JP6324730B2 - Method for distinguishing lymph nodes in tissue fragments obtained by lymph node dissection - Google Patents
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Description
本発明は、リンパ節郭清により得られた組織片中のリンパ節判別方法、及び該方法に用いられる処理剤に関する。 The present invention relates to a method for discriminating lymph nodes in a tissue piece obtained by lymph node dissection, and a treatment agent used in the method.
がん組織の切除手術においては、がん細胞のリンパ行性転移に対する処置として、がん組織のみならず、その周辺組織（所属リンパ節含む）ごと除去（郭清）することが行われる。そして、除去されたリンパ節について、がん細胞の転移の有無が調べられる。 In excision of a cancer tissue, as a treatment for lymphatic metastasis of cancer cells, not only the cancer tissue but also its surrounding tissues (including regional lymph nodes) are removed (dissection). The removed lymph nodes are then examined for the presence of cancer cell metastasis.
この転移の有無や、転移しているリンパ節の数などは、切除したがん組織のステージ及びその後の化学療法戦略を決定する重要な要因となる。したがって、これらの決定をより正確に行うという観点から、リンパ節郭清により得られた組織片から、できるだけ多くのリンパ節について調べることが重要である。 The presence or absence of this metastasis and the number of metastatic lymph nodes are important factors that determine the stage of the resected cancer tissue and the subsequent chemotherapy strategy. Therefore, from the viewpoint of making these determinations more accurately, it is important to examine as many lymph nodes as possible from the tissue pieces obtained by lymph node dissection.
しかしながら、リンパ節はリンパ節郭清により得られた組織片中に埋もれた状態で存在するので、リンパ節を調べるためには、まず該組織片からリンパ節を判別して取り出すことが必要となる。通常、この判別は医師の触診によって行われており、多くの労力と時間を要する。 However, since the lymph node is buried in the tissue piece obtained by lymph node dissection, it is necessary to first identify and remove the lymph node from the tissue piece in order to examine the lymph node. . Usually, this determination is performed by a doctor's palpation, and requires a lot of labor and time.
このような問題を解決するために、リンパ節郭清により得られた組織片からmRNAを抽出し、遺伝子増幅反応によってがん細胞の存在の有無を判定する方法が報告されている（非特許文献1〜3）。 In order to solve such a problem, a method has been reported in which mRNA is extracted from a tissue fragment obtained by lymph node dissection and the presence or absence of cancer cells is determined by a gene amplification reaction (Non-Patent Document). 1-3).
しかしながら、この方法では、組織片中のがん細胞の有無の情報しか得ることができず、極一部のリンパ節のみへの転移なのか、或いは大半のリンパ節への転移なのかといった情報を得ることができない。したがって、がん組織のステージ及びその後の化学療法戦略を決定するための情報としては不十分である。 However, this method can only obtain information on the presence or absence of cancer cells in the tissue fragment, and it can be used to determine whether the metastasis is to only some lymph nodes or to most lymph nodes. Can't get. Therefore, there is insufficient information to determine the stage of cancer tissue and subsequent chemotherapy strategy.
本発明は、リンパ節郭清により得られた組織片中のリンパ節を、簡便且つ効率的に判別する方法を提供することを目的とする。また、リンパ節をより多く判別することも目的とする。さらに、この判別を、リンパ節の構造が保たれた状態で行うことをも目的とする。 An object of the present invention is to provide a method for easily and efficiently discriminating lymph nodes in a tissue piece obtained by lymph node dissection. Another object is to identify more lymph nodes. It is another object of the present invention to make this determination while maintaining the lymph node structure.
本発明者等は鋭意研究した結果、リンパ節郭清により得られた組織片を、タンパク質分解酵素及び脂肪分解酵素からなる群より選択される少なくとも１種の酵素で処理することによって、該組織片からリンパ節や脈管以外の余分な組織の大半が除かれることを見出した。処理後の組織片からは、目視により容易にリンパ節を判別することができた。さらに、上記処理剤で処理した後でも、リンパ節の構造は破壊されずに残っていることをも見出した。これらの知見に基づいてさらに研究した結果、本発明が完成した。 As a result of intensive studies, the present inventors treated the tissue piece obtained by lymph node dissection with at least one enzyme selected from the group consisting of a proteolytic enzyme and a lipolytic enzyme. It was found that most of the extra tissues other than lymph nodes and vessels were removed from. From the treated tissue piece, lymph nodes could be easily discriminated visually. Furthermore, it has also been found that the structure of the lymph node remains without being destroyed even after the treatment with the treatment agent. As a result of further studies based on these findings, the present invention has been completed.
即ち、本発明は、下記の態様を包含する。 That is, the present invention includes the following aspects.
項１．
タンパク質分解酵素及び脂肪分解酵素からなる群より選択される少なくとも１種の酵素を有効成分として含む、リンパ節郭清により得られた組織片中のリンパ節判別に用いられる当該組織片の処理剤。
Item 1.
A treating agent for a tissue piece used for discrimination of a lymph node in a tissue piece obtained by lymph node dissection, which contains at least one enzyme selected from the group consisting of a proteolytic enzyme and a lipolytic enzyme as an active ingredient.
