TW201707713A - Composition and method for inhibiting histone deacetylase - Google Patents

Composition and method for inhibiting histone deacetylase Download PDF

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TW201707713A
TW201707713A TW105123920A TW105123920A TW201707713A TW 201707713 A TW201707713 A TW 201707713A TW 105123920 A TW105123920 A TW 105123920A TW 105123920 A TW105123920 A TW 105123920A TW 201707713 A TW201707713 A TW 201707713A
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俊麟 王
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幹細胞生物科技股份有限公司
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Abstract

A method of inhibiting a histone deacetylase activity, comprising administering to a recipient subject in need thereof a composition that contains an effective amount of small somatic stem cells that are greater than 2 micrometers and less than 6 micrometers in size, wherein the small somatic stem cells include CD349+ somatic stem cells and Lgr5+ somatic stems cells.

Description

用於抑制組蛋白去乙醯酶之組成物及方法 Composition and method for inhibiting histone deacetylase 相關申請案之交叉引用 Cross-reference to related applications

本申請案請求全部均於2015年7月28日提申之美國臨時申請案第62/197,742號、第62/197,745號、第62/197,753號、第62/197,757號、第62/197,760號、第62/197,770號以及第62/197,782號之優先權。所有的先前申請案之內容，完整地在此併入本案以為參考。 U.S. Provisional Application Nos. 62/197,742, 62/197,745, 62/197,753, 62/197,757, and 62/197,760, both of which are incorporated herein by reference. Priority 62/197, 770 and 62/197, 782. The contents of all of the prior applications are hereby incorporated by reference in its entirety.

本發明係有關於用於抑制組蛋白去乙醯酶之組成物及方法。 The present invention relates to compositions and methods for inhibiting histone deacetylase.

發明背景 Background of the invention

幹細胞具有自我更新產生更多的幹細胞，還能分化成其它類型之細胞之能力。因此其等可用於治療各種疾病。 Stem cells have the ability to self-renew to produce more stem cells and to differentiate into other types of cells. Therefore, they can be used to treat various diseases.

組蛋白去乙醯酶已顯示為包括癌症、免疫病症以及神經退化性病症之病症的藥物標靶。可抑制組蛋白去乙醯酶活性之組成物，具有治療此等病症之巨大的潛力。 Histone deacetylase has been shown to be a drug target for diseases including cancer, immune disorders, and neurodegenerative disorders. A composition that inhibits histone deacetylase activity has enormous potential for treating such conditions.

發明概要 Summary of invention

在一態樣方面，在此描述的是一種抑制組蛋白去乙醯酶活性之方法。該方法包括對一有需要的受試者投予一組成物，該組成物含有一有效量之尺寸大於2微米以及小於6微米之小型成體幹細胞，其中該小型成體幹細胞包括CD349+成體幹細胞以及Lgr5+成體幹細胞。該方法可另外包括一在該投予步驟之前或之後(或二者)測定該受試者中組蛋白去乙醯酶活性之步驟。 In one aspect, described herein is a method of inhibiting histone deacetylase activity. The method comprises administering to a subject in need thereof a composition comprising an effective amount of small adult stem cells having a size greater than 2 microns and less than 6 microns, wherein the mini adult stem cells comprise CD349+ adult stem cells And Lgr5+ adult stem cells. The method can additionally comprise the step of determining the activity of histone deacetylase in the subject before or after the administration step (or both).

該受試者可能有自體免疫病症、糖尿病、癌症、神經退化性病症或病毒感染。例如，該受試者可能有失智症、帕金森氏症、關節炎、僵直性脊椎炎或糖尿病。 The subject may have an autoimmune disorder, diabetes, cancer, a neurodegenerative disorder, or a viral infection. For example, the subject may have dementia, Parkinson's disease, arthritis, ankylosing spondylitis, or diabetes.

在一具體例中，在該組成物之全部的細胞中，大於95%(如，大於99%或99.99%)可為小型細胞。該小型細胞可包括一或多種類型之細胞，包括血小板、Lgr5(+)細胞、CD349(+)細胞、CD133(+)細胞、CD34(+)以及CD66e(+)細胞中之任一種。血小板可占該組成物之小型細胞的75%至85%。全部的小型細胞中，大於4%(如，大於5%或4.5%至10%之間)可為Lgr5+成體幹細胞。CD349(+)成體幹細胞可占大於該組成物之全部的小型細胞的4%(如，大於5%或4.5%至10%之間)。該小型細胞中，小於2%(如，小於1%或0.5%)可為CD133(+)細胞以及CD34(+)細胞之組合。該小型細胞中，小於6%(如小於5%或4.5%)可為CD66e(+)細胞。 In one embodiment, greater than 95% (eg, greater than 99% or 99.99%) of the cells of the composition may be small cells. The minicell may comprise one or more types of cells, including platelets, Lgr5(+) cells, CD349(+) cells, CD133(+) cells, CD34(+), and CD66e(+) cells. Platelets can comprise from 75% to 85% of the small cells of the composition. More than 4% (eg, greater than 5% or between 4.5% and 10%) of all small cells may be Lgr5+ adult stem cells. CD349(+) adult stem cells can account for 4% (eg, greater than 5% or between 4.5% and 10%) of the small cells that are greater than all of the composition. In this small cell, less than 2% (eg, less than 1% or 0.5%) may be a combination of CD133(+) cells and CD34(+) cells. Less than 6% (e.g., less than 5% or 4.5%) of the small cells may be CD66e(+) cells.

此外，該組成物可實質上排除尺寸大於6微米之大型細胞。例如，大型細胞可占小於該組成物之細胞總數目的5%(如，小於1%、0.5%或0.01%)。 In addition, the composition can substantially exclude large cells having a size greater than 6 microns. For example, large cells may comprise less than 5% (eg, less than 1%, 0.5%, or 0.01%) of the total number of cells of the composition.

在一具體例中，該組成物是一種靜脈投予之注射溶液。其亦可含二價陽離子螯合劑(如，乙二胺四乙酸)。該注射溶液可用包括下列之程序製得：提供一含有血液樣本以及二價陽離子螯合劑之混合物；將該混合物貯存在2℃至12℃間之溫度下3至72個小時，因此該混合物會分出上層以及下層，其中該上層含有小型成體幹細胞；以及收集該上層，因此製得該注射溶液。在該程序中使用之血液樣本，可從該受試者或供體獲得。 In one embodiment, the composition is an intravenously administered injection solution. It may also contain a divalent cation chelating agent (e.g., ethylenediaminetetraacetic acid). The injection solution can be prepared by the following procedure: providing a mixture containing a blood sample and a divalent cation chelating agent; storing the mixture at a temperature between 2 ° C and 12 ° C for 3 to 72 hours, so the mixture is divided The upper layer and the lower layer, wherein the upper layer contains small adult stem cells; and the upper layer is collected, thereby preparing the injection solution. A blood sample for use in the procedure can be obtained from the subject or donor.

任擇地，在獲得用於製備該注射溶液之血液樣本之前，可先在該受試者或供體上進行增加幹細胞數目之動作。例如，該動作可為投予褐藻醣膠(fucoidan)或顆粒球群落刺激因子(granulocyte-colony stimulating factor)。 Optionally, prior to obtaining a blood sample for preparing the injectable solution, an action of increasing the number of stem cells can be performed on the subject or donor. For example, the action can be administration of fucoidan or granulocyte-colony stimulating factor.

在另一態樣方面，在此所述的是一種用於減少一受試者中組蛋白去乙醯酶活性之注射組成物。該組成物含有一有效量之尺寸大於2微米以及小於6微米之小型成體幹細胞，該小型成體幹細胞包括CD349+成體細胞以及Lgr5+成體幹細胞。 In another aspect, described herein is an injection composition for reducing histone deacetylase activity in a subject. The composition comprises an effective amount of small adult stem cells having a size greater than 2 microns and less than 6 microns, the mini-body stem cells comprising CD349+ adult cells and Lgr5+ adult stem cells.

一或多個具體例之詳細內容述於下面的附圖以及說明書中。該等具體例之其它的特徵、目的以及優點，在參考說明書與圖式以及申請專利範圍後，將變得清楚明瞭。 The details of one or more specific examples are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and appended claims.

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圖1是描述根據本發明之一具體例，用於製備幹細胞混合物之流程圖。 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow chart depicting the preparation of a stem cell mixture in accordance with one embodiment of the present invention.

圖2是顯示褐藻醣膠補充物(每粒)之成份資料表。 Figure 2 is a table showing the composition of the fucoidan supplement (per capsule).

圖3是描述根據本發明之一具體例，用於幹細胞活化、純化以及濃縮之方法的流程圖。 Figure 3 is a flow diagram depicting a method for stem cell activation, purification, and concentration, in accordance with one embodiment of the present invention.

圖4是顯示六個受試人在口服褐藻醣膠補充物之前與之後所獲得之幹細胞數據圖表。 Figure 4 is a graph showing stem cell data obtained before and after oral administration of fucoidan supplements by six subjects.

圖5A是顯示在一個顆粒球刺激因子(GCSF或G-CSF)注射療程之前所獲得之流動式細胞測量術之數據圖。 Figure 5A is a graph showing data of flow cytometry obtained prior to a particle ball stimulation factor (GCSF or G-CSF) injection course.

圖5B是顯示在一個顆粒球刺激因子(GCSF或G-CSF)注射療程之後所獲得之流動式細胞測量術之數據圖。 Figure 5B is a graph showing data of flow cytometry obtained after a particle ball stimulation factor (GCSF or G-CSF) injection course.

圖6A是顯示前向散射係數(FSC)對側向散射係數(SSC)之流動式細胞測量術點圖。 Figure 6A is a flow cytometry dot plot showing forward scatter coefficient (FSC) versus side scatter coefficient (SSC).

圖6B是從流動式細胞測量術分析法獲得之螢光直方圖。 Figure 6B is a fluorescence histogram obtained from flow cytometry analysis.

圖6C是FSC對SSC流動式細胞測量術之點圖。 Figure 6C is a dot plot of FSC versus SSC flow cytometry.

圖7A是FSC對SSC流動式細胞測量術之點圖。 Figure 7A is a dot plot of FSC versus SSC flow cytometry.

圖7B是由流動式細胞測量術分析法獲得之CD61螢光直方圖。 Figure 7B is a CD61 fluorescence histogram obtained by flow cytometry analysis.

圖7C是由流動式細胞測量術分析法獲得之CD133螢光直方圖。 Figure 7C is a CD133 fluorescence histogram obtained by flow cytometry analysis.

圖7D是由流動式細胞測量術分析法獲得之CD34螢光直方圖。 Figure 7D is a CD34 fluorescence histogram obtained by flow cytometry analysis.

圖7E是由流動式細胞測量術分析法獲得之CD66e螢光直方圖。 Figure 7E is a CD66e fluorescence histogram obtained by flow cytometry analysis.

圖7F是由流動式細胞測量術分析法獲得之CD349螢光直方圖。 Figure 7F is a CD349 fluorescence histogram obtained by flow cytometry analysis.

圖7G是從流動式細胞測量術分析法獲得之Lgr5螢光直方圖。 Figure 7G is a Lgr5 fluorescence histogram obtained from flow cytometry analysis.

圖8是表2中所示之數據之散布圖。 Figure 8 is a scatter diagram of the data shown in Table 2.

較佳實施例之詳細說明 Detailed description of the preferred embodiment

意外地發現含有某些小型成體幹細胞(如，Lgr5(+)以及CD349(+)細胞)之組成物，在活體內會抑制組蛋白去乙醯酶(HDAC)之活性。據此，在此所述的是用於抑制組蛋白去乙醯酶之方法以及組成物。 It has been unexpectedly found that compositions containing certain small adult stem cells (eg, Lgr5 (+) and CD349 (+) cells) inhibit the activity of histone deacetylase (HDAC) in vivo. Accordingly, described herein are methods and compositions for inhibiting histone deacetylase.

