Method for preparing samples for the quantification of biofactors

Abstract

The object of the present invention is to provide a method for preparing a sample suitable for quantifying biofactors selected from the group consisting of neurotransmitters and lipid mediators, from tissues in which inflammatory cells produce and / or accumulate, and for quantifying the tissue content of said biofactors. [Solution] A method for preparing a sample to quantify the content of a biofactor selected from the group consisting of neurotransmitters and lipid mediators in tissue produced and / or accumulated by inflammatory cells, comprising: a tissue fixation step of fixing the tissue by contacting it with methanol at -15°C or below before collecting cells from the tissue; a cell collection step of collecting cells from the fixed tissue; and a disruption step of ultrasonically disrupting the obtained cells.

Description

Technical Field

[0001] The present invention relates to a method for preparing a sample for quantifying a biological factor. More specifically, the present invention relates to a method for preparing a sample for quantifying a biological factor selected from the group consisting of neurotransmitters and lipid mediators in bone marrow.

Background Art

[0002] As a sampling method for quantifying biological factors in tissue, a method of homogenizing a target tissue using a phosphate buffer solution is common (for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, when the present inventors independently examined the quantitative values of biological factors obtained by conventional sampling methods, they encountered the problem that biological factors such as neurotransmitters and lipid mediators tend to have their values fluctuate due to stimulation during cell collection from a living body. Specifically, when prostaglandin E2, a type of lipid mediator, was quantified using LC-MS / MS, it was found that for femur bone marrow samples, when the bone marrow was extruded using a syringe and needle in a 4°C phosphate buffer solution and subjected to several rounds of pipetting and agitation in a tube, the quantitative value varied from about 60 pg to about 480 pg depending on the mouse. In other words, the content of biological factors contained in bone marrow samples sampled by conventional methods cannot be said to stably reflect the in vivo content of the biological factors.

[0005] Thus, the reasons for the stability issues in the quantitative determination of biofactors in bone marrow samples are thought to be: (i) because bone marrow tissue is rich in inflammatory cells such as neutrophils, monocytes, and macrophages, the lipid metabolic pathway cannot be stopped in a 4°C phosphate-buffered environment against the stimulation during sample collection; and (ii) even pipetting acts as a stimulus, causing the aforementioned lipid metabolic pathway to progress. For this reason, even with high-performance measuring instruments, if the above problems in sampling persist, it is impossible to accurately determine the content of biofactors in body tissues. This problem is common not only to sampling from bone marrow but also to sampling from tissues that are rich in inflammatory cells, similar to bone marrow.

[0006] Therefore, the object of the present invention is to provide a method for preparing a sample suitable for quantifying a biofactor selected from the group consisting of neurotransmitters and lipid mediators, from tissues in which inflammatory cells produce and / or accumulate, and which is suitable for quantifying the content of said biofactor in body tissues. [Means for solving the problem]

[0007] As a result of diligent research, the inventors have found that by fixing the tissue with cold methanol before cell collection, the reaction of biofactors is stopped without activating or inactivating them. Subsequently, by collecting the cells and then performing sonication, the quantitative values ​​of the obtained sample are stabilized. In other words, by combining tissue fixation with cold methanol with subsequent cell collection and sonication, it is possible to extract biofactors contained in the cells constituting the tissue while suppressing fluctuations in their quantity. The present invention was completed by further research based on this finding. That is, the present invention provides the invention in the following embodiments.

[0008] Item 1. A method for preparing a sample to quantify the content of a biofactor selected from the group consisting of neurotransmitters and lipid mediators in body tissues produced and / or accumulated by inflammatory cells, A tissue fixation step is performed by fixing the tissue by contacting it with methanol at -15°C or below before collecting cells from the tissue. The cell harvesting process involves collecting cells from fixed tissue, A preparation method comprising a disruption step of ultrasonically disrupting cells in collected tissue. Item 2. If the aforementioned tissue is bone marrow, In the tissue fixation step, the bone marrow is fixed in the bone by injecting methanol at -15°C or below into the bone marrow cavity. In the cell collection process, the fixed bone marrow is drained outside the bone. The preparation method described in item 1. Item 3. The preparation method according to Item 2, wherein the tissue fixation step and the cell collection step are performed by injecting from one end of the bone and pushing out the bone marrow from the other end by the injection pressure. Item 4. The preparation method according to any one of items 1 to 3, wherein the crushing step is performed at a temperature greater than 0°C and 6°C or lower. Item 5. A quantitative sample obtained by the preparation method described in any of Items 1 to 4. Item 6. A quantitative sample as described in Item 5, stored at -40°C or below. Item 7. A method for quantifying the content of a biofactor in body tissue, comprising the step of measuring the biofactor using a quantitative sample obtained by the preparation method described in any of Items 1 to 4. [Effects of the Invention]

