570823 玖、發明說明 (發明說明應敘明：發明所屬之技術領域、先前技術、內容、實施方式及圖式簡單說明) ㈠發明所屬之技術領域 本發明係關於一種巨分子導入細胞之方法，特別地指一 種直接將巨分子施打在活體組織上，在注入組織之同時施 加傳送巨分子進入細胞所需電壓之新穎方法，以及藉以實 現此方法之注射器裝置。 ㈡先前技術 查傳統生物技術領域中，欲將核酸（基因）、或帶有功 能的蛋白質分子植入細胞中，一般而言，分子量在3000以 下之有機分子，採用浸泡方式可被細胞直接吸收，分子量 若超過3000以上之有機分子（即所謂巨分子），則無法直 接被細胞所吸收，而須施加外力或另行處理方可爲細胞所 吸收，唯困難在於它非常的微小，無法使用一般器械達成 之。 現行廣泛採用一種”電穿孔技術”，即利用電場原理刺激 細胞膜瞬間開口，使巨分子滲透入細胞膜內。使用方法大 致上例如第1圖所示，先將目標個體之組織取出，打碎後 篩選游離的單一細胞浸泡在含有巨分子溶液中，再將放電 儀之正、負電極與該溶液接通產生間歇性電擊；誠如第2 圖所示，細胞在含有巨分子溶液中受直形波週期性脈衝放 電下，通電時細胞膜表面產生無數的開口，斷電時又密合 ，如此在斷、續通電下成週期性地收縮、與舒張，猶如吸 球一般將細胞膜外的巨分子吸入。上述處理過程中，非但 一 5- 570823 手續繁瑣，且往往因環境因素而導致細胞死亡，成功率不 盡理想。 另外，在遺傳工程和基因療法之硏究中，對於活體目標 組織進行基因轉殖時，同樣地必須將含有所欲導入之巨分 子、或救援基因等分子先行注入組織中，再以電極穿刺分 兩步驟進行。惟，目前使用之裝置，電極並無絕緣處理， 而是正、負電極整支裸露通電，因此電場範圍擴散，影響 目標部位組織以外的正常組織細胞，故僅能針對目標個體 進行表層使用，而無法適用於較深層、或內臟器官、或較 小的組織等進行電轉殖。 ㈢發明內容 本發明之主要目的在於提供一種細胞電壓注射方法，可 對活體目標將巨分子導入組織細胞中，或對病兆組織進行 藥物導入作醫療用途等。 根據本發明方法，其係將一電極針帶有正電極之針部， 及另一裝塡有巨分子藥液之注射筒其針頭並夾帶有負電極 之注射針，穿刺入目標個體之目標部位組織中，在注入巨 分子藥液至組織時施加傳送巨分子進入細胞所需之電壓， 一次同時完成。 本發明之另一目的在於提供一種細胞電壓注射器，可對 活體目標之較小組織、或涂層組織、或特疋組織、甚或內 臟器官組織等進行巨分子電破導入。 爲了達成上述目的，其包括：一電極針帶有正電極之針 部經絕緣處理，僅尖端裸露；另一注射巨分子之注射筒針 一 6 - 570823 頭中夾帶有負電極之注射針，表面且經絕緣處理，僅尖端 裸露’利用尖端放電使電場集中在目標組織之局部位置， 而不影響目標部位組織以外的正常組織細胞。 ㈣實施方式 以下將配合實施例對本發明技術特點作進一步地說明， 該實施例僅爲較佳的範例並非用來限定本發明之實施範圍 ’謹藉由參考附圖結合下列詳細說明而獲致最好的理解。 首先請參考第3圖並對照第4圖，本發明細胞電壓注射方 法，其執行步驟包括： 準備一電極針10，其針部101帶有正電極； 準備另一裝塡有巨分子藥液之注射筒20，其注射針21 自針頭20a內夾帶有負電極211並延伸至尖端； 將電極針1 0及注射針2 1穿刺入目標個體30之目標部位 組織中； 在注入巨分子藥液至組織時，在電極針1 〇之正電極1 〇 1 、及注射針2 1之負電極2 1 1施加瞬間高電壓，刺激細胞膜 開孔，使藥液滲入細胞內。 如此，注入巨分子藥液至組織之同時施予電壓於正、負 電極針，使執行巨分子進入細胞所需之作業一次完成，故 可對活體目標將巨分子導入組織細胞中，或對病兆組織進 行藥物導入作醫療用途等。 根據本發明，爲了能夠針對活體目標之較小組織、或較 深組織、或特定組織、甚或內臟器官組織等進行巨分子電 破導入，而不影響其他正常細胞，本發明同時提供了一種 -Ί 一 570823 細胞電壓注射器1，誠如第4圖所示，其包括：一注射筒2 ο ，具備如習知構造之容納室2 0 1，用以容納巨分子藥液， 一端設有推進藥液之栓塞裝置202 (剖面圖中未示出已省 略），另一端包含有一注射針2 1，該注射針2 1自針頭2 0 a 內夾帶有一負電極211，並延伸至尖端。另外，一電極針1〇 ，一端包含有一柄部10a，及一帶有正電極之針部101。一 放電儀40 ’經控制可筒壓瞬間放電，輸出端之負極連接在 負電極2 1 1之遠端，正極則與電極針1 〇之遠端相連接。 根據本發明，電極針1 0之針部1 0 1表面包覆有絕緣物1 〇丨a 胃 例如鐵弗龍等，僅尖端裸露；及，注射筒20之注射針2 1 表面包覆有絕緣物2 1 a例如塑膠材料等，僅尖端裸露。如 此’當將電極針1 0針部101、及注射針21穿刺入目標個 體之組織部位中，利用尖端放電使電場集中在目標組織之 局部位置’而不影響目標部位組織以外的正常組織細胞。 再如第5及6圖所示，根據本發明爲了操作之方便性， 上述注射器1可設置在一支持架50上，該支持架50包含 · 有一固著裝置5 1，例如習知的活動環扣件、及挾持件等， 可固定注射器1之注射筒20及電極針1 〇等；一距離調整 裝置5 2，例如習知的螺桿微調裝置，可調整注射針2 1與 電極針10針部101間的距離（誠如第5圖所示）；及，一 角度調整裝置5 3，例如習知的螺桿微調裝置，近針尖之一 端定位、利用遠端微調等，可調整注射針21與電極針1 0 針部101間的角度等（誠如第6圖所示）。此外，上述注 射器1之電極針1 0、及注射針2 1等，可直接聯結在線性 一 8 - 570823 波放電器、或指數型非線性波放電器上，因此適用範圍非 常廣泛，在產業上可對動物、或水生動物等的生殖巢進行 基因轉殖，或基因疫苗、及救援基因的導入進行基因治療 等，甚至能應用在醫療用途上，例如對病變之癌細胞導入 β 帶有核酸（自殺基因）、或治療藥劑等分子。 以上，僅爲本發明的較佳實施例，並不侷限本發明的實 施形式及範圍，熟習此項技術者應可以理解的是，上述注 射器依操作上之需要，亦可設置在一習知的遙控機械手臂 上，由遙控機械手臂來執行穿刺以及藥液的推進等；亦即 ，不偏離本發明申請專利範圍所作之均等變化與修飾，應 仍屬本發明之涵蓋範圍。 綜上所述，利用本發明細胞電壓注射方法及注射器，在 注入巨分子藥液至組織之同時施予傳送巨分子進入細胞所 需之電壓一次完成，不僅可將巨分子導入活體目標組織細 胞中，或對病兆組織.進行藥物導入作醫療用途等，更能夠 針對活體目標之較小組織、或較深組織、或特定組織、甚 φ 或內臟器官組織等進行巨分子電破導入，而不影響其他正 常細胞；實爲一新穎、進步且具產業利用性之發明。 ㈤圖式簡單說明 第1圖爲傳統將巨分子導入細胞的方法及裝置之示意圖 〇 第2圖爲第1圖之電破過程中細胞週期性變化之示意圖 〇 第3圖爲本發明細胞電壓注射方法之立體示意圖。 -9- 570823 第4圖爲本發明細胞電壓注射器之示意圖，其中局部已 剖開並放大。 第5圖爲本發明細胞電壓注射器之第二種實施例。 .第6圖爲本發明細胞電壓注射器之第三種實施例。 主要部分之代表符號說明 1 .. · · · 本發明注射器 10 電極針 10a . .柄部 101 ... .針部/正電極 101a .絕緣物 20 _注射筒 20a . .針頭 201 . .容納室 202 . .栓塞裝置 2 1 .... .注射針 21 a . .絕緣物 21 1 . ...負電極 30 .目標個體 4 0 .放電儀 5 0 .支持架 5 1 .固著裝置 5 2 · ·· .距離調整裝置 5 3 ... .