201031746 六、發明說明： 【發明所屬之技術領域】 本發㈣’-種治療_檢方法以及椎間盤培養方法 根據本發明之治療劑篩檢方法可用以建立活 治療劑篩選平台。 ’ 【先前技術】 椎間盤位於兩個脊椎體之間，係由柔軟富膠質的髓核(nucleus m PUlp〇SUS)以及堅勃富纖維質的纖維外環(―fibrosus)所構成， 像是-個包果馨的甜甜圈。椎間盤容許脊椎體之間的活動， 當作-個脊椎體間的避震器，用以分散脊椎的壓力及扭曲力。 以人類為例，在年輕的時候，髓核裡有許多具有分裂及分化 能力的細胞，可以不斷製造新細胞和髓核讓椎間盤維持正常的功 能。然而，隨著年齡漸漸增長，椎間盤就會慢慢退化（大約從3〇 歲開始），髓核裡的細胞會慢慢凋亡而無法製造新髓核，致使 間盤内含的水分漸漸減少，同時喪失其承受形變的彈性。不僅如 此’纖維外環的包覆性也會隨著脊椎退化而減弱。 Φ 退化性椎間盤疾病(Degenerative Disc Disease, DDD)是一種椎 間盤老化的現象。除了前述的自然老化之外’因工作或活動所、生 成的重複性壓力及勞累也會減弱結缔組織對椎間盤的支持，、 椎間盤密度下降或改變。DDD最常發生在下脊部(腰椎）:再 脖子(頸椎）’但DDD可以發生在脊椎的任何部位。於實務中疋 DDD通常經由病例顯示，配合X光或其他檢查被診斷出來。 目前’ DDD治療的方法主要可分為三種：非手術性治 理治療、休息、抗發炎藥物）、手術治療(如果非手術療法不'能 輕症狀’可在確定痛源後予以手術治療)以及注射治療。 201031746 至』見固，非類固醇消炎止痛劑投藥 能纾緩病患的疼痛，對、、曰人工關即置換術。然而’止痛藥僅 置換則屬於大型手術、DDD本身並沒有幫助，而人工關節 的患者需要長時間的復Z手=過程的風險較大’且經過此手術 常需要再進行手術，置換後的幾相，病患通 期待能要找出闕「nm人員再度投人關節退化的基礎研究， φ 說，部分研究者透過床上、n」』Authentic施㈣。舉例來 方式來治療軟骨缺損，並且心t培養、移植、…等 究者將目標放在找出合適的治n夕成功的報告。此外’也有研 由於椎間盤組織屬於軟骨組織， $況下，組織所需之養份完全依賴渗透^1理 織適合在無血管供應養分_間盤組 關治療劑的筛檢。月/凡卜取出體外進仃組織培養與相 由於豬的基因與脊椎結構都與 用豬隻(如，迷你豬）當做試驗對象。m目=療劑筛檢常 ^研究人員通常會將豬隻分組並果t信 驗時’往往需要犧牲許多動物’也會耗複性， 空間及照顧之人力。此外，因為豬隻個體間差異^^本、飼養 呈現再現性會增加其困難度。 "大，试驗結果要 【發明内容】 因此本發明之一範轉在於提供一種治療劑餘格*ir、+ ra 建立治療麟選之平台，並解決先前技術巾帥題。法’用以 根據-具體實施例，本發明之治療劑篩檢方法包含下列步 201031746 ;該=盤自接，並以-第-無菌溶液處 中，並持續培養—預言J時間ϋ椎間盤置於-無菌培養環境 盤的影響。 、最後，砰估該待測治療劑對該椎間 種活=以=種 ❹ ❹ 椎間盤組射模擬體内退化性椎__，並聽治療m之 自一 Ui動以!發3椎養：t首先’ 菌溶液包含無菌生理養，間盤。其中該第一無 該破酒的比例介於】食二=及f之酉間並且該無菌生理食鹽水與 ===為獨立事件’成為每組試驗中二個個體，因 門及心之的猪隻數量’也因此可節省飼養成本、飼養空 以及每組重複之健 ，丄可大 取下至少十節-之 二量:高時:组間差異變小； ^ 複上發大明二療實 邊椎體肖及關樣肖_ ’模擬椎間健_境，因此載體外2 201031746 =大批次培養’作為高效率之促進制盤再 5 進行藥物有效性與安全性之測試 關 式得到轉輯可㈣㈣下的發舞述及所附 【實施方式】 圖 用以治療劑 在於触-餘療綱檢方法，201031746 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a method for screening a therapeutic agent according to the present invention. [Prior Art] The intervertebral disc is located between two vertebral bodies, consisting of a soft, medullary nucleus (nucleus m PUlp〇SUS) and a sturdy fibrous outer ring (-fibrosus), like a A sweet doughnut. The intervertebral disc allows for movement between the vertebral bodies and acts as a shock absorber between the vertebral bodies to dissipate the pressure and distortion of the spine. In humans, for example, at a young age, there are many cells in the nucleus with division and differentiation ability, which can continuously create new cells and nucleus pulposus to maintain normal function of the intervertebral disc. However, as the age increases, the intervertebral disc will slowly degenerate (about 3 years old), and the cells in the nucleus will slowly become apoptotic and will not be able to make a new nucleus, resulting in a gradual decrease in the water contained in the disc. At the same time, it loses its elasticity of deformation. Not only does the coating of the outer fiber ring weaken as the spine degenerates. Φ Degenerative Disc Disease (DDD) is a phenomenon of intervertebral disc aging. In addition to the aforementioned natural aging, repetitive stress and fatigue caused by work or activity can also weaken the support of the connective tissue to the intervertebral disc, and the density