JP6644395B2 - Methods for improving the ability of normal human hepatocytes to remove ammonia - Google Patents
Methods for improving the ability of normal human hepatocytes to remove ammonia Download PDFInfo
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Description
本発明は、培地で培養した移植用のヒト肝細胞に関する。 The present invention relates to human hepatocytes for transplantation cultured in a medium.
肝臓移植は肝不全に対して極めて有効な治療法であるが、ドナー不足は深刻で現状では解決の糸口は見いだせない。そこで肝臓移植に代わり肝細胞移植が考慮されるが、ヒト肝細胞移植の臨床研究プロトコール作成はまだ十分に解明されておらず、骨髄細胞移植のプロトコールをそのままあてはめることはできない。 Liver transplantation is an extremely effective treatment for liver failure, but the shortage of donors is severe and no clue has been found at present. Therefore, hepatocyte transplantation is considered instead of liver transplantation. However, the preparation of a clinical research protocol for human hepatocyte transplantation has not yet been sufficiently elucidated, and the protocol for bone marrow cell transplantation cannot be directly applied.
肝臓は生体内では高い再生能力を持っていることが知られており、生体部分肝移植はこの高い再生能力を応用した治療といえるが、その一方で分離培養した肝細胞は例え正常肝から分離された肝細胞であってもその増殖能がin vivoと比較して劣っている。また更には分離後において、細胞のアンモニア除去能が低下することもある。 It is known that the liver has a high regenerative ability in vivo, and it can be said that partial liver transplantation is a treatment that applies this high regenerative capacity, but on the other hand, hepatocytes separated and cultured are separated from normal liver Even proliferated hepatocytes have inferior proliferative ability as compared to in vivo. Furthermore, after the separation, the ability of the cells to remove ammonia may decrease.
特許文献1には、免疫不全マウスに単離したヒト肝細胞を注入するステップと、該肝細胞を増大させるステップと、該ヒト肝細胞を採取するステップと、を含む移植用のヒト肝細胞を増殖させる方法が記載されている。 Patent Document 1 discloses that a human hepatocyte for transplantation comprises a step of injecting an isolated human hepatocyte into an immunodeficient mouse, a step of expanding the hepatocyte, and a step of collecting the human hepatocyte. Methods for growing are described.
また非特許文献1には、肝幹前駆細胞をラミニン含有培地にて培養するヒトiPS細胞からの肝細胞への分化誘導・培養方法が記載されている。 Non-patent Document 1 describes a method for inducing and culturing differentiation of human iPS cells into hepatocytes by culturing hepatic stem progenitor cells in a laminin-containing medium.
しかし、上述の技術は、移植用のヒト肝細胞数を十分に増大させるものではなく、また培養後における肝細胞の機能維持面においても十分であるとはいえない。 However, the above-mentioned technique does not sufficiently increase the number of human hepatocytes for transplantation, and cannot be said to be sufficient in terms of maintaining the function of hepatocytes after culture.
本発明はかかる問題点に鑑みてなされたものであって、効率的に増殖可能であるとともに、培養後においても肝細胞機能が十分に担保されたヒト肝細胞を提供することを目的とする。 The present invention has been made in view of such a problem, and an object of the present invention is to provide a human hepatocyte that can be efficiently proliferated and has a sufficient hepatocyte function even after culture.
本発明にかかるヒト肝細胞は、ミリセチンを含有する培地にて培養したヒト肝細胞である。 The human hepatocytes according to the present invention are human hepatocytes cultured in a medium containing myricetin.
本発明によれば、効率的に増殖可能であるとともに、培養後においても肝細胞機能が十分に担保されたヒト肝細胞が得られる。 ADVANTAGE OF THE INVENTION According to this invention, while being able to proliferate efficiently, the human hepatocyte which the hepatocyte function was fully ensured also after culture | cultivation is obtained.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating the understanding of the principle of the present invention, and the scope of the present invention is described below. The present invention is not limited to the embodiments, and other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.