成體幹細胞 Adult stem cell

目前有各種類型的成體幹細胞，包括全能幹細胞、多功能幹細胞、多潛能幹細胞以及前趨幹細胞(亦稱作單潛能幹細胞)。***球樣幹細胞(BLSCs)是全能或多功能成體幹細胞。極小型胚胎樣幹細胞(VSELs)是多功能成體幹細胞。SB細胞是多功能或多潛能成體幹細胞。間葉幹細胞(MSCs)以及造血幹細胞(HSC)是多潛能成體幹細胞。 There are various types of adult stem cells, including pluripotent stem cells, pluripotent stem cells, pluripotent stem cells, and pre-existing stem cells (also known as singular potential stem cells). Mitochondrial stem cells (BLSCs) are totipotent or multifunctional adult stem cells. Very small embryonic stem cells (VSELs) are multifunctional adult stem cells. SB cells are multifunctional or pluripotent adult stem cells. Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSC) are pluripotent adult stem cells.

在此使用之諸如幹細胞之細胞尺寸(Z)，意指(1)細胞生物學領域或幹細胞領域中，對細胞之尺寸以及具代表性長度之慣用的定義、(2)細胞之直徑，特別是當細胞為實質上球形時、(3)細胞長軸之長度，特別是當細胞為實質 上橢球形時、(4)細胞之寬度，當細胞之形狀具大致上正方形時、(5)細胞之長度，當細胞之形狀為大致上方形時或(6)細胞之最大橫截面或橫向尺度。尺寸(Z)，不管是直徑、長度、寬度或最大橫截面或橫向尺度，可使用例如從光學顯微鏡或從電子顯微鏡(如，掃描式電子顯微鏡(SEM))獲得之細胞影像，或使用從流動式細胞測量術獲得之細胞的數據(如，二維點圖、等高線或密度圖)決定或測得。從光學顯微鏡或電子顯微鏡獲得之影像，可為細胞之二維(2D)橫截面或三維(3D)結構。舉例而言，細胞之尺寸(Z)，可藉由測量從光學顯微鏡或電子顯微鏡(如，SEM)獲得之2D橫截面影像之最大橫截面或橫向尺寸而得。 As used herein, cell size (Z) of stem cells means (1) the definition of cell size and representative length in the field of cell biology or stem cells, (2) the diameter of cells, especially When the cell is substantially spherical, (3) the length of the long axis of the cell, especially when the cell is substantial When the upper ellipsoid is spherical, (4) the width of the cell, when the shape of the cell is substantially square, (5) the length of the cell, when the shape of the cell is substantially square or (6) the largest cross-section or transverse dimension of the cell . Dimensions (Z), whether diameter, length, width or maximum cross-section or lateral dimension, can be obtained, for example, from an optical microscope or from an electron microscope (eg, scanning electron microscope (SEM)), or from a flow Data from cells obtained by cytometry (eg, two-dimensional dot plots, contours, or density plots) are determined or measured. An image obtained from an optical microscope or an electron microscope may be a two-dimensional (2D) cross-section or a three-dimensional (3D) structure of a cell. For example, the size (Z) of a cell can be obtained by measuring the largest cross-sectional or lateral dimension of a 2D cross-sectional image obtained from an optical microscope or an electron microscope (eg, SEM).

術語“小型細胞”(如，小型成體幹細胞)意指尺寸小於6微米之細胞(如，2.0至6.0微米之間)。術語“大型細胞”意指尺寸大於6微米之細胞。 The term "small cells" (eg, small adult stem cells) means cells that are less than 6 microns in size (eg, between 2.0 and 6.0 microns). The term "large cell" means a cell having a size greater than 6 microns.

CD349(+)SB細胞是多功能或多潛能成體幹細胞。CD349(+)SB細胞亦可為CD9(+)、Oct4(+)與Nanog(+)以及CD133(-)、CD90(-)、CD34(-)與Sox2(-)。CD349(+)SB細胞各具有尺寸等於或小於4、5或6微米，諸如0.1至6.0微米之間、0.5至6.0微米之間、1.0至6.0微米之間、2.0至6.0微米之間、0.1至5.0微米之間、0.5至5.0微米之間、1.0至5.0微米之間、0.1至4.0微米之間、0.5至4.0微米之間或1.0至4.0微米之間。較佳地，該尺寸大於2微米以及小於6微米。 CD349(+)SB cells are multifunctional or pluripotent adult stem cells. CD349(+)SB cells can also be CD9(+), Oct4(+) and Nanog(+) as well as CD133(-), CD90(-), CD34(-) and Sox2(-). CD349(+)SB cells each have a size equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 2.0 and 6.0 microns, and between 0.1 and Between 5.0 microns, between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns or between 1.0 and 4.0 microns. Preferably, the size is greater than 2 microns and less than 6 microns.

Lgr5(+)SB細胞是多功能或多潛能成體幹細胞。其等亦可為Oct4(+)與Nanog(+)以及CD133(-)、 CD66e(-)、CD4(-)、CD8(-)、CD9(-)、CD10(-)、CD11(-)、CD16(-)、CD17(-)、CD18(-)、CD19(-)、CD20(-)、CD21(-)、CD31(-)、CD42(-)、CD63(-)、CD34(-)、Lin(-)、CD38(-)、CD90(-)、CD45(-)、CD349(-)以及Sox2(-)。Lgr5(+)SB細胞之尺寸可等於或小於4、5或6微米，諸如0.1至6.0微米之間、0.5至6.0微米之間、1.0至6.0微米之間、2.0至6.0微米之間、0.1至5.0微米之間、0.5至5.0微米之間、1.0至5.0微米之間、0.1至4.0微米之間、0.5至4.0微米之間或1.0至4.0微米之間。較佳地，Lgr5(+)SB細胞之尺寸大於2微米以及小於6微米。 Lgr5(+)SB cells are multifunctional or pluripotent adult stem cells. They can also be Oct4(+) and Nanog(+) and CD133(-), CD66e(-), CD4(-), CD8(-), CD9(-), CD10(-), CD11(-), CD16(-), CD17(-), CD18(-), CD19(-), CD20(-), CD21(-), CD31(-), CD42(-), CD63(-), CD34(-), Lin(-), CD38(-), CD90(-), CD45(-), CD349(-) and Sox2(-). The size of the Lgr5(+)SB cells may be equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 2.0 and 6.0 microns, and between 0.1 and Between 5.0 microns, between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns or between 1.0 and 4.0 microns. Preferably, the Lgr5(+)SB cells are larger than 2 microns and less than 6 microns in size.

***球樣幹細胞(BLSCs)是CD66e(+)全能或多功能成體幹細胞。其等各具有尺寸等於或小於4、5或6微米，諸如0.1至6.0微米之間、0.5至6.0微米之間、1.0至6.0微米之間、2.0至6.0微米之間、0.1至5.0微米之間、0.5至5.0微米之間、1.0至5.0微米之間、0.1至4.0微米之間、0.5至4.0微米之間或1.0至4.0微米之間。例如，BLSC可具有大於2微米以及小於6微米之尺寸。 Mitochondrial stem cells (BLSCs) are CD66e (+) totipotent or multifunctional adult stem cells. Each of them has a size equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 2.0 and 6.0 microns, and between 0.1 and 5.0 microns. Between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns or between 1.0 and 4.0 microns. For example, the BLSC can have a size greater than 2 microns and less than 6 microns.

極小型胚胎樣幹細胞(VSELs)是多功能成體幹細胞，其可為CD133(+)或CD34(+)。VSEL亦可為CD45(-)以及Lin(-)。例如，VSEL可為CD133(+)、CD45(-)以及Lin(-)，或CD34(+)、CD45(-)以及Lin(-)。VSEL之尺寸可等於或小於4、5或6微米，諸如0.1至6.0微米之間、0.5至6.0微米之間、1.0至6.0微米之間、2.0至6.0微米之間、0.1至5.0微米之間、0.5至5.0微米之間、1.0至5.0微米之間、0.1至4.0微米之間、 0.5至4.0微米之間或1.0至4.0微米之間。VSEL之尺寸可大於2微米以及小於6微米。 Very small embryonic stem cells (VSELs) are multifunctional adult stem cells that can be CD133(+) or CD34(+). The VSEL can also be CD45 (-) and Lin (-). For example, the VSEL can be CD133(+), CD45(-), and Lin(-), or CD34(+), CD45(-), and Lin(-). The size of the VSEL may be equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 2.0 and 6.0 microns, between 0.1 and 5.0 microns, Between 0.5 and 5.0 microns, between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, Between 0.5 and 4.0 microns or between 1.0 and 4.0 microns. The size of the VSEL can be greater than 2 microns and less than 6 microns.

間葉幹細胞(MSCs)是多潛能成體幹細胞。MSC可表達細胞表面標記CD13、CD29、CD44、CD73、CD90以及CD105中之一或多個。MSCs構成一非常異質的群組。一些類型的MSCs之尺寸可等於或小於4、5或6微米，諸如0.1至6.0微米之間、0.5至6.0微米之間、1.0至6.0微米之間、0.1至5.0微米之間、0.5至5.0微米之間、1.0至5.0微米之間、0.1至4.0微米之間、0.5至4.0微米之間或1.0至4.0微米之間。其它類型的MSCs之尺寸可大於6、7或10微米。 Mesenchymal stem cells (MSCs) are pluripotent adult stem cells. MSCs can express one or more of the cell surface markers CD13, CD29, CD44, CD73, CD90, and CD105. MSCs constitute a very heterogeneous group. Some types of MSCs may be equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 0.1 and 5.0 microns, and between 0.5 and 5.0 microns. Between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns or between 1.0 and 4.0 microns. Other types of MSCs can be larger than 6, 7, or 10 microns in size.

造血幹細胞(HSCs)是多潛能成體幹細胞。其等可為CD34(+)、cKit(-)、CD38(-)、Lin(-)細胞，或CD150(+)、CD244(-)與CD48(-)細胞。HSCs之尺寸可等於或小於4、5或6微米，諸如0.1至6.0微米之間、0.5至6.0微米之間、1.0至6.0微米之間、0.1至5.0微米之間、0.5至5.0微米之間、1.0至5.0微米之間、0.1至4.0微米之間、0.5至4.0微米之間或1.0至4.0微米之間。 Hematopoietic stem cells (HSCs) are pluripotent adult stem cells. These may be CD34(+), cKit(-), CD38(-), Lin(-) cells, or CD150(+), CD244(-) and CD48(-) cells. The size of the HSCs may be equal to or less than 4, 5 or 6 microns, such as between 0.1 and 6.0 microns, between 0.5 and 6.0 microns, between 1.0 and 6.0 microns, between 0.1 and 5.0 microns, between 0.5 and 5.0 microns, Between 1.0 and 5.0 microns, between 0.1 and 4.0 microns, between 0.5 and 4.0 microns or between 1.0 and 4.0 microns.

