201103945 六、發明說明： _ 【發明所屬之技術領域j 本發明係有關於〜種膠原蛋白製備方法。 【先前技術】 膠原蛋白係種纖維蛋白（fibrous protein)，其可在軟 月、筋鍵、真皮組織以及其它結締組織中發現。膠原蛋白 已廣泛運用於工業以及醫藥。 膠原蛋白一般係藉由可將非膠原蛋白物質移除的反覆 I谷或鹽析處理’而由結締組織純化出來。此種處理需重 i數夂以改善膠原蛋白之純度。重複處理不僅會拉長純化 製程，更會導致膠原蛋白產率低。 因而需要一種新穎的高純度膠原蛋白製備方法。 【發明内容】 法，1此，本發明的特徵為一種高純度膠原蛋白製備方 係先由一結締組織（例如真皮組織或筋腱）製造一 ^方法ΐ土質，然後再由基質萃取出膠原蛋白。詳細言之， 20平方下列步驟：⑴提供一結締組織，其表面積為 公分）.&釐至2平方公尺(較佳為25平方公釐至900平方 分之五U)以一第一酸液使該結締組織之體積至少膨脹百 、 （^)产較佳為1〇0%至500%)而形成一膨脹結締組織； 一膠該膨脹結締組織以除去非膠原蛋白物質藉此形成 一白基質；以及(iv)利用一萃取液從該膠原蛋白基 3 201103945 質萃取出膠原蛋白，藉此製造一含有膠原蛋白之溶液。 上述的膨脹步驟可藉由將結締組織浸泡於該第一酸液 而達成。較佳地，上述的浸泡步驟係藉由同時將一液體喷 射至結締組織或同時以一超音波處理結缔組織而達成。上 述的結締組織係源自真皮組織或筋腱。該第一酸液的pH 值為1至6(較佳為2至4)，且該第一酸液實質上不包含鹽 類，也就是說溶液中沒有鹽類成分，或者是鹽類濃度非常 低，使得溶液的離子強度不大於0.005M。上述的酸液係可 由甲酸、羧酸、草酸、醋酸、檸檬酸、乳酸、蘋果酸、硼 酸、磷酸或其混合物來製備。較佳的是，上述酸液為0.1-6M 的醋酸溶液。 在上述的膨脹步驟之後，將膨脹後的結締組織加以清 洗以除去非膠原蛋白物質，藉此製備出一膠原蛋白基質。 上述的清洗步驟可藉由將該膨脹結締組織浸泡於一清洗液 而達成，該清洗液可以進一步包含清潔劑、蛋白水解酶或 其混合物；在上述浸泡或清洗步驟中，可將該膨脹結締組 織以超音波處理或以液體噴射處理。 然後將前述膠原蛋白基質浸於一萃取液中，藉此形成 一含有膠原蛋白之溶液。該萃取液可以是含有弱有機酸（例 如甲酸、羧酸、草酸、醋酸、擰檬酸、乳酸、蘋果酸、硼 酸、磷酸或其混合物）之酸液，該酸液之pH值係適於溶 解膠原蛋白（較佳為小於4)。此外，該萃取液可以是含有 鹽（例如氯化鈉或氯化鉀）的中性溶液（例如0.05M的Tris 缓衝溶液），該鹽之濃度係適於溶解膠原蛋白（例如大於 1M)。在一實施例中，膠原蛋白係藉由下列步驟從該膠原 201103945 蛋白基質萃取出：研磨該膠原蛋白基質而製得膠原蛋白粉 -末，以及將該膠原蛋白粉末與該萃取液混合而製得該含有 膠原蛋白之溶液。該研磨步驟以及該混合步驟可同時進行。 接著，可以習用方法將膠原蛋白由該含有膠原蛋白之 溶液中沈澱。在一實施例中，該膠原蛋白係藉由透析法沈 澱。在另一實施例中，係將該含有膠原蛋白之溶液與/發 以1M至4M的濃度混合來使膠原蛋白沈澱；如此獲得的朦 原蛋白較佳需進行脫鹽。在又一實施例中，係將該含有膠 原蛋白之溶液的pH值調整至4.5到8來使膠原蛋白沈臌° 製得之膠原蛋白可加以冷凍乾燥、空氣乾燥或真空乾 燥而形成膠原蛋白粉末、膠原蛋白綿狀物(sponge)、膠原蛋 白片狀物或膠原蛋白膜。製得之膠原蛋白粉末可分散於一 酸液中而形成一膠原蛋白分散溶液，製得之膠原蛋白粉末 亦可以蛋白水解酶處理以製造無端肽膠原蛋白 (atelopeptide collagen)。蛋白水解酶之例子包含，但不限 於，胃蛋白酶(pepsin)、鳳梨蛋白酶(bromelain)、木瓜凝乳 蛋白酶（chymopapain)、膜凝乳蛋白酶(chymotrypsin)、膠 原蛋白酶(collagenase)、無花果蛋白酶(ficin)、木瓜蛋白酶 (papain)、胜肽酶（peptidase )、蛋白酶 A ( proteinase A )、 蛋白酶K ( proteinase K )、騰蛋白酶(trypsin)、微生物蛋白 酶（microbial proteases)及其混合物。 下文特舉本發明一些實施例作詳細說明如後。本發明 之其他目的或特徵將呈現於後述實施例以及後附之申請專 利範圍。 201103945 【實施方式】 膠原蛋白長約300奈米（nm)，直徑約1.5奈米（nm)。大 量的膠原蛋白分子形成膠原蛋白纖維絲，而一束膠原蛋白 纖維絲形成一膠原蛋白纖維。膠原蛋白分子之間以及膠原 蛋白分子内部存在著共價的交聯，藉此在結缔組織内形成 了纖維狀的網絡。 在此所述為一種高純度膠原蛋白製備方法，其係直接 先由一結締組織製造一膠原蛋白基質，然後再由該膠原蛋 白基質萃取出膠原蛋白。 製備膠原蛋白基質 起始材料（亦即結締組織）可取自於動物，例如牛、 緒、馬、羊、雞、鴨、火雞、鹤、絲魚或者鯊魚等畜類或 魚類。適用於本發明方法的結締組織包含，但不限於，真 皮組織、皮下組織、韌帶、筋腱、腱膜、軟骨、骨頭組織。 假如需要的話，可先用人工（例如藉由粗略的切開（gross dissection))或機械清理結締組織以除去不需要的材料，例 如脂肪及脂質。在一實施例中，真皮組織係取自新鮮的動 物表皮，將動物表皮除去脂質、以鹽水(saline)清洗表皮數 次後，以一植皮刀（dermatome)除去動物表皮的表面層即可 獲得真皮組織。所得的真皮組織可用磷酸鹽緩衝溶液進一 步清洗。 假如需要，結締組織可以先用一適合的有機溶劑或者 是一有機溶劑與水的混合液進行處理，使有機溶劑可以滲 透入結締組織。有機溶劑包含，但不限於醇、酮、丙酮、 乙腈、氯仿、N，N-二曱基曱醯胺、二曱亞颯或其混合物。 201103945 當使用有機溶劑與水的混合液時’有機溶劑與水的比例高 於 1 : 4(例如 1: 1、2. 1 或 4. 1)。 當結締組織含有毛髮或毛根時’可先以一微驗溶液或 蛋白水解酶處理以除去毛髮或毛根，蛋白水解酶可為，例 如中性蛋白酶（dispase)、胰蛋白酶(trypsin)、木瓜蛋白酶 (papain)、胃蛋白酶(pepsin)、胰凝乳蛋白酶(chymotrypsin)、 胤梨蛋白酶(bromelain)、無花果蛋白酶(ficin)或其混合物。 接著，將上述任一結締組織浸泡於適量酸液中，經過 適當的時間後，使得結締組織膨脹到所需的標準，亦即比 原厚度至少增加50°/❶的厚度(較佳為原厚度的2-10倍）。上 述的酸液可以一有機酸來製備，有機酸可為，例如曱酸、 幾酸、草酸、醋酸、擰檬酸、乳酸、蘋果酸、棚酸、碟酸 或其混合物。在一實施例中，酸液可為濃度〇1〜6M(較佳 為0.1〜2M或0.5〜1.25M)的醋酸。為了獲得較佳的膨脹效 果’本發明所使用的酸液實質上不包含鹽類。 在上述的膨脹步驟中，結締組織是懸浮於以上所述的 酸液中。假如需要的話，可利用一或數道液體來處理結締 組織，促使酸液滲透入結締組織並縮短膨脹結締組織至所 需標準的時間。上述的液體可以由—容器的喷嘴或喷孔來 喷出，上述的酸液及結締組織係置放於該容器内。 或者，或此外，可將超音波（由一超音波震動裝置產 生）或高頻率水波（藉由一電磁場產生）施加於浸泡於酸 液中的結締組織，以促使酸液滲透入結締組織。 使用一清洗液來清洗上述膨脹步驟所製備的膨脹結締 組織’以實質上除去上述膨脹m織中的非膠原蛋白物 201103945 質，藉此以製備一膠原蛋白基質。所述的清洗液可含有清 潔劑、螯合劑、蛋白酶或其混合物。 用以製備清洗液的範例清潔劑包含，但不限於十二烷 基硫酸納（sodium dodecyl sulphate，SDS)、Tego 化合物（例 如介面活性劑Tween-80、非離子清潔劑TWR (Triton W. R· 1339)、對-異辛烷基聚氧-乙烯苯酚聚合物 (p-isooctylpolyoxy-ethylene phenol polymer)與四 丁齡酸 (Triton A20)表面活性劑）、氣化十六烷基吡啶 (cetylpyridinium chloride)、溴化十六燒基三甲基銨 (cetyltrimethyl ammonium bromide)、二辛烧鋼續基破珀酸 酉旨（dioctyl sodium sulphosuccinate)、購自曰本 Kao Soap 有 限公司品名為「Emasol 4130」的聚氧基乙烯山梨糖醇油酸 西旨（polyoxyethylene sorbitan monoleate)介面活性劑、購自英 國帝國化學工業有限公司品名為「Lubrol W」的非離子介 面活性劑、購自歐洲Sigma公司品名為「Nonidet P40」的 非離子介面活性劑在一實施例中，使用一含有SDS濃度 0.01%至10%的清洗液，在4°C至45°C溫度下處理所述膨脹 結締組織1小時至150小時。 