項２．
有効成分がタンパク質分解酵素、又はタンパク質分解酵素及び脂肪分解酵素である、項１に記載の処理剤。
Item 2.
Item 2. The treatment agent according to Item 1, wherein the active ingredient is a proteolytic enzyme, or a proteolytic enzyme and a lipolytic enzyme.
項３．
前記タンパク質分解酵素がコラゲナーゼ及びトリプシンからなる群より選択される少なくとも１種の酵素である、項１又は２に記載の処理剤。
Item 3.
Item 3. The treating agent according to Item 1 or 2, wherein the proteolytic enzyme is at least one enzyme selected from the group consisting of collagenase and trypsin.
項４．
前記脂肪分解酵素がリパーゼである、項１〜３のいずれかに記載の処理剤。
Item 4.
Item 4. The treatment agent according to any one of Items 1 to 3, wherein the lipolytic enzyme is lipase.
項５．
有効成分がトリプシン及びリパーゼからなる群より選択される少なくとも１種の酵素並びにコラゲナーゼである、請求項１〜４のいずれかに記載の処理剤。
Item 5.
The treatment agent according to any one of claims 1 to 4, wherein the active ingredient is at least one enzyme selected from the group consisting of trypsin and lipase, and collagenase.
項６．
（ａ）リンパ節郭清により得られた組織片と項１〜５のいずれかに記載の処理剤とを接触させる工程、及び
（ｂ）工程（ａ）後の組織片中の球形部分をリンパ節として判別する工程
を含む、リンパ節郭清により得られた組織片中のリンパ節判別方法。
Item 6.
(A) a step of contacting a tissue piece obtained by lymph node dissection with the treatment agent according to any one of Items 1 to 5, and (b) a spherical portion in the tissue piece after step (a) is lymphatic A method for discriminating a lymph node in a tissue piece obtained by lymph node dissection, comprising a step of discriminating as a node.
項７．
（ａ）リンパ節郭清により得られた組織片と項１〜５のいずれかに記載の処理剤とを接触させる工程、
（ｂ）工程（ａ）後の組織片中の球形部分をリンパ節として判別する工程、及び
（ｃ）工程（ｂ）で判別されたリンパ節中のがん細胞の有無を判定する工程
を含む、リンパ節中のがん細胞の検査方法。
Item 7.
(A) a step of bringing the tissue piece obtained by lymph node dissection into contact with the treatment agent according to any one of Items 1 to 5,
(B) including a step of discriminating a spherical portion in the tissue piece after step (a) as a lymph node, and a step of (c) determining the presence or absence of cancer cells in the lymph node determined in step (b). A method for examining cancer cells in lymph nodes.
本発明によれば、リンパ節郭清により得られた組織片中のリンパ節を、簡便且つ効率的に判別することができる。また、より多くのリンパ節を判別できるので、判別されたリンパ節へのがん細胞の転移を調べることによって、切除したがん組織のステージ及びその後の化学療法戦略をより正確に決定することができる。さらに、リンパ節の構造が保たれた状態で判別できるので、病理学的診断へは影響を与えない。 According to the present invention, lymph nodes in a tissue piece obtained by lymph node dissection can be easily and efficiently discriminated. In addition, because more lymph nodes can be identified, it is possible to more accurately determine the stage of the resected cancer tissue and the subsequent chemotherapy strategy by examining the metastasis of cancer cells to the identified lymph nodes. it can. Furthermore, since it can be discriminated while maintaining the lymph node structure, it does not affect the pathological diagnosis.
１．リンパ節郭清により得られた組織片中のリンパ節判別に用いられる当該組織片の処理剤
本発明は、リンパ節郭清により得られた組織片中のリンパ節判別に用いられる当該組織片の処理剤（以下、単に「処理剤」と略記することもある）に関する。
1. Treatment agent for tissue piece used for lymph node discrimination in tissue piece obtained by lymph node dissection The present invention relates to a treatment agent for the tissue piece used for lymph node discrimination in tissue piece obtained by lymph node dissection. The present invention relates to a treating agent (hereinafter sometimes simply referred to as “treating agent”).
「リンパ節郭清」とは、がん組織周囲のリンパ節への転移の有無を調べるために、これらのリンパ節を除去することである。通常、リンパ節は単独で存在しているのではなく、他の組織（リンパ管、脂肪組織等）と連結した状態或いは他の組織と付着した状態で存在している。したがって、「リンパ節郭清により得られた組織片」には、リンパ節のみならず他の組織も含まれる。 “Lymph node dissection” is the removal of these lymph nodes in order to check for metastasis to lymph nodes surrounding the cancer tissue. Usually, lymph nodes do not exist alone, but exist in a state of being connected to other tissues (lymphatic vessels, adipose tissue, etc.) or in a state of being attached to other tissues. Therefore, “a tissue piece obtained by lymph node dissection” includes not only lymph nodes but also other tissues.