用於增加幹細胞之動作 Used to increase the action of stem cells

在此所使用之動作(X)是一種可在活體內，諸如受試人或非人受試者內，有效的增加一或多種幹細胞之數量之動作。動作(X)可包括：1.服用藥物，諸如人工合成藥物或源於自然的化合物；2.服用藥草或中藥，諸如冬蟲夏草、人參、枸杞、靈 芝(lingzhi)、牛樟芝和/或巴西蘑菇；3.服用營養品或膳食補充物，諸如營養丸或粉，包括下列材料或元素：維生素(維生素A、B、B群、B12、D、D3、E等等)、巨量和/或微量礦物質(如，鈣、鈉、鉀、氟、溴、鉻、碘、矽、砷、鈹、鋰、鈷、釩和/或鎳)、多醣類、高分子量含岩藻糖之糖蛋白、海藻(包括綠藻、藍綠藻、褐藻等等)、岩藻糖、褐藻醣膠(褐藻之主要組份)、小分子褐藻醣膠、藻類、含褐藻醣膠之褐藻(例如，在日本沖繩島生長以及生產的褐藻)、日本水雲、綠藻、藍綠藻(或藍藻)、褐藻(包括水雲、昆布、裙帶菜、羊棲菜、裙帶菜等等)、植物性化合物(如，異黃酮或植物***)、茄紅素、表沒食子兒茶素沒食子酸酯(EGCG)、綠茶素、醣質營養素(如，木醣、半乳糖、葡萄糖、甘露糖N-乙醯麩胺酸、N-乙醯半乳糖胺或N-乙醯神經胺酸)、魚油、香樁(toona sinensis)和/或從植物、葉子、果實、蔬菜、魚、海藻或藻類萃取而得之營養素；4.吃素；5.服用或食用健康食品或有機食品；6.採用替代(非傳統)醫學；7.接受替代療法或治療，諸如Gerson療法或Breuss癌症療法；8.接受針灸治療；9.接受按摩治療，諸如腳部按摩；10.運動，諸如走路、慢跑、跳舞、體操、瑜珈、有氧運動和/或太極拳(Chinese shadow exercise)； 11.睡覺(為測量睡眠品質之目的)；12.冥想；13.實施由個人、醫療專家人員或醫生擬定的健康改善計劃或疾病治療計劃；14.服用某些用於改善身體中某些器官之健康之營養素，例如，服用茄紅素，用以改善***之健康；15.採取用以醫治受傷或用以醫治手術所導致之傷口或用以治癒疾病之修護計劃；16.服用，如，將一種中藥材或多種中藥材浸在烈酒或果酒中一段時間製得之藥酒，諸如，將人參浸在高酒精濃度之米酒中一個月製得之人參酒；17.服用一或多種經政府部門或機構，諸如美國食品藥品管理局(U.S.FDA)核准，可用於治療特定疾病(如，一種癌症、皮膚病、腎病和/或等等)之藥物；18.採用或接受經政府部門核准可用於治療特定疾病(如，一種癌症、皮膚病或腎病)之治療或療法；19.參與宗教活動，諸如祈求平靜或拜神；20.直接或間接照日光(早上，例如日出前10分鐘以及日出後50分鐘之間(含有大量的紅外(IR)線))；或中午時分，例如，11：30 AM至12：30 PM之間(含有大量的紫外(UV)線)；或下午，例如，日落前50分鐘以及日落後10分鐘(含有大量的紅外線(IR)線))；21.照射燈光或發光二極體(LED)燈，其可包括整個可見光光譜、IR線、紅光、綠光、藍光或紫外線，或以上光 線中一個以上之組合；22.執行或接受用於改善身體自我修復之計劃、治療法、方法、裝置和/或系統，例如，受傷或手術後用於改善自我修復之方法或治療法(如，高壓氧治療法)；23.喝咖啡，諸如黑咖啡；24.喝茶，諸如綠茶、紅茶或茉莉花茶；25.喝紅酒；26.服用褪黑激素；27.聽音樂，諸如莫札特或貝多芬之交響曲；28.注射含有褐藻醣膠或小分子褐藻醣膠之物質(如，營養品或補充物)；29.服用荷爾蒙補充劑或接受荷爾蒙注射；30.注射顆粒球群落刺激因子(G-CSF或GCSF)，其是一種醣蛋白；31.接受一個GCSF注射療程；以及32.服用含有下列之營養素、營養品、營養液、營養飲料、營養液、營養食品(1)各種胺基酸(諸如精胺酸、組胺酸、離胺酸、天門冬胺酸、麩胺酸、絲胺酸、蘇胺酸、天門冬醯胺酸、麩醯胺酸、半胱胺酸、纈胺酸、脯胺酸、甘胺酸、硒半胱胺酸、丙胺酸、異白胺酸、白胺酸、***酸、甲硫胺酸、酪胺酸或色胺酸)、(2)平衡型胺基酸或(3)9種人體必需的胺基酸(如，組胺酸、異白胺酸、白胺酸、離胺酸、甲硫胺酸、***酸、蘇胺酸、色胺酸以及纈胺酸)。例如：(a)由紅、綠、黑豆之發酵產生或萃取而得之產物；(b)由水 果或水果組合，諸如甜菜、蘋果、芭樂、奇異果、葡萄、鳳梨、火龍果(龍果)、青木瓜、蕃茄和/或酪梨等等之發酵產生之液體或飲料；(c)如將一種中藥材或多種中藥材浸在烈酒或果酒中一段時間製得之藥酒，諸如，將人參浸在高酒精濃度之米酒中一個月製得之人參藥酒。 The action (X) used herein is an action that can effectively increase the number of one or more stem cells in a living body, such as a subject or a non-human subject. Action (X) may include: 1. taking a drug, such as a synthetic drug or a natural-derived compound; 2. taking a herb or a Chinese medicine, such as Cordyceps sinensis, ginseng, medlar, lingzhi, burdock and/or Brazilian mushroom; Taking nutrients or dietary supplements, such as nutritional pills or powders, including the following materials or elements: vitamins (vitamin A, B, B, B 12 , D, D 3 , E, etc.), macro and/or trace Minerals (eg, calcium, sodium, potassium, fluorine, bromine, chromium, iodine, antimony, arsenic, antimony, lithium, cobalt, vanadium and/or nickel), polysaccharides, high molecular weight fucose-containing glycoproteins, Seaweed (including green algae, blue-green algae, brown algae, etc.), fucose, fucoidan (major component of brown algae), small molecule fucoidan, algae, brown algae containing fucoidan (for example, in Okinawa, Japan) Island growth and production of brown algae), Japanese water cloud, green algae, blue-green algae (or cyanobacteria), brown algae (including water clouds, kelp, wakame, sarcophagus, wakame, etc.), botanical compounds (eg, isoflavones) Or phytoestrogens), lycopene, epigallocatechin gallate (EGCG), green tea, Nutrients (eg, xylose, galactose, glucose, mannose N-acetamide, N-acetylgalactosamine or N-acetamidine), fish oil, toona sinensis and / Or nutrients extracted from plants, leaves, fruits, vegetables, fish, algae or algae; 4. vegetarian; 5. taking or eating healthy or organic foods; 6. using alternative (non-traditional) medicine; Therapy or treatment, such as Gerson therapy or Breuss cancer therapy; 8. Receiving acupuncture treatment; 9. Receiving massage therapy, such as foot massage; 10. Exercise, such as walking, jogging, dancing, gymnastics, yoga, aerobics and/or Chinese Shadow Exercise; 11. Sleeping (for the purpose of measuring sleep quality); 12. Meditation; 13. Implementing a health improvement plan or disease treatment plan prepared by an individual, medical professional or doctor; 14. Taking certain Nutrients used to improve the health of certain organs in the body, for example, taking lycopene to improve the health of the prostate; 15. Take repairs to treat wounds or to treat wounds caused by surgery or to cure diseases Protection plan 16. Taking, for example, a Chinese herbal medicine or a variety of Chinese herbal medicines dipped in spirits or fruit wine for a period of time, such as ginseng immersed in high alcoholic rice wine for one month to produce ginseng wine; Take one or more drugs approved by government agencies or agencies, such as the US Food and Drug Administration (USFDA), for the treatment of specific diseases (eg, a cancer, skin disease, kidney disease, and/or the like); Approved by the government to treat or treat a particular disease (eg, a cancer, skin disease, or kidney disease); 19. Participate in religious activities, such as praying for peace or worship; 20. Direct or indirect sunlight (in the morning, such as the day) Between the first 10 minutes and 50 minutes after sunrise (containing a large amount of infrared (IR) lines); or at noon, for example, between 11:30 AM and 12:30 PM (containing a large amount of ultraviolet (UV) Line); or afternoon, for example, 50 minutes before sunset and 10 minutes after sunset (containing a large amount of infrared (IR) lines); 21. illuminating a light or a light-emitting diode (LED) lamp, which may include the entire visible spectrum, IR line, red light, green light, blue light or ultraviolet light, a combination of more than one of the above light; 22. performing or receiving a plan, treatment, method, device, and/or system for improving the body's self-healing, for example, a method or treatment for improving self-repair after injury or surgery ( For example, hyperbaric oxygen therapy); 23. drinking coffee, such as black coffee; 24. drinking tea, such as green tea, black tea or jasmine tea; 25. drinking red wine; 26. taking melatonin; 27. listening to music, such as Moza Symphony of special or Beethoven; 28. Injection of substances containing fucoidan or small molecule fucoidan (eg, nutraceuticals or supplements); 29. taking hormone supplements or receiving hormone injections; 30. Factor (G-CSF or GCSF), which is a glycoprotein; 31. receives a GCSF injection course; and 32. takes the following nutrients, nutrients, nutrient solution, nutrient drink, nutrient solution, nutritious food (1) Amino acids (such as arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, aspartic acid, glutamic acid, cysteine, Proline, valine, glycine, selenocysteine Acid, alanine, isoleucine, leucine, phenylalanine, methionine, tyrosine or tryptophan), (2) balanced amino acids or (3) 9 essential amino groups Acids (eg, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine). For example: (a) a product produced or extracted from the fermentation of red, green, black beans; (b) a combination of fruits or fruits, such as beets, apples, guava, kiwi, grapes, pineapples, dragon fruit (dragon fruit) Liquid or beverage produced by fermentation of green papaya, tomato and/or avocado, etc.; (c) medicinal liquor obtained by immersing a Chinese herbal medicine or a plurality of traditional Chinese medicines in spirits or fruit wine for a period of time, such as ginseng Ginseng medicinal liquor prepared in one month in a high alcohol concentration rice wine.

含幹細胞之組成物 Composition containing stem cells

含幹細胞之組成物(如，含幹細胞之溶液)，可使用圖1中所示之流程圖所描述之例示性方法製得。 Compositions containing stem cells (e.g., solutions containing stem cells) can be prepared using the exemplary methods described in the flow chart shown in Figure 1.

參照圖1，於步驟10中，受試者採用或接受以上所述之動作(X)中之一個動作(X)的處理。該受試者為，例如，人(如，小孩、青少年、成人或老年人)或非人之動物。非人之動物之例子包括靈長類動物(如，猴子或猩猩)、狗、囓齒動物(如，老鼠或天竺鼠)、貓、馬、乳牛、牛、綿羊、豬、雞、鴨、鵝、鳥以及大象。 Referring to Figure 1, in step 10, the subject takes or accepts the processing of one of the actions (X) of action (X) described above. The subject is, for example, a human (eg, a child, adolescent, adult, or elderly) or a non-human animal. Examples of non-human animals include primates (eg, monkeys or orangutans), dogs, rodents (eg, mice or guinea pigs), cats, horses, cows, cows, sheep, pigs, chickens, ducks, geese, birds. And the elephant.

例如，在步驟10中，該受試者可服用幹細胞動員劑，諸如含褐藻醣膠之化合物。該含褐藻醣膠之化合物可為褐藻補充物。圖2示出褐藻補充物之成份。一粒褐藻補充物含有80%之水雲粉、15%之結晶纖維素、3%之蔗糖脂肪酸酯以及2%之微或細矽粉(含有二氧化矽)。從生長在日本沖繩島附近海域中之水雲褐藻(一種海藻)中，可萃取得水雲粉。之後將水雲粉與結晶纖維素、蔗糖脂肪酸酯以及微或細矽粉(含二氧化矽)混合，以形成褐藻補充丸，其含有0.1克的褐藻醣膠。在步驟10中，受試者可服用20或更多粒(如，至少30粒)之褐藻補充物，或2克或更多(諸如至少3克)之褐 藻醣膠。在另一範例中，於步驟10中，可於受試者身上注射顆粒球群落刺激因子(GCSF)，即，動員劑，或可使其接受一個GCSF注射療程。 For example, in step 10, the subject can take a stem cell mobilizer, such as a compound containing fucoidan. The compound containing fucoidan may be a brown algae supplement. Figure 2 shows the composition of the brown algae supplement. A brown algae supplement contains 80% water cloud powder, 15% crystalline cellulose, 3% sucrose fatty acid ester, and 2% micro or fine powder (containing cerium oxide). Water cloud powder can be extracted from the water cloud brown algae (a seaweed) grown in the sea near Okinawa, Japan. The water cloud powder is then mixed with crystalline cellulose, sucrose fatty acid ester, and micro or fine mash (containing cerium oxide) to form a brown algae supplement pill containing 0.1 gram of fucoidan. In step 10, the subject may take 20 or more (eg, at least 30) brown algae supplements, or 2 grams or more (such as at least 3 grams) of brown Alginose gum. In another example, in step 10, a particulate ball community stimulating factor (GCSF), ie, a mobilizing agent, can be injected into the subject, or it can be subjected to a GCSF injection course.