角度調整裝置570823 发明 Description of the invention (The description of the invention should state: the technical field to which the invention belongs, the prior art, the content, the embodiments, and the drawings briefly) ㈠The technical field to which the invention belongs The present invention relates to a method for introducing macromolecules into cells, particularly Land refers to a novel method of directly applying macromolecules to living tissue, and applying the voltage required to transport the macromolecules into the cell while injecting the tissue, and a syringe device for realizing this method. ㈡In the prior art, in the field of traditional biotechnology, nucleic acids (genes) or protein molecules with functions are to be implanted into cells. Generally speaking, organic molecules with a molecular weight of less than 3000 can be directly absorbed by the cells by soaking. Organic molecules with molecular weights above 3000 (so-called macromolecules) cannot be directly absorbed by the cells, and must be applied by external forces or additional treatments. The only difficulty is that they are very small and cannot be achieved with ordinary equipment. Of it. Currently, a kind of "electroporation technology" is widely used, that is, the principle of electric field is used to stimulate the cell membrane to open instantly, so that macromolecules penetrate into the cell membrane. The method of use is roughly as shown in Figure 1. First, the target individual's tissue is removed, and the single cells are broken and screened to soak in a solution containing macromolecules, and then the positive and negative electrodes of the discharge meter are connected to the solution to produce Intermittent electric shock; as shown in Figure 2, under the periodic pulse discharge of a linear wave in a solution containing a macromolecule, the cell membrane surface has numerous openings when it is energized, and closes when it is powered off. Under electricity, it periodically contracts and relaxes, as if sucking a ball, sucking macromolecules outside the cell membrane. In the above-mentioned treatment process, not only the process of 5-570823 is cumbersome, but often the cells die due to environmental factors, the success rate is not ideal. In addition, in the research of genetic engineering and gene therapy, in the case of transgenic living tissues, it is also necessary to inject molecules containing macromolecules to be introduced or rescue genes into tissues first, and then puncture them with electrodes. Two steps. However, in the currently used devices, the electrodes are not insulated, but the entire positive and negative electrodes are exposed to electricity. Therefore, the electric field spreads and affects normal tissue cells other than the target tissue. Therefore, it can only be used on the surface of the target individual and cannot be used. It is suitable for deeper, internal organs, or smaller tissues for electrotransplantation. ㈢ Summary of the Invention The main object of the present invention is to provide a method for cell voltage injection, which can introduce macromolecules into tissue cells for living targets, or introduce drug into diseased tissues for medical purposes. According to the method of the present invention, one electrode needle is provided with a positive electrode needle portion, and the other injection syringe is filled with a macromolecular drug solution, and the needle is inserted into the injection needle with a negative electrode to penetrate the target site of the target individual. In tissues, when the macromolecular liquid is injected into the tissue, the voltage required to transport the macromolecules into the cells is applied at the same time. Another object of the present invention is to provide a cell voltage injector, which can introduce macromolecular electrolysis into small tissues, coated tissues, special tissues, or even internal organ tissues of a living target. In order to achieve the above purpose, it includes: an electrode needle with a positive electrode is insulated, and only the tip is exposed; another injection of a macromolecular syringe needle 6-570823, an injection needle