所述清潔液所含的螯合劑包含，但不限於乙二胺四乙 酸（ethylene diamine tetra_acetic acid, EDTA)、1，4,7，10—四 氮雜環十二烷基一 1,4,7，10’一四乙酸 (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA)、1，4,7，10—四氮雜環十二烷基—ι，4,7,10，一四亞甲 基磷酸 (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene 201103945 phosphonic acid)，DOTP)、環己二胺四醋酸 (trans-1,2-diaminocyclohexantetra-acetic acid, CDTA) ' 4,5 一二經基苯—1，3 —二績酸201103945 VI. Description of the invention: _ [Technical field to which the invention pertains] The present invention relates to a method for preparing collagen. [Prior Art] Collagen is a fibrous protein which can be found in soft months, tendons, dermal tissues, and other connective tissues. Collagen has been widely used in industry as well as medicine. Collagen is typically purified from connective tissue by a reverse I valley or salting out treatment that removes non-collagen material. This treatment requires a certain amount of 夂 to improve the purity of collagen. Repeated processing not only lengthens the purification process, but also leads to low collagen yields. There is therefore a need for a novel method of preparing high purity collagen. SUMMARY OF THE INVENTION The present invention is characterized in that a high-purity collagen preparation method is first made of a connective tissue (for example, dermal tissue or tendon) to produce a soil, and then the collagen is extracted from the matrix. . In detail, 20 squares of the following steps: (1) providing a connective tissue having a surface area of centimeters). & centimeter to 2 square meters (preferably 25 square centimeters to 900 square centimeters of five U) to a first acid The liquid causes the volume of the connective tissue to expand at least 100%, preferably (1% to 0% to 500%) to form an expanded connective tissue; the gel expands the connective tissue to remove non-collagen material thereby forming a white a matrix; and (iv) extracting collagen from the collagen group 3 201103945 using an extract to thereby produce a collagen-containing solution. The above expansion step can be achieved by immersing the connective tissue in the first acid solution. Preferably, the soaking step described above is accomplished by simultaneously ejecting a liquid to connective tissue or simultaneously treating the connective tissue with an ultrasonic wave. The connective tissue described above is derived from dermal tissue or tendons. The first acid solution has a pH of 1 to 6 (preferably 2 to 4), and the first acid solution does not substantially contain a salt, that is, there is no salt component in the solution, or the salt concentration is very high. Low, so that the ionic strength of the solution is not more than 0.005M. The above acid solution can be prepared from formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid or a mixture thereof. Preferably, the acid solution is a 0.1-6 M acetic acid solution. After the expansion step described above, the expanded connective tissue is washed to remove non-collagen material, thereby preparing a collagen matrix. The cleaning step may be achieved by immersing the expanded connective tissue in a cleaning solution, which may further comprise a detergent, a proteolytic enzyme or a mixture thereof; in the soaking or washing step, the expanded connective tissue may be Ultrasonic treatment or liquid jet treatment. The aforementioned collagen matrix is then immersed in an extract to form a collagen-containing solution. The extract may be an acid containing a weak organic acid such as formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid or a mixture thereof, and the pH of the acid solution is suitable for dissolution. Collagen (preferably less than 4). Further, the extract may be a neutral solution containing a salt such as sodium chloride or potassium chloride (e.g., a 0.05 M Tris buffer solution) at a concentration suitable for dissolving collagen (e.g., greater than 1 M). In one embodiment, the collagen is extracted from the collagen 201103945 protein matrix by grinding the collagen matrix to produce a collagen powder, and