「リンパ節郭清により得られた組織片」は、上記のような組織片である限り特に限定されない。例えば、大腸がん、小腸がん、胃がん、食道がん、肝臓がん、胆嚢がん、膵臓がん、乳がん、口腔がん、頭頸部がん、卵巣がん、子宮頚がん、子宮内膜がん、腎細胞がん、膀胱がん、前立腺がん、肺がんなどのがん組織からのがん細胞の転移が疑われるリンパ節を含む組織片が挙げられる。これらの中でも、本発明の効果をより確実に発揮させるという観点からは、好ましくは大腸がん、小腸がん、胃がん、及び乳がん、より好ましくは大腸がん、小腸がん、及び胃がん、さらに好ましくは大腸がん、よりさらに好ましくは結腸直腸がんのがん組織からのがん細胞の転移が疑われるリンパ節を含む組織片が挙げられる。 The “tissue piece obtained by lymph node dissection” is not particularly limited as long as it is the above-described tissue piece. For example, colorectal cancer, small intestine cancer, stomach cancer, esophageal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, oral cancer, head and neck cancer, ovarian cancer, cervical cancer, intrauterine Examples include tissue fragments including lymph nodes suspected of metastasizing cancer cells from cancer tissues such as membrane cancer, renal cell cancer, bladder cancer, prostate cancer, and lung cancer. Among these, from the viewpoint of more reliably exerting the effect of the present invention, preferably colorectal cancer, small intestine cancer, stomach cancer, and breast cancer, more preferably colorectal cancer, small intestine cancer, and stomach cancer, more preferably Includes a tissue fragment containing a lymph node suspected of metastasizing cancer cells from colorectal cancer, more preferably colorectal cancer tissue.
「がん組織からのがん細胞の転移が疑われるリンパ節を含む組織」は、がんの発生した部位毎に、及びがんの進行度に従って定められている。例えば、大腸がんの場合は、大腸癌治療ガイドライン2010年版に定められている。 “A tissue including a lymph node in which cancer cell metastasis from cancer tissue is suspected” is determined for each site where cancer has occurred and according to the degree of progression of cancer. For example, in the case of colorectal cancer, it is defined in the colorectal cancer treatment guidelines 2010 edition.
「リンパ節郭清により得られた組織片中のリンパ節判別に用いられる」とは、リンパ節と他の組織が混在する「リンパ節郭清により得られた組織片」の中から、リンパ節を判別する目的で用いられることを意味する。このようにして判別されたリンパ節は、がん細胞の転移の有無について調べられる。 “Used for lymph node discrimination in tissue pieces obtained by lymph node dissection” means “tissue pieces obtained by lymph node dissection” in which lymph nodes and other tissues coexist. It is used for the purpose of discriminating. The lymph nodes thus discriminated are examined for the presence or absence of cancer cell metastasis.
本発明の処理剤は、この目的のために、「リンパ節郭清により得られた組織片」を処理するための剤であり、その限りにおいて使い方は特に限定されない。 The treatment agent of the present invention is an agent for treating “a tissue piece obtained by lymph node dissection” for this purpose, and the method of use is not particularly limited as long as it is treated.
本発明の処理剤は、有効成分として、タンパク質分解酵素及び脂肪分解酵素からなる群より選択される少なくとも1種の酵素を含む。 The treatment agent of the present invention contains at least one enzyme selected from the group consisting of proteolytic enzymes and lipolytic enzymes as an active ingredient.
タンパク質分解酵素としては、プロテアーゼ活性を有するものであれば特に限定されない。例えば、コラゲナーゼ、トリプシン、キモトリプシン、エラスターゼ、カルボキシぺプチダーゼA、カルボキシぺプチダーゼB、アミノぺプチダーゼN、ペプシン、パンクレアチン、パパイン、ズブチリシン等が挙げられる。これらの中でも、リンパ節判別をより簡便に行えるという観点からは、コラゲナーゼ及びトリプシンが挙げられる。タンパク質分解酵素は1種単独であってもよいし、2種以上の組み合わせであってもよい。 The proteolytic enzyme is not particularly limited as long as it has protease activity. For example, collagenase, trypsin, chymotrypsin, elastase, carboxypeptidase A, carboxypeptidase B, aminopeptidase N, pepsin, pancreatin, papain, subtilisin and the like can be mentioned. Among these, collagenase and trypsin are mentioned from a viewpoint that lymph node discrimination | determination can be performed more simply. One type of proteolytic enzyme may be used, or a combination of two or more types may be used.
脂肪分解酵素としては、例えばリパーゼが挙げられる。リパーゼとしては、各種生物由来のものを用いることができる。例えば、ヒト、サル、マウス、ラット、ブタ等の哺乳類の膵臓から抽出されたリパーゼを用いることができる。脂肪分解酵素は1種単独であってもよいし、2種以上の組み合わせであってもよい。 Examples of the lipolytic enzyme include lipase. As the lipase, those derived from various organisms can be used. For example, lipase extracted from the pancreas of mammals such as humans, monkeys, mice, rats, and pigs can be used. One type of lipolytic enzyme may be used, or a combination of two or more types may be used.