with a negative electrode in the head, and After the insulation treatment, only the tip is exposed. The tip discharge is used to concentrate the electric field at a local position of the target tissue without affecting normal tissue cells other than the target tissue. ㈣Implementation The technical characteristics of the present invention will be further described below with reference to an embodiment. This embodiment is only a good example and is not intended to limit the scope of the present invention. Understanding. First, please refer to FIG. 3 and compare with FIG. 4. The cell voltage injection method according to the present invention includes the following steps: preparing an electrode needle 10 with a positive electrode 101 on its needle portion; preparing another one containing a macromolecular liquid Syringe 20, the injection needle 21 of which includes a negative electrode 211 inside the needle 20a and extends to the tip; pierce the electrode needle 10 and the injection needle 21 into the target site tissue of the target individual 30; and inject the macromolecular liquid to During tissue organization, an instantaneous high voltage is applied to the positive electrode 1 0 1 of the electrode needle 10 and the negative electrode 2 1 1 of the injection needle 21 to stimulate the opening of the cell membrane to allow the drug solution to penetrate into the cell. In this way, when the macromolecular liquid is injected into the tissue and voltage is applied to the positive and negative electrode needles, the task required to perform the entry of the macromolecule into the cell is completed at one time. Therefore, the macromolecule can be introduced into the tissue cell for the living target, or the disease Mega organizations conduct drug introduction for medical purposes, etc. According to the present invention, in order to enable the introduction of macromolecular electrolysis for small tissues, deep tissues, or specific tissues, or even internal organ tissues of a living target without affecting other normal cells, the present invention also provides a -Ί A 570823 cell voltage syringe 1, as shown in FIG. 4, includes: a syringe 2 ο, equipped with a well-known accommodating chamber 2 0 1 for accommodating a macromolecular liquid, and one end is provided with a propelling liquid The embolization device 202 (not shown in the sectional view and omitted) has an injection needle 21 at the other end. The injection needle 21 has a negative electrode 211 inside the needle 20 a and extends to the tip. In addition, an electrode needle 10 includes a handle portion 10a at one end and a needle portion 101 with a positive electrode. A discharge meter 40 ′ can be controlled to perform instantaneous discharge by controlling the cylinder pressure. The negative terminal of the output terminal is connected to the distal end of the negative electrode 2 1 1, and the positive electrode is connected to the distal end of the electrode pin 10. According to the present invention, the surface of the needle portion 10 of the electrode needle 10 is covered with an insulator 1 〇a a stomach such as Teflon and only the tip is exposed; and the surface of the injection needle 2 1 of the syringe 20 is covered with insulation Objects 2 1 a, such as plastic materials, have bare tips only. In this way, when the electrode needle 10 and the injection needle 21 and the injection needle 21 are penetrated into the tissue site of the target individual, the tip discharge is used to concentrate the electric field at the local position of the target tissue without affecting normal tissue cells other than the target tissue. As shown in Figs. 5 and 6, according to the present