mixing the collagen powder with the extract. The collagen-containing solution. The grinding step and the mixing step can be carried out simultaneously. Next, collagen can be precipitated from the collagen-containing solution by a conventional method. In one embodiment, the collagen is precipitated by dialysis. In another embodiment, the collagen-containing solution is mixed with / at a concentration of 1 M to 4 M to precipitate collagen; the prion protein thus obtained preferably desalinates. In still another embodiment, the pH of the collagen-containing solution is adjusted to 4.5 to 8 to cause collagen to precipitate. The collagen obtained can be freeze-dried, air dried or vacuum dried to form collagen powder. , collagen sponge, collagen sheet or collagen membrane. The prepared collagen powder can be dispersed in an acid solution to form a collagen dispersion solution, and the obtained collagen powder can also be treated with a proteolytic enzyme to produce an atelopeptide collagen. Examples of proteolytic enzymes include, but are not limited to, pepsin, bromelain, chymopapain, chymotrypsin, collagenase, and ficin. , papain, peptidase, proteinase A, proteinase K, trypsin, microbial proteases, and mixtures thereof. Some embodiments of the invention are described in detail below. Other objects and features of the present invention will be apparent from the following description and the appended claims. 201103945 [Embodiment] Collagen is about 300 nanometers (nm) long and has a diameter of about 1.5 nanometers (nm). A large amount of collagen molecules form collagen filaments, and a bundle of collagen filaments form a collagen fiber. There is a covalent cross-linking between the collagen molecules and within the collagen molecules, thereby forming a fibrous network within the connective tissue. Here, a method for preparing high-purity collagen is to directly produce a collagen matrix from a connective tissue, and then extract collagen from the collagen matrix. Preparation of the collagen matrix The starting material (i.e., connective tissue) can be obtained from animals such as cattle, silk, horses, sheep, chickens, ducks, turkeys, cranes, silkfish or sharks. Connective tissue suitable for use in the methods of the invention includes, but is not limited to, dermal tissue, subcutaneous tissue, ligaments, tendons, aponeurosis, cartilage, bone tissue. If desired, the connective tissue can be manually removed (e.g., by gross dissection) or mechanically to remove unwanted materials such as fats and lipids. In one embodiment, the dermal tissue is obtained from a fresh animal epidermis, the animal epidermis is removed from the skin, and the epidermis is washed several times with saline, and the surface layer of the animal epidermis is removed by a dermatome to obtain the dermis. organization. The resulting dermal tissue can be further washed with a phosphate buffer solution. If necessary, the connective tissue can be treated with a suitable organic solvent or a mixture of an organic solvent and water to allow the organic solvent to permeate into the connective tissue. The organic solvent includes, but is not limited to, an alcohol, a ketone, acetone, acetonitrile, chloroform, N,N-didecylguanamine, diterpenoid or a mixture thereof. 201103945 When using a mixture of organic solvent and water, the ratio of organic solvent to water is higher than 1:4 (for example, 1: 1, 2. 1 or 4. 