有効成分は、リンパ節判別をより簡便に行えるという観点からは、好ましくはタンパク質分解酵素、又はタンパク質分解酵素及び脂肪分解酵素であることができ、より好ましくはトリプシン及びリパーゼからなる群より選択される少なくとも１種の酵素並びにコラゲナーゼであることができる。 The active ingredient is preferably a proteolytic enzyme, or a proteolytic enzyme and a lipolytic enzyme, more preferably selected from the group consisting of trypsin and lipase from the viewpoint that lymph node discrimination can be performed more easily. It can be at least one enzyme as well as collagenase.
本発明の処理剤において、タンパク質分解酵素の含有割合については、タンパク質分解酵素の種類に応じて適宜設定される。例えば、処理剤の総量に対して、タンパク質分解酵素が総量で0.001〜10 w/v％、好ましくは0.01〜3 w/v％、より好ましくは0.05〜1 w/v％、さらに好ましくは0.1〜0.5 w/v％であることができる。 In the treatment agent of the present invention, the content ratio of the proteolytic enzyme is appropriately set according to the type of the proteolytic enzyme. For example, the total amount of proteolytic enzyme is 0.001 to 10 w / v%, preferably 0.01 to 3 w / v%, more preferably 0.05 to 1 w / v%, still more preferably 0.1 to It can be 0.5 w / v%.
本発明の処理剤において、脂肪分解酵素の含有割合については、脂肪分解酵素の種類に応じて適宜設定される。例えば、処理剤の総量に対して、脂肪分解酵素が総量で0.001〜20 w/v％、好ましくは0.01〜10 w/v％、より好ましくは0.05〜5 w/v％、さらに好ましくは0.1〜3 w/v％であることができる。 In the treatment agent of the present invention, the content ratio of the lipolytic enzyme is appropriately set according to the type of lipolytic enzyme. For example, the total amount of lipolytic enzyme is 0.001 to 20 w / v%, preferably 0.01 to 10 w / v%, more preferably 0.05 to 5 w / v%, still more preferably 0.1 to Can be 3 w / v%.
本発明の処理剤のpHは、有効成分であるタンパク質分解酵素や脂肪分解酵素の至適pHから大きく外れない限り特に限定されない。例えば、有効成分がトリプシン（至滴pHは通常pH 8〜9）及びコラゲナーゼ（至滴pHは通常、中性付近）である場合は、処理剤のpHはpH 6〜9、好ましくは6.5〜8程度であることができる。 The pH of the treatment agent of the present invention is not particularly limited as long as it does not greatly deviate from the optimum pH of proteolytic enzymes and lipolytic enzymes which are active ingredients. For example, when the active ingredients are trypsin (droplet pH is usually pH 8-9) and collagenase (droplet pH is usually near neutral), the pH of the treatment agent is pH 6-9, preferably 6.5-8. Can be about.
本発明の処理剤は、有効成分以外にも、塩、緩衝剤等の他の成分を含んでいてもよい。また、有効成分である酵素の種類によっては、カルシウムイオンやマグネシウムイオン等の金属イオンをさらに含んでいることが好ましい。 The treatment agent of the present invention may contain other components such as salts and buffering agents in addition to the active ingredients. Moreover, it is preferable that metal ions, such as a calcium ion and a magnesium ion, are further included depending on the kind of enzyme which is an active ingredient.
塩としては、主に処理剤を細胞内液と等張にするために用いられるものである限り特に限定されず、例えば塩化ナトリウム、塩化カリウム等が挙げられる。 The salt is not particularly limited as long as it is mainly used to make the treatment agent isotonic with the intracellular solution, and examples thereof include sodium chloride and potassium chloride.
緩衝剤は、公知の緩衝剤である限り特に限定されず、有効成分である酵素の至適pHに従って適宜選択される。例えば、リン酸緩衝剤、トリス緩衝剤、ホウ酸緩衝剤、クエン酸緩衝剤、酢酸緩衝剤等や、HEPES等のグッド緩衝剤等が挙げられる。 The buffer is not particularly limited as long as it is a known buffer, and is appropriately selected according to the optimum pH of the enzyme that is an active ingredient. For example, a phosphate buffer, a Tris buffer, a borate buffer, a citrate buffer, an acetic acid buffer, a good buffer such as HEPES, and the like can be given.
本発明の処理剤の剤形は、液状であることが好ましい。液状の中でも水性液状が好ましい。本発明の処理剤は、剤形に応じた公知の方法に従って製造することができる。例えば水性液状にする場合、蒸留水、常水、精製水、滅菌精製水等の水に、上記した有効成分や他の成分を溶解することにより製造することができる。また、有効成分を、予め緩衝剤や塩等を含む溶液（PBS等）に溶解することによっても製造することができる。 The dosage form of the treatment agent of the present invention is preferably liquid. Among the liquids, an aqueous liquid is preferable. The treatment agent of the present invention can be produced according to a known method corresponding to the dosage form. For example, when making it into aqueous liquid form, it can manufacture by melt | dissolving the above-mentioned active ingredient and other components in water, such as distilled water, normal water, purified water, and sterilized purified water. The active ingredient can also be produced by dissolving in advance in a solution (PBS or the like) containing a buffering agent, salt or the like.