本実施形態にかかるヒト肝細胞は、ミリセチンを含有する培地にて培養したヒト肝細胞である。 The human hepatocytes according to the present embodiment are human hepatocytes cultured in a medium containing myricetin.
ミリセチン(myricetin)は3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)- 4-chromenoneであり、下記式1で示される構造を有しており、フラボノイドである天然フラボノールの一種である。 Myricetin (myricetin) is 3,5,7-Trihydroxy-2- (3,4,5-trihydroxyphenyl) -4-chromenone, has a structure represented by the following formula 1, and is a natural flavonol that is a flavonoid. It is a kind.
培地は特に限定されるものではないが、例えばウシ胎児血清、インスリン、デキサメタゾン、ペニシリン、及び、ストレプトマイシを含有する培地であり、例えばWilliam’s E培地を用いることが可能である。 The medium is not particularly limited, but is, for example, a medium containing fetal bovine serum, insulin, dexamethasone, penicillin, and streptomyces, and for example, William's E medium can be used.
培地中におけるミリセチンの濃度は特に限定されるものではないが、例えば1〜30μモル/Lである。 The concentration of myricetin in the medium is not particularly limited, but is, for example, 1 to 30 μmol / L.
ミリセチンを含有する培地にて培養するヒト肝細胞の種類は特に限定されるものではなく、例えば肝実質細胞、全肝細胞、間葉系幹細胞やiPS細胞を分化させた肝細胞等を使用することが可能であるが、好ましくは肝実質細胞である。 The type of human hepatocytes cultured in a medium containing myricetin is not particularly limited, and for example, hepatocytes, whole hepatocytes, mesenchymal stem cells, hepatocytes differentiated from iPS cells, and the like may be used. However, it is preferably a hepatocyte.
細胞採取源として、自己細胞又は他己細胞の何れであっても好適に用いることが可能であり、また保存状態においても新鮮又は凍結の何れであっても好適に用いることが可能である。凍結肝細胞を使用する場合は、保存臍帯血移植、保存自己幹細胞移植のように解凍後保存液ごと輸注するか、遠心洗浄の後に輸液に懸濁して輸注するか何れであっても好適に用いることが可能である。凍結肝細胞の方が、いつでも使用することが出来また凍結している間に細菌汚染などの品質チェックができるので利点がある。 Either an autologous cell or another autologous cell can be suitably used as a cell collection source, and either fresh or frozen, even in a stored state, can be suitably used. When using frozen hepatocytes, it is preferable to inject either the preserved cord blood transplant or the preserved autologous stem cell transplant and then inject the whole preservation solution after thawing, or to suspend and inject the transfusion solution after centrifugal washing. It is possible. Frozen hepatocytes have the advantage that they can be used at any time and that they can be checked for quality such as bacterial contamination while frozen.
本実施形態にかかるヒト肝細胞は移植用であり、移植先としては特に限定されるものではないが例えば先天代謝異常症、慢性肝不全症、又は、急性肝不全症の患者に対する移植とすることが可能である。先天代謝異常症は、家族性高コレステロール血症、α1-アンチトリプシン欠損症、クリグラー・ナジャール症候群、第7因子欠損症、糖原病、レフサム病(パーオキシゾーム病)、進行性家族性肝内胆汁鬱滞症等である。慢性肝不全症例はウィルス性肝炎等である。急性肝不全症は、薬剤性、ウィルス性等の肝不全症である。移植の場は、先天代謝異常では門脈経由であるが、慢性肝不全及び急性肝不全では脾内に移植されることも可能である。 The human hepatocytes according to the present embodiment are for transplantation, and the transplantation destination is not particularly limited.For example, inborn error of metabolism, chronic liver failure, or transplantation to a patient with acute liver failure Is possible. Inborn errors of metabolism include familial hypercholesterolemia, α1-antitrypsin deficiency, Crigler-Najal syndrome, factor deficiency, glycogen disease, Refsum disease (peroxisome disease), progressive familial intrahepatic disease Cholestatic disease and the like. Chronic liver failure cases include viral hepatitis. Acute hepatic failure is hepatic failure such as drug-induced or viral. The place of transplantation is via the portal vein for inborn errors of metabolism, but it is also possible to transplant into the spleen for chronic liver failure and acute liver failure.
