2 2 2 A7 __B7_ 五、發明説明（1 ) 技術分野 本發明係關於腫瘤免疫學的分野。若要特定則本發明 係關於，具抗腫瘤以及/或抗癌活性的免疫治療劑進行篩 選之物質以及其方法。若進一步特定則本發明係關於，對 in vitro而言，判斷是否獲得in vivo的L A K細胞（ Lymphokine Activated Killed Cell :淋巴激活素活性化殺傷 細胞）是否具活性增強效果的篩選物質以及其方法。 過去技術 關於腫瘤免疫學的基礎槪念，已知腫瘤細胞具有腫瘤 抗原。腫瘤細胞所具有的腫瘤抗原，其於正常細胞中未發 現僅於腫瘤細胞中存在之腫瘤特異抗原（TSA:Tumor Specific Antigen )，或正常細胞中存在者極微量而隨著細 胞的癌化而增強的腫瘤關連抗原（TAA:Tumor Associated Antigen )。如此的腫瘤抗原雖隨著正常細胞的癌化產生的 基因變異而初次被發現，或隨著該變異因表現調節的變化 而被表現。作爲具有異常的抗原性的腫瘤細胞治療法，免 疫治療法爲最普通的方法，其中一例子爲，將被檢體以腫 瘤抗原進行免疫，使用增強被檢體的免疫機能的藥劑。已 知一般破壞腫瘤細胞的活性，以免疫系細胞中亦以N K ( 自然殺傷細胞）爲高，又N K細胞的活性因免疫治療法而 被增強。NK細胞於正常個體中存在的非T非B細胞障礙 性淋巴系細胞，對於腫瘤細胞、病毒感染細胞等不被 Μ H C抗原限制，對不表現Μ H C等級1分子或表現能力 本紙張尺度適用中國國家標準（CNS)A4規格（210x297公釐） -4- (請先閱讀背面之注意事項再填寫本頁) 餘 --線- 經濟部智慧財產局員工消費合作社印製 2 2 2 A7 __B7___ 五、發明說明（2 ) 降低的細胞已知顯示活性障礙。然而現今，亦存在者不被 NK細胞殺傷的腫瘤細胞。 美國國立癌症硏究所（NCI:National Cancer Institute ) 的S. Rosenberg將末稍淋巴球與I L 一 2 (人類間白素）共 同培養時，發現對於含有自身癌症的廣範圍的目標癌細胞. 而言可誘導顯示細胞障礙性的細胞，此細胞可殺傷N K細 胞不可能殺傷的癌細胞亦可殺傷（參考特開昭 62—116518號）。此殺傷細胞取名爲淋巴激活素 活性化細胞（L A K 細胞：Lymphokine Activated Killer Cell ) °LAK細胞於細菌學上已知其非爲均一集團，而 係爲N K細胞系或殺傷T細胞系的細胞集團。近年來，來 自被檢體的末稍淋巴球於細胞培養系中使用I L - 2使其 活化後，顯示抗腫瘤活性的L A K細胞再次被移至被檢體 體內，進行細胞免疫治療法（L A K治療法）。重複投與 此L A K細胞的細胞免疫治療法，有末期癌縮小或增加抑 制的例子被報告。然而，L A K治療法的效果有個體上的 差異，亦有幾乎無效果的狀態。又，有著必須由被檢體分 離大量的白血球細胞而造成被檢體肉體上的負擔，而對於 分離後的白血球細胞必須大量培養造成經濟上的大負擔等 種種問題點。且，進行IL一2直接投與的LAK治療法 時，因必須投與高濃度的I L - 2而產生嚴重的副作用。 具體而言’使用I L 一 2進行LAK細胞免疫治療法 時，會產生全身倦怠、寒冷、發燒、低白蛋白、貧血、好 氧球增加等症狀之副作用，已知這些副作用比單獨使用 ^紙張尺度適用中國國家標準(CNS)A4規格（210 X 297公釐) ~ ' -------T------·%--- (請先閱讀背面之注意事項再填寫本頁) 訂-Jr i線. 經濟部智慧財產局員工消費合作社印製 1222515 A7 B7 五、發明說明（3 ) (請先閱讀背面之注意事項再填寫本頁) IL-2時還強。且値得注意的事爲，對於產生幾項重大 副作用而言，可考慮其可能原因爲LAK細胞對於正常細 胞具有活性傷害。有報告指出此LAK細胞所造成的造血 幹細胞傷害，除產生貧血或血小板減少外，亦對淋巴球、 巨噬細胞或對血管內皮細胞的in vitro產生傷害。又，I L - 2經口投與之吸收較差，現今以直接投與的方式進行注 射投與爲主要方式。 因此，效果未顯著的狀態下，不進行有可能產生副作 用的LAK治療法，而對直接投與是否能使LAK活性增 強，重新對m vitro系作篩選被期待著。然而，使用I L 一 2於in vitro進行篩選有費用過高的缺點。 --線. 經濟部智慧財產局員工消費合作社印製 自過去’知道細菌類或食品等具有抗癌作用。由具此 抗癌作用的細菌類或食品等副作用較少且具安全性的事看 來’可探索來自這些細菌類或食品等的抗癌作用物質，例 如有報告指出使用沙雷氏菌與溶連菌的培養濾液之Coley's 毒素（1964) 、BCG的白血病治療（Mathe，G，Adv. Cancer Res·，14，1，1971)以及 molmot 對癌腫瘤的退縮（Zbar， B·，et al·，J· Natl· Cancer Inst·，48, 83 1，1971 )，以及對酵 母菌多醣體投與之肉瘤1 8 0等移植癌具有效性。 特別關於多醣體，酵母葡聚糖、酵母甘露聚糖、其他 菌體的多醣體、地衣類以及擔子菌類的多醣體等對於抗癌 的效果’已有很多硏究報告。這些中作爲抗癌免疫增強藥 ’現今被販賣的有，擔子菌類的靈芝科之來自河原菇培養 菌絲體的乙酸間甲苯酯（吳羽化學，三共製藥：宿主的免 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）-6 - 2 2 2 A7 ___B7____ 五、發明說明（4) 疫機能賦活劑）以及香菇多醣類的香菇多醣以及末廣菇多 醣體等。 又，香链（Lentinusedodes)爲日本與中國的代表性 食用菇，於日本早在3 0 0年前便實行人工栽培，其藥理 效果與藥效成分僅於最近才解明，例如有報告指出，對於 白老鼠的大腸與肝臟等的移植腫瘤細胞之其具增値抑制效 果（Sugan〇，N. et al·，Cancer Letter，27: 1，1985 ;鈴木康 將等，日本大腸肛門病會誌，43 : 178、1990) 以及促進細胞***劑效果（T a b a t a. T. e t a 1.， Immunopharmacology, 24:57, 199 2; Hibino, et al., Immunopharmacology, 28: 77, 1994 .)等 ° 本發明者，注意到香菇所具有的L A K活性增強效果 (抗腫瘤以及/或抗癌活性），香菇菌絲體萃取物直接投 與於生物體內時於in vivo的L A K活性增強效果，本發明 提供一種於in vitro的篩選，其篩選物質以及其方法作爲課 題。 於過去的方法中，實際上將L A K活性增強物質投與 於生物體，或大量調製生物體內的淋巴球，此於in vitro中 以L A K活性增強物質進行活化，此後還原實際於生物體 內活性化的淋巴球，而可確認是否有L A K活性增強效果 。此方法有因治療費用過高的缺點，其他亦有因存在著被 檢體的肉體負擔等問題。然而，實際上L A K活性增強物 質於生物體內是否有效果可由in vitro的簡便篩選，對於被 檢體的肉體、金錢上的負擔則大大減輕。 ---f--— HI —----· I I (請先閱讀背面之注意事項再填寫本頁) 幻· 線. 經濟部智慧財產局員工消費合作社印製 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -7 - 1222515 A7 __ B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（5 ) 發明的摁示 本發明者對於香菇的生活史之可食用型態的子實體前 型態，其菌絲中萃取成分裡，發現其子實體中具有驚人的 免疫賦活活性與抗腫瘤活性以及/或抗癌活性。而且當該 萃取物作爲I L 一 2的代替物於in vitro中作爲L A K活性 誘導物質使用時，將當該萃取物直接投與於生物體內時， 對於in vivo的抗腫瘤作用以及/或抗癌作用，特別對 L A K活性增強作用，發現可於in Vitr0中篩選，於是完成 本發明。 更具體而言，本發明者將含有香菇菌絲萃取物的抗腫 瘤物質或抗癌物質，特別爲L A K活性增強物質直接投與 於生物體內時對於in vivo細胞傷害活性，發現與被檢體中 調製出的淋巴球於in vitro中因前述L A K活性增強物質而 活化時的細胞傷害活性有著正相關性。以下爲本發明的步 驟： 包含 (a )採樣被檢體的末稍血，由此進行淋巴球部分的 調製； (b )調製前述淋巴球部分中添加本發明的篩選物質 之L A K誘導樣品，與無添加篩選物質的對照組樣品； (c )對於前述誘導樣品與前述對照組樣品，互相比 較其測定出的L A K活性結果，而決定對當該被檢體篩選 物質於in vitro中L. A K活性增強作用； (請先閱讀背面之注意事項再填寫本頁) ¾ 訂-I-· --線· 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）-8 - 1222515 A7 ___ B7 五、發明說明（6) 之提供適合被檢體而具有L A K活性增強作用之物質 於m vitro可決定的方法。本發明又提供一種使用前述匕 vitro的篩選方法，於in vivo可篩選是否獲得增強的l a K 活性之含有香菇菌絲體萃取物的篩選物質。因此，本發明 係關於’ L A K活性增強物質於in vivo是否能期待l A K 活性增強效果，於L A K活性增強物質投與前可於in Vitr0 中判斷之含有香菇菌絲體萃取物的篩選物質以及篩選方法 ，不僅用於人類亦可使用於家畜類。 .本說明書中，所謂「L A K活性」爲不能攻擊擔任 N K活性的淋巴球所不能辨認的腫瘤，且意味著自身的正 常細胞幾乎不影響細胞障礙性T淋巴球所示的抗腫瘤活性 。「L A K活性增強」係意味著L A K活性的增強，與增 強由淋巴球誘導L AK細胞或既有的L AK細胞之抗腫瘤 活性。 增強L A K活性使L A K細胞的抗腫瘤活性提高，結 果爲細胞性免疫系的提高。因此，既可期待抗腫瘤活性提 高之治療，亦可使用於以提高免疫系爲目的的治療方法。 實施發明的最佳形態： 本發明爲提供一種含有本發明的香菇菌絲體萃取物的 抗腫瘤劑或抗癌劑，特別爲對增強L A K活性的製劑’因 直接投與於生物體內故對是否可期待L A K活性的增強效 果，可於製劑投與前in vitro中判斷，含有香菇菌絲體萃取 物的篩選物質以及其篩選方法。對於本發明所謂「篩選物 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱）冬 — T- — !丨！ ·丨| (請先閱讀背面之注意事項再填寫本頁) 訂· 經濟部智慧財產局員工消費合作社印製2 2 2 A7 __B7_ V. Description of the invention (1) Technical field This invention is a field of tumor immunology. To specify, the present invention relates to a substance for screening an immunotherapeutic agent having antitumor and / or anticancer activity and a method for the same. If it is more specific, the present invention relates to a screening substance for judging whether an in vivo L A K cell (Lymphokine Activated Killed Cell) has an activity-enhancing effect and its method for in vitro. Past technology Regarding the basic concepts of tumor immunology, tumor cells are known to have tumor antigens. Tumor antigens possessed by tumor cells. No tumor-specific antigens (TSA: Tumor Specific Antigen) found in tumor cells are found in normal cells, or a very small amount is present in normal cells, which increases with the canceration of the cells. Tumor-associated antigen (TAA: Tumor Associated Antigen). Such