斯かる本発明に処理剤を、下記「２．リンパ節郭清により得られた組織片中のリンパ節判別方法」のように用いることによって、リンパ節郭清により得られた組織片中のリンパ節を、簡便且つ効率的に、より多く判別することが可能となる。 By using the treatment agent according to the present invention as described in “2. Method for discriminating lymph nodes in tissue pieces obtained by lymph node dissection” below, lymph in tissue pieces obtained by lymph node dissection is used. More nodes can be discriminated more easily and efficiently.
２．リンパ節郭清により得られた組織片中のリンパ節判別方法
本発明は、リンパ節郭清により得られた組織片中のリンパ節判別方法（以下、単に「判別方法」と略記することもある）に関する。
2. The present invention relates to a method for discriminating lymph nodes in a tissue piece obtained by lymph node dissection (hereinafter sometimes simply referred to as “discrimination method”). )
本発明の判別方法は、下記工程（ａ）及び工程（ｂ）を含む。
工程（ａ）：リンパ節郭清により得られた組織片と本発明の処理剤とを接触させる工程
工程（ｂ）：工程（ａ）後の組織片中の球形部分をリンパ節として判別する工程
工程（ａ）
「リンパ節 郭清により得られた組織片」、及び「本発明の処理剤」については、上記「１．リンパ節郭清により得られた組織片中のリンパ節判別に用いられる当該組織片の処理剤」の記載と同様である。
The discriminating method of the present invention includes the following steps (a) and (b).
Step (a): The step of contacting the tissue piece obtained by lymph node dissection with the treatment agent of the present invention Step (b): The step of discriminating the spherical portion in the tissue piece after step (a) as a lymph node
Step (a)
Regarding “the tissue piece obtained by lymph node dissection” and “the treatment agent of the present invention”, the above-mentioned “1. The tissue piece used for discrimination of the lymph node in the tissue piece obtained by lymph node dissection” This is the same as the description of the “treatment agent”.
接触は、リンパ節郭清により得られた組織片と本発明の処理剤とが接する態様であれば特に限定されない。典型的には、該組織片を本発明の処理剤に浸すことによって行われる。また、該組織片と本発明の処理剤とが均一に混合されるように、撹拌することが好ましい。撹拌は、有効成分である酵素が失活しない程度の速度で行えばよい。撹拌によってより効率的に接触させることが出来るが、必ずしも必要ではない。 The contact is not particularly limited as long as the tissue piece obtained by lymph node dissection is in contact with the treatment agent of the present invention. Typically, this is performed by immersing the tissue piece in the treatment agent of the present invention. Moreover, it is preferable to stir so that this tissue piece and the processing agent of this invention may be mixed uniformly. Stirring may be performed at a speed that does not deactivate the enzyme that is an active ingredient. Although it can contact more efficiently by stirring, it is not always necessary.
リンパ節郭清により得られた組織片と、本発明の処理剤との比率としては、該処理剤中の有効成分が該組織片全体に作用できる限り特に限定されない。例えば、該組織片1 g当たり、該処理剤0.1〜10 ml程度の比率が挙げられる。 The ratio of the tissue piece obtained by lymph node dissection to the treatment agent of the present invention is not particularly limited as long as the active ingredient in the treatment agent can act on the entire tissue piece. For example, a ratio of about 0.1 to 10 ml of the treatment agent can be mentioned per 1 g of the tissue piece.
接触中の温度は、有効成分である酵素が活性を有する限り特に限定されない。例えば15〜50℃、好ましくは20〜40℃程度であることができる。 The temperature during the contact is not particularly limited as long as the enzyme which is an active ingredient has activity. For example, it can be about 15-50 ° C, preferably about 20-40 ° C.
接触時間は、有効成分である酵素が活性を十分に発揮できる限り特に限定されない。例えば、酵素の濃度が比較的低く、接触中の温度が比較的低温（20℃程度）である場合は、1〜8時間程度であることができる。また、酵素の濃度が比較的高く、接触中の温度が比較的高温（40℃程度）である場合は、5〜20分程度であることができる。 The contact time is not particularly limited as long as the enzyme as an active ingredient can sufficiently exhibit the activity. For example, when the enzyme concentration is relatively low and the temperature during contact is relatively low (about 20 ° C.), it can be about 1 to 8 hours. Moreover, when the enzyme concentration is relatively high and the temperature during contact is relatively high (about 40 ° C.), it can be about 5 to 20 minutes.
斯かる処理によって、リンパ節郭清により得られた組織片から、余分な組織（脂肪組織等）の大半を分離させることができる。処理後は、リンパ節を含む組織片、分離された余分な組織片、及び本発明の処理剤の混合物が得られるので、該混合物から、水分を除去し、さらに分離された余分な組織を取り除いておくとよい。 By such treatment, most of the excess tissue (adipose tissue or the like) can be separated from the tissue piece obtained by lymph node dissection. After the treatment, a tissue piece containing lymph nodes, a separated excess tissue piece, and a mixture of the treatment agent of the present invention are obtained, so that water is removed from the mixture, and the separated excess tissue is removed. It is good to keep.
工程（ｂ）
工程（ａ）後の組織片においてはリンパ節周囲の余分な組織が除去されているので、該組織片中の球形部分を、リンパ節として判別することができる。
Step (b)
In the tissue piece after step (a), excess tissue around the lymph node is removed, so that the spherical portion in the tissue piece can be identified as a lymph node.