移植細胞数は、特に限定されるものではなく病型、患者の年齢等によって異なるが、例えば1回投与量において4x107個〜3.9x1010個とすることが可能である。 Transplanted cell number, particularly limited as not disease type varies by the patient's age and the like, for example may be a 4x10 7 cells ~3.9X10 10 pieces in a single dose.
本実施形態にかかるヒト肝細胞の製造方法は、当初からミリセチンを含有する培地にて例えば肝実質細胞を培養することも可能であるが、ミリセチンを含有しない培地で例えば肝実質細胞を18〜22時間培養後、その培地にミリセチンを添加して、ミリセチンを含有する培地にて培養することも可能である。ミリセチンを含有する培地における培養日数は特に限定されるものではなく、例えば5〜10日である。 In the method for producing human hepatocytes according to the present embodiment, for example, hepatic parenchymal cells can be cultured in a medium containing myricetin from the beginning. After culturing for a period of time, it is also possible to add myricetin to the medium and culture in a medium containing myricetin. The number of culture days in a medium containing myricetin is not particularly limited, and is, for example, 5 to 10 days.
本発明にかかるヒト肝細胞はミリセチンを含有する培地にて培養したヒト肝細胞であるが、このような実施形態に限定されるものではなく、ミリセチン誘導体を含有する培地にて培養したヒト肝細胞とすることも可能である。 The human hepatocytes according to the present invention are human hepatocytes cultured in a medium containing myricetin, but are not limited to such embodiments, and human hepatocytes cultured in a medium containing a myricetin derivative It is also possible to use
ミリセチン誘導体はミリセチンを基本骨格としてそこから誘導される化合物であり、特に限定されるものではないが具体的には下記式2で表される。 The myricetin derivative is a compound derived from the myricetin as a basic skeleton and is not particularly limited, but is specifically represented by the following formula 2.
ここで、Xa、Xb、Xc、Xd、Xe、Xf、X1及び2は、それぞれ独立してO又はSであり、
Ra、Rb、Rc、Rd、Re及びRfは、それぞれ独立してH、1〜5つのRyで置換された炭素数1〜10のアルキル基、又は、炭素数2〜20のジアルキルエーテル基であり、ただし該ジアルキルエーテル基の各炭素数1〜10のアルキルは1〜5つのRyで置換されており、
Ryは、Rq、あるいは、炭素数2〜10のアルキル基、炭素数3〜10のシクロアルキル基、炭素数3〜10のシクロアルキル基アルキル基、炭素数8〜14のトリシクロアルキル基アルキル基、フェニル基、ナフチル基又は炭素数14の芳香族基であり、
R1、R2、R3及びR4は、それぞれ独立してH又は炭素数1〜10のアルキル基、炭素数2〜10のアルケニル基、炭素数2〜10のアルキニル基、炭素数3〜10のシクロアルキル基、フェニル基、ナフチル基、炭素数14の芳香族基であり、
Rqは、CN、OH又はハロゲン原子である。
Here, Xa, Xb, Xc, Xd, Xe, Xf, X1 and 2 are each independently O or S,
Ra, Rb, Rc, Rd, Re and Rf are each independently H, an alkyl group having 1 to 10 carbon atoms substituted with 1 to 5 Ry, or a dialkyl ether group having 2 to 20 carbon atoms. Wherein each alkyl having 1 to 10 carbon atoms of the dialkyl ether group is substituted with 1 to 5 Ry,
Ry is Rq, or an alkyl group having 2 to 10 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms, or a tricycloalkyl group alkyl group having 8 to 14 carbon atoms. A phenyl group, a naphthyl group or an aromatic group having 14 carbon atoms,
R1, R2, R3 and R4 each independently represent H or an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, an alkynyl group having 2 to 10 carbon atoms, or a cycloalkyl having 3 to 10 carbon atoms. Group, a phenyl group, a naphthyl group, an aromatic group having 14 carbon atoms,
Rq is CN, OH or a halogen atom.
