlying muscle and peritoneum, reveals the liver, which is dark brown, large, and almost unmistakable, however an inexperienced student might mis-identify the coecum (appendix, quite large in rats) as the liver. If the student gets into the rat quickly (opened within 2-3 min of decapitation) and cuts through the sternum, opening the chest, the heart can be seen still beating. The liver should be chilled immediately by pouring a generous amount (100 ml) of ice-cold 0.9% NaCl into the peritoneal cavity. It can be removed in pieces or removed, intact, by cuting it off at the base, and then should be dropped into a second beaker of ice-cold saline solution to continue to reduce the temperature. Further steps in the isolation should all be done at "ice-bucket" temperatures.

The liver should be cubed, weighed, and divided into two equal portions, each weighing 3 to 5 grams. For homogenization, the ratio of homogenizing medium to tissue is imporant, as is the depth of liquid in the container when using a Polytron type homogenizer. We have had great success by mincing the tissue 'dry' in a 50 ml plastic disposable beaker, adding 20 ml homogenizing medium, mixing to suspend the mince, and homogenizing at a setting of '40' for 10 sec.

In our lab we use 0.25M sucrose, 5 mM HEPES buffer, and 1 mM EDTA, pH 7.2 for homogenizing. We use a shearing type homogenizer called 'Tissuemizer,' with T25 stainless steel shaft, made by Tekmar, Inc. (Cincinnatti, Oh). A teflon-on-glass (Potter-Elvehjem) homogenizer can also be used, but it takes longer. The Polytron is fast but prolonged or overly vigorous homogenization can be damaging to mitochondria. Both methods yield good preparations from rat liver.

To remove large cell and tissue fragments and cell nuclei (the 'nuclear pellet'), we centrifuge the homogenate at 500 x g for 10 minutes (2600 rpm in a Sorvall SS-34 centrifuge), using a Sorvall SS-34 centifuge tube and rotor, or the equivalent. Before centrifugation, we top off the homogenate with medium to fill each tube. To bring down the mitochondrial pellet we pour the supernatant into a clean centrifuge tube, and without topping off we centrifuge at 11,000 rpm (9400 x g) for 10 min.

When the supernatant is poured off, the loose upper part of the mitochondrial pellet may come off as well. Most of the pellet, containing the healthy mitochondria, is dense enough to remain behind. The white foamy material near the top of the tube consists of lipids, which must be kept from contact with the mitochondria. They can be removed by wiping the inside of the tube with a lab wiper. Any mixing of lipids with the mitochondria suspension will cause them to uncouple (lose their ability to maintain a chemiosmotic gradient) quickly. After using a pasteur pipet to remove the last bit of liquid, a glass rod should be used to stir the remaining pellet into a smooth paste. We keep the centrifuge tube on ice while stirring, and try not to introduce air into the suspension.

Mitochondria keep best when concentrated, to minimize exposure to oxygen. They remain dormant until diluted into an oxygen-rich respiration medium. The paste should be transferred to an eppendorf tube, and air spaces prevented by careful pipetting with a yellow tip micropipettor. Use of a pasteur pipet at this point results in loss of much of the pellet, since it sticks readily to glass. Once transferred to an eppendorf tube the preparation is ready for use.