JP2008261633A - Administration agent for recovering colon cell, colon cell recovery method, colon cancer examination method and colon cell recovery kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell recovery method from fecal matters enhanced in cell recovery and not affected by a subject, a cell recovery kit, a colon cancer detection method and a colon cancer detection kit. <P>SOLUTION: A result of the earnest investigation of the cell recovery method from fecal matter enhanced in cell recovery and not affected by the subject proves that an administration agent for recovering colon cells which contains a substance containing a component having intestinal disorder medicating action should be used. The component having the intestinal disorder medicating action is food fiber more preferably pectin. In this method, the subject ingests the substance containing the component having the intestinal disorder medicating action before fecal matter is sampled. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to an administration agent for recovering cells from stool, a cell recovery method, and a colorectal cancer detection method.

  In the field of medical diagnosis, for example, a method of extracting and analyzing a nucleic acid for the purpose of detecting a pathogen or early detection of a disease is used. However, when nucleic acids are extracted from blood, biological tissue, feces, urine, etc., purification of cells and nucleic acids is necessary to prevent false positive results due to contamination. Therefore, for example, one or several types of cells to be subjected to genetic testing are specifically collected from a specimen containing cells. Nucleic acids are extracted from the cells thus collected, and genetic diagnosis is performed by amplifying the nucleic acids by polymerase chain reaction (PCR) or the like to detect gene mutations.

  In particular, one of the cell recovery methods existing in stool is a method of capturing cells on a solid support such as a magnetic bead that specifically binds to the cells to be examined, and removing the impurities (for example, Patent Document 1). There is. Moreover, as a magnetic bead used for cell collection | recovery, there exists a magnetic bead (Dynabeads Epithelial Enrich, the product made from Dynal) couple | bonded with Ber-EP4 antibody.

  In Patent Document 2 below, cells are collected from stool, and the amount of DNA separated from the stool, or the DNA index of stool (the value of nanograms of separated DNA divided by the value of grams of stool weight). It can be determined whether the subject has colorectal cancer, the colorectal is in a precancerous state, or is healthy.

JP 2005-46065 A Japanese National Patent Publication No. 11-511982

  However, conventional cell recovery using a solid support such as magnetic beads has a problem that the cell recovery rate is low depending on the subject.

  Furthermore, the cell recovery rate for recovering cells using a solid carrier such as magnetic beads is 20% to 100% when determining whether colorectal cancer is present from the amount of DNA separated from cells recovered from stool or from the DNA index. There was a problem that accurate colorectal cancer determination could not be made.

  Therefore, in view of these problems, the present invention aims to provide an administration agent for obtaining a stool having a high cell recovery rate and not affected by a subject, a method for recovering cells, and a method for detecting colorectal cancer. did.

  In order to solve the above-mentioned problems, the inventors have conducted intensive studies on an administration agent for obtaining a stool that has a high cell recovery rate and is not affected by a subject, a method for recovering cells, and a method for detecting colorectal cancer. It came to complete. The administration agent for collecting colon cells of the present invention is characterized mainly by containing a substance containing a component having an intestinal regulating action. In addition, the cell collection method of the present invention is mainly characterized in that an administration agent for collecting colon cells is administered before stool collection. Hereinafter, the present invention will be specifically described.

  The administration agent for collecting colon cells of the present invention is characterized by containing a substance containing a component having an intestinal regulating action. Since the content of components that inhibit cell recovery in the stool can be reduced by administering the component having an intestinal regulating function before collecting the stool, cell recovery can be performed with high cell recovery rate and variation Less cell recovery can be performed. In addition, the effect of the agent for 48 hours after administration is more effectively expressed.

  Moreover, the above-mentioned administration agent for collecting colon cells is characterized by being used for a colon cancer test. Since the cell recovery rate is high and the cells can be recovered with little variation, false positives and false negatives can be suppressed and can be suitably used for colorectal cancer tests.

  Furthermore, in the above-mentioned administration agent for collecting colon cells, the component having an intestinal regulating action is preferably dietary fiber. Since dietary fiber is not decomposed by digestive enzymes, it is discharged out of the body together with stool, so that the content of components that inhibit cell recovery in stool can be reduced. More preferably, it is an insoluble dietary fiber. Encourage defecation activity.

  Furthermore, in the above-mentioned administration agent for collecting colon cells, the dietary fiber is more preferably pectin. Pectin is contained in fruits and can be consumed in large quantities. Apple pectin is more preferable. Bad bacteria that inhibit cell recovery compared to other pectin can be further suppressed.