[0009] The present invention provides a method for preparing a sample suitable for quantifying a biofactor selected from the group consisting of neurotransmitters and lipid mediators, from tissue in which inflammatory cells are produced and / or accumulate, and for quantifying the content of said biofactor in body tissue. [Brief explanation of the drawing]

[0010] [Figure 1] The results of dopamine level measurement for the quantitative sample in Example 1 are shown. [Figure 2] The results of dopamine level measurement for the quantitative sample in Comparative Example 1 are shown. [Figure 3] The measurement results of monoamine (neurotransmitter) levels in the quantification sample of Example 2 are shown. [Figure 4] The measurement results of lipid mediator levels in the quantification sample of Example 2 are shown. [Figure 5] The measurement results of lipid mediator levels in the quantification sample of Example 2 are shown. [Figure 6] The measurement results of monoamine (neurotransmitter) levels in the quantification sample of Example 5 are shown. [Figure 7] The measurement results of lipid mediator levels in the quantification sample of Example 5 are shown. [Mode for Carrying Out the Invention]

[0011] 1. Method for preparing samples for the quantification of biofactors The method for preparing a sample for quantifying a biological factor of the present invention is a method for preparing a sample for quantifying the content of a biological factor selected from the group consisting of neurotransmitters and lipid mediators (hereinafter also referred to as "predetermined biological factor") in a body tissue where inflammatory cells are produced and / or accumulated, and the method includes a predetermined tissue fixation step, a tissue acquisition step, and a disruption step.

[0012] 1-1.Tissue fixation process In the tissue fixation step, the tissue is fixed by bringing methanol at a temperature of -15°C or lower (hereinafter also referred to as "cold methanol") into contact with the tissue where inflammatory cells are produced and / or accumulated. By performing tissue fixation before collecting the cells of the tissue, it is considered that biological reactions can be instantaneously stopped, and the activation (progression of metabolic pathways) or inactivation of the predetermined biological factor due to stimulation during cell collection can be suppressed.

[0013] Examples of the inflammatory cells include neutrophils, monocytes, macrophages and the like. The tissue where inflammatory cells are produced and / or accumulated may be any tissue that is rich in inflammatory cells. Examples of such a tissue include tissues containing a large amount of blood cells such as bone marrow, spleen and liver, and inflammatory tissues where hematopoietic cell infiltration has occurred.

[0014] The animal from which these tissues are derived is not particularly limited, and examples include humans and non-human animals (mammals, birds, amphibians, reptiles, etc.).

[0015] The temperature of cold methanol may be -15°C or lower, preferably -40°C to -18°C, more preferably -30°C to -20°C.

[0016] The tissue is subjected to this step while retaining its tissue morphology. For example, when bone marrow is selected as the tissue, the bone marrow is subjected to this step in a state where it is present in the bone marrow cavity of a bone collected from the body. When the spleen, liver, or other inflamed tissue is selected as the tissue, the tissue is subjected to the step immediately after being collected from the body.

[0017] The method for bringing cold methanol into contact with the cells of the tissue is not particularly limited, and typical examples include a method of injecting cold methanol into the tissue or immersing the tissue in cold methanol.

[0018] For example, when bone marrow is selected as the tissue, a preferred method for fixing the bone marrow includes a method of injecting cold methanol into the bone marrow by puncturing from one epiphysis of a bone removed from a living body. When the spleen, liver, or other inflamed tissue is selected as the tissue, a method for fixing these tissues includes a method of immersing the tissue in cold methanol immediately after collecting the tissue from the body.