1). When the connective tissue contains hair or hair roots, it can be treated with a micro-test solution or proteolytic enzyme to remove hair or hair roots. Proteolytic enzymes can be, for example, dispase, trypsin, papain ( Papain), pepsin, chymotrypsin, bromelain, ficin or mixtures thereof. Next, immersing any of the above connective tissues in an appropriate amount of acid solution, and after a suitable period of time, expanding the connective tissue to a desired standard, that is, a thickness of at least 50°/❶ more than the original thickness (preferably the original thickness) 2-10 times). The above acid solution may be prepared as an organic acid such as capric acid, acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, shed acid, dish acid or a mixture thereof. In one embodiment, the acid solution may be acetic acid having a concentration of 〜1 to 6 M (preferably 0.1 to 2 M or 0.5 to 1.25 M). In order to obtain a better expansion effect, the acid used in the present invention does not substantially contain a salt. In the above expansion step, the connective tissue is suspended in the above-described acid solution. If desired, one or more fluids may be used to treat connective tissue, causing the acid to penetrate into the connective tissue and shorten the time it takes to swell the connective tissue to the desired standard. The liquid can be ejected from the nozzle or orifice of the container, and the acid and connective tissue are placed in the container. Alternatively, or in addition, ultrasonic waves (produced by an ultrasonic vibration device) or high frequency water waves (produced by an electromagnetic field) may be applied to connective tissue soaked in the acid to promote penetration of the acid into the connective tissue. The swelled connective tissue prepared by the above expansion step is washed with a cleaning solution to substantially remove the non-collagen material in the above-mentioned expanded m woven fabric, thereby preparing a collagen matrix. The cleaning solution may contain a cleaning agent, a chelating agent, a protease, or a mixture thereof. Exemplary cleaners for preparing cleaning solutions include, but are not limited to, sodium dodecyl sulphate (SDS), Tego compounds (eg, surfactant Tween-80, nonionic detergent TWR (Triton W. R. 1339), p-isooctylpolyoxy-ethylene phenol polymer and tetratonic acid (Triton A20) surfactant, cetylpyridinium chloride , cetyltrimethyl ammonium bromide, dioctyl sodium sulphosuccinate, purchased from Sakamoto Kao Soap Co., Ltd. under the name "Emasol 4130" Polyoxyethylene sorbitan monoleate surfactant, non-ionic surfactant purchased from Imperial Chemical Industries Ltd. under the name "Lubrol W", purchased from European Sigma Company under the name "Nonidet P40" In one embodiment, the non-ionic surfactant is treated with a cleaning solution containing a SDS concentration of 0.01% to 10%, and the expanded connective is treated at a temperature of 4 ° C to 45 ° C. Woven from 1 to 150 hours. The chelating agent contained in the cleaning liquid includes, but is not limited to, ethylene diamine tetra-acetic acid (EDTA), 1,4,7,10-tetraazacyclododecyl-1,4,7 , 10'-tetraacetate (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA), 1,4,7,10-tetraazacyclododecyl-ι, 4,7,10,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene 201103945 phosphonic acid), DOTP), cyclohexanediaminetetraacetic acid (trans -1,2-diaminocyclohexantetra-acetic acid, CDTA) ' 4,5-di-diphenylbenzene-1,3-dibasic acid
(4,5-dihydroxybenzene-l,3-disulphonic acid，Tiron)、疏腺 (thiourea)、8-羥基噎淋-5-績酸 (8-hydroxyquinoline-5-sulphonic acid)、3,6-二續基-1，8-二經 基萘（3,6-disulpho-1,8-dihydroxy- naphthalene)、 Eriochromeschwarz T(l-(1·羥基-2-萘基偶氮)-2-羥基-5-硝 基-4-萘續酸)(Eriochromeschwarz T (l-(l-hydroxy-2-naphthylazo)-2-hydroxy-5— nitro-4-naphthalene sulphonic acid))、紅紫酸銨（ammonium purpurate)等。較佳的螯合劑為〇.〇lrn]V[至l〇〇mM的EDTA。 或者，或此外，清洗液可包含一或多種蛋白水解酶， 例如無花果蛋白酶(ficin)、胃蛋白酶(pepsin)、胰蛋白酶 (trypsin)以及溶血素（hermolysin)，以除去連接於細胞外基 質的蛋白質、其它非膠原蛋白的蛋白質與膠原蛋白分子的 末端肽(telopeptide)。在此技術領域中，有限度的酵素切割 (亦即使非膠原蛋白的蛋白f降解但仍保持#原蛋白纖^ 的完整性）的條件是眾所皆知的。 在'月洗步驟中’可將所述膨脹的結締組織浸泡於前面 所提到酸液之巾的任H適當的時間 。在一實施例 ’將-❹道㈣自一喷嘴或纽噴射至所述的結缔組 進雜原蛋白物f的移除 為水 酵素的，在另-實施例中； 洗效率。π/包於清洗液中的膨脹結締組織，可改善清 201103945 習知用以將非膠原蛋白物質由結締組織移除的方法 (例如美國專利第7,498,412、5,993,844以及5,374,539號） 亦可用於本發明。 可將已經進行過上述清洗步驟的膠原蛋白基質在液態 氮中冷凍乾燥法進行保存。或者，膠原蛋白基質可浸泡於 一磷酸鹽緩衝溶液中並儲存於4°C的溫度環境。