1.ヒト肝細胞培養
正常ヒト凍結肝細胞(Lot# FLO, EJW)はCelsis、In Vitro Technologies(Baltimore, MD)から購入した。常法に従い解凍後、肝細胞1×106個を1 micro-mol/L dexamethasone, 1 micro-mol/L insulin, 2 mmol/L L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 10%ウシ胎児血清を含むWilliam’s E培地(Sigma、W1878)2 mLに懸濁し、6穴培養プレート(BD Biocoat、type I collagen-coated 6-well plate、Becton Dickinson、Cat code 2352227)に播種、5%CO2、37℃で培養した。20時間培養後、20mmoL/Lの濃度でdimethylsulfoxideに溶かしたミリセチン(Sigma-Aldrich、M6760)を、最終濃度で1、3、10、30micro-mol/Lとなるように培地に添加し、7日間培養した(図1A、B)。図1Aは、ミリセチン存在下で培養する前のヒト肝実質細胞の形態であり、Ctrl;対照1(ミリセチン無添加)、DMSO;対照2(ミリセチン添加時の溶媒であるDMSOのみを添加)、1μM;ミリセチン1micro-mol/L添加、3μM;ミリセチン3micro-mol/L添加、10μM;ミリセチン10micro-mol/L添加、30μM;ミリセチン30micro-mol/L添加である。図1Bは、ミリセチン存在下で7日間培養したときのヒト肝実質細胞の形態であり、Ctrl;対照1(ミリセチン無添加)、DMSO;対照2(ミリセチン添加時の溶媒であるDMSOのみを添加)、1μM;ミリセチン1micro-mol/L添加、3μM;ミリセチン3micro-mol/L添加、10μM;ミリセチン10micro-mol/L添加、30μM;ミリセチン30micro-mol/L添加である。
1. Human hepatocyte culture Normal human frozen hepatocytes (Lot # FLO, EJW) were purchased from Celsis, In Vitro Technologies (Baltimore, MD). After thawing according to a conventional method, 1 × 10 6 hepatocytes were subjected to 1 micro-mol / L dexamethasone, 1 micro-mol / L insulin, 2 mmol / L L-glutamine, 100 U / ml penicillin, 100 U / ml streptomycin, Suspended in 2 mL of William's E medium (Sigma, W1878) containing 10% fetal bovine serum, and seeded on a 6-well culture plate (BD Biocoat, type I collagen-coated 6-well plate, Becton Dickinson, Cat code 2352227), 5 Cultured at 37 ° C.,% CO 2 . After culturing for 20 hours, add myricetin (Sigma-Aldrich, M6760) dissolved in dimethylsulfoxide at a concentration of 20 mmoL / L to the culture medium to a final concentration of 1, 3, 10, 30 micro-mol / L, and add it for 7 days. The cells were cultured (FIGS. 1A, B). FIG. 1A shows the morphology of human hepatic parenchymal cells before culturing in the presence of myricetin; Ctrl; control 1 (no addition of myricetin), DMSO; 1 micro-mol / L myricetin, 3 μM; 3 micro-mol / L myricetin, 10 μM; 10 micro-mol / L myricetin, 30 μM; 30 micro-mol / L myricetin. FIG. 1B shows the morphology of human hepatic parenchymal cells when cultured for 7 days in the presence of myricetin; Ctrl; control 1 (no addition of myricetin); DMSO; control 2 (only addition of DMSO, the solvent when adding myricetin) 1 μM; myricetin 1 micro-mol / L, 3 μM; myricetin 3 micro-mol / L, 10 μM; myricetin 10 micro-mol / L, 30 μM; myricetin 30 micro-mol / L.
2.細胞の特性解析
2−1.アンモニア除去能
上記培地に最終濃度で2mmol/LとなるようにNH4Clを加え、1時間おきに4時間まで培地中のアンモニア濃度を測定(ブロモクレゾールグリーン法)し、濃度の減少直線の傾きから細胞のアンモニア除去能(fmol/hr/cell)を求めた(図2)。
2. Cell characteristic analysis 2-1. The NH 4 Cl so as to 2 mmol / L at a final concentration of ammonia removal capacity above medium was added, the ammonia concentration in the medium up to 4 hours every hour was measured (bromocresol green method), the slope of the decreasing linear density Was used to determine the ability of the cells to remove ammonia (fmol / hr / cell) (FIG. 2).