リンパ節の直径は、通常0.2〜15 mm程度である。したがって、この点も考慮に入れることにより、より正確にリンパ節を判別することができる。 The diameter of the lymph node is usually about 0.2 to 15 mm. Therefore, by taking this point into consideration, the lymph node can be more accurately determined.
判別は、上記のように形状や大きさに基づいて行うことができるので、通常、目視によって行われる。 Since the determination can be performed based on the shape and size as described above, it is usually performed visually.
斯かる本発明の判別方法によれば、リンパ節郭清により得られた組織片中のリンパ節を、簡便且つ効率的に、より多く判別することが可能となる。 According to such a discrimination method of the present invention, it is possible to more easily and efficiently discriminate more lymph nodes in a tissue piece obtained by lymph node dissection.
判別されたリンパ節は、がん細胞の転移の有無を調べるために、通常、工程（ａ）後の組織片から分離される。リンパ節中のがん細胞の有無は下記「３．リンパ節中のがん細胞の検査方法」に従って行うことができる。 The determined lymph nodes are usually separated from the tissue piece after step (a) in order to examine the presence or absence of cancer cell metastasis. The presence or absence of cancer cells in lymph nodes can be determined according to the following “3. Method for examining cancer cells in lymph nodes”.
３．リンパ節中のがん細胞の検査方法
本発明は、リンパ節中のがん細胞の検査方法（以下、単に「検査方法」と略記することもある）に関する。
3. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for examining cancer cells in a lymph node (hereinafter sometimes simply referred to as “test method”).
本発明の検査方法は、下記工程（ａ）、工程（ｂ）、及び工程（ｃ）を含む。
工程（ａ）：リンパ節郭清により得られた組織片と本発明の処理剤とを接触させる工程
工程（ｂ）：工程（ａ）後の組織片中の球形部分をリンパ節として判別する工程
工程（ｃ）：工程（ｂ）で判別されたリンパ節中のがん細胞の有無を判定する工程
工程（ａ）及び工程（ｂ）
工程（ａ）及び工程（ｂ）については、上記「２．リンパ節郭清により得られた組織片中のリンパ節判別方法」の記載の同様である。
The inspection method of the present invention includes the following step (a), step (b), and step (c).
Step (a): The step of contacting the tissue piece obtained by lymph node dissection with the treatment agent of the present invention Step (b): The step of discriminating the spherical portion in the tissue piece after step (a) as a lymph node Step (c): A step of determining the presence or absence of cancer cells in the lymph node determined in step (b).
Step (a) and Step (b)
Step (a) and step (b) are the same as described in “2. Method for discriminating lymph nodes in tissue piece obtained by lymph node dissection” above.
工程（ｃ）
リンパ節中のがん細胞の有無の判定は、公知の方法に従って行うことができる。一般的にはリンパ節の切片をヘマトキシリンエオジン染色し、染色像を観察することによりがん細胞の有無を判定する。この判定は、例えば大腸癌取り扱い規約第8版2013年度に記載の基準に従って行うことができる。
Step (c)
Determination of the presence or absence of cancer cells in lymph nodes can be performed according to a known method. Generally, lymph node sections are stained with hematoxylin and eosin, and the presence or absence of cancer cells is determined by observing the stained images. This determination can be made, for example, according to the criteria described in the Colorectal Cancer Handling Regulations 8th Edition 2013.
斯かる本発明の検査方法によれば、リンパ節郭清により得られた組織片から、より多くのリンパ節中のがん細胞の有無を、簡便且つ効率的に検査することができる。 According to such an inspection method of the present invention, the presence or absence of cancer cells in more lymph nodes can be easily and efficiently inspected from a tissue piece obtained by lymph node dissection.
以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.
実施例１：リンパ節郭清により得られた組織片の酵素処理、及びリンパ節の判別
図1に示す方法に従って、リンパ節郭清により得られた組織片からリンパ節を判別し、その数を測定した。具体的には次のように行った。
Example 1: Enzymatic treatment of tissue pieces obtained by lymph node dissection and discrimination of lymph nodes According to the method shown in Fig. 1, lymph nodes are discriminated from tissue pieces obtained by lymph node dissection, and the number thereof is determined. It was measured. Specifically, it was performed as follows.
（１）組織片の調製
結腸直腸がん手術を受けた10名の被験者それぞれから、倫理規定に従った書面によるインフォームド・コンセントを受けた上で、該手術中のリンパ節郭清により得られた腸間膜組織片を取得した（計10検体）。該組織片の大半は脂肪組織から構成されており、その中にリンパ節が埋め込まれた状態で存在している。外科医が該組織片を触診することによってリンパ節を判別し、判別されたリンパ節を除去した。リンパ節が除去された組織片を2つに分割した。
(1) Preparation of tissue piece Obtained by lymph node dissection during surgery after receiving written informed consent from each of 10 subjects who had undergone colorectal cancer surgery. The obtained mesenteric tissue pieces were obtained (10 samples in total). Most of the tissue pieces are composed of adipose tissue, and the lymph nodes are embedded therein. The surgeon discriminated the lymph node by palpating the tissue piece, and the discriminated lymph node was removed. The tissue piece from which the lymph node was removed was divided into two.