當有需要 時，可藉由一般的化學或物理方法使上述的膠原蛋白基質 交聯化。用於使上述膠原蛋白基質交聯化的交聯劑包含戊 二酸(glutaraldehyde)、甲路(formaldehyde)、碳二醯胺 (carbodiimide)或其它聚環氧樹脂化合物(p〇ly ep0Xy compound)，例如乙二醇二缩水甘油醚(glycol diglycidyl ether)、聚 多缩水甘油謎(p〇iy〇i p〇iygiyCidyl ether)、二幾 酸二缩水甘油 S旨（dicarboxylic acid diglycidyl ester)。 前述用以製備膠原蛋白基質的方法在至少兩個面向上 不同於習知的方法。第一，本發明之方法不需要嚴苛的物 理或化學處理法(例如研磨、均質化或酸/鹼降解），這些方 法都會破壞結締組織中膠原蛋白纖維的網絡結構。第二， 用以膨脹結締組織的酸液實質上不包含鹽類，而一般習知 方法是利用鹽類來穩定膠原蛋白纖維。 從膠原蛋白基質萃取出膠原蛋白 以前述方法製備之膠原蛋白基質可以被研磨，例如以 震盪、授拌、均質化、絞碎、撕裂、切割、磨碎、切或其 混合方式。將膠原蛋白基質（完整的或研磨過的）浸於一 萃取液中一段適當期間，以允許溶解最大量的膠原蛋白。 在一實施例中’係將研磨過的膠原蛋白基質與萃取液在溫 和的機械動作（例如震盪、攪動或攪拌）下混合以促進膠 201103945 原蛋白溶解。 該萃取液可以是一酸液或含有鹽的中性溶液，其pH值 或鹽濃度係適於溶解膠原蛋白。適用於製備萃取液之酸包 含，但不限於，甲酸、羧酸、草酸、醋酸、檸檬酸、乳酸、 蘋果酸、硼酸、磷酸或其混合物。當使用醋酸時，其濃度 為0.1-6厘(較佳為0.1-2]^或〇.5-1.25]^)。例示的鹽包含氯化 鈉以及氯化鉀，其濃度為0.1-2M (較佳為1M)。例示的中 性溶液包含磷酸緩衝溶液(PBS)以及Tris緩衝溶液。當使用 pH值7-8的中性緩衝溶液，可加入一種或多種中性鹽（例如 1M氣化鉀或氯化鈉）以增加膠原蛋白在緩衝溶液中的溶解 度。其它適用於製備萃取液之緩衝溶液包含，但不限於， glycine-HCl 緩衝溶液、Clark and Lubs 緩衝溶液、citric acid-Na2HP04 缓衝溶液、Britton-Robinson 緩衝溶液、 citric acid-sodium citrate 緩衝溶液、 beta:beta'-dimethylglutaric acid_NaOH 緩衝溶液、sodium acetate-sodium citrate 緩衝溶液、succinic acid-NaOH緩衝 溶液、sodium cacodylate-HCl 緩衝溶液、sodium hydrogen maleate-NaOH 緩衝溶液、Na2HP04-NaH2P04 緩衝溶液、 sodium bicarbonate_5% C02 缓衝溶液、imidazole (glyoxaline)-HCl 緩衝溶液、2,4,6-trimethylpyridine (collidine)缓衝溶液、triethanolamine hydrochloride-NaOH 緩衝溶液、sodium 5,5’-diethyl barbiturate 緩衝溶液、 dimethylleucylglycine 緩衝溶液以及 N-ethylmoirpholine-HCl 緩衝溶液。 萃取後’將不溶物移除（例如藉由離心或過濾）可得 含有膠原蛋白之溶液。如有必要，可以用相同的萃取液萃 201103945 取不溶物一次或數次，上清液（soluble fraction)可以與該 含有膠原蛋白之溶液混合在一起。 以習用方法將膠原蛋白由該含有膠原蛋白之溶液中沈 澱。在一實施例中，該膠原蛋白係藉由透析法沈澱。在另 一實施例中’係將該含有膠原蛋白之溶液與一鹽以1M至4M 的濃度混合來使膠原蛋白沈澱；如此獲得的膠原蛋白較佳 需進行脫鹽。在又一實施例中，係將該含有膠原蛋白之溶 液的pH值調整至4.5到8來使膠原蛋白沈澱。 該含有膠原蛋白之溶液可以蛋白水解酶水解(digestion) 處理以除去末端肽(telopeptide)，藉此製造無端肽膠原蛋白 (atelopeptide collagen )。適用於此水解之蛋白水解酶包 含’但不限於，胃蛋白酶(pepsin)、鳳梨蛋白酶(bromeiain)、 木瓜凝乳蛋白酶（chymopapain )、膜凝乳蛋白酶 (chymotrypsin)、膠原蛋白酶(collagenase)、無花果蛋白酶 (ficin)、木瓜蛋白酶(papain)、胜肽酶（peptidase)、蛋白酶 A ( proteinase A )、蛋白酶K ( proteinase K )、胰蛋白酶 (trypsin)、微生物蛋白酶(microbial proteases)及其混合物。 水解反應的條件視所使用的酶而定。例如，當使用胃蛋白 酶時，反應混合物之pH值約為2-5，酶濃度約為待處理膠原 蛋白的0.001-10 wt% (較佳為〇.l-l〇g/l)。 膠原蛋白可由前述含有膠原蛋白之溶液中沈搬而進_ 步純化。此沈澱製程可重複直至達到所要的純度。在一實 施例中，該膠原蛋白係藉由透析法沈澱，其係將前述含有 膠原蛋白之溶液以一透析管或透析袋（截留分子量約為 12,000-14,000)對著一缓衝溶液透析。在另一實施例中， 係在該含有膠原蛋白之溶液中加入鹽（例如鹼金屬_化物 12 201103945 (例如氣化鈉））至1M至4M的濃度來使膠原蛋白沈澱，然 後離心收集膠原蛋白並以超過滤(ultrafiltration)、透析或稀 釋酸液脫鹽。在又一實施例中，係將該含有膠原蛋白之溶 液的pH值調整至膠原蛋白不溶的pH值來使膠原蛋白沈 澱。參見WO/2004/096384。膠原蛋白的純化也可藉由前述 方法的組合而達成。沈澱之膠原蛋白可以使其重新懸浮 (resuspended)並利用超過遽膜進行缓衝液交換。 以前述任一方法製得之膠原蛋白可在真空下冷;東乾 燥。此外，亦可以使膠原蛋白重新懸浮於一適當溶液。 咸信在此技術領域中的熟悉技藝者基於前述的描述， 都可將本發明充分的利用而不需進一步的詳細描述。因 此，下文所舉實施例僅僅只是作為範例，並非用以在任何 方面限制本發明。在此引用的所有文獻係併入本案以為參 照。 實施例1 :製備膠原蛋白基質 自豬身上取得皮膚組織，除去脂質後，以鹽水清洗表 皮數次，以植皮刀（dermatome)除去表皮的表面層以獲得一 厚度為0.3毫米（mm)的真皮组織。進一步以磷酸鹽緩衝溶液 清洗所述的真皮組織。在清洗過後，將所有緩衝溶液殘餘 物自真皮組織的表面完全的除去。 、 將真皮組織置入一裝有濃度0.5M醋酸溶液的容器内， 並在37°C定溫放置一天半來使真皮組織膨脹至厚度^c mm。在定溫放置期間’容器是置於一震動器上，以伟 組織懸浮。 吏真皮 接著將膨脹後的真皮組織浸泡於室溫下含有〇 5〇 SDS與0.5mM的EDTA的溶液中兩小時，以除去非膠 ^原蛋白 201103945 物質而產生一膠原蛋白基質。以一經過殺菌處理的磷酸鹽 緩衝溶液清洗上述的膠原蛋白基質，以除去殘餘的SDS與 EDTA。 ’、 實施例2:由膠原蛋白基質萃取膠原蛋白 將以實施例1所述方法製備之膠原蛋白基質浸於0.5M 醋酸溶液12-24小時並加以攪拌。將產生的混合物以2〇〇〇 rpm(700 xg)離心1小時後，收集上清液並儲存在代。 將含有純化膠原蛋白之上清液以胃蛋白酶⑼2mg/ml) 處理24小時以製造無端肽膠原蛋白。 實施例3:以透析法純化膠原蛋白 將膠原蛋白利用實施例2所述的步驟由膠原蛋白基質 萃取出來並製備含有膠原蛋白之溶液。將該溶液以一纖維 素膜（MWC0 12-14,〇〇〇)對著一0.02M磷酸二氫鈉緩衝液 透析後’在8000 X g離心1小時。收集peuet，以冷MiiHQ水 沖洗數次’然後將pellet重新懸浮在冷MilliQ水中。將懸浮 液在8000 xg離心1小時。 將產生的膠原蛋白pellet重新懸浮在0.1M的醋酸中。 實施例4 :以鹽析法(saiting-out)或改變pH來純化膠原 蛋白 將膠原蛋白利用實施例2所述的步驟由膠原蛋白基質 萃取出來並製備含有膠原蛋白之溶液。 在該溶液中逐漸加入氣化鈉至最終濃度為2.5m。收集 沈澱的膠原蛋白並以蒸餾水清洗後，將其重新懸浮在 的醋酸中。 此外，在該含有膠原蛋白之溶液中加入1M氫氧化鈉， 將其pH值調整至7。將混合物置於冷房中攪拌3小時讓膠原 201103945 蛋白沈澱。之後，將混合物在4°C離心1小時後，將膠原蛋 白pellet重新懸浮在去離子水中。將懸浮液的pH值以0.1M 鹽酸調整至低於3.5用以讓膠原蛋白溶解。 其他實施例 本發明所揭露之所有特徵應可以任何結合方式實現。 本發明所揭露之每一特徵應可以相同、均等或相似目的的 取代物所取代。因此，除非有明確的指定，否則所揭露的 每一個特徵僅僅只是均等物或相似特徵的一個種類的一實 施例。 由上述内容可知，任何在此技術領域中具有通常知識 者，將可輕易從本發明之揭露中了解到本發明之特徵， 在不偏離后附申請專利範圍所界定之本發明的精神與範 圍下，當可在此進行各種改變、取代以及修正。因此， 其他實施例亦落於后附申請專利範圍内。 