在步驟10之後，進行步驟11，讓受試者等候一段時間(如，一段預定的時間)，諸如15分鐘至60分鐘之間、20分鐘至100分鐘之間、30分鐘至4個小時之間、60分鐘至90分鐘之間、0.5個小時至3個小時之間、1個小時至6個小時之間、1個小時至12個小時之間、12個小時至36個小時之間或36個小時至50個小時之間。步驟10與11可使一或多種特定類型之成體幹細胞，諸如SB細胞(即，CD349(+)以及Lgr5(+)SB細胞)，從如受試者之骨髓移動進入受試者的周邊血液。該受試者之周邊血液因此變成富含該一或多種特定類型之成體幹細胞。該一或多個特定類型的成體幹細胞可為或可包括例如以上所述之成體幹細胞中之一或多種。例如，該一或多種特定類型的成體幹細胞可為或可包括尺寸小於6微米，更佳地尺寸大於2微米之成體幹細胞，諸如CD349(+)成體幹細胞和/或Lgr5(+)成體幹細胞。 After step 10, step 11 is performed to allow the subject to wait for a period of time (eg, a predetermined period of time), such as between 15 minutes and 60 minutes, between 20 minutes and 100 minutes, between 30 minutes and 4 hours. Between 60 minutes and 90 minutes, between 0.5 hours and 3 hours, between 1 hour and 6 hours, between 1 hour and 12 hours, between 12 hours and 36 hours or 36 Hours to 50 hours. Steps 10 and 11 may cause one or more specific types of adult stem cells, such as SB cells (ie, CD349(+) and Lgr5(+) SB cells), to move from the bone marrow of the subject into the peripheral blood of the subject. . The peripheral blood of the subject thus becomes enriched in the one or more specific types of adult stem cells. The one or more particular types of adult stem cells can be or can include, for example, one or more of the adult stem cells described above. For example, the one or more specific types of adult stem cells can be or can include adult stem cells having a size of less than 6 microns, more preferably greater than 2 microns, such as CD349(+) adult stem cells and/or Lgr5(+). Somatic stem cells.

步驟10以及11是任擇的。換句話說，欲製造含幹細胞之組成物，可在沒有先在受試者上進行任何的動作(X)之情況下，從該受試者身上取得血液樣本。 Steps 10 and 11 are optional. In other words, to make a composition containing stem cells, a blood sample can be taken from the subject without first performing any action (X) on the subject.

在步驟11後立即接著的步驟12中(假如有進行步驟10以及11的話)，從該受試者之周邊血液中提取、抽出、取出、獲得、收集或衍生出血液樣本，然後置於一或多種含有二價陽離子螯合劑之容器中(如，袋、一或多種針筒或 一或多種試管)。使血液樣本與容器中之二價陽離子螯合劑混合，形成一混合物。該二價陽離子螯合劑，如，抗凝血劑，可為乙二胺四乙酸(EDTA)，諸如K2 EDTA抗凝血劑或K3 EDTA抗凝血劑，具有重量，如，大於70mg，諸如90至900mg之間、120至450mg之間或150至400mg之間。選擇性地，該二價陽離子螯合劑可為具有重量，如，大於70mg，諸如90至900mg之間、120至450mg之間或150至400mg之間之檸檬酸。該血液樣本含有數種細胞，包括尺寸小於6微米之小型細胞以及大於6微米之大型細胞。該小型細胞含有，例如，血小板以及尺寸小於6微米之小型成體幹細胞。例如，該小型成體幹細胞含有一或多種特定類型之成體幹細胞(即，例如SB細胞)、BLSCs(即，CD66e(+)成體幹細胞)以及VSELs(如，CD133(+)成體幹細胞與CD34(+)成體幹細胞)。該大型細胞含有，例如，尺寸大於6微米之大型成體幹細胞以及諸如紅血球細胞與白血球細胞之細胞系。該血液樣本之體積可大於或等於45毫升，諸如60至500毫升之間、80至250毫升之間或100至200毫升之間。在一範例中，每毫升之血液樣本可混合與1.5mg或更多，諸如1.6至2.0mg之間，之二價陽離子螯合劑(諸如K2 EDTA、K3 EDTA或檸檬酸)，於容器中形成混合物。 Immediately after step 11 in step 12 (if steps 10 and 11 are performed), blood samples are extracted, extracted, removed, obtained, collected or derived from the peripheral blood of the subject and then placed in one or a variety of containers containing divalent cation chelating agents (eg, bags, one or more syringes or One or more test tubes). The blood sample is mixed with a divalent cation chelating agent in the container to form a mixture. The divalent cation chelating agent, such as an anticoagulant, may be ethylenediaminetetraacetic acid (EDTA), such as a K2 EDTA anticoagulant or a K3 EDTA anticoagulant, having a weight, such as greater than 70 mg, such as 90. To between 900 mg, between 120 and 450 mg or between 150 and 400 mg. Alternatively, the divalent cation chelating agent can be citric acid having a weight, such as greater than 70 mg, such as between 90 and 900 mg, between 120 and 450 mg, or between 150 and 400 mg. The blood sample contains several cells, including small cells smaller than 6 microns in size and large cells larger than 6 microns. The minicell contains, for example, platelets and small adult stem cells of less than 6 microns in size. For example, the mini adult stem cells contain one or more specific types of adult stem cells (ie, such as SB cells), BLSCs (ie, CD66e (+) adult stem cells), and VSELs (eg, CD133 (+) adult stem cells and CD34 (+) adult stem cells). The large cells contain, for example, large adult stem cells larger than 6 micrometers and cell lines such as red blood cells and white blood cells. The volume of the blood sample can be greater than or equal to 45 milliliters, such as between 60 and 500 milliliters, between 80 and 250 milliliters, or between 100 and 200 milliliters. In one example, each milliliter of blood sample can be mixed with 1.5 mg or more, such as between 1.6 and 2.0 mg, of a divalent cation chelating agent (such as K2 EDTA, K3 EDTA or citric acid) to form a mixture in a container. .

接著，步驟13，將該混合物處理成含幹細胞之溶液。該處理可包括幹細胞活化以及純化/分離之步驟，諸如圖3中所述之步驟21以及22。在此所使用之術語“純化”或“分離”意指實質的分開小型細胞(如，尺寸大於2微米以及小 於6微米之細胞)與大型細胞(如，尺寸大於6微米之細胞)，如，藉由移除混合物或血液樣本中之大型細胞，獲得該混合物或血液樣本中之小型細胞之動作。 Next, in step 13, the mixture is processed into a solution containing stem cells. This treatment may include steps of stem cell activation and purification/separation, such as steps 21 and 22 as described in FIG. The term "purified" or "isolated" as used herein, means substantially separate small cells (eg, larger than 2 microns in size and small). The action of the small cells in the mixture or blood sample is obtained by removing cells or large cells (e.g., cells larger than 6 micrometers in size), for example, by removing large cells from the mixture or blood sample.

參照圖3，於步驟21中，將在步驟12中形成之混合物貯存在2℃至12℃之間之溫度下，更佳地2℃至7℃之間，或在4℃下，適合的設備中(如，冰箱或其它用於保持東西冷度之裝置)歷時一段預定的時間。該段時間可在3個小時至72個小時之間，更佳地3個小時至6個小時之間、6個小時至72個小時之間、6個小時至48個小時之間、16個小時至72個小時之間、16個小時至48小時之間、36個小時至60個小時之間、48個小時至72個小時之間或大約48個小時。在該混合物已經貯存一段預定的時間之後，用二價陽離子螯合劑(諸如K2 EDTA、K3 EDTA或檸檬酸)活化該混合物中一或多種特定類型之成體幹細胞(如，SB細胞)，即，活化該一或多種特定類型之成體幹細胞之細胞周期，使從G0進入G1。該活化有關二價陽離子螯合劑抑制p53的功能之能力(假設藉由螯合Zn2+)，從而使該一或多種特定類型之成體幹細胞(如，SB細胞)離開其細胞周期之G0靜止階段，進入G1階段。因為p53蛋白需要Zn2+才可適當折疊而形成功能蛋白，所以利用二價陽離子螯合劑螯合Zn2+，可能是活化該一或多種特定類型之成體幹細胞(如，SB細胞)之關鍵步驟。也有可能是，二價陽離子螯合劑螯合其它二價離子(如，Ca2+)，從而活化該一或多種特定類型之成體幹細胞，迫使其等增生以及擴張。 Referring to Figure 3, in step 21, the mixture formed in step 12 is stored at a temperature between 2 ° C and 12 ° C, more preferably between 2 ° C and 7 ° C, or at 4 ° C, suitable equipment Medium (eg, a refrigerator or other device for maintaining the coolness of things) for a predetermined period of time. This period can be between 3 hours and 72 hours, preferably between 3 hours and 6 hours, between 6 hours and 72 hours, between 6 hours and 48 hours, 16 Hours to 72 hours, 16 hours to 48 hours, 36 hours to 60 hours, 48 hours to 72 hours, or approximately 48 hours. After the mixture has been stored for a predetermined period of time, one or more specific types of adult stem cells (eg, SB cells) in the mixture are activated with a divalent cationic chelating agent (such as K2 EDTA, K3 EDTA or citric acid), ie, The cell cycle of the one or more specific types of adult stem cells is activated to enter G1 from G0. This activation is related to the ability of the divalent cation chelating agent to inhibit the function of p53 (assuming sequestration of Zn 2+ ) such that the one or more specific types of adult stem cells (eg, SB cells) leave the G0 of their cell cycle. Stage, enter the G1 stage. Because p53 protein requires Zn 2+ to fold properly to form a functional protein, chelation of Zn 2+ with a divalent cation chelating agent may be the key to activation of one or more specific types of adult stem cells (eg, SB cells). step. It is also possible that the divalent cation chelating agent sequesters other divalent ions (e.g., Ca2 + ) to activate the one or more specific types of adult stem cells, forcing them to proliferate and expand.

在步驟21中，該混合物因重力而分開成多個各別層，包括上層以及下層。該上層，或上清液，可具有體積在20至250毫升之間、40至125毫升之間或50至100毫升之間。該上層含有血小板、血清以及一或多種特定類型之小型成體幹細胞(即，例如SB細胞)、BLSCs(即，CD66e(+)成體幹細胞)以及VSELs(如，CD133(+)成體幹細胞以及CD34(+)成體幹細胞)。血液樣本中，大部分含大型細胞之品系以及大型成體幹細胞，如該血液樣本中，大於95%、98%或99%之大型細胞，是在下層中。上清液之體積對血液樣本之體積之比，範圍可從，例如，1/3至一半。 In step 21, the mixture is divided into a plurality of individual layers by gravity, including an upper layer and a lower layer. The upper layer, or supernatant, may have a volume between 20 and 250 milliliters, between 40 and 125 milliliters, or between 50 and 100 milliliters. The upper layer contains platelets, serum, and one or more specific types of small adult stem cells (ie, such as SB cells), BLSCs (ie, CD66e (+) adult stem cells), and VSELs (eg, CD133 (+) adult stem cells, and CD34 (+) adult stem cells). In blood samples, most of the large cell-containing lines and large adult stem cells, such as large blood cells in the blood sample, greater than 95%, 98%, or 99%, are in the lower layer. The ratio of the volume of the supernatant to the volume of the blood sample can range, for example, from one third to one half.