  Furthermore, in the above-mentioned administration agent for collecting colon cells, it is preferable that the substance containing a component having an intestinal regulating action is fruits, sugar beet, sunflower and processed products thereof. It contains a large amount of components having an intestinal action, and a large amount of ingredients having an intestinal action can be administered orally. More preferably it is an apple. Apples are easy to administer in large amounts orally and can consume higher amounts of dietary fiber. In addition, it is preferable to administer 50 g or more of apples orally. A large amount of ingredients having an intestinal regulating action can be ingested. More preferably, 100 g or more of apple is orally administered. Ingredients with an intestinal regulating action can be ingested in larger amounts.

  Furthermore, in the above-mentioned administration agent for collecting colon cells, it is preferable that the administration is effective once. It can be used simply and easily as compared with an administration agent that is administered continuously or intermittently. Further, when used for a test such as a colorectal cancer test, the number of times of administration is one, and the test cost can be reduced as compared with continuous or intermittent administration.

  The method for recovering colon cells of the present invention is characterized by using the above-mentioned administration agent for cell recovery. Cells can be stably recovered with high yield.

  Moreover, in the said cell collection method, it is preferable that it is a colon cancer cell made into collection object. A large number of colorectal cancer cells are considered to exist in stool compared to normal cells, and can be preferably used.

  Furthermore, in the above cell recovery method, it is preferable to use an individual carrier for cell recovery. Cells can be collected easily. More preferably, the solid carrier is a magnetic bead. It can be easily recovered in response to a magnetic field generated by a magnet or the like.

The colorectal cancer testing method of the present invention is characterized by using the above-described cell recovery method.
Cells can be stably recovered with high yield, and accurate colorectal cancer determination can be performed.

  The colon cell collection kit of the present invention is characterized by comprising the above colon cell collection administration agent and an individual carrier. By simultaneously using the administration agent for collecting colon cells and the individual carrier, colon cells can be easily collected. Furthermore, a kit containing a reagent used for cell recovery is preferred. Colon cells can be collected more easily.

  The colorectal cancer test kit of the present invention comprises the above-mentioned administration agent for collecting colon cells and an individual carrier. A colon cancer test can be easily performed by simultaneously using an administration agent for collecting colon cells and an individual carrier. Furthermore, a kit containing a reagent used for a colorectal cancer test is preferable. A colorectal cancer test can be performed more easily.

  According to the present invention, an administration agent for obtaining a stool having a high cell recovery rate and not affected by a subject, a method and kit for recovering cells, a method and kit for accurately detecting colorectal cancer without being affected by individual differences Can be provided.

(Administration agent for collecting colon cells containing a substance containing a component having an intestinal regulating action)
The administration agent for collecting colon cells of the present invention contains a substance containing a component having an intestinal action. The form of the administration agent for collecting colon cells may be either solid or liquid.

  The administration agent for collecting large intestine cells may be administered with a component having an intestinal action before collecting a stool, and a component having an intestinal action may be administered alone, or may be administered simultaneously with other components. As the administration method, oral administration is desirable, but other methods may be used. Administration of the colon cell recovery administration agent is performed before stool collection, and the administration may be performed only once, and colon cell recovery and colon cancer screening can be easily performed without imposing a burden on the subject. Needless to say, the administration agent for collecting colon cells can be administered continuously or intermittently.

  The administration agent for collecting colon cells is preferably administered within 48 hours before collecting the stool. Administration of the colon cell collection agent is preferably within 37 hours before stool collection, and more preferably at dinner on the day before stool collection, and the stool is collected the next morning.

  Although it is desirable to administer 1 g or more of a substance containing a component having an intestinal regulating action, it is preferable to administer about 0.01 g or more.

  The substance containing a component having an intestinal regulating action is preferably dietary fiber. Dietary fiber can be either water-soluble or insoluble. Among dietary fibers, pectin is contained in fruits and is preferable because it can be easily administered in large amounts. In addition, apple pectin can be preferably used because it can further suppress bad bacteria considered to inhibit cell recovery compared to other pectin.

  The substance containing a component having an intestinal regulating action is desirably fruits, sugar beet, sunflower, and the like, and processed products thereof. Fruits are general fruits, for example, fruits described in the “Fiveth Supplement Japanese Food Standard Ingredients Table”. In addition, apples are desirable because they are easy to administer in large amounts orally and contain a large amount of dietary fiber. Further, when orally administering fruits, sugar beet, sunflower, etc. as a substance containing a component having an intestinal regulating action, it is desirable to administer 150 g or more, but it is sufficient to administer about 20 g or more.