【圖式簡單說明】 15(4,5-dihydroxybenzene-l,3-disulphonic acid,Tiron), thiourea, 8-hydroxyquinoline-5-sulphonic acid, 3,6-two continued 3,6-disulpho-1,8-dihydroxy-naphthalene, Eriochromeschwarz T(l-(1·hydroxy-2-naphthylazo)-2-hydroxy-5- (Nitrochromeschwarz T (l-(l-hydroxy-2-naphthylazo)-2-hydroxy-5-nitro-4-naphthalene sulphonic acid)), ammonium purpurate, and the like. A preferred chelating agent is ED.rrn]V [to 1 mM EDTA. Alternatively, or in addition, the cleaning solution may comprise one or more proteolytic enzymes, such as ficin, pepsin, trypsin, and hemolysin, to remove proteins linked to the extracellular matrix. Other non-collagen proteins and telopeptides of collagen molecules. In this technical field, the conditions for limited enzyme cleavage (even if the protein f of non-collagen is degraded, but the integrity of the original protein fiber is maintained) are well known. The expanded connective tissue may be soaked in the 'month wash step' at any time appropriate for the H of the previously mentioned acid liquid. In one embodiment, the removal of the helium (4) from a nozzle or nozzle to the connective group of protoproteins f is hydroenzyme, in another embodiment; washing efficiency. π/Expanded connective tissue in the cleaning solution can be improved. The method for removing non-collagen material from connective tissue is also known in the art (for example, U.S. Patent Nos. 7,498,412, 5,993,844 and 5,374,539). The collagen matrix which has been subjected to the above washing step can be stored by freeze-drying in liquid nitrogen. Alternatively, the collagen matrix can be immersed in a phosphate buffer solution and stored at a temperature of 4 °C. When necessary, the above collagen matrix can be crosslinked by general chemical or physical methods. The crosslinking agent for crosslinking the above-mentioned collagen matrix comprises glutaraldehyde, formaldehyde, carbodiimide or other polyepoxy compound (p〇ly ep0Xy compound). For example, glycol diglycidyl ether, polypyridyl ether (p〇iy〇ip〇iygiyCidyl ether), dicarboxylic acid diglycidyl ester. The foregoing method for preparing a collagen matrix differs from conventional methods in at least two orientations. First, the method of the present invention does not require harsh physical or chemical treatments (e.g., grinding, homogenization, or acid/base degradation) which destroy the network structure of collagen fibers in connective tissue. Second, the acid used to swell the connective tissue does not substantially contain salts, and conventional methods use salts to stabilize the collagen fibers. Extraction of collagen from collagen matrix The collagen matrix prepared as described above can be ground, for example, by shaking, mixing, homogenizing, mashing, tearing, cutting, grinding, cutting or mixing thereof. The collagen matrix (complete or milled) is immersed in an extract for a suitable period of time to allow dissolution of the maximum amount of collagen. In one embodiment, the ground collagen matrix is mixed with the extract under mild mechanical action (e.g., shaking, agitation or agitation) to promote dissolution of the gel 201103945 proprotein. The extract may be an acid solution or a neutral solution containing a salt, the pH or salt concentration of which is suitable for dissolving collagen. Suitable acids for the preparation of the extract include, but are not limited to, formic acid, carboxylic acid, oxalic acid, acetic acid, citric acid, lactic acid, malic acid, boric acid, phosphoric acid or mixtures thereof. When acetic acid is used, its concentration is from 0.1 to 6% (preferably from 0.1 to 2) or 〇.5-1.25]. The exemplified salts comprise sodium chloride and potassium chloride in a concentration of from 0.1 to 2 M (preferably 1 M). The exemplified neutral solution comprises a phosphate buffer solution (PBS) and a Tris buffer solution. When a neutral buffer solution