接著，在步驟22中，可收集實際上所有的上層，或將其轉移至液體容器中，諸如袋、針筒或玻璃瓶，用以產生含幹細胞之溶液或幹細胞混合物。該上層，如，含幹細胞之溶液，含有小型細胞，其包含血小板以及小型成體幹細胞。小型成體幹細胞於該含幹細胞之溶液中之數目，可大於或等於1千萬個(如，大於或等於3千萬個、大於或等於5千萬個、1千萬至5億個之間、2千5百萬至3億個之間或3千萬至5億個之間)。該含幹細胞之溶液亦可含二價陽離子螯合劑(如，EDTA)和/或生長因子。 Next, in step 22, virtually all of the upper layers can be collected or transferred to a liquid container, such as a bag, syringe or glass bottle, to produce a solution containing stem cells or a mixture of stem cells. The upper layer, for example, a solution containing stem cells, contains small cells including platelets and small adult stem cells. The number of small adult stem cells in the stem cell-containing solution may be greater than or equal to 10 million (eg, greater than or equal to 30 million, greater than or equal to 50 million, and between 10 and 500 million) Between 25 and 300 million or between 30 and 500 million). The stem cell-containing solution may also contain a divalent cation chelating agent (eg, EDTA) and/or a growth factor.

此外，該含幹細胞之溶液幾乎不包括或實質上排除大型細胞(如，大型成體幹細胞以及細胞系)。例如，大型細胞可占小於該含幹細胞之溶液之總數目的5%(如，小於1%、0.5%或0.01%)。例如，該含幹細胞之溶液(如，收集到的上層)中，紅血球之數目可小於105或104個/毫升。較佳地， 每毫升該含幹細胞之溶液中紅血球之數目小於103個。每毫升該含幹細胞之溶液中白血球之數目可小於104個(如，小於103個)。較佳地，每毫升該含幹細胞之溶液中白血球之數目小於102個。 Furthermore, the stem cell-containing solution hardly includes or substantially excludes large cells (eg, large adult stem cells and cell lines). For example, large cells may comprise less than 5% (eg, less than 1%, 0.5%, or 0.01%) of the total number of solutions of the stem cells. For example, in the solution containing stem cells (e.g., the collected upper layer), the number of red blood cells may be less than 10 5 or 10 4 /ml. Preferably, the number of red blood cells per ml of the stem cell-containing solution is less than 10 3 . The number of white blood cells per ml of the stem cell-containing solution may be less than 10 4 (e.g., less than 10 3 ). Preferably, the number of white blood cells per ml of the stem cell-containing solution is less than 10 2 .

該含幹細胞之溶液之全部的細胞中，大於95%(如，99%或99.99%)可為小型細胞。該小型細胞可包括血小板、Lgr5(+)細胞、CD349(+)細胞、CD133(+)細胞、CD34(+)以及CD66e(+)細胞。血小板可占該含幹細胞之溶液中之幹細胞的75%至85%。該全部的小型細胞中，大於4%(如，大於5%或4.5%至10%之間)可為Lgr5+成體幹細胞。CD349(+)成體幹細胞可占大於該含幹細胞之溶液之全部的小型細胞中的4%(如，大於5%或4.5%至10%之間)。該小型細胞中，小於2%(如，小於1%或0.5%)可為CD133(+)細胞以及CD34(+)細胞之組合。該小型細胞中，小於6%(如，小於5%或4.5%)可為CD66e(+)細胞。 More than 95% (e.g., 99% or 99.99%) of the cells of the stem cell-containing solution may be small cells. The minicells may include platelets, Lgr5(+) cells, CD349(+) cells, CD133(+) cells, CD34(+), and CD66e(+) cells. The platelets may comprise from 75% to 85% of the stem cells in the solution containing the stem cells. More than 4% (eg, greater than 5% or between 4.5% and 10%) of all small cells may be Lgr5+ adult stem cells. CD349(+) adult stem cells may account for 4% (e.g., greater than 5% or between 4.5% and 10%) of the smaller cells than the total solution of the stem cells. In this small cell, less than 2% (eg, less than 1% or 0.5%) may be a combination of CD133(+) cells and CD34(+) cells. Less than 6% (eg, less than 5% or 4.5%) of the small cells may be CD66e(+) cells.

使用流動式細胞測量術或其它習知的技術(如，抗體為基礎的技術，諸如抗體-共軛珠)，亦可進一步分離出任一種特定的小型細胞。 Any particular small cell can be further isolated using flow cytometry or other conventional techniques (eg, antibody-based techniques, such as antibody-conjugated beads).

該收集到的上層可用作為含幹細胞之溶液(如，投予至受試者或貯存起來)，或進一步處理。例如，可進一步將其純化(如，過濾)或混合與一或多種額外的組份。可於該收集到的上層中加入體積，如大於400毫升，諸如500至900毫升之間之適合的無Ca2+細胞介質或溶液，用以製造含幹細胞之溶液。該適合的無Ca2+介質或溶液，諸如含NaCl 溶液，可進一步不含任何的二價離子，包括Mg2+。該含NaCl溶液，例如，可為食鹽水(如，具0.90% w/v之NaCl，約300mOsm/L或每公升9.0克之溶液)。 The collected upper layer can be used as a solution containing stem cells (e.g., administered to a subject or stored), or further processed. For example, it can be further purified (eg, filtered) or mixed with one or more additional components. A volume, such as greater than 400 ml, such as between 500 and 900 ml of a suitable Ca 2+ free cell medium or solution may be added to the collected upper layer to produce a solution containing stem cells. The suitable Ca 2+ free medium or solution, such as a solution containing NaCl, may be further free of any divalent ions, including Mg 2+ . The NaCl-containing solution, for example, may be a saline solution (e.g., a solution having 0.90% w/v NaCl, about 300 mOsm/L or 9.0 g per liter).

可將該含幹細胞之溶液貯存在冷凍貯存溫度下，如，等於或小於-70℃或-80℃(如，-75℃至-85℃之間)，歷時一段很長的時間(如，超過一周、一個月或一年)。當準備使用時，可快速地解凍該冷凍的含幹細胞之溶液，以及任擇地混合與之前所述之適合的無Ca2+介質或溶液(如，0.9% NaCl)。 The stem cell-containing solution can be stored at a freezing storage temperature, for example, at or below -70 ° C or -80 ° C (eg, between -75 ° C and -85 ° C) for a long period of time (eg, exceeding One week, one month or one year). When ready for use, the frozen stem cell-containing solution can be quickly thawed, and optionally mixed with a Ca 2+ free medium or solution (e.g., 0.9% NaCl) as described previously.

抑制組蛋白去乙醯酶(HDAC)之活性 Inhibition of histone deacetylase (HDAC) activity

用以上所述之程序產生的含幹細胞之溶液或組成物，可用於抑制HDAC活性。 A solution or composition containing stem cells produced by the procedures described above can be used to inhibit HDAC activity.

例如，可於受試者身上投予該含幹細胞之組成物(如，靜脈注射)，用以抑制或減少該受試者之HDAC活性。該治療可，例如，減少該受試者之HDAC活性至少20%，如，至少30%、40%、50%、60%、70%或更高。於受試者身上，可投予自體或異體成體幹細胞。 For example, the stem cell-containing composition (e.g., intravenously) can be administered to a subject to inhibit or reduce HDAC activity in the subject. The treatment can, for example, reduce the HDAC activity of the subject by at least 20%, such as at least 30%, 40%, 50%, 60%, 70% or more. Autologous or allogeneic adult stem cells can be administered to the subject.

HDACs在後生的調節中扮演關鍵的角色，其涉及許多生物學過程以及疾病。後生修飾是可逆的。然而，其等在某些疾病中可能會不平衡或異常。HDACs已顯示出可為許多疾病之有前途的藥物標靶，包括癌症、神經退化性病症、免疫病症、糖尿病以及心血管疾病。見，如，Arguelles et al.,Drug Discovery Today,21(3)：499-509(2015)：Abel and Zukin,Curr.Opin.Pharmacol.,8(1)：57-64(2008)； Irwin et al.,Drug Development Research,77：109-123(2016)；以及Huang,Journal of Cellular Physiology,209：611-616(2006)。因為含幹細胞之溶液是HDAC抑制劑，其可用於調整此等過程以及治療該等疾病。 HDACs play a key role in epigenetic regulation, involving many biological processes and diseases. Epigenetic modifications are reversible. However, they may be unbalanced or abnormal in certain diseases. HDACs have been shown to be promising drug targets for many diseases, including cancer, neurodegenerative disorders, immune disorders, diabetes, and cardiovascular disease. See, for example, Arguelles et al., Drug Discovery Today, 21(3): 499-509 (2015): Abel and Zukin, Curr. Opin. Pharmacol., 8(1): 57-64 (2008); Irwin et al., Drug Development Research, 77: 109-123 (2016); and Huang, Journal of Cellular Physiology, 209:611-616 (2006). Because the solution containing stem cells is an HDAC inhibitor, it can be used to modulate such processes and treat such diseases.

例如，譜系重編程(亦稱作轉分化)是一種過程，其中一種成熟的成體細胞可在沒有經歷中間多功能狀態或前趨細胞類型之情況下，轉形成另一種成熟的成體細胞。該過程涉及不同後生狀態之轉變。因此，後生的調節在譜系重編程方面，扮演關鍵的角色。因此於受試者身上投予在此所述之含幹細胞之溶液，可調節該受試者身上之譜系重編程。例如，含幹細胞之溶液可操控第一類型之終末分化細胞回轉或轉形成(i)該第一類型之終末分化細胞上游的成體細胞，或(ii)不同類型的終末分化細胞。 For example, lineage reprogramming (also known as transdifferentiation) is a process in which a mature adult cell can be transformed into another mature adult cell without undergoing an intermediate multifunctional state or a pre-existing cell type. This process involves a shift in different epigenetic states. Therefore, epigenetic regulation plays a key role in pedigree reprogramming. Thus, administration of a solution containing stem cells as described herein to a subject can modulate lineage reprogramming in the subject. For example, a solution containing stem cells can manipulate a terminally differentiated cell of the first type to rotate or transform into (i) adult cells upstream of the terminally differentiated cells of the first type, or (ii) different types of terminally differentiated cells.

據此，在此所述之含幹細胞之組成物可用作為HDAC抑制劑，用於治療，例如，免疫病症(如，自體免疫病症或發炎性病症)、神經退化性或神經系統病症、病毒感染、癌症或糖尿病。 Accordingly, the stem cell-containing composition described herein can be used as an HDAC inhibitor for the treatment, for example, an immune disorder (eg, an autoimmune disorder or an inflammatory disorder), a neurodegenerative or neurological disorder, a viral infection. , cancer or diabetes.

自體免疫病症或發炎性病症包括，但不限於，全身性紅斑性狼瘡、類風濕性關節炎、鳩氏症候群、僵直性脊椎炎、血小板減少性紫瘢病、橋本氏甲狀腺炎、格雷氏病、Grover氏病、多發性硬化症、發炎性皮膚病以及發炎性腸病。 Autoimmune or inflammatory conditions include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, sputum syndrome, ankylosing spondylitis, thrombocytopenic purpura, Hashimoto's thyroiditis, Gracies disease , Grover's disease, multiple sclerosis, inflammatory skin disease, and inflammatory bowel disease.