[0019] 1-2.Cell collection process In the cell collection step, the cells of the tissue fixed in the tissue fixation step are collected.

[0020] By being instantaneously fixed in the tissue fixation step, the activation or deactivation of a predetermined biological factor in the cells of the tissue is suppressed, so that the content of the biological factor against stimulation is stabilized. Therefore, the collection of the tissue cells can be performed by any method involving stimulation. That is, the cells can be dispersed by stirring or homogenizing the collected tissue in a liquid (for example, in methanol) (hereinafter also simply referred to as stirring or the like).

[0021] When bone marrow is selected as the tissue, the injection pressure of the cold methanol injected during the tissue fixation process can be used to push the bone marrow out from the end opposite the puncture site of the bone, thereby collecting bone marrow cells, and the collected bone marrow can be agitated or otherwise processed. When the spleen, liver, or other inflammatory tissue is selected as the tissue, the fixed tissue can be agitated or otherwise processed.

[0022] 1-3. Crushing process In the disruption step, the cells of the tissue collected in the cell collection step are ultrasonically disrupted. As described above, since the activation or inactivation of predetermined biofactors is suppressed, ultrasonic disruption allows for the selection of conditions that enable the elution of predetermined biofactors within the cells without any particular restrictions.

[0023] For example, in the crushing process, ultrasound may be applied continuously or intermittently. The total time for ultrasonic crushing can be, for example, 50 to 500 minutes, 80 to 200 minutes, or 100 to 300 minutes. When ultrasound is applied intermittently, a cycle of, for example, 5 to 20 minutes (preferably 10 to 30 minutes) of ultrasonic crushing followed by, for example, 5 to 20 minutes (preferably 10 to 30 minutes) of ultrasound stopping can be repeated.

[0024] There are no particular restrictions on the temperature during the crushing process, but preferred temperatures are greater than 0°C and 6°C or less, 2 to 6°C, or 4 to 6°C.

[0025] 1-4. Other processes The method for preparing a sample for the quantification of biofactors according to the present invention may include, or may not include, any other steps in addition to the tissue fixation step, cell collection step, and disruption step described above, as long as they do not impair the effects of the present invention.

[0026] Other steps include a purification step and / or a solvent removal step. If the preparation method of the present invention includes other steps, these other steps may be performed after the crushing step.

[0027] The purification process can be performed for the purpose of removing unwanted components and / or solubilizing the biofactors to be measured, and specifically includes one or more selected from solid-liquid separation, extraction, solvent addition, etc.

[0028] Solid-liquid separation can be performed to remove insoluble matter (such as cellular impurities) generated during the crushing process. The method of solid-liquid separation is not particularly limited and can be appropriately determined by those skilled in the art depending on the type of insoluble matter to be removed, but examples include filtration and centrifugation.

[0029] Extraction can be performed to elute the biofactor to be measured. Extraction can be performed, for example, before solid-liquid separation. The elution solvent can be appropriately selected according to the type of biofactor to be measured and its solubility.

[0030] Solvent addition can be performed to solubilize the biofactor to be measured. The solubilizing solvent can be appropriately selected according to the type of biofactor to be measured and its solubility.

[0031] The solvent removal step can be performed to increase the concentration of the biofactor to be measured. The solvent removal method may include concentration or drying (e.g., freeze-drying).

[0032] 1-4. Samples for Quantitative Analysis The quantitative sample obtained by the preparation method of the present invention (hereinafter also referred to as "quantitative sample of the present invention") is useful as a sample for quantifying the content of a predetermined biofactor in body tissues where inflammatory cells produce and / or accumulate.

[0033] The predetermined biofactors to be measured are selected from the group consisting of neurotransmitters and lipid mediators. Examples of neurotransmitters include monoamines, specifically norepinephrine, dopamine, serotonin, and adrenaline, with norepinephrine, dopamine, and serotonin being preferred. Examples of lipid mediators include PGE2, PGD2, 2S-HHT, AA, 15-Deoxy-delta12,14-PGJ2, PGF2a, 5-HEPE, 12-HEPE, 15-HEPE, 18-HEPE, HYA+HYC, 5-HETE, 12-HETE, 15-HETE, 4-HDHA, 7-HDHA, 14-HDHA, 17-HDHA, EPA, DHA, HYB, ketoA, ketoB, etc. Preferably, 15-Deoxy-delta12,14-PGJ2, PGF2a, 18-HEPE, HYA+HYC, 5-HETE, 15-HETE, ketoA, and ketoB are included.