a tumor antigen is discovered for the first time with a genetic mutation caused by canceration of normal cells, or it is expressed with a change in expression regulation. As a method for treating tumor cells with abnormal antigenicity, immunotherapy is the most common method. One example is immunizing a subject with a tumor antigen and using an agent that enhances the immune function of the subject. It is known that the activity of tumor cells is generally destroyed. N K (natural killer cells) is also high in immune system cells, and the activity of N K cells is enhanced by immunotherapy. Non-T and non-B cell dysfunction lymphocytes in NK cells in normal individuals are not restricted by MHC antigens for tumor cells, virus-infected cells, etc., and do not exhibit MHC class 1 molecules or expression capabilities. This paper applies to China National Standard (CNS) A4 Specification (210x297 mm) -4- (Please read the notes on the back before filling out this page) Yu--line-Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 2 2 2 A7 __B7___ V. DESCRIPTION OF THE INVENTION (2) Reduced cells are known to show a disorder of activity. Today, however, there are also tumor cells that are not killed by NK cells. When S. Rosenberg of the National Cancer Institute (NCI: National Cancer Institute) co-cultured peripheral lymphocytes with IL-2 (human interleukin), he found a wide range of target cancer cells containing his own cancer. And It can induce cells showing dysfunction of cells, which can kill cancer cells that NK cells cannot kill, as well (see JP 62-116518). This killer cell is called a Lykokine Activated Killer Cell (LAK cell) ° LAK cells are known bacteriologically to be a non-homogeneous group, which is a cell group of NK cell line or killer T cell line . In recent years, after the terminal lymphocytes from the subject were activated with IL-2 in a cell culture line, LAK cells showing anti-tumor activity were again moved into the subject and subjected to cellular immunotherapy (LAK treatment law). Cellular immunotherapy with repeated administration of this L A K cell has reported examples of shrinkage or increased suppression of terminal cancer. However, there are individual differences in the effects of the L A K treatment, and there are also almost no effects. In addition, there are various problems such as that a large amount of white blood cells must be separated by the subject, which causes a physical burden on the subject, and that a large number of cultured white blood cells must be cultured to cause a large economic burden. In addition, in the case of the LAK treatment method in which IL-2 is directly administered, a high concentration of IL-2 is required to cause severe side effects. Specifically, 'When using IL-2 for LAK cell immunotherapy, side effects such as general burnout, cold, fever, low albumin, anemia, and increased aerobic globules may occur. These side effects are known to be higher than when used alone ^ paper size Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) ~ '------- T ------ ·% --- (Please read the precautions on the back before filling this page) Order-Jr i line. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1222515 A7 B7 V. Invention Description (3) (Please read the precautions on the back before filling this page) IL-2 is still strong. It is important to note that for several major side effects, the possible cause can be considered to be that LAK cells have active damage to normal cells. It has been reported that in addition to the hematopoietic stem cell damage caused by this LAK cell, in addition to anemia or thrombocytopenia, it also causes damage to lymphocytes, macrophages, or vascular endothelial cells in vitro. In addition, I L-2 is poorly absorbed by oral administration. Nowadays, direct injection is the main method of injection administration. Therefore, in a state where the effect is not significant, LAK treatments that may cause side effects are not performed, and whether direct administration can enhance LAK activity is expected to re-screen the m vitro line. However, the use of IL-2 for in vitro screening has the disadvantage of being too expensive. --Line. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. From the past, I know that bacteria or foods have anti-cancer effects. From the point of view of safety and fewer side effects such as bacteria and foods with anticancer effects, it is possible to explore anticancer substances from these bacteria and foods. For example, reports of the use of Serratia and lysobacteria Coley's toxin from culture filtrate (1964), leukemia treatment of BCG (Mathe, G, Adv. Cancer Res ·, 14, 1, 1971), and tumor shrinkage of tumors by molmot (Zbar, B ·, et al ·, J · Natl. Cancer Inst., 48, 83 1, 1971) and transplanted cancers such as sarcoma 1 800 administered to yeast polysaccharides. In particular, there have been many research reports on anticancer effects of polysaccharides, yeast dextran, yeast mannan, polysaccharides of other bacterial cells, polysaccharides of lichens and basidiomycetes, and the like. Among these are currently being sold as anti-cancer immune-enhancing drugs, and the ganoderma lucidaceae of the basidiomycete family is derived from m-toluyl acetate from cultured mycelia of Kawaguchi mushrooms (Wu Yu Chemical, Sankyo Pharmaceutical: Host-free paper standard applicable to China) Standard (CNS) A4 specification (210 X 297 mm) -6-2 2 2 A7 ___B7____ V. Description of the