神經退化性疾病包括，例如，精神***症、阿茲海默症、帕金森氏症、杭丁頓氏舞蹈症、多發性硬化症以 及肌肉萎縮性脊髓側索硬化症(ALS)。 Neurodegenerative diseases include, for example, schizophrenia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis. And amyotrophic lateral sclerosis (ALS).

可治療之癌症可為，如，淋巴瘤(諸如皮膚T細胞淋巴瘤、周邊T細胞淋巴瘤、何杰金氏淋巴瘤、非何杰金氏淋巴瘤或彌漫性大型B細胞淋巴瘤)、肺癌(諸如非小細胞肺癌或支氣管肺癌)、乳癌、卵巢癌、***癌、結直腸癌、多發性骨髓瘤、肝癌(諸如肝細胞癌或肝內膽管癌)、腎癌、胃癌、皮膚癌(諸如黑色素癌)、甲狀腺癌、食道癌、腦癌、胰臟癌、口腔癌、咽喉癌(諸如咽癌或喉癌)、子宮頸癌、骨癌、膀胱癌、白血病或原位癌。 The treatable cancer can be, for example, a lymphoma (such as cutaneous T-cell lymphoma, peripheral T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or diffuse large B-cell lymphoma), lung cancer. (such as non-small cell lung cancer or bronchial lung cancer), breast cancer, ovarian cancer, prostate cancer, colorectal cancer, multiple myeloma, liver cancer (such as hepatocellular carcinoma or intrahepatic cholangiocarcinoma), kidney cancer, stomach cancer, skin cancer ( Such as melanoma), thyroid cancer, esophageal cancer, brain cancer, pancreatic cancer, oral cancer, throat cancer (such as pharyngeal or laryngeal cancer), cervical cancer, bone cancer, bladder cancer, leukemia or carcinoma in situ.

病毒感染(或病毒感染引起之疾病)可為HIV感染、後天免疫缺乏症候群(AIDS)、疱疹病毒感染(如，HCMV、HSV或EBV感染)或HBV感染。 The viral infection (or the disease caused by the viral infection) may be HIV infection, acquired immunodeficiency syndrome (AIDS), herpes virus infection (eg, HCMV, HSV or EBV infection) or HBV infection.

含幹細胞之組成物可用於治療第I型糖尿病(或稱作糖尿病I型)或第II型糖尿病(或稱作糖尿病II型)。 The stem cell-containing composition can be used to treat Type I diabetes (or called Type I Diabetes) or Type II Diabetes (or Type II Diabetes).

利用針對特殊疾病或病症之標準診斷技術，可辨識出可接受以上所述之疾病或病症治療之受試者。“治療”意指於患有疾病或病症或具有發生該疾病或病症風險之受試人身上，投予組成物、劑或物質(如，含幹細胞之溶液)，目的為治癒、減輕、解除、改正、延遲發生、預防或改善該疾病或病症、該疾病或病症之症狀、該疾病或病症繼發之疾病狀態或易患該疾病或病症之傾向。“有效量”意指在治療的受試者身上能夠產生所欲的(醫療)結果之組成物、劑或物質之數量。該治療方法可單獨進行，或結合其它藥物或治療法一起進行。 Subjects that are eligible for treatment of the diseases or conditions described above can be identified using standard diagnostic techniques for a particular disease or condition. "Treatment" means administration of a composition, agent or substance (eg, a solution containing stem cells) to a subject having a disease or condition or at risk of developing the disease or condition for the purpose of healing, mitigating, relieving, Correcting, delaying, preventing or ameliorating the disease or condition, the condition of the disease or condition, the disease state secondary to the disease or condition, or the predisposition to the disease or condition. By "effective amount" is meant the amount of a composition, agent or substance that is capable of producing a desired (medical) result in a subject being treated. The method of treatment can be performed alone or in combination with other drugs or therapies.

在投予該含幹細胞之組成物至受試者之前或之後(或二者)，評估從該受試者身上獲得之樣本(如，周邊血液樣本或受欲治療之疾病影響之組織樣本)中之HDAC活性。例如，可分析從樣本製得之核抽取物或HDAC蛋白中之HDAC活性。投予後HDAC活性減少(如，至少20%、30%、40%、50%、60%、70%或更高)指出，該含幹細胞之溶液具抑制受試者身上之HDAC活性之效力。HDAC活性減少亦可意指，該治療能有效的治療以上所述之病症中之一者。在投予該含幹細胞之組成物之後或一個投藥療程期間，受試者身上之HDAC位準亦可用於做治療決定，如繼續或中止治療，或決定治療頻率。用於分析HDAC活性之技術以及試劑是業界已知的。HDAC活性分析套組亦可從商業獲得。 Evaluating a sample obtained from the subject (eg, a peripheral blood sample or a tissue sample affected by the disease to be treated) before or after administration of the composition comprising the stem cell to the subject (or both) HDAC activity. For example, the HDAC activity in a nuclear extract or HDAC protein prepared from a sample can be analyzed. A decrease in HDAC activity (e.g., at least 20%, 30%, 40%, 50%, 60%, 70% or higher) after administration indicates that the stem cell-containing solution has an effect of inhibiting HDAC activity in a subject. A decrease in HDAC activity may also mean that the treatment is effective in treating one of the conditions described above. The HDAC level in the subject may also be used to make a treatment decision, such as continuing or discontinuing treatment, or determining the frequency of treatment, after administration of the composition comprising the stem cells or during a course of administration. Techniques and reagents for analyzing HDAC activity are known in the art. HDAC activity assay kits are also commercially available.

以下之具體範例僅作為例示說明之解釋，無論如何不能以任何方式用來限制揭示內容之剩餘部分。一般認為，根據在此之說明書，熟悉此技藝之人士可在沒有進一步的詳細敘述之情況下，使用本揭示內容至其最大程度。在此所引述之所有的公開文獻之全部，均在此併入本案以為參考。 The following specific examples are merely illustrative, and are not intended to limit the remainder of the disclosure in any way. It is to be understood that in the light of the description, the disclosure of the invention may be All of the publications cited herein are incorporated herein by reference.

範例 example 範例1：幹細胞動員劑之作用 Example 1: The role of stem cell mobilizer

使用以上所述之程序，使用從六位受試人身上取得之周邊血液樣本，製備含幹細胞之溶液。亦可見圖1以及圖3。 A solution containing stem cells was prepared using the peripheral blood samples taken from six subjects using the procedure described above. See also Figure 1 and Figure 3.

受試人C、L、M、W以及Y各口服20粒圖2中所 述之褐藻補充物。也就是說，受試者C、L、M、W以及Y各口服至少2克的褐藻醣膠。受試人P作為對照組，沒有服用褐藻補充物。採取受試者服用該等丸劑之前以及之後之樣本。 Subjects C, L, M, W, and Y each took 20 tablets orally. Said brown algae supplement. That is, subjects C, L, M, W, and Y each took at least 2 grams of fucoidan. Subject P was used as a control group and no brown algae supplement was taken. Subjects were taken before and after taking the pills.

如圖4所示，發現在四個受試者中，服用褐藻補充物之後1.5個小時之SB細胞(即，CD349(+)SB細胞以及Lgr5(+)SB細胞)之數目顯著的增加。在對照受試者中沒有發現增加。數據顯示出，服用褐藻醣膠，如褐藻補充物，可動員SB細胞(如，CD349(+)SB細胞以及Lgr5(+)SB細胞)進入周邊血液(或血流)並使周邊血液富含SB細胞。在圖4中，0小時代表受試人C、L、M、W以及Y“服用褐藻醣膠之前”，且代表受試人P“對照組測試開始之時間點”；1.5小時代表受試人C、L、M、W以及Y“服用褐藻醣膠後1.5個小時之時間點”，且亦代表受試者P“對照組測試開始後1.5個小時之時間點”；24小時代表“受試人C、L、M、W以及Y“服用褐藻醣膠後24個小時之時間點”，且亦代表受試者P“對照組測試開始後24個小時之時間點”。 As shown in Figure 4, a significant increase in the number of SB cells (i.e., CD349(+) SB cells and Lgr5(+) SB cells) 1.5 hours after taking the brown algae supplement was found in the four subjects. No increase was found in the control subjects. The data show that taking fucoidan, such as brown algae supplements, mobilizes SB cells (eg, CD349(+)SB cells and Lgr5(+)SB cells) into peripheral blood (or bloodstream) and enriches peripheral blood with SB cell. In Fig. 4, 0 hours represents the subjects C, L, M, W, and Y "before taking fucoidan", and represents the subject P "time point of the start of the control test"; 1.5 hours represents the subject C, L, M, W, and Y "1.5 hours after taking fucoidan", and also represents the subject P "1.5 hours after the start of the control group test"; 24 hours means "tested People C, L, M, W, and Y "24 hours after taking fucoidan", and also represent the subject "P 24 hours after the start of the control group test."

亦評估GCSF在SB細胞之動員上的影響。連續5天，在受試人身上注射單劑5微克/kg/天之GCSF。在第一次注射之前，從受試者身上取得第一周邊血液樣本，而在最後注射之後3.5個小時，從受試者身上取得第二周邊血液樣本。相較於第一樣本，在第二樣本中發現Lgr5+ SB細胞之數目顯著的增加。 The effect of GCSF on mobilization of SB cells was also assessed. A single dose of 5 μg/kg/day of GCSF was injected into the subject for 5 consecutive days. A first peripheral blood sample is taken from the subject prior to the first injection, and a second peripheral blood sample is taken from the subject 3.5 hours after the last injection. A significant increase in the number of Lgr5+ SB cells was found in the second sample compared to the first sample.

二個樣本之流動式細胞測量術數據示於圖5A以 及5B中。在二個圖式中，區域Q5-LR中之黑點代表Lgr5(+)細胞。在第一周邊血液樣本方面，如圖5A所示，區域Q5-LR中Lgr5(+)細胞之數目為區域Q5-UL、Q5-UR、Q5-LL以及Q5-LR中細胞總數目之1.6%。在第二周邊血液樣本方面，如圖5B所示，區域Q5-LR中Lgr5(+)細胞之數目為區域Q5-UL、Q5-UR、Q5-LL以及Q5-LR中細胞總數目之8%。因此，GCSF可動員SB細胞(特別是Lgr5(+)細胞)進入周邊血液(或血流)，以及使周邊血液富含SB細胞。 The flow cytometry data for the two samples is shown in Figure 5A. And 5B. In both figures, the black dots in the region Q5-LR represent Lgr5(+) cells. In terms of the first peripheral blood sample, as shown in Fig. 5A, the number of Lgr5(+) cells in the region Q5-LR is 1.6% of the total number of cells in the regions Q5-UL, Q5-UR, Q5-LL, and Q5-LR. . In terms of the second peripheral blood sample, as shown in FIG. 5B, the number of Lgr5(+) cells in the region Q5-LR is 8% of the total number of cells in the regions Q5-UL, Q5-UR, Q5-LL, and Q5-LR. . Therefore, GCSF can mobilize SB cells (especially Lgr5(+) cells) into peripheral blood (or bloodstream) and enrich peripheral blood with SB cells.

以上所述的數據顯示，服用褐藻補充物或接受一個GCSF注射療程，可動員SB細胞進入受試者的周邊血液。 The data described above shows that taking a brown algae supplement or receiving a GCSF injection course can mobilize SB cells into the peripheral blood of the subject.

範例2：含幹細胞之溶液中之細胞 Example 2: Cells in a solution containing stem cells

依照以上所述以及圖1與圖3中例示說明之方法，使用從受試者身上取得之周邊血液樣本，製備含幹細胞之溶液。分析該含幹細胞之溶液中之細胞含量。 A solution containing stem cells is prepared using the peripheral blood sample taken from the subject in accordance with the methods described above and illustrated in Figures 1 and 3. The amount of cells in the solution containing the stem cells was analyzed.