2−2.遺伝子発現
培養プレートから細胞を回収しRNAを抽出、逆転写によりcDNAを調製した。このcDNAを用い、肝関連遺伝子(アルブミン、CYP3A4、α1アンチトリプシン、チロシンアミノトランスフェラーゼ、トリプトファン2,3ジオキシゲナーゼ、HNF4a、サイトケラチン18、クレアチンリン酸合成酵素1、オルニチントランスカルバミラーゼ)および内部標準としてGAPDH遺伝子の発現を定量PCRによって調べた(図3)。
2-2. Gene expression Cells were collected from the culture plate, RNA was extracted, and cDNA was prepared by reverse transcription. Using this cDNA, liver related genes (albumin, CYP3A4, α1 antitrypsin, tyrosine aminotransferase, tryptophan 2,3 dioxygenase, HNF4a, cytokeratin 18, creatine phosphate synthase 1, ornithine transcarbamylase) and internal standard The expression of the GAPDH gene was examined by quantitative PCR (FIG. 3).
3.マウス肝への移植実験
免疫不全SCIDマウス(C.B-17/IcrHsd-Prkdcscid、日本エスエルシー)オスとSCIDマウス化したオルニチントランスカルバミラーゼ欠損マウス(spf-ash、熊本大学、国立成育医療研究センター)メスを交配し、産仔を得た。ポリメラーゼチェーンリアクション(PCR)法によってオルニチントランスカルバミラーゼ欠損を確認した後、生後4日齢の新生仔肝に、ミリセチン5 micro-mol/L存在下で10日間培養したヒト肝細胞2×105個(生理的食塩水懸濁液として20 micro-L)を直接穿針して移植した(27ゲージ注射針使用)。対照として、生理的食塩水のみ、あるいはミリセチンを加えない培地で10日間培養したヒト肝細胞を投与した。その後、マウスの生存日数を調べた。結果を表1に示す。
3. Transplantation experiment into mouse liver Immunodeficient SCID mouse (CB-17 / IcrHsd-Prkdcscid, Japan SLC) male and SCID mouse ornithine transcarbamylase deficient mouse (spf-ash, Kumamoto University, National Center for Child Health and Development) female Were crossed to obtain offspring. After confirming ornithine transcarbamylase deficiency by the polymerase chain reaction (PCR) method, 2 × 10 5 human hepatocytes cultured for 4 days in the presence of 5 micro-mol / L myricetin in 4-day-old neonatal liver (20 micro-L as a physiological saline suspension) was directly penetrated and implanted (using a 27 gauge injection needle). As controls, human hepatocytes cultured in physiological saline alone or in a medium without the addition of myricetin for 10 days were administered. Thereafter, the survival days of the mice were determined. Table 1 shows the results.
図4Aは、オルニチントランスカルバミラーゼ欠損免疫不全マウス(OTCD-C.B-17/IcrHsd-Prkdcscid)肝にヒト肝細胞を移植した場合のマウス外観の変化を示す図であり、ヒト肝細胞移植後3週間目における状態を示す。図4Bは、オルニチントランスカルバミラーゼ欠損免疫不全マウス肝にヒト肝細胞を移植した場合の肝組織内ヒト肝細胞生着像を示す図であり、肝組織写真内の四角はそれぞれ右側の視野を示す。ここで、Ctrl;無処置マウス(陰性対照)、Non treated hHPCs;ミリセチン無添加培養ヒト肝細胞の移植、Treated hHPCs;ミリセチン添加培養ヒト肝細胞の移植である。 FIG. 4A is a diagram showing changes in mouse appearance when human hepatocytes were transplanted into the liver of an ornithine transcarbamylase-deficient immunodeficient mouse (OTCD-CB-17 / IcrHsd-Prkdcscid), three weeks after human hepatocyte transplantation. This shows the condition in the eyes. FIG. 4B is a diagram showing human hepatocyte engraftment images in liver tissue when human hepatocytes are transplanted into the liver of an ornithine transcarbamylase-deficient immunodeficient mouse. Each square in the liver tissue photograph indicates the right field of view. . Here, Ctrl; untreated mice (negative control), Non treated hHPCs; transplantation of cultured human hepatocytes without myricetin, Treated hHPCs; transplantation of cultured human hepatocytes with myricetin.