(Cell recovery)
As shown in FIG. 1, which is a standard protocol for the cell recovery method of the present invention, it has a step of administering a colon cell recovery administration agent.

  For cell recovery, it is desirable to suspend stool in a medium and adsorb it on a solid carrier after filtration. If the medium contains fetal bovine serum (FBS), the cells can be recovered more stably. In addition, it is desirable to use magnetic beads as the solid carrier, and it is desirable to collect cells captured on the magnetic beads by a substance or device that generates a magnetic field such as a magnet. The magnetic beads may be any type of magnetic beads as long as they respond to a magnetic field. However, since a solution containing feces has a high viscosity, it is desirable to contain Fe particles having a high magnetic field response. Furthermore, in order to ensure corrosion resistance, it must be coated with Ti, Si, oxides thereof, nitrides, carbides, and carbon, or with multiple layers of the above substances. Is more desirable. Alternatively, after freezing the stool, the cells may be collected after separating the stool surface.

  By using the method of the present invention, colon epithelial cells and cells detached from the colon epithelium can be collected. In order to detect colorectal cancer after cell recovery, the target cell is desirably a colorectal cancer cell, but it goes without saying that it can also be used to recover normal cells.

  Cells collected by the method of the present invention can be used for colorectal cancer detection or colorectal cancer detection because cells can be collected without high stool dependence but with high yield and small cell recovery rate. It is. Extract DNA and RNA from the collected cells, and determine whether the subject has colorectal cancer, colorectal precancerous condition, or health based on the extracted DNA amount or DNA index It can be used in various methods such as genetic analysis.

  It is desirable to use a substance containing a component having an intestinal regulating action and an individual carrier as a colon cell collection kit. The kit may also contain reagents such as medium. Furthermore, it can also be used as a kit for colorectal cancer detection.

  Hereinafter, embodiments according to the present invention will be described in detail. However, the present invention is not necessarily limited by these examples.

Examples used in the present invention and comparative examples thereof are shown below. In addition, stool was collected from healthy subjects and all from the same subject.
Example 1
Apples were orally administered in an amount of 200 g with the skin peeled, and stool was collected after 13 hours. The collected stool was used after 1 hour at room temperature and 2 hours at 4 ° C. 1 g of the collected stool was collected in a 50 mL centrifuge tube, and 30 mL of 10% FBS-containing L-15 Medium (Sigma) was added and stirred. After stirring, the suspension was filtered using a filter. Ten thousand cells of a rectal cancer cell line (HT-29 cells) were added to the filtrate, and the mixture was vortexed for 2 seconds. 80 μL of a commercially available magnetic bead for recovering epithelial cells (Dynabeads Epithelial Enrich, Dynal), to which a commercially available Ber-Ep4 antibody is immobilized, was mixed at room temperature for 30 minutes using a mix rotor, and colon cancer was detected using the magnetic beads for recovering epithelial cells. Cells were captured.

  The centrifuge tube was placed on a magnetic stand (MultiSep Magnetic Separator, Polyscience) and stirred for 30 minutes using a seesaw type stirrer to magnetically collect the magnetic beads capturing the cells, and the supernatant was removed. After washing with 10% FBS-containing L-15 Medium, the centrifuge tube was removed from the magnetic stand, 1000 μL of 10% FBS-containing L-15 Medium was suspended in magnetic beads, and the magnetic bead suspension was transferred to a 1.5 mL Eppendorf tube. The supernatant was removed using a 1.5 mL Eppendorf magnetic stand (MPC-S, Dynal), and 600 μL of PBS (phosphate buffer) was added and suspended.

  The solution in which the magnetic beads capturing the cells are suspended is stained using a cell double staining kit (Donjin Chemical Co., Ltd.) and observed with a plankton calculation plate (Matsunami Glass Co., Ltd.) using a fluorescence microscope. The number of viable cells was counted by counting the number of viable cells. The cell recovery rate was determined by dividing the number of living cells by the number of cells added (10000 cells) and multiplying by 100. The results are shown in Table 1 and FIG.

(Example 2)
Cells were collected in the same manner as in Example 1 except that 200 g of apple was orally administered with the skin peeled, and stool was collected after 37 hours. The results are shown in Table 1.