（２）リンパ節の判別
（２−１）再度の触診によるリンパ節判別
分割された組織片の一方から、外科医による再度の触診によりリンパ節を判別した。判別されたリンパ節の数を測定した。
(2) Identification of lymph nodes
(2-1) Lymph node discrimination by repeated palpation From one of the divided tissue pieces, lymph nodes were discriminated by repeated palpation by a surgeon. The number of identified lymph nodes was measured.
（２−２）酵素処理後、目視によるリンパ節判別
もう一方の組織片はポリ袋に入れ、そこへ組織片3 g当たり1 mlの酵素処理剤（0.25％トリプシン溶液（25200072; Life technologies, Carlsbad, CA, USA）にコラゲナーゼ（C6885; Sigma-Aldrich, St.Louis, MO, USA）を1 mg/mlになるように溶解した溶液）を添加した。ポリ袋を密閉し、40℃で10分間、100 rpmの速度で回転させた。回転後、ポリ袋から組織片を取り出し、乾燥させた。酵素処理前の組織片の写真、及び酵素処理後、乾燥した組織片の写真を図2に示す。図2に示すように、酵素処理前の組織片には多量の脂肪等の組織が付着しており、目視でリンパ節を判別することはできなかった（図2左側）。一方、酵素処理後の組織片からは余分な組織が除かれており、目視で容易にリンパ節及びリンパ管を判別することができた（図2右側）。判別されたリンパ節の数を測定した。
(2-2) After the enzyme treatment, lymph node discrimination by visual observation The other tissue piece was placed in a plastic bag, and 1 ml of enzyme treatment agent (0.25% trypsin solution (25200072; Life technologies, Carlsbad) , CA, USA) was added collagenase (C6885; Sigma-Aldrich, St. Louis, MO, USA) dissolved to 1 mg / ml). The plastic bag was sealed and rotated at 40 rpm for 10 minutes at a speed of 100 rpm. After rotation, the tissue piece was removed from the plastic bag and dried. A photograph of the tissue piece before the enzyme treatment and a photograph of the dried tissue piece after the enzyme treatment are shown in FIG. As shown in FIG. 2, tissues such as a large amount of fat adhered to the tissue pieces before the enzyme treatment, and the lymph nodes could not be visually determined (left side of FIG. 2). On the other hand, excess tissue was removed from the tissue pieces after the enzyme treatment, and the lymph nodes and lymph vessels could be easily discriminated visually (right side of FIG. 2). The number of identified lymph nodes was measured.
（３）結果
上記「（２）リンパ節の判別」で判別されたリンパ節数を下記表1に示す。触診により判別されたリンパ節数と、酵素処理後、目視により判別されたリンパ節数の測定値を、ウィルコクソンの符号順位検定で統計解析したところ、P値は0.004であった。また、図3に、表1の結果の箱髭図を示す。図3中、ボックスから上方向に伸びた髭の上端は90パーセンタイル値を示し、ボックスから下方向に伸びた髭の下端は10％パーセンタイル値を示す。
(3) Results The number of lymph nodes discriminated in “(2) Discrimination of lymph nodes” is shown in Table 1 below. When the number of lymph nodes determined by palpation and the measured value of the number of lymph nodes visually determined after enzyme treatment were statistically analyzed by Wilcoxon's sign rank test, the P value was 0.004. FIG. 3 shows a box diagram of the results of Table 1. In FIG. 3, the upper end of the ridge extending upward from the box indicates the 90th percentile value, and the lower end of the ridge extending downward from the box indicates the 10% percentile value.
前述したように、酵素処理の場合は、短時間（10分間）の処理により、目視で容易にリンパ節を判別することができた（図2）。また、表1及び図3に示されるように、触診により判別した場合に比べて、酵素処理後に目視により判別した場合の方が、より多くのリンパ節を判別できた。 As described above, in the case of enzyme treatment, lymph nodes could be easily discriminated visually by treatment for a short time (10 minutes) (FIG. 2). In addition, as shown in Table 1 and FIG. 3, more lymph nodes were discriminated when visually discriminated after the enzyme treatment than when discriminated by palpation.
実施例２：酵素処理条件の検討
表2に示す条件で、実施例1と同様に組織片の酵素処理を行った。酵素処理剤の調製は、酵素としてトリプシンを含まない場合は、DPBS（14190250; Life technologies）にコラゲナーゼ及び／又はリパーゼ（MP Bio, 100817）を溶解して作成し、酵素としてトリプシンを含む場合は0.25％トリプシン溶液コラゲナーゼ及び／又はリパーゼを溶解して作成した。酵素処理後、目視によるリンパ節の判別のし易さを3段階で評価した。評価基準は、「1」が「酵素処理前と判別し易さは変らない」という評価、「3」が「目視により容易にリンパ節を判別できる」という評価、「2」は1と3との中間の評価とした。
Example 2: Examination of enzyme treatment conditions Tissue pieces were subjected to enzyme treatment in the same manner as in Example 1 under the conditions shown in Table 2. The preparation of the enzyme treatment agent is prepared by dissolving collagenase and / or lipase (MP Bio, 100817) in DPBS (14190250; Life technologies) when trypsin is not included as an enzyme, and 0.25 when trypsin is included as an enzyme. % Trypsin solution was prepared by dissolving collagenase and / or lipase. After enzyme treatment, the ease of visual discrimination of lymph nodes was evaluated in three stages. Evaluation criteria are: “1” is an evaluation that “easiness to distinguish from pre-enzyme treatment does not change”, “3” is an evaluation that “the lymph node can be easily discriminated by visual inspection”, and “2” is 1 and 3. An intermediate evaluation was made.