having a pH of 7-8 is used, one or more neutral salts (e.g., 1 M potassium carbonate or sodium chloride) may be added to increase the solubility of collagen in the buffer solution. Other buffer solutions suitable for the preparation of the extract include, but are not limited to, glycine-HCl buffer solution, Clark and Lubs buffer solution, citric acid-Na2HP04 buffer solution, Britton-Robinson buffer solution, citric acid-sodium citrate buffer solution, beta :beta'-dimethylglutaric acid_NaOH buffer solution, sodium acetate-sodium citrate buffer solution, succinic acid-NaOH buffer solution, sodium cacodylate-HCl buffer solution, sodium hydrogen maleate-NaOH buffer solution, Na2HP04-NaH2P04 buffer solution, sodium bicarbonate_5% C02 Flush solution, imidazole (glyoxaline)-HCl buffer solution, 2,4,6-trimethylpyridine (collidine) buffer solution, triethanolamine hydrochloride-NaOH buffer solution, sodium 5,5'-diethyl barbiturate buffer solution, dimethylleucylglycine buffer solution, and N- Ethylmoirpholine-HCl buffer solution. After the extraction, the insoluble matter is removed (for example, by centrifugation or filtration) to obtain a solution containing collagen. If necessary, the same extract can be used. 201103945 Insoluble matter can be taken once or several times, and the soluble fraction can be mixed with the collagen-containing solution. Collagen is precipitated from the collagen-containing solution by a conventional method. In one embodiment, the collagen is precipitated by dialysis. In another embodiment, the collagen-containing solution is mixed with a salt at a concentration of 1 M to 4 M to precipitate collagen; the collagen thus obtained is preferably desalted. In still another embodiment, the pH of the collagen-containing solution is adjusted to 4.5 to 8 to precipitate collagen. The collagen-containing solution can be subjected to proteolytic digestion treatment to remove a telopeptide, thereby producing an atelopeptide collagen. Proteolytic enzymes suitable for this hydrolysis include, but are not limited to, pepsin, bromeiain, chymopapain, chymotrypsin, collagenase, and ficinase. (ficin), papain, peptidase, proteinase A, proteinase K, trypsin, microbial proteases, and mixtures thereof. The conditions of the hydrolysis reaction depend on the enzyme used. For example, when pepsin is used, the pH of the reaction mixture is about 2 to 5, and the enzyme concentration is about 0.001 to 10% by weight (preferably 〇.l-l〇g/l) of the collagen to be treated. Collagen can be purified by sinking from the aforementioned collagen-containing solution. This precipitation process can be repeated until the desired purity is achieved. In one embodiment, the collagen is precipitated by dialysis by dialysis of the aforementioned collagen-containing solution against a buffer solution in a dialysis tube or dialysis bag (with a molecular weight cutoff of about 12,000 to 14,000). In another embodiment, a salt (eg, alkali metal hydride 12 201103945 (eg, sodium carbonate)) is added to the collagen-containing solution to a concentration of 1 M to 4 M to precipitate collagen, and then collagen is collected by centrifugation. It is desalted by ultrafiltration, dialysis or diluted acid. In still another embodiment, the pH of the collagen-containing solution is adjusted to a collagen insoluble pH to precipitate collagen. See WO/2004/096384. Purification of collagen can also be achieved by a combination of the foregoing methods. The precipitated collagen can be resuspended and buffer exchanged over the ruthenium membrane. The collagen obtained by any of the foregoing methods can be cooled under vacuum; dried in the east. In addition, collagen can also be resuspended in a suitable solution. The present invention will be fully utilized by those skilled in the art based on the foregoing description without further detailed description. Accordingly, the