invention (4) Epidemic function activator), lentinan polysaccharides of lentinan and polysaccharides of Morinaki mushroom. In addition, Lentinusedodes is a representative edible mushroom in Japan and China. It was artificially cultivated as early as 300 years ago in Japan. Its pharmacological effects and medicinal ingredients have only been explained recently. For example, some reports indicate that Transplanted tumor cells from the large intestine and liver of white mice have an inhibitory effect (Sugan 0, N. et al., Cancer Letter, 27: 1, 1985; Yasuhiro Suzuki, et al., Japan Journal of Colorectal and Anal Diseases, 43 : 178, 1990) and the effect of promoting cell division (T abat a. T. eta 1., Immunopharmacology, 24:57, 199 2; Hibino, et al., Immunopharmacology, 28: 77, 1994.) etc. Note that the LAK activity enhancement effect (anti-tumor and / or anti-cancer activity) possessed by Lentinula edodes is noticed. The LAK activity enhancement effect of Lentinus edodes mycelium extract in vivo is directly provided in the body. The present invention provides a In vitro screening, screening of substances and methods thereof are subject. In the past methods, LAK activity-enhancing substances were actually administered to living organisms, or a large number of lymphocytes in the body were modulated. This was activated in vitro with LAK activity-enhancing substances, and thereafter reductions of those actually activated in vivo were reduced. Lymphocytes can be confirmed whether there is an effect of enhancing LAK activity. This method has the disadvantage of high treatment costs, and other problems such as the physical burden of the subject. However, in fact, whether the L A K activity-enhancing substance is effective in vivo can be easily screened in vitro, which greatly reduces the physical and financial burden on the subject. --- f --— HI —---- · II (Please read the notes on the back before filling this page) Magic line. The paper printed by the Intellectual Property Bureau Staff Consumer Cooperatives of the Ministry of Economic Affairs applies Chinese national standards ( CNS) A4 specification (210 X 297 mm) -7-1222515 A7 __ B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 5. Description of the invention (5) The invention shows the edibleness of the inventor's life history of shiitake mushrooms. The preform of this type of fruiting body was extracted from its hyphae and found that its fruiting body had amazing immune-activating activity and anti-tumor activity and / or anti-cancer activity. Moreover, when the extract is used as a substitute for IL-2 as a LAK activity-inducing substance in vitro, the antitumor effect and / or anticancer effect on in vivo when the extract is directly administered into a living body In particular, the effect of enhancing LAK activity was found to be screened in in Vitr0, and the present invention was completed. More specifically, the present inventors found that the antitumor substance or anticancer substance, especially the LAK activity-enhancing substance, containing the extract of Lentinus edodes mycelium, had an in vivo cell damaging activity when it was directly administered to a living body, and found that The prepared lymphocytes have a positive correlation with the cytotoxic activity when activated by the aforementioned LAK activity enhancing substance in vitro. The steps of the present invention are as follows: (a) sampling the peripheral blood of the subject to perform the modulation of the lymphosphere; (b) modulating the LAK-induced sample to which the screening substance of the present invention is added to the aforementioned lymphosphere, and Control sample without added screening substance; (c) Comparing the results of the LAK activity measured between the aforementioned induction sample and the aforementioned control sample with each other to determine the L. AK activity of the screening substance in vitro in the subject Enhancement effect; (Please read the notes on the back before filling in this page) ___ B7 V. Description of the invention (6) The method for determining the LAK activity-enhancing substance suitable for the subject can be determined in vitro. The present invention further provides a screening method using the aforementioned in vitro method, which can be used to screen in vivo whether a screening substance containing an extract of lentinus edodes mycelium with enhanced 1 a K activity is obtained. Therefore, the present invention relates to whether the LAK activity enhancing substance can be expected to enhance the AK activity enhancing effect in vivo. Before the LAK activity enhancing substance is administered, it can be judged in Vitr0 that the screening substance contains the mushroom mycelium extract and the screening. The method can be used not only for humans but also for livestock. In the present specification, "LAK activity" refers to a tumor that cannot be recognized by lymphocytes that act as NK activity, and means that its own normal cells hardly affect the antitumor activity shown by cytolytic T-lymphocytes. "Enhanced L A K activity" means an increase in L A K activity and an increase in the antitumor activity of L AK cells or existing L AK cells induced by lymphocytes. Increasing the activity of L A K increased the anti-tumor activity of L A K cells, with the result that the cellular immune system was improved. Therefore, both anti-tumor activity and anti-tumor activity can be expected, and it can also be used for the purpose of improving the immune system. The best mode for carrying out the invention: The present invention is to provide an anti-tumor agent or anti-cancer agent containing the extract of shiitake mushroom mycelium of the present invention, and particularly to a preparation that enhances the activity of LAK. The enhancement effect of LAK activity can be expected, and it can be judged in vitro before the preparation administration that the screening substance contains the mushroom mycelium extract and the screening method thereof. For the so-called "screen size of the screening material applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 public love) winter-T--! 丨! · || Please read the precautions on the back before filling this page) · Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs
2 2 2 I A7 ___B7____ 五、發明説明（8 ) m 1。本發明的香菇菌絲體萃取物添加於培養細胞前或對 末稍血進行直接添加前，以丙酮進行無菌處理較佳。 作爲使用本發明的篩選物質進行篩選的方法，雖依照 高木們的方法（臨床免疫，1 9 : 245 - 249， 1 9 8 7 )，但代替I L — 2，篩選物質例如使用本發明， 的香菇菌絲體萃取物，此點來看不同於過去的方法。即， 本發明的L A K活性增強篩選方法，爲以下步驟：其含有 (a )採樣被檢體的末稍血，由此進行淋巴球部分的 調製； (b)調製前述淋巴球部分中添加本發明的篩選物質 之L A K誘導樣品，與無添加篩選物質的對照組樣品； (c )對於前述誘導樣品與前述對照組樣品，互相比 較其測定出的L A K活性結果，而決定對當該被檢體篩選 物質於in vitro中L A K活性增強作用；之提供適合被檢體 而具有L A K活性增強作用之物質於in vitro可決定的方法 〇 爲使誘導LAK細胞，由被檢驗者的末稍血中分離淋 巴球。 自被驗者中所採血的末稍血加上肝素，使用Ficoll-Conary液（s · g ·=1 · 077)的比重離心分離方 法使得界面的單核球分離。被分離的單核球以P B S ( ΡΗ7 · 4，不含Ca以及Mg)洗淨2至3次，爲使其 成爲1 X 1 0 6 /m 1懸濁於培養液（較佳爲於 RPMI 1640培養基（Gibco )中適宜地添加FBS( (請先閱讀背面之注意事項再填寫本頁) 訂·- --線- 經濟部智慧財產局員工消費合作社印製 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -11 - 1222515 Α7 Β7 經濟部智慧財產局員工消費合作社印製 五、發明說明（9) 失活性化血淸）或抗生物質等）中。將此以自身血淸（血 漿）於3 7°C下處理1 5分鐘後移至培養皿中，於3 7°C 下培養1小時。回收非附著性細胞，作爲淋巴球部分。 L A K誘導樣品係由例如由以下的方法所調製出。即 ，由上述的方法得到的淋巴球部分調製至細胞之最終濃度. 爲lxl05/ml〜lxl06/ml ，浮游於培養液中 ，1洞穴添加1 0 0 # 1的細胞懸濁培養液，使其細胞數 爲 lxl04/we 1 1 〜lxl05/we 1 1 ，每 1 w e 1 1的細胞數，對於斯業者爲可以使用的功能細胞（ effects細胞）的活性，對目標細胞的功能細胞之感受性等 而適宜地決定。該浮游液中，依照實驗步驟作爲篩選物質 加入最終濃度爲範圍的香菇菌絲體萃取物。 如此以被檢體的種種淋巴球濃度（含濃度爲零的濃度 )於本發明的香菇菌絲體萃取物存在下培養作爲功能細胞 。對於本發明的說明書，所謂功能細胞，即指進行上述培 養處理3天的細胞而言，包含與LAK活性增強物質共同 培養的淋巴球（L A K活性增強物質添加處理的淋巴球） 以及僅培養於不含L A K活性增強物質的培養液中之淋巴 球（無添加誘導處理之淋巴球）兩者。 對照組爲代替添加的篩選物質使用滅菌的重組I L -2 (rIL-2;2000U/ml)之外，其他調整成 與調製L A K誘導樣品的方法相同。 L A K活性的測定爲進行5 1 C r釋出測定法、〔3 Η 〕尿定法等方法。對於本發明，由簡便性以及客觀性的觀 (請先閱讀背面之注意事項再填寫本頁) 訂· --線· -J. 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）-12- 經濟部智慧財產局員工消費合作社印製 1222515 A7 B7 五、發明説明（10) 點看來，使用5 1 C Γ釋出測定法較佳。5 1 C r釋出測定法 爲測定經L A K活性增強物質處理的淋巴球，其所誘導出 的L AK細胞所引起的目標細胞傷害於in vitro下測定之方 法之一。5 1 C r釋出測定法爲以下的步驟： (i )目標細胞中添加5 1 C r所標識的酪酸鈉而標識 目標細胞。 (ϋ )前述的目標細胞與，篩選物質或作爲對照的以 r I L - 2刺激後的功能細胞（例如，殺傷Τ細胞或 L A K細胞）進行反應； (iii )目標細胞被功能細胞破壞時，於細胞培養液的 上淸液中測出釋出的5 1 C r量； 又上述所成的以功能細胞對目標細胞作細胞傷害活性 的測定方法。 對於5 1 C I·釋出測定，使用作爲目標細胞的連續培養 細胞，較佳爲使用Daudi細胞或Raji細胞。目標細胞的培養 爲，回收培養錐形瓶中的細胞，以5 1 C 1：標識後分裝於微 量滴定培養皿（micro titer plate )中進行。作爲目標細胞 的培養液，爲增殖使用的細胞使用適當的用具，例如 RPMI 1640等培養液中適宜地添加血淸、抗生物 質等而使用。 目標細胞的標識爲，培養後的目標細胞1 0 6個添加 100〜150//Ci的51Cr -酪酸鈉，仔細攪拌後於 3 7 °C下培養1至2小時。培養細胞爲以P B S進行3次 的洗淨後，爲使成1 X 1 0 6/m 1而使用添加1 0 % 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） TTTr " ---,------------- (請先閱讀背面之注意事項再填寫本頁) 訂· 1222515 經濟部智慧財產局員工消費合作社印製 A7 ___B7__ 五、發明說明（ιυ F B S懸濁於R Ρ Μ I 1 6 4 0培養基中者。標識後， 使用培養時所用的培養液或磷酸緩衝液（P B S )洗淨， 最後以含有1 0 %的牛胎血淸（F B S )或小牛血淸（ F C S )培養液調製成1 X 1 〇 6 /m 1 ，作用測定使用。 目標細胞爲微量滴定培養皿的各穴中其細胞數能成5 X , 1 04/m 1而分別注入50// 1。 爲測定細胞傷害活性的化驗法中，分注上述的目標細 胞的各穴中，最大解離則再添加1N - HC 1 1〇〇 # 1 ，自然解離則僅添加培養液1 0 0 // 1 ，於是再添加 實驗解離用的種種濃度之含有本發明香菇菌絲體萃取物， 或對照用的2 0 0 0 U /m 1的r I L 一 2所刺激的功能 細胞之培養液1 0 0 // 1。其次，微量滴定培養皿以板式 離心分離機，於800rpm、5分鐘離心處理的細胞集 中於穴底部後，以5 % C〇2培養器於3 7 °C下培養3 · 5 小時。 5 1 C r -酪酸鈉的化驗法中，目標細胞傷害活性可由 以下式子表示算出： τ Λ κ，壬性η/ 實驗解離（QOT7)-自然解離（Q咖）、叫η/Ί L A κ活性％ =最大解離(_)-自然ϋίίφ⑻x100 此式所算出的L A K活性，誘導樣品與對照樣品的値 互相比較下，篩選物質於in vitro中可測出L A K活性誘導 對於上述的最大解離、自然解離以及實驗解離所需求 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） _ 14 - -------------壤 i, (請先閱讀背面之注意事項再填寫本頁) 訂i --線· 1222515 A7 B7____ 五、發明說明（12) (請先閱讀背面之注意事項再填寫本頁) 的步驟，目標細胞的培養於5 % C Ο 2，3 7 °C下進行’培 養時間、實驗目的、使用細胞的數目等其他條件’斯業者 可適宜地決定，但本發明爲3 · 5小時。 培養上淸液所釋出的5 1 C r之放射線量，可由使用閃 煉計數管等測出。 . 本發明的較佳狀態爲，雖以實施如下的各步驟’但如 何適切的變更及修飾爲斯業者所認知的。 採取來自培養後的培養皿的各穴培養上淸液，以閃煉 計數管等測出放射活性。 以述的篩選法於in vitro中確認的L A K誘導活性之香 藤菌絲體萃取物於，3 6 0 0m g/天的7天間投與於生 物體中，於in vitro中誘導L A K活性。已知使用述淋巴球 回收方法所取出的淋巴球部分，與上述的L AK誘導樣品 相同的條件下測定L A K活性％ ，於此所獲得的in vivo的 L A K活性增強作用爲於in vitro所得的結果顯示有著正相 關。 經濟部智慧財產局員工消費合作社印製 雖本發明於以下的實施例更詳細說明，但僅能代表一 部份的例子，並不能限制本發明的範圍。且不脫離本發明 的精神，對本發明可作種種變更與修飾爲斯業者可瞭解的 事。 實施例 實施例1 :調製香菇菌絲體萃取物 蔗渣9 0重量份，米糠1 0重量份所成的固體培養基 ϋ張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱Υ"""· 15· 1222515 A7 B7 五、發明說明（I3) 使其含適當水後，接種香菇種菌，放置於調整溫度以及濕 度的培養室內，使菌絲體增殖。菌絲體於固體培養基下聚 集後，解開蔗渣基材的纖維素束，使其成爲1 2篩子可通 過份量爲24重量％以下。此解束之後的培養基1 · Ok g中，純水3 · 5L以及純化的纖維酵素2 · Og，以邊. 保持固體培養基爲4 0 °C邊加入，成爲含有培養基的混合 物。 其次，含有培養基的混合物附有變速齒輪的幫浦一邊 將其轉動，一邊將固體培養基於齒輪部分進行粉碎以及禱 爛作用2 0 0分鐘的程度，使其蔗渣纖維能約以8 0重量 %作爲1 2篩子的通過份量，含有培養基的混合物進行粉 碎與禱爛爲，邊徐徐上升該混合物的溫度邊實施。其後， 含有培養基的混合物更加溫至9 0 °C使酵素失去活性同時 進行滅菌，於9 0 °C下放置3 0分鐘。所得的含有培養基 混合液以6 0篩子濾布過濾作爲香菇菌絲體萃取物，濃縮 後得到凍結乾燥粉末。 經濟部智慧財產局員工消費合作社印製 -------·Ί--------- (請先閱讀背面之注意事項再填寫本頁) --線· 如此所得的香菇菌絲體萃取物以苯酚-硫酸法進行糖 値分析得知含有糖値爲2 5 · 3 % (重量/重量）、以羅 里法進行蛋白質分析得知含有蛋白質1 9 · 7 % (重量/ 重量），以沒食子酸爲基準的Folon-Denis法得知含有聚苯 酚2 · 6 % (重量/重量）。香菇菌絲體萃取物中亦含有 其他粗脂肪8 % 、粗灰份2 2 % ，糖質以外的可溶性無氨 物約2 0 % 。其中香菇菌絲體萃取物中所構成的糖組成物 如以下所示。 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱^ :16- 2 2 2 A7 __B7______ 五、發明說明（14) 實施例2 : L A K活性測定 首先，分別採血來自被檢體A、B以及C的香菇菌絲 體萃取物服用前之末稍血，以及各被檢體以香菇菌絲體萃 取物1 2 0 0 m g每日3次，經過1星期投與後的末稍血， .