以圖6A與6B中所示之數據為基礎，計算每毫升該含幹細胞之溶液中紅血球之數目。圖6A是用流動式細胞測量術分析10微升(μl)之該含幹細胞之溶液所獲得的。紅血球細胞(即，CD235a(+)細胞)顯示在圖6A之區域R3中；圖6A之區域R3中全部的細胞數目為25000個。圖6B顯示圖6A之區域R3中全部的細胞中CD235a之螢光強度的分佈。在圖6B中，螢光直方圖被垂直線2a(即，參考值)分成具低螢光強度之區域V3-L以及具高螢光強度之區域V3-R。圖6B之區域V3-R代表CD235a染色為陽性之細胞，即，紅血球細胞；圖6B之區域V3-L代表CD235a染色為陰性之細胞。圖6B之結果 指出，圖6A之區域R3中之紅血球細胞之數目對圖6A之區域R3中全部的細胞數目之百分比為0.1%。將區域R3中全部的細胞數目，即，25000個，乘以圖6A之區域R3中之紅血球細胞之數目對圖6A之區域R3中全部的細胞數目之百分比，即0.1%，可得到在10微升之該含幹細胞之溶液中之紅血球細胞之數目為25個。從10微升之該含幹細胞之溶液中之紅血球細胞之數目，計算每毫升該含幹細胞之溶液中之紅血球細胞之數目，計算值等於2500。 Based on the data shown in Figures 6A and 6B, the number of red blood cells per ml of the stem cell-containing solution was calculated. Fig. 6A is obtained by analyzing 10 μl of the stem cell-containing solution by flow cytometry. Red blood cells (i.e., CD235a (+) cells) are shown in region R3 of Figure 6A; the total number of cells in region R3 of Figure 6A is 25,000. Fig. 6B shows the distribution of the fluorescence intensity of CD235a in all cells in the region R3 of Fig. 6A. In FIG. 6B, the fluorescence histogram is divided into a region V3-L having a low fluorescence intensity and a region V3-R having a high fluorescence intensity by a vertical line 2a (ie, a reference value). In the region of Fig. 6B, V3-R represents cells positive for CD235a staining, i.e., red blood cells; and region V3-L of Fig. 6B represents cells negative for CD235a staining. Figure 6B results It is noted that the percentage of the number of red blood cells in the region R3 of Fig. 6A to the total number of cells in the region R3 of Fig. 6A is 0.1%. The number of all cells in the region R3, that is, 25,000, multiplied by the number of red blood cells in the region R3 of FIG. 6A to the total number of cells in the region R3 of FIG. 6A, that is, 0.1%, can be obtained at 10 micro. The number of red blood cells in the solution containing the stem cells was 25. The number of red blood cells per ml of the stem cell-containing solution was calculated from 10 microliters of the number of red blood cells in the stem cell-containing solution, and the calculated value was equal to 2,500.

以圖6C中流動式細胞測量術數據為基礎，計算每毫升該含幹細胞之溶液中白血球之數目。圖6C中之流動式細胞測量術數據，是分析50微升(μl)該含幹細胞之溶液所獲得的。在圖6C中，區域R1是白血球細胞(WBC)圈選。以圖6C中流動式細胞測量術數據為基礎，計算每50微升該含幹細胞之溶液中之白血球細胞之數目，計算值等於30個。因此，從50微升該含幹細胞之溶液中之白血球細胞之數目，計算每毫升該含幹細胞之溶液中之白血球細胞之數目，計算值等於600個。 Based on the flow cytometry data in Figure 6C, the number of white blood cells per ml of the stem cell-containing solution was calculated. The flow cytometry data in Fig. 6C was obtained by analyzing 50 microliters (μl) of the stem cell-containing solution. In Figure 6C, region R1 is a white blood cell (WBC) circle. Based on the flow cytometry data in Fig. 6C, the number of white blood cells per 50 microliters of the stem cell-containing solution was calculated, and the calculated value was equal to 30. Therefore, the number of white blood cells per ml of the stem cell-containing solution was calculated from 50 microliters of the number of white blood cells in the stem cell-containing solution, and the calculated value was equal to 600.

圖7A顯示前向散射(FSC)對側向散射(SSC)之流動式細胞測量術點圖，其是以流動式細胞測量術分析5.6微升(μl)之該含幹細胞之溶液而得。在圖7A中，區域R5代表尺寸小於6微米及大於2微米之細胞。也就是說，區域R5中全部的細胞之尺寸均大於2微米以及小於6微米。圖7B顯示圖7A之區域R5中全部的細胞中CD61螢光強度之分佈。在圖7B中，螢光直方圖被一垂直線(即，參考值)分成具有低螢 光強度之區域V1-L以及具有高螢光強度之區域V1-R。圖7B之區域V1-R代表CD61染色為陽性之細胞，即，血小板；圖7B之區域V1-L代表CD61染色為陰性之細胞。7B之結果指出，區域R5中血小板之數目，為區域5中全部的細胞之80.5%。 Figure 7A shows a flow cytometry dot plot of forward scatter (FSC) versus side scatter (SSC) obtained by flow cytometry analysis of 5.6 microliters (μl) of the stem cell-containing solution. In Figure 7A, region R5 represents cells that are less than 6 microns in size and greater than 2 microns in size. That is, the size of all cells in the region R5 is greater than 2 microns and less than 6 microns. Fig. 7B shows the distribution of CD61 fluorescence intensity in all cells in the region R5 of Fig. 7A. In FIG. 7B, the fluorescence histogram is divided into a low-fluorescence by a vertical line (ie, a reference value). The region of light intensity V1-L and the region V1-R having high fluorescence intensity. Region V1-R of Figure 7B represents cells stained positive for CD61, i.e., platelets; and region V1-L of Figure 7B represents cells negative for CD61 staining. The result of 7B indicates that the number of platelets in the region R5 is 80.5% of all the cells in the region 5.

圖7C顯示圖7A之區域R5中全部的細胞中之CD133螢光強度之分佈。圖7C中，螢光直方圖被一垂直線(即，參考值)分成具有低螢光強度之區域V3-L以及具有高螢光強度之區域V3-R。圖7C之區域V3-R代表CD133染色為陽性之細胞，即VSELs；圖7C之區域V3-L代表CD133染色為陰之細胞。圖7C之結果指出，區域R5中CD133(+)細胞之數目之百分比，為區域R5中全部的細胞之數目的0.3%。圖7D顯示圖7A之區域R5中全部的細胞中CD34螢光強度之分佈。在圖7D中，螢光直方圖被一垂直線(即，參考值)分成具有低螢光強度之區域V1-L以及具有高螢光強度之區域V1-R。圖7D之區域V1-R代表CD34染色為陽性之細胞，即VSELs；圖7D之區域V1-L代表CD34染色為陰性之細胞。圖7D之結果指出，區域R5中CD34(+)細胞之數目為該區域中全部的細胞之數目的0.4%。 Fig. 7C shows the distribution of CD133 fluorescence intensity in all cells in the region R5 of Fig. 7A. In Fig. 7C, the fluorescence histogram is divided into a region V3-L having a low fluorescence intensity and a region V3-R having a high fluorescence intensity by a vertical line (i.e., a reference value). In the region of Figure 7C, V3-R represents cells positive for CD133 staining, i.e., VSELs; and region V3-L of Figure 7C represents cells stained with CD133 as negative cells. The results of Fig. 7C indicate that the percentage of the number of CD133(+) cells in the region R5 is 0.3% of the total number of cells in the region R5. Figure 7D shows the distribution of CD34 fluorescence intensity in all cells in region R5 of Figure 7A. In Fig. 7D, the fluorescence histogram is divided into a region V1-L having a low fluorescence intensity and a region V1-R having a high fluorescence intensity by a vertical line (i.e., a reference value). Region V1-R of Figure 7D represents cells positive for CD34 staining, i.e., VSELs; and region V1-L of Figure 7D represents cells negative for CD34 staining. The results of Figure 7D indicate that the number of CD34(+) cells in region R5 is 0.4% of the total number of cells in the region.

圖7E顯示圖7A之區域R5中全部的細胞中CD66e螢光強度之分佈。在圖7E中，螢光直方圖被一垂直線(即，參考值)分成具有低螢光強度之區域V2-L以及具有高螢光強度之區域V2-R。圖7E之區域V2-R代表CD66e染色為陽性之細胞，即BLSCs；圖7E之區域V2-L代表CD66e染色為陰 性之細胞。圖7E之結果指出，區域R5中CD66e(+)細胞之數目為該區域中全部的細胞之數目的4%。 Figure 7E shows the distribution of CD66e fluorescence intensity in all cells in region R5 of Figure 7A. In FIG. 7E, the fluorescence histogram is divided into a region V2-L having a low fluorescence intensity and a region V2-R having a high fluorescence intensity by a vertical line (ie, a reference value). In the region of Fig. 7E, V2-R represents a cell positive for CD66e staining, that is, BLSCs; and in the region of Fig. 7E, V2-L represents a staining of CD66e for yin. The cell of sex. The results of Figure 7E indicate that the number of CD66e(+) cells in region R5 is 4% of the total number of cells in the region.

圖7F顯示圖7A之區域R5中全部的細胞中CD349螢光強度之分佈。在圖7F中，螢光直方圖被一垂直線(即，參考值)分成具有低螢光強度之區域V3-L以及具有高螢光強度之區域V3-R。圖7F之區域V3-R代表CD349染色為陽性之細胞，即SB-1細胞；圖7F之區域V3-L代表CD349染色為陰性之細胞。圖7F之結果指出，區域R5中CD349(+)細胞之數目為該區域中全部的細胞之數目的5.6%。 Figure 7F shows the distribution of CD349 fluorescence intensities in all cells in region R5 of Figure 7A. In FIG. 7F, the fluorescence histogram is divided into a region V3-L having a low fluorescence intensity and a region V3-R having a high fluorescence intensity by a vertical line (ie, a reference value). Region V3-R of Figure 7F represents cells stained positive for CD349, i.e., SB-1 cells; and region V3-L of Figure 7F represents cells negative for CD349 staining. The results of Figure 7F indicate that the number of CD349(+) cells in region R5 is 5.6% of the total number of cells in the region.

圖7G顯示圖7A之區域R5中全部的細胞中Lgr5螢光強度之分佈。在圖7G中，螢光直方圖被一垂直線(即，參考值)分成具有低螢光強度之區域V2-L以及具有高螢光強度之區域V2-R。圖7G之區域V2-R代表Lgr5染色為陽性之細胞，即SB-2細胞；圖7G之區域V2-L代表Lgr5染色為陰性之細胞。圖7G之結果指出，區域R5中Lgr5(+)細胞之數目為該區域中全部的細胞之數目的5.4%。 Fig. 7G shows the distribution of Lgr5 fluorescence intensity in all cells in the region R5 of Fig. 7A. In Fig. 7G, the fluorescence histogram is divided into a region V2-L having a low fluorescence intensity and a region V2-R having a high fluorescence intensity by a vertical line (i.e., a reference value). The region V2-R of Fig. 7G represents a cell positive for Lgr5 staining, i.e., SB-2 cells; and the region V2-L of Fig. 7G represents a cell negative for Lgr5 staining. The results of Figure 7G indicate that the number of Lgr5(+) cells in region R5 is 5.4% of the total number of cells in the region.