評価結果を表2の最右列に示す。この結果より、コラゲナーゼやトリプシンなどのタンパク質分解酵素やリパーゼ等の脂肪分解酵素を単独で用いることにより（条件1〜3）、リンパ節の判別がより容易になることが示された（評価「2」）。さらに、タンパク質分解酵素や脂肪分解酵素を2種類以上組み合わせることにより（条件4〜8）、それぞれ単独で用いるよりもより容易にリンパ節を判別できることが示された（評価「3」）。 The evaluation results are shown in the rightmost column of Table 2. From these results, it was shown that the use of a proteolytic enzyme such as collagenase or trypsin or a lipolytic enzyme such as lipase alone (Conditions 1 to 3) facilitated the discrimination of lymph nodes (Evaluation “2”). "). Furthermore, it was shown that by combining two or more types of proteolytic enzymes and lipolytic enzymes (conditions 4 to 8), lymph nodes can be distinguished more easily than using them alone (evaluation “3”).
実施例３：酵素処理後に判別されたリンパ節の観察
リンパ節へのがん細胞の転移を調べるためには、リンパ節が破壊されていないことが重要である。リンパ節が破壊されていると、検査中にがん細胞がリンパ節から抜け落ちる可能性があるので、正確にリンパ節転移の有無を調べることができなくなるからである。そこで、実施例1及び2で判別されたリンパ節の切片像を観察し、破壊の有無を調べた。具体的には次のように行った。
Example 3: Observation of lymph nodes determined after enzyme treatment In order to examine the metastasis of cancer cells to lymph nodes, it is important that the lymph nodes are not destroyed. This is because if the lymph node is destroyed, cancer cells may fall out of the lymph node during the examination, so it is not possible to accurately check for the presence of lymph node metastasis. Therefore, the slice images of the lymph nodes discriminated in Examples 1 and 2 were observed to examine the presence or absence of destruction. Specifically, it was performed as follows.
実施例1及び2において判別されたリンパ節から、定法に従って、パラフィン切片を得て、該切片をヘマトキシリン・エオジン染色した。染色後の切片を光学顕微鏡で観察した。触診により判別した場合のリンパ節の観察像を図4に左側に示し、実施例1において酵素処理後に判別した場合の観察像を図4の右側に示す。 Paraffin sections were obtained from the lymph nodes identified in Examples 1 and 2 according to a conventional method, and the sections were stained with hematoxylin and eosin. The stained section was observed with an optical microscope. The observation image of the lymph node when discriminated by palpation is shown on the left side in FIG. 4, and the observation image when discriminated after the enzyme treatment in Example 1 is shown on the right side of FIG.
図4に示すように、酵素処理後に判別した場合であっても、触診により判別された場合と同様に、リンパ節の構造は破壊されていなかった。このことから、酵素処理後にリンパ節を判別する方法を用いても、該リンパ節へのがん細胞の転移を正確に調べられることが示された。 As shown in FIG. 4, even when the determination was made after the enzyme treatment, the structure of the lymph node was not destroyed as in the case of the determination by palpation. From this, it was shown that cancer cell metastasis to the lymph node can be accurately examined even if a method for discriminating the lymph node after enzyme treatment is used.
Claims (7)
（ｂ）工程（ａ）後の組織片中の球形部分をリンパ節として判別する工程
を含む、リンパ節郭清により得られた組織片中のリンパ節判別方法。 (A) a step of contacting a tissue piece obtained by lymph node dissection with a treatment agent containing at least one enzyme selected from the group consisting of a proteolytic enzyme and a lipolytic enzyme as an active ingredient; and (b) ) A method for discriminating a lymph node in a tissue piece obtained by lymph node dissection, comprising the step of discriminating a spherical portion in the tissue piece after step (a) as a lymph node.
（ｂ）工程（ａ）後の組織片中の球形部分をリンパ節として判別する工程、及び
（ｃ）工程（ｂ）で判別されたリンパ節中のがん細胞の有無を判定する工程
を含む、リンパ節中のがん細胞の検査方法。 (A) a step of contacting a tissue piece obtained by lymph node dissection with a treatment containing at least one enzyme selected from the group consisting of a proteolytic enzyme and a lipolytic enzyme as an active ingredient;
(B) including a step of discriminating a spherical portion in the tissue piece after step (a) as a lymph node, and a step of (c) determining the presence or absence of cancer cells in the lymph node determined in step (b). A method for examining cancer cells in lymph nodes.
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