，使用這些末稍血以下述方法進行分離所得的淋巴球部分 ，對於本發明的萃取物於生物體內之使淋巴球活性化能力 與，使用萃取物淋巴球於in vitro活性化的能力的相關性可2 2 2 I A7 ___B7____ 5. Description of the invention (8) m 1. The lentinella mycelium extract of the present invention is preferably sterilized with acetone before being added to cultured cells or directly added to the terminal blood. As a method for screening using the screening substance of the present invention, although the method of Takagi (Clinical Immunology, 19: 245-249, 1 9 8 7) is used, instead of IL-2, the screening substance uses the mushroom of the present invention. The mycelium extract is different from the past in this respect. That is, the LAK activity-enhancing screening method of the present invention includes the following steps: (a) sampling of the terminal blood of the subject, thereby modulating the lymphosphere portion; (b) modulating the aforementioned lymphosphere portion, and adding the present invention The LAK-induced sample of the screening substance is compared with the control sample without the added screening substance; (c) The measured LAK activity results of the aforementioned induction sample and the aforementioned control sample are compared with each other, and it is decided to screen the subject. The substance enhances the LAK activity in in vitro; provides a method that can be determined in vitro for substances that have LAK activity-enhancing effects suitable for the subject. In order to induce LAK cells, the lymphocytes are separated from the terminal blood of the subject. . Heparin was added to the final blood collected from the subjects, and the mononuclear spheres at the interface were separated using a specific gravity centrifugation method using Ficoll-Conary solution (s · g · = 1.077). The separated mononuclear spheres were washed 2 to 3 times with PBS (pH 7.4, Ca and Mg-free) to make it 1 X 1 0 6 / m 1 suspended in culture medium (preferably in RPMI 1640). FBS is appropriately added to the culture medium (Gibco) ((Please read the precautions on the back before filling out this page). Order---Line-Printed by the Intellectual Property Bureau Staff Consumer Cooperatives of the Ministry of Economic Affairs This paper applies Chinese national standards (CNS) A4 specifications (210 X 297 mm) -11-1222515 Α7 Β7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (9) Inactivated blood pupa) or anti-biomass, etc.). This was treated with autologous blood pupa (plasma) at 37 ° C for 15 minutes, then transferred to a petri dish, and cultured at 37 ° C for 1 hour. Non-adherent cells were recovered as a lymphoblastic fraction. The L A K-induced sample is prepared, for example, by the following method. That is, the lymphosphere fraction obtained by the method described above is adjusted to the final concentration of the cells. It is lxl05 / ml to lxl06 / ml, which floats in the culture solution, and 1 cave is added with the cell suspension culture solution of 100 # 1 The number of cells is lxl04 / we 1 1 to lxl05 / we 1 1. The number of cells per 1 we 1 1 is for the activity of functional cells (effect cells) that can be used by practitioners, and the sensitivity of functional cells to target cells. Appropriately decided. To this planktonic solution, a mushroom mycelium extract having a final concentration in a range was added as a screening substance according to an experimental procedure. In this way, various lymphocyte concentrations (including zero concentration) of the subject are cultured as functional cells in the presence of the mushroom mycelium extract of the present invention. In the description of the present invention, the term "functional cell" refers to a cell that has been subjected to the above-mentioned culture treatment for 3 days. Both lymphocytes in culture medium containing LAK activity-enhancing substances (lymphocytes without added induction treatment). The control group was adjusted to the same method as that used to prepare the L A K-induced sample, except that sterilized recombinant IL-2 (rIL-2; 2000 U / ml) was used instead of the added screening substance. The measurement of L A K activity is performed by a method such as a 5 1 C r release measurement method, a [3 Η] urination method, and the like. For the present invention, from the perspective of simplicity and objectivity (please read the notes on the back before filling out this page). -Line · -J. This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 (Mm) -12- Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1222515 A7 B7 V. Description of the invention (10) From the point of view, it is better to use the 5 1 C Γ release method. 5 1 C r release assay As one of the methods for measuring lymphocytes treated with L A K activity-enhancing substances, target cells caused by L AK cells induced in vitro are measured in vitro. The 5 1 C r release assay involves the following steps: (i) The target cell is identified by adding sodium caseinate identified by 5 1 C r to the target cell. (ii) the aforementioned target cells react with the screening substance or functional cells stimulated with r IL-2 (for example, killer T cells or LAK cells); (iii) when the target cells are destroyed by functional cells, The amount of 5 1 C r released was measured in the supernatant of the cell culture fluid; and the method for measuring the cytotoxic activity of the functional cells on the target cells formed as described above. For the 5 1 C I release assay, continuous cultured cells are used as target cells, preferably Daudi cells or Raji cells. The target cells were cultured. The cells in the culture flask were recovered and labeled