[0034] In the preparation method of the present invention, the tissue fixation step is performed before cell collection, which suppresses the activation or inactivation of predetermined biofactors due to stimulation during cell collection, enabling cell collection and sonication. Furthermore, sonication allows for the stable and sufficient release of predetermined biofactors, thereby suppressing variability in measurement values. Therefore, the content of predetermined biofactors in the quantitative sample of the present invention can reflect the content in body tissues, and variability in measurement values ​​between samples can be suppressed.

[0035] Furthermore, since the quantitative sample of the present invention has a stable content of biofactors against stimuli, exposure to stimuli such as stirring is not strictly prohibited but is permissible.

[0036] Furthermore, the quantitative sample of the present invention can be stored for a long period of time if cooled. Therefore, the quantitative sample of the present invention is preferably stored under cooling. The cooling temperature can be, for example, -40°C or lower, preferably -75°C or lower. The storage period under cooling can be, for example, 1 week to 6 months, or 1 to 3 months.

[0037] 2. Method for quantifying the content of biofactors in body tissues The quantitative sample of the present invention described in "1-4. Quantitative Sample" above is useful as a sample for quantifying the content of a predetermined biofactor in body tissues where inflammatory cells are produced and / or accumulate. Accordingly, the present invention also provides a method for quantifying the content of a biofactor in body tissues, which includes the step of measuring the biofactor using the quantitative sample of the present invention described in "1-4. Quantitative Sample" above.

[0038] Regarding the measurement method, a person skilled in the art can appropriately determine a method that allows for trace analysis, depending on the type of biofactor to be measured. For example, monoamines, which are neurotransmitters, can be measured by high-performance liquid chromatography-electrochemical detection (HPLC-ECD), and lipid mediators can be measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS), but the method is not limited to these. [Examples]

[0039] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0040] [Example 1] As wild-type (normal) mice, strain C57BL / 6, 8-10 weeks old, were prepared. These mice were wild-type mice produced by the inventors by crossing JAK2V617F Tg mice (genetically modified mice in which the JAK2V617F gene mutation (mouse JAK2 human type V617F) was inserted into cells throughout the body) with wild-type mice. Femurs were collected from these wild-type mice, and the following treatment was performed on each femur. Using a syringe with a needle containing methanol (-30 to -20°C), one end of the femur was punctured, and 1 mL of methanol was injected into the bone marrow tissue in the medullary cavity of the femur (tissue fixation step). Along with the injection of methanol, the injection pressure pushed the bone marrow from the opposite end of the femur into a sample tube (cell collection step). The bone marrow cells collected in the sample tube were ultrasonically disrupted in a cooling water (4-6°C) bath using Bioruptor BR-II. Ultrasonic disruption was performed for a total of 300 seconds (conditions: Time ON 30 sec / OFF 30 sec: 10 cycles, power high) (disruption process). Subsequently, centrifugation was performed, and the supernatant was obtained as a quantitative sample. The quantitative sample was stored at -80°C, and the amount of dopamine (amount of dopamine obtained from one femur) was measured by HPLC-ECD.

[0041] Figure 1 shows the results of dopamine level measurements in the quantitative samples. As shown in Figure 1, the dopamine measurements for most samples showed a distribution where they were around the mean value. These distributions showed less variability compared to the samples in Comparative Example 1, which will be described later. In other words, the quantitative samples in Example 1 provided stable quantitative results that reflected the bone marrow content of the neurotransmitter dopamine.

[0042] [Comparative Example 1] A quantitative sample (Comparative Example 1) was prepared in the same manner as in Example 1, except that ultrasonic disruption was not performed. After storage at -80°C, the amount of dopamine (amount of dopamine obtained from one femur) was measured by HPLC-ECD.