following examples are merely illustrative and are not intended to limit the invention in any way. All documents cited herein are incorporated herein by reference. Example 1: Preparation of collagen matrix Skin tissue was obtained from pigs. After removing lipids, the epidermis was washed several times with saline, and the surface layer of the epidermis was removed with a dermatome to obtain a dermis group having a thickness of 0.3 mm (mm). Weaving. The dermal tissue is further washed with a phosphate buffer solution. After washing, all buffer solution residues were completely removed from the surface of the dermal tissue. The dermal tissue was placed in a container containing a 0.5 M acetic acid solution and allowed to stand at 37 ° C for one and a half days to expand the dermal tissue to a thickness of ^ cm. During the constant temperature placement, the container is placed on a vibrator to suspend the tissue.吏 Dermis The dermal tissue was then immersed in a solution containing 〇 5〇 SDS and 0.5 mM EDTA for two hours at room temperature to remove the non-gelatin 201103945 substance to produce a collagen matrix. The collagen matrix was washed with a sterilized phosphate buffer solution to remove residual SDS and EDTA. Example 2: Extraction of Collagen from Collagen Matrix The collagen matrix prepared by the method described in Example 1 was immersed in a 0.5 M acetic acid solution for 12-24 hours and stirred. After the resulting mixture was centrifuged at 2 rpm (700 x g) for 1 hour, the supernatant was collected and stored in the generation. The supernatant containing purified collagen was treated with pepsin (9) 2 mg/ml for 24 hours to produce an endopeptide collagen. Example 3: Purification of collagen by dialysis The collagen was extracted from the collagen matrix by the procedure described in Example 2 and a solution containing collagen was prepared. The solution was dialyzed against a 0.02 M sodium phosphate monobasic buffer with a cellulose membrane (MWC0 12-14, 〇〇〇) and centrifuged at 8000 X g for 1 hour. The peuett was collected and rinsed several times with cold MiiHQ water' and the pellet was then resuspended in cold MilliQ water. The suspension was centrifuged at 8000 xg for 1 hour. The resulting collagen pellet was resuspended in 0.1 M acetic acid. Example 4: Purification of collagen by salting-out or changing pH The collagen was extracted from the collagen matrix by the procedure described in Example 2 and a collagen-containing solution was prepared. Sodium vaporate was gradually added to the solution to a final concentration of 2.5 m. The precipitated collagen was collected and washed with distilled water and resuspended in acetic acid. Further, 1 M sodium hydroxide was added to the collagen-containing solution, and the pH was adjusted to 7. The mixture was placed in a cold room and stirred for 3 hours to precipitate collagen 201103945 protein. Thereafter, after the mixture was centrifuged at 4 ° C for 1 hour, the collagen pellet was resuspended in deionized water. The pH of the suspension was adjusted to less than 3.5 with 0.1 M hydrochloric acid to dissolve the collagen. Other Embodiments All of the features disclosed in the present invention should be implemented in any combination. Each feature disclosed in the present invention should be replaced by a substitute of the same, equal or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is merely an embodiment of the one of the From the above, it will be apparent to those skilled in the art that the present invention can be easily understood from the scope of the present invention without departing from the spirit and scope of the invention as defined by the appended claims. , when various changes, substitutions, and corrections can be made here. Therefore, other embodiments are also within the scope of the appended claims. [Simple description of the diagram] 15