with 5 1 C 1: labeled in a micro titer plate. As the culture medium of the target cells, a suitable device is used for the cells used for proliferation. For example, culture medium such as RPMI 1640 is appropriately added with blood pupa, antibiotics, and the like. The target cells are labeled as follows: 106 target cells after culture are added with 51 ~ 150 / Ci of 51Cr-sodium caseinate and carefully stirred for 1 to 2 hours at 37 ° C. The cultured cells were washed 3 times with PBS, and then added to 10% in order to make 1 X 1 0 6 / m 1. This paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) TTTr " ---, ------------- (Please read the notes on the back before filling this page) Order · 1222515 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 ___B7__ V. Invention Instructions (ιυ FBS suspended in R ΡΜΙ 1640 medium. After labeling, wash with culture medium or phosphate buffered saline (PBS) used during cultivation, and finally use 10% bovine fetal blood 淸(FBS) or calf blood cockroach (FCS) culture solution is prepared to 1 X 1 0 6 / m 1, and the function is used for measurement. The target cell is 5 X, 1 04 / m in each well of a microtiter culture dish. 1 and inject 50 // 1 respectively. In the assay method for measuring cell injury activity, in each of the above-mentioned target cells, 1N-HC 1 1〇〇 # 1 was added for the maximum dissociation, and only natural dissociation was added. The culture solution 1 0 0 // 1 is then added with various concentrations of the mushroom mycelium extract of the present invention for experimental dissociation, or Control 2 000 U / m 1 r IL-2 stimulated functional cell culture medium 1 0 // // 1. Second, the microtiter culture dish was centrifuged at 800 rpm for 5 minutes. After concentrating the cells at the bottom of the acupoint, the cells were cultured in a 5% C02 incubator at 37 ° C for 3 · 5 hours. In the 5 1 C r -sodium caseinate assay, the target cell injury activity can be calculated by the following formula : Τ Λ κ, non-native η / experimental dissociation (QOT7)-natural dissociation (Q coffee), called η / Ί LA κ activity% = maximum dissociation (_)-natural ϋίφ⑻x100 The LAK activity calculated by this formula induces the sample and The comparison of the control sample with each other, the LAK activity can be measured in the in vitro screening material. Induction is required for the above-mentioned maximum dissociation, natural dissociation, and experimental dissociation. This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297). %) _ 14-------------- Soil i, (Please read the notes on the back before filling this page) Order i-line · 1222515 A7 B7____ V. Description of the invention (12) (Please read the precautions on the reverse side before filling out this page). Other conditions such as "cultivation time, experimental purpose, number of cells used, etc." may be appropriately determined at 5% C 02, 37 ° C, but the present invention is 3.5 hours. The amount of 5 1 C r released from the culture supernatant can be measured by using a flash counting tube or the like. The preferred state of the present invention is that the following steps are performed, but how appropriate changes and modifications are recognized by the practitioners. The supernatant was collected from each well of the cultured petri dish, and the radioactivity was measured by a flashing counting tube or the like. The L. A. mycorrhizal mycelium extract of the L A K-inducing activity confirmed in vitro by the screening method described above was administered to a living body at 7600 mg / day for 7 days to induce L A K activity in in vitro. It is known that the percentage of LAK activity measured under the same conditions as the above-mentioned L AK-induced sample is used for the lymphosphere portion taken out by the lymphocyte recovery method. The in vivo LAK activity enhancement effect obtained here is the result obtained in in vitro. The displays are positively correlated. Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Although the present invention is described in more detail in the following examples, it can only represent a part of the examples and should not limit the scope of the present invention. Without departing from the spirit of the present invention, various changes and modifications to the present invention can be made by those skilled in the art. EXAMPLES Example 1: Preparation of 90 parts by weight of bagasse from shiitake mushroom mycelium, and 10 parts by weight of rice bran. The solid medium was prepared using the Chinese National Standard (CNS) A4 specification (210 X 297). · 15 · 1222515 A7 B7 V. Description of the invention (I3) After making it contain appropriate water, inoculate shiitake mushroom seed fungus and place it in a culture room with temperature and humidity adjustment to proliferate the mycelium. The mycelium is under a solid medium After the aggregation, the cellulose bundles of the bagasse base material were unraveled to make the 12 sieve passable amount less than 24% by weight. After this unbundling, the culture medium 1 · Ok g, pure water 3. 