(Example 3)
For cell recovery, iron particles were coated with titania, and magnetic beads coated with silica were added with streptavidin (Wako Pure Chemical Industries, Ltd.) and bound to magnetic beads, and biotinylated VU-1D9 antibody (Biomeda) The cells were collected in the same manner as in Example 1 except that magnetic beads bound with the same method were used. The results are shown in Table 1.

(Comparative Example 1)
Cells were collected in the same manner as in Example 1 except that stool collected from a subject who did not administer apples for 3 days before collection was used. The results are shown in Table 1 and FIG.

(Comparative Example 2)
Cells were collected in the same manner as in Example 2 except that stool collected from subjects who did not administer apples for 3 days before collection was used. The results are shown in Table 1.

  No. in Table 1 1 and no. 2 is the difference in the date of the experiment, stool collected without administration of apples for 3 days or more was used in Comparative Example 1 or Comparative Example 2, and 200 g of apple was administered after dinner on that day, and stool collected the next morning Was used in Example 1 or Example 3. No. For stool 1, stool collected after one day was used in Example 2. The experiment date is No. 1 and No. A period of one month was left between the two to eliminate the effects of apple administration.

  As clearly shown in Table 1, in the stool collected from a subject who did not administer apples for 3 days prior to collection, which is a comparative example, the cell recovery rate was as low as 60% or less regardless of antibody and magnetic beads, and extremely low at 24%. In some cases, the cell recovery rate was low. The cell recovery rate is 24% to 58%, and the cell recovery rate is greatly affected by the difference in the stool collection date, that is, the difference in stool. On the other hand, stool collected after 13 hours or 37 hours after administration of the apples of the Examples showed a high cell recovery rate of 65% or more regardless of the antibody, magnetic beads, and stool collection date, and the cell recovery rate increased. ing. Moreover, the cell recovery rate is 65% to 89%, and the variation in the cell recovery rate is also decreased, indicating that the influence on the cell recovery rate due to the difference in stool is reduced. In other words, it was shown that the administration agent for collecting colon cells of the present invention works effectively, and that the cell recovery method of the present invention is useful as a method for recovering cells from stool.

Example 4
Apple and magnetic beads were packed in the same box to make a colon cancer test kit.

(Example 5)
Apple and magnetic beads and 10% FBS-containing L-15 Medium were packed in the same box to make a colon cancer test kit.

It is a flowchart which shows the outline of the protocol which orally administers the substance containing the component which has an intestinal regulating action before stool collection, and collect | recovers cells from the collected stool. It is the graph which compared the cell recovery rate of Example 1 and Comparative Example 2. FIG.

Claims (16)

  An agent for collecting colon cells, comprising a substance containing a component having an intestinal regulating action.   The administration agent for collecting colon cells according to claim 1, which is used for a colon cancer test.   The administration agent for collecting colon cells according to claim 1 or 2, wherein the component having an intestinal regulating action is dietary fiber.   The said dietary fiber is pectin, The administration agent for colon cell collection | recovery in any one of Claims 1-3 characterized by the above-mentioned.   The said pectin is an apple pectin, The administration agent for colon cell collection | recovery in any one of Claims 1-4 characterized by the above-mentioned.   The agent for collecting large intestine cells according to claim 1 or 2, wherein the substance containing a component having an intestinal regulating action is fruits, sugar beet, sunflower, and processed products thereof.   The administration agent for collecting colon cells according to any one of claims 1 to 6, wherein the administration is effective at one administration.   A method for recovering colon cells, comprising using the administration agent for recovering colon cells according to any one of claims 1 to 7.   The method for recovering colon cells according to claim 8, wherein the colon cells are colon cancer cells.   A method for recovering colon cells, comprising using the administration agent for recovering colon cells according to any one of claims 1 to 7 and an individual carrier.   The method according to claim 10, wherein the solid carrier is a magnetic bead.   A method for examining colorectal cancer, comprising using the method according to claim 8.   A kit for collecting colon cells, comprising the administration agent for collecting colon cells according to any one of claims 1 to 7 and an individual carrier.   A kit for collecting colon cells, which comprises the administration agent for collecting colon cells according to any one of claims 1 to 7, an individual carrier, and a reagent.   A test kit for colorectal cancer comprising the administration agent for recovering colorectal cells according to any one of claims 1 to 7 and an individual carrier.   A test kit for colorectal cancer comprising the administration agent for collecting colon cells according to any one of claims 1 to 7, an individual carrier, and a reagent.

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