範例3：用於抑制HDAC活性之含幹細胞之溶液 Example 3: Stem-containing solution for inhibiting HDAC activity

用十個受試人身上採取的周邊血液樣本製備含幹細胞之溶液。請受試者各吃20粒圖2中所述之褐藻補充物。在吃完後1.5個小時，從各受試者身上抽取150毫升之周邊血液樣本。各血液樣本取20毫升(之後稱作“20毫升血液樣本”)進行流動式細胞測量法之分析，以獲得樣本中特定成體幹細胞之數目。該特定成體幹細胞是尺寸大於2微米以及小於6微米之小型成體幹細胞，包括Lgr5(+)成體幹細胞、 CD349(+)成體幹細胞、CD66e(+)成體幹細胞、CD133(+)成體幹細胞以及CD34(+)成體幹細胞。依照以上所述之程序處理各剩下的周邊血液樣本，以獲得約65毫升之含幹細胞之溶液。各含幹細胞之溶液含有該小型成體幹細胞。以對應的20毫升血液樣本中成體幹細胞之數目為基礎，計算或評估各含幹細胞之溶液中成體幹細胞之數目。 A solution containing stem cells was prepared using peripheral blood samples taken from ten subjects. Subjects were asked to ate each of the 20 brown algae supplements described in Figure 2. 150 ml of peripheral blood samples were taken from each subject 1.5 hours after eating. 20 ml of each blood sample (hereinafter referred to as "20 ml blood sample") was subjected to flow cytometry analysis to obtain the number of specific adult stem cells in the sample. The specific adult stem cells are small adult stem cells having a size greater than 2 microns and less than 6 microns, including Lgr5(+) adult stem cells, CD349(+) adult stem cells, CD66e (+) adult stem cells, CD133 (+) adult stem cells, and CD34 (+) adult stem cells. Each of the remaining peripheral blood samples was processed according to the procedure described above to obtain approximately 65 ml of a solution containing stem cells. Each of the stem cell-containing solutions contains the small adult stem cells. The number of adult stem cells in each stem cell-containing solution is calculated or evaluated based on the number of adult stem cells in the corresponding 20 ml blood sample.

使各含幹細胞之溶液混合與500毫升之食鹽水。在該十位受試人身上，靜脈注射其等各自的含幹細胞之溶液。表1顯示各該含幹細胞之溶液中成體幹細胞之數目。 Each solution containing stem cells was mixed with 500 ml of saline. In each of the ten subjects, their respective solutions containing stem cells were intravenously administered. Table 1 shows the number of adult stem cells in each of the stem cell-containing solutions.

分析從各該十位受試人之周邊血液樣本獲得之純化的有核細胞中之HDAC活性。從針對各受試者進行之二個相同的實驗，計算出平均HDAC活性。如以下表2所示，發現各受試者在靜脈注射該含幹細胞之溶液後48個小時，HDAC活性顯著的減少(至少22%)。 The HDAC activity in purified nucleated cells obtained from peripheral blood samples of each of the ten subjects was analyzed. Mean HDAC activity was calculated from two identical experiments performed on each subject. As shown in Table 2 below, each subject was found to have a significant reduction (at least 22%) in HDAC activity 48 hours after intravenous injection of the stem cell-containing solution.

圖8是表2中所示之平均HDAC活性的散布圖(帶線性回歸線)。如圖8所示，靜脈注射該含幹細胞之溶液之 前的平均HDAC活性，與注射後48個小時之對應的HDAC活性減少之百分比之間之判定係數(即，R2)大如0.7346，其表明二個變數之間具很大的相關性。該圖顯示出，注射前HDAC活性越高，注射後48個小時之HDAC活性的減少百分比越高。 Figure 8 is a scatter plot (with linear regression line) of the average HDAC activity shown in Table 2. As shown in Figure 8, the coefficient of determination (i.e., R 2 ) between the mean HDAC activity prior to intravenous injection of the solution containing the stem cells and the percentage reduction in HDAC activity corresponding to 48 hours after injection is as large as 0.7346, which indicates There is a great correlation between the two variables. The figure shows that the higher the HDAC activity before injection, the higher the percentage reduction in HDAC activity 48 hours after injection.

以上所述之數據指出，該含幹細胞之溶液可顯著地減少受試人中之HDAC活性。 The above data indicates that the stem cell-containing solution can significantly reduce HDAC activity in the subject.

範例4：用於抑制病人中HDAC活性之含幹細胞之溶液 Example 4: Stem-containing solution for inhibiting HDAC activity in a patient

對各個具有神經退化性病症、自體免疫病症或糖尿病之病人，以其等各自之使用以上所述之程序製備的含幹細胞之溶液治療。 For each patient having a neurodegenerative disorder, autoimmune disorder, or diabetes, they are treated with a solution containing stem cells prepared using the procedures described above.

針對各個病人，取得在治療之前以及之後之HDAC活性以及臨床評估。結果示於以下表3、4以及5中。結果顯示出，在投予含幹細胞之溶液後，HDAC活性有減少， 且病人的臨床評估有對應的改善。 HDAC activity and clinical evaluation before and after treatment were obtained for each patient. The results are shown in Tables 3, 4 and 5 below. The results showed that HDAC activity was reduced after administration of the solution containing stem cells. And the patient's clinical evaluation has a corresponding improvement.

其它具體例 Other specific examples

所有在此說明書中所揭示之特徵，可以任意組合方式進行結合。在此該明書中所揭示之各個特徵，可用提供相同、相等或相似目的之替代特徵取代。因此，除非有特別的表示，否則所揭示之各個特徵僅是一系列相等或相似特徵之例子。 All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features that provide the same, equivalent or similar purpose. Therefore, unless expressly stated otherwise, the various features disclosed are merely illustrative of a series of equivalent or similar features.

從以上之說明，熟悉此技藝之人士可輕易地弄清所述的具體例之主要特徵，以及在不逸離其思想範圍之情 況下，對具體例做出各種的改變以及修改，以便使其適合各種用途以及條件。因此，其它具體例亦落在本申請專利範圍內。 From the above description, those skilled in the art can easily ascertain the main features of the specific examples described, as well as without departing from the scope of their thoughts. In this case, various changes and modifications are made to the specific examples to make them suitable for various uses and conditions. Therefore, other specific examples are also within the scope of the present patent application.

Claims (22)

一種抑制組蛋白去乙醯酶活性之方法，其包含對一有需要的受試者投予一組成物，該組成物含有一有效量之尺寸大於2微米以及小於6微米之小型成體幹細胞，其中該小型成體幹細胞包括CD349(+)成體幹細胞以及Lgr5(+)成體幹細胞。 A method of inhibiting histone deacetylase activity, comprising administering to a subject in need thereof a composition comprising an effective amount of small adult stem cells having a size greater than 2 microns and less than 6 microns, Wherein the mini adult stem cells include CD349(+) adult stem cells and Lgr5(+) adult stem cells. 如請求項1之方法，其進一步包含在該投予步驟之前，測定該受試者中之組蛋白去乙醯酶活性。 The method of claim 1, further comprising determining the histone deacetylase activity in the subject prior to the administering step. 如請求項2之方法，其進一步包含在該投予步驟之後，測定該受試者中之組蛋白去乙醯酶活性。 The method of claim 2, further comprising determining the histone deacetylase activity in the subject after the administering step. 如請求項1之方法，其進一步包含在該投予步驟之前以及之後，測定該受試者中之組蛋白去乙醯酶活性。 The method of claim 1, further comprising determining histone deacetylase activity in the subject before and after the administering step. 如請求項4之方法，其中該測定步驟之進行，是藉由測量從該受試者獲得之周邊血液樣本中有核細胞中之組蛋白去乙醯酶活性。 The method of claim 4, wherein the measuring step is performed by measuring histone deacetylase activity in nucleated cells in a peripheral blood sample obtained from the subject. 如請求項1-5中任一項之方法，其中該組成物是經靜脈投予之注射溶液。 The method of any one of claims 1 to 5, wherein the composition is an intravenously administered injection solution. 如請求項6之方法，其中該組成物含有1千萬至5億個該小型成體幹細胞。 The method of claim 6, wherein the composition contains from 10 to 500 million of the small adult stem cells. 如請求項7之方法，其中該組成物含有二價陽離子螯合劑。 The method of claim 7, wherein the composition comprises a divalent cation chelating agent. 如請求項8之方法，其中該注射溶液是以包括下列之程序製得： 提供一含有血液樣本以及二價陽離子螯合劑之混合物；將該混合物貯存在2℃至12℃間之溫度下3至72個小時，藉此該混合物分離成上層以及下層，其中該上層含有該小型成體幹細胞；以及收集該上層，藉此製得該注射溶液。 The method of claim 8, wherein the injection solution is prepared by the following procedure: Providing a mixture comprising a blood sample and a divalent cation chelating agent; storing the mixture at a temperature between 2 ° C and 12 ° C for 3 to 72 hours, whereby the mixture is separated into an upper layer and a lower layer, wherein the upper layer contains the small The adult stem cells; and the upper layer are collected, thereby preparing the injection solution. 如請求項9之方法，其中該血液樣本是從該受試者或一供體獲得。 The method of claim 9, wherein the blood sample is obtained from the subject or a donor. 如請求項10之方法，其中在獲得該血液樣本之前，在該受試者或供體上進行增加幹細胞數目之動作。 The method of claim 10, wherein the act of increasing the number of stem cells is performed on the subject or donor prior to obtaining the blood sample. 如請求項11之方法，其中該動作是投予褐藻醣膠(fucoidan)或顆粒球群落刺激因子(granulocyte-colony stimulating factor)。 The method of claim 11, wherein the action is administration of fucoidan or granulocyte-colony stimulating factor. 如請求項9之方法，其中於該血液樣本中加入每毫升之該血液樣本1.5至2.0mg之二價陽離子螯合劑，以獲得該混合物。 The method of claim 9, wherein 1.5 to 2.0 mg of the divalent cation chelating agent per ml of the blood sample is added to the blood sample to obtain the mixture. 如請求項13之方法，其中該二價陽離子螯合劑是乙二胺四乙酸(EDTA)。 The method of claim 13, wherein the divalent cation chelating agent is ethylenediaminetetraacetic acid (EDTA). 如請求項9之方法，其中該用於製備該注射溶液之程序進一步包括，在收集該上層之後，於該收集到的上層中加入一藥學上可接受之賦形劑。 The method of claim 9, wherein the procedure for preparing the injectable solution further comprises, after collecting the upper layer, adding a pharmaceutically acceptable excipient to the collected upper layer. 如請求項15之方法，其中該藥學上可接受之賦形劑是食鹽水溶液。 The method of claim 15, wherein the pharmaceutically acceptable excipient is an aqueous salt solution. 如請求項6之方法，其中該小型成體幹細胞包括CD133(+) 細胞、CD34(+)細胞以及CD66e(+)細胞。 The method of claim 6, wherein the mini adult stem cells comprise CD133(+) Cells, CD34(+) cells, and CD66e(+) cells. 如請求項6之方法，其中該受試者具有自體免疫病症、糖尿病、癌症、神經退化性病症或病毒感染。 The method of claim 6, wherein the subject has an autoimmune disorder, diabetes, cancer, a neurodegenerative disorder, or a viral infection. 如請求項18之方法，其中該受試者具有失智症、帕金森氏症、關節炎、僵直性脊椎炎或糖尿病。 The method of claim 18, wherein the subject has dementia, Parkinson's disease, arthritis, ankylosing spondylitis or diabetes. 一種用於減少一受試者中之組蛋白乙醯酶活性之注射組成物，其中該組成物含有一有效量之尺寸大於2微米以及小於6微米之小型成體幹細胞，該小型成體幹細胞包括CD349(+)成體細胞以及Lgr5(+)成體幹細胞。 An injection composition for reducing histone acetylase activity in a subject, wherein the composition comprises an effective amount of small adult stem cells having a size greater than 2 microns and less than 6 microns, the small adult stem cells comprising CD349(+) adult cells and Lgr5(+) adult stem cells. 如請求項20之注射組成物，其中該受試者具有自體免疫病症、糖尿病、癌症、神經退化性病症或病毒感染。 The injectable composition of claim 20, wherein the subject has an autoimmune disorder, diabetes, cancer, a neurodegenerative disorder, or a viral infection. 如請求項21之注射組成物，其中該受試者具有失智症、帕金森氏症、關節炎、僵直性脊椎炎或糖尿病。 The injection composition of claim 21, wherein the subject has dementia, Parkinson's disease, arthritis, ankylosing spondylitis or diabetes.
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