5L and purified cellulase 2 · Og, add while keeping the solid culture medium at 40 ° C to form a mixture containing the medium. Next, the pump containing the gear with gears attached to the mixture containing the culture medium is rotated while the solid medium is placed on the gear part. The crushing and rotten effect is about 200 minutes, so that its bagasse fiber can pass about 80% by weight as the passing amount of the 12 sieve. The mixture containing the culture medium is pulverized and rotted. The temperature of the mixture was increased by Xu. Thereafter, the mixture containing the culture medium was further warmed to 90 ° C to deactivate the enzyme while sterilizing, and left at 90 ° C for 30 minutes. The resulting mixture containing the culture medium was heated at 6 The 0 sieve filter cloth is used as the extract of shiitake mushroom mycelium and concentrated to obtain a freeze-dried powder. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs ------- · ------------- (Please (Please read the notes on the back before filling this page) --- Line · The lenticular mushroom mycelium extract thus obtained was analyzed by the phenol-sulfuric acid method for glycocalyx. It was found that the content of glycocalyx was 2 5 · 3% (w / w), Protein analysis by Rory method showed that it contained 19.7% (w / w) protein, and Folon-Denis method based on gallic acid showed that it contained polyphenols (2.6% (w / w)). Shiitake The mycelium extract also contains 8% of other crude fats, 22% of crude ash, and about 20% of soluble ammonia-free substances other than sugars. Among them, the sugar composition of the mushroom mycelium extract is as follows The paper size is applicable to Chinese National Standard (CNS) A4 (21 0 X 297 public love ^: 16- 2 2 2 A7 __B7______ 5. Description of the invention (14) Example 2: LAK activity measurement First, blood was collected from the mushroom mycelium extracts from subjects A, B and C before taking At the end of the blood, and each subject with shiitake mushroom mycelium extract 1 200 mg 3 times a day, after the 1 week of administration, the terminal blood was used. The correlation between the obtained lymphocyte portion and the ability of the extract of the present invention to activate the lymphocytes in vivo and the ability of the extract lymphocytes to be activated in vitro
CBB 師进。 首先，所採取這些末稍血加入肝素，使用Ficoll-Conary液（s. g.=1.077)以比重離心分離法 進行界面單核球的分離，再將分離的單核球以P B S ( P h 7 · 4，不含Ca以及Mg)洗淨2次，欲使其成 lxl06/ml將10% FBS (失活牛胎血淸）添加於 培養液中於R Ρ Μ I 1 6 4 0培養基（Gibco )中懸濁。 以上述方法分離的細胞，重新移至使用自身血淸（血漿） 於3 7 °C下處理1 5分鐘塗佈後培養皿中，3 7 °C下培養 1小時後，採取非附著性的細胞作爲淋巴球部分。 1 0 % F B S添加的R Ρ Μ I 1 6 4 0培養液中培 養目標細胞之連續培養細胞（Daudi細胞）以離心分離方式 回收，106細胞中添加100〜150eCi的51Cr —酪酸鈉（New England Nuclear )，5% C〇 2 培養器中 3 7 °C下培養1小時。以5 1 C r標識的培養細胞以P B S 洗淨3次後，欲成1 x 1 〇 6 / πι 1而懸濁於添加1 〇 % 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）· 17 _ -------1---------- (請先閲讀背面之注意事項再填寫本頁) 訂·1、 --線- 經濟部智慧財產局員工消費合作社印製 1222515 A7 15/ 五、發明說明（I5) FBS的RPMI 1640培養液。 微量滴定培養皿的各穴中以上述的方法標識的目標細 胞分別注入50//1 (5xl04/well)，而且最大 解離群（陽性對照組）爲分別注入1 〇 〇 # 1的1 N — H C 1 ’自然解離群（陰性對照組）爲分別注入1 〇 〇 A 1的添加10% FBS的RPMI 1640培養液， 於是實驗解離群中以1 〇 // g/m 1濃度的本發明之香菇 菌絲體萃取物或作爲對照組以2 0 0 0 U /m 1濃度刺激 的功能細胞（分別爲1〇〇#1 (lxl〇4/well) )注入之。培養皿離心分離機以8 0 0 r p m，5分鐘離 心處理之細胞聚集於穴的底部後，於5 % C〇2培養器中 3 7 °C下培養3 · 5小時。 由培養的培養基中以S Ο K E N — Ρ Ε Τ Σ — 9 6 採取各穴的培養上淸液，7 -閃煉計數管測定放射活性。 L A K活性由以下的表所算出 τ Amo/ 實驗解離自然解離(Q啦）、/ipp a κ活性％ =最大解離㈣-自然解ϊγ(_χ1〇〇 (請先閱讀背面之注意事項再填寫本頁) € •線. 經濟部智慧財產局員工消費合作社印製 結果於表1與圖1所示 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）_ 18 1222515 A7 B7 五、發明説明（16) 表1 : (請先閱讀背面之注意事項再填寫本頁) 實驗號碼 LAK活性CBB learns. First, heparin was added to these terminal blood, and Ficoll-Conary solution (sg = 1.077) was used to separate the mononuclear spheres at the interface by gravity centrifugation. Then the separated mononuclear spheres were treated with PBS (P h 7 · 4, (Ca-free and Mg-free) was washed twice, to make it 1xl06 / ml. 10% FBS (inactivated bovine fetal blood pupa) was added to the culture medium and suspended in R P M I 1640 medium (Gibco). turbidity. The cells isolated in the above method were re-transferred to a plated petri dish treated with autologous blood plasma (plasma) at 37 ° C for 15 minutes, and cultured at 37 ° C for 1 hour, and non-adherent cells were collected. As part of the lymphosphere. Continuous cultured cells (Daudi cells) for cultivating target cells in R PBS with 10% FBS added were recovered by centrifugation, and 106 ~ 100eCi of 51Cr-sodium caseinate (New England Nuclear ), Incubate at 37 ° C for 1 hour in a 5% CO2 incubator. After washing the cultured cells labeled with 5 1 C 3 times in PBS, the cells were suspended in the presence of 1 x 1 〇6 / π 1 and added with 10%. The paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) · 17 _ ------- 1 ---------- (Please read the precautions on the back before filling out this page) Order1, --Line-Intellectual Property of the Ministry of Economic Affairs 1222515 A7 15 / Printed by the Bureau's Consumer Cooperative. V. Invention Description (I5) FBS's RPMI 1640 medium. 50 // 1 (5xl04 / well) of the target cells identified by the above method were injected into each well of the microtiter culture dish, and the largest dissociation group (positive control group) was 1 N — HC injected with 100 # 1 respectively. 1 'Natural dissociation group (negative control group) was injected with 100A 1 of RPMI 1640 medium supplemented with 10% FBS, so the experimental dissociation group was at a concentration of 10 // g / m 1 The silk body extract or functional cells stimulated at a concentration of 2000 U / m 1 (100 # 1 (l × 104 / well)) were injected as a control group. The cells were centrifuged in a petri dish centrifuge at 800 r pm for 5 minutes to accumulate at the bottom of the hole, and then cultured in a 5% CO 2 incubator at 37 ° C for 3.5 hours. From the culture medium, S 0 K E N — ΡΕΤ Σ — 9 6 was taken from the culture supernatant of each hole, and the radioactivity was measured by a 7-flash counting tube. LAK activity is calculated from the following table: τ Amo / experimental dissociation natural dissociation (Q 啦), / ipp a κ activity% = maximum dissociation ㈣-natural dissociation ϊ (_χ1〇〇 (Please read the precautions on the back before filling in this page ) € • Line. The results printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs are shown in Table 1 and Figure 1. This paper size applies the Chinese National Standard (CNS) A4 (210 X 297 mm) _ 18 1222515 A7 B7 V. Description of the Invention (16) Table 1: (Please read the precautions on the back before filling this page) Experiment Number LAK Activity