また、免疫不全SCIDマウス新生仔肝にミリセチン5 micro-mol/L存在下で10日間培養したヒト肝細胞あるいはミリセチン非存在下で10日間培養したヒト肝細胞2×105個を移植し、経日的に犠死させ血清および肝を採取、肝組織内のヒト肝細胞(図5A)と血清中のヒトアルブミン量(図5B)を調べた。図5Aは、免疫不全マウス(C.B-17/IcrHsd-Prkdcscid)肝内に移植したヒト肝細胞の生着評価を示す図であり、写真内の四角はそれぞれ右側の視野を示す。ここで、Ctrl;無処置マウス肝組織(陰性対照)、hLiver;ヒト肝組織(陽性対照)、Non treated hHPCs;ミリセチン無添加培養ヒト肝細胞の移植、Treated hHPCs;ミリセチン添加培養ヒト肝細胞の移植である。免疫組織染色の手法は、肝組織を10%中性ホルマリンで固定したのち切片を作成し、ウサギ抗ヒトアルブミン抗体(Inter-Cell Technologies, Jupiter, FL)と反応させ、horseradish peroxidase (HRP)-conjugated二次抗体で検出するものであった。図5Bは、免疫不全マウス(C.B-17/IcrHsd-Prkdcscid)肝内にヒト肝細胞を移植した場合のマウス血中ヒトアルブミン量を示す。ヒトアルブミン量の測定は、Albumin ELISA Quantitation Kit, Human(Bethyl, E80-129)によった。図5Aに示すように、ミリセチン添加培養ヒト肝細胞は移植後の生着が優れていた。また図5Bに示すように、ミリセチン添加培養ヒト肝細胞は移植後においてアルブミン量の増加において優れていた。 In addition, human hepatocytes cultured for 10 days in the presence of 5 micro-mol / L myricetin or 2 × 10 5 human hepatocytes cultured for 10 days in the absence of myricetin were transplanted into immunodeficient SCID mouse neonatal liver, Sera were sacrificed daily and serum and liver were collected, and human hepatocytes in liver tissue (FIG. 5A) and the amount of human albumin in serum (FIG. 5B) were examined. FIG. 5A is a diagram showing the evaluation of engraftment of human hepatocytes transplanted into the liver of an immunodeficient mouse (CB-17 / IcrHsd-Prkdcscid). Each square in the photograph indicates a visual field on the right side. Here, Ctrl; untreated mouse liver tissue (negative control), hLiver; human liver tissue (positive control), Non treated hHPCs; transplantation of cultured human hepatocytes without myricetin, Treated hHPCs; transplantation of cultured hepatocytes with myricetin It is. Immunohistochemical staining involves fixing liver tissue with 10% neutral formalin, making sections, reacting with rabbit anti-human albumin antibody (Inter-Cell Technologies, Jupiter, FL), and horseradish peroxidase (HRP) -conjugated. It was detected with a secondary antibody. FIG. 5B shows the amount of human albumin in mouse blood when human hepatocytes were transplanted into the liver of an immunodeficient mouse (CB-17 / IcrHsd-Prkdcscid). The measurement of the amount of human albumin was performed using the Albumin ELISA Quantitation Kit, Human (Bethyl, E80-129). As shown in FIG. 5A, the cultured human hepatocytes supplemented with myricetin exhibited excellent engraftment after transplantation. Further, as shown in FIG. 5B, the cultured human hepatocytes added with myricetin were superior in increasing the amount of albumin after transplantation.
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