invention, for the convenience of operation, the above-mentioned syringe 1 may be disposed on a support frame 50. The support frame 50 includes a fixing device 51, such as a conventional movable ring. Fasteners and holders can fix the syringe 20 and electrode needle 10 of the syringe 1; a distance adjustment device 52, such as the conventional screw fine adjustment device, can adjust the injection needle 21 and the electrode needle 10 needles The distance between 101 (as shown in Figure 5); and, an angle adjustment device 53, such as the conventional screw fine adjustment device, positioning near one end of the needle tip, and using remote adjustment, can adjust the injection needle 21 and the electrode The angle between the needles 10 and 101 (as shown in Figure 6). In addition, the electrode needle 10 and the injection needle 21 of the syringe 1 can be directly connected to a linear 8-570823 wave arrester or an exponential non-linear wave arrester. Therefore, it has a wide range of applications and is industrially applicable. It can be used to transgene the germ nest of animals or aquatic animals, or to introduce gene vaccines and rescue genes for gene therapy. It can even be used in medical applications, such as introducing β-nucleic acid to diseased cancer cells ( Suicide genes), or therapeutic agents. The above are only the preferred embodiments of the present invention, and do not limit the implementation form and scope of the present invention. Those skilled in the art should understand that the above-mentioned syringes can also be set in a conventional On the remote control robotic arm, the remote control robotic arm performs puncture and advancing of the medicinal solution; that is, equal changes and modifications made without departing from the scope of the patent application for the present invention should still fall within the scope of the present invention. In summary, by using the cell voltage injection method and syringe of the present invention, the macromolecular liquid is injected into the tissue and the voltage required to transport the macromolecules into the cell is given at one time, which can not only introduce the macromolecules into the living target tissue cells Or for the introduction of medicines into diseased tissues for medical purposes, etc., it is more able to introduce macromolecular electrolysis for small or deep tissues of living targets, or specific tissues, or even internal organ tissues, without Affects other normal cells; it is a novel, progressive, and industrially usable invention. ㈤Schematic description. Figure 1 is a schematic diagram of the traditional method and device for introducing macromolecules into cells. Figure 2 is a schematic diagram of the periodic changes of cells during the electrolysis process of Figure 1. Figure 3 is the cell voltage injection of the present invention. Schematic illustration of the method. -9-570823 Fig. 4 is a schematic diagram of a cell voltage injector of the present invention, in which a part has been cut away and enlarged. Figure 5 is a second embodiment of the cell voltage injector of the present invention. Figure 6 shows a third embodiment of the cell voltage injector of the present invention. Description of the main symbols of the main parts 1. The electrode 10a of the syringe 10 of the present invention. The handle 101 ... The needle / positive electrode 101a. The insulator 20 _injection cylinder 20a .. the needle 201. 202. Embolization device 2 1... Needle 21 a.. Insulator 21 1... Negative electrode 30. Target individual 40. Discharger 50. Support frame 5 1. Fixing device 5 2 ···. Distance adjustment device 5 3 .... Angle adjustment device