被檢體A 被檢體B 被檢體C 服用前 13% 27% 14% 萃取物的篩選 (最終濃度：10 // g/ml) 21% 34% 15% 服用萃取物後 40% 43% 15% 產業的可利用性 經濟部智慧財產局員工消費合作社印製 對被檢體A以及B而言，實際上以香菇菌絲體萃取物 經口投與時，可確認in vivo中LAK活性的增強（參考表 1、實驗3 )。對於此使用本發明的萃取物於in vitro的篩 選中，由被檢體A以及B的末稍血中採取的淋巴球，以使 用本發明的萃取物刺激時，與本發明的萃取物直接投與於 被檢體A以及B時所得之L A K活性增強效果，確認其顯 示正相關的L A K活性增強效果（參照表1、實驗2 )。 因此，本發明的香菇菌絲體萃取物經口投與時L A K活性 的增強效果，判斷以in vitro的篩選可預測。 另一方面，對於被檢體C而言，實際上含有香菇菌絲 體的物質經口投與時，及使於in vivo中可確認L A K活性 並無增強（參考表1、實驗3 )。對於此使用本發明的萃 取物於in vitro篩選中，及使由被檢體C的末稍血所採取的 淋巴球以使用本發明的萃取物刺激之，並不能確認本發明 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐) 一 經濟部智慧財產局員工消費合作社印製 1222515 A7 B7 五、發明說明（17) 的萃取物以直接投與時所之L A K活性有增強效果（參考 表1、實驗2 )。因此，由此例中亦可得本發明的香菇菌 絲體萃取物經口投與時於in vivo的LAK活性增強效果， 判斷可由in vitro中篩選而預測之。 因此，可知本發明的L A K活性增強物質經口投與於 生物體時，於in viuo中的LAK活性增強效果，可由in vhro中的篩選結果正確的預測出。於是，本發明的香菇菌 絲體萃取物直接投與時於in vivo的L A K活性增強效果， 可於當該L A K活性增強物質被直接投與前，於in vitro中 簡單地判斷出，不僅對於期待L A K活性增強效果的被檢 體而言，LAK活性增強物質的具效果的投與可迅速進行 ，連對不期待L A K活性增強效果的被檢體而言，可防止 L A K活性增強物質的無用途投與。 又，本發明的方法，不僅無須由被檢體的血液中取出 大量的淋巴球，且L A K活性增強物質於in vivo的治療效 果，可於in vitro中篩選出，顯示可減輕被檢體的肉體負擔 〇 圖面的簡單說明 圖1爲表示使用本發明的香菇俊絲體萃取物篩選 L A K活性增強的結果，其數據以柱狀圖形表示。 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）· 20 - ---•丨 _ijj---------- (請先閱讀背面之注意事項再填寫本頁) 訂· --線.Subject A Subject B Subject C 13% 27% 14% Screening of extracts (final concentration: 10 // g / ml) 21% 34% 15% 40% 43% after taking extract 15 % Industrial availability Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. For subjects A and B, when the oral administration of the mushroom mycelium extract was orally administered, it was confirmed that the LAK activity in in vivo was enhanced. (Refer to Table 1, Experiment 3). In this in vitro screening using the extract of the present invention, the lymphocytes collected from the peripheral blood of the subjects A and B are stimulated with the extract of the present invention, and directly injected with the extract of the present invention. It was confirmed that the LAK activity enhancement effect obtained in the subjects A and B showed a positive correlation with the LAK activity enhancement effect (refer to Table 1, Experiment 2). Therefore, the enhancement effect of L A K activity when the mushroom mycelium extract of the present invention is administered orally is judged to be predictable by in vitro screening. On the other hand, in the case of the subject C, when the substance containing mycelia of mushrooms was orally administered, it was confirmed that L A K activity was not enhanced in vivo (see Table 1 and Experiment 3). The use of the extract of the present invention in in vitro screening and the stimulation of lymphocytes taken from the terminal blood of the subject C with the extract of the present invention does not confirm that the paper size of the present invention is applicable to China National Standard (CNS) A4 (210 X 297 mm) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1222515 A7 B7 V. Description of the invention (17) The extract when directly administered has enhanced LAK activity (Refer to Table 1, Experiment 2). Therefore, in this example, the LAK activity-enhancing effect of the Lentinus edodes mycelium extract of the present invention when administered orally can also be obtained, and it can be predicted by screening in vitro. Therefore, it can be seen that when the LAK activity-enhancing substance of the present invention is administered orally to a living body, the LAK activity-enhancing effect in inviuo can be accurately predicted from the screening results in in vhro. Therefore, the in vivo LAK activity enhancement effect of the Lentinus edodes mycelium extract of the present invention can be simply judged in in vitro before the LAK activity enhancement substance is directly administered. For subjects with LAK activity-enhancing effects, the effective administration of LAK activity-enhancing substances can be performed quickly, and even for subjects who do not expect LAK activity-enhancing effects, it can prevent the use of LAK activity-enhancing substances from being used unnecessarily. versus. In addition, the method of the present invention not only does not need to take out a large number of lymphocytes from the blood of the subject, but also the therapeutic effect of the LAK activity-enhancing substance in in vivo can be screened in in vitro, showing that the body of the subject can be reduced Brief Description of the Drawings FIG. 1 shows the results of screening for enhanced LAK activity using the shiitake mushroom jujube extract of the present invention, and the data are shown as bar graphs. This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) · 20---- • 丨 _ijj ---------- (Please read the precautions on the back before filling in this Page) Order-line.