[0043] Figure 2 shows the results of dopamine level measurements in the quantitative samples. As shown in Figure 2, the dopamine measurements for most samples were significantly lower than the average values ​​shown in Figure 1, and a distribution with large variability was observed. In other words, the quantitative samples in Comparative Example 1 did not reflect the bone marrow content of the neurotransmitter dopamine.

[0044] [Example 2] Wild-type mice (WT) and vav-NUP98 / HOXD13 Tg myelodysplastic syndrome model mice (Tg) created by genetically modifying wild-type mice were prepared. Femurs were collected from the WT mice and Tg mice produced by crossing these WT mice with Tg mice, and quantitative samples were obtained in the same manner as in Example 1.

[0045] In the obtained quantitative samples, the monoamines (norepinephrine, dopamine, serotonin), which are neurotransmitters, were measured by HPLC-ECD as predetermined biofactors, and the lipid mediators (15-Deoxy-delta12,14-PGJ2, PGF2a, 18-HEPE, HYA+HYC, 5-HETE, 15-HETE, ketoA, ketoB) were measured by LC-MS / MS. It is known that the expression of these predetermined biofactors is not affected by the predetermined gene modification; that is, the expression levels do not change between WT mice and Tg mice. The measurement results for monoamines are shown in Figure 3, and the measurement results for lipid mediators are shown in Figures 4 and 5.

[0046] As shown in Figures 3 to 5, neurotransmitters (monoamines) and lipid mediators showed similar distributions in both WT mice and Tg mice. In other words, the quantitative samples from Example 2 provided stable quantitative results that reflected the bone marrow content of neurotransmitters and lipid mediators.

[0047] [Example 3] After the cell collection process in Example 2, the tissue was agitated by repeatedly aspirating and discharging it using the syringe with the same needle, and then measured in the same manner. As a result, it was confirmed that the measured values ​​remained stable at the levels shown in Figures 3 to 5.

[0048] [Example 4] The quantitative samples prepared in Example 2 were stored at -80°C for 2 months, and then measured in the same manner. As a result, it was confirmed that the measured values ​​remained stable, as shown in Figures 3 to 5.

[0049] [Example 5] Wild-type mice (C57BL / 6 strain, purchased from CLEA Japan Co., Ltd., aged 6 weeks, 26 weeks, 52 weeks, 78 weeks, 104 weeks, and 130 weeks) were prepared. Femurs were collected from each of these mice, and quantitative samples were obtained in the same manner as in Example 2.

[0050] In the obtained quantitative samples, some of the neurotransmitters (monoamines) and lipid mediators measured in Example 2 (norepinephrine, HYA+HYC, ketoA, ketoB) were measured as predetermined biofactors. The results are shown in Figures 6 and 7.

[0051] As shown in Figures 6 and 7, these biological factors tended to be depleted in the bone marrow of aging mice (104 weeks of age and older).

Claims

1. A method for preparing a sample to quantify the content of a biofactor selected from the group consisting of neurotransmitters and lipid mediators in body tissues produced and / or accumulated by inflammatory cells, A tissue fixation step is performed by fixing the tissue by contacting it with methanol at -15°C or below before collecting cells from the tissue. The cell harvesting process involves collecting cells from fixed tissue, A preparation method comprising a disruption step of ultrasonically disrupting cells in collected tissue.

2. The aforementioned tissue is bone marrow, In the tissue fixation step, the bone marrow is fixed in the bone by injecting methanol at -15°C or below into the bone marrow cavity. In the cell collection process, the fixed bone marrow is drained outside the bone. The preparation method according to claim 1.

3. The preparation method according to claim 2, wherein the tissue fixation step and the cell collection step are performed by injecting from one end of the bone and pushing out the bone marrow from the other end due to the injection pressure.

4. The preparation method according to claim 1, wherein the crushing step is carried out at a temperature greater than 0°C and 6°C or less.

5. A quantitative sample obtained by the preparation method described in claim 1.

6. A quantitative sample according to claim 5, stored at -40°C or below.

7. A method for quantifying the content of a biofactor in body tissue, comprising the step of measuring the biofactor using a quantitative sample obtained by the preparation method described in claim 1.

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