or change of the intervertebral disc. DDD occurs most often in the lower spine (lumbar spine): the neck (cervical spine) but DDD can occur anywhere in the spine. In practice, DDD is usually diagnosed by case, with X-ray or other tests. At present, 'DDD treatment methods can be mainly divided into three types: non-surgical treatment, rest, anti-inflammatory drugs), surgical treatment (if non-surgical treatment is not 'can be mild symptoms' can be treated after the determination of the source of pain) and injection treatment. 201031746 To "see solid, non-steroidal anti-inflammatory analgesic drugs can relieve the pain of patients, right, and artificial closure. However, the replacement of painkillers is a major surgery, DDD itself does not help, and patients with artificial joints need a long time to re-Z hands = the risk of the process is large, and after this operation, surgery is often required, after the replacement Phase, the patient is expected to find out "the basic research of nm personnel re-investing in joint degeneration, φ said that some researchers through the bed, n" Authentic Shi (four). Examples of ways to treat cartilage defects, and heart t culture, transplantation, ... etc. The goal is to find a suitable report on the success of the treatment. In addition, there is also research. Since the intervertebral disc tissue belongs to cartilage tissue, the nutrients required for tissue are completely dependent on the osmotic ^1 texture suitable for screening in the avascular supply of nutrients. Month/Fanbu was taken out of the in vitro tissue culture and phase. Because of the pig's gene and spine structure, pigs (eg, mini pigs) were used as test subjects. M-mesh = screening of therapeutic agents often ^ Researchers usually group pigs and test their results. 'There is often a need to sacrifice many animals'. It also consumes manpower, space and care. In addition, because of the differences between pigs, the reproducibility of feeding and breeding will increase its difficulty. " large, test results to be [invention] Therefore, one of the inventions is to provide a therapeutic agent, *ir, + ra to establish a platform for treatment of lining, and to solve the problem of the prior art towel. The method according to the specific embodiment, the therapeutic agent screening method of the present invention comprises the following step 201031746; the disk is self-connected, and is in the -first-sterile solution, and continues to culture - predicting J time ϋ intervertebral disc placement - The effect of sterile culture dishes. Finally, it is estimated that the therapeutic agent to be tested for the intervertebral seed activity = with the type of ❹ ❹ intervertebral disc group to simulate the degenerative vertebrae in the body __, and listen to the treatment of m from a Ui movement! t First 'bacteria solution contains sterile physiology, disc. Among them, the ratio of the first no-breaking wine is between the two of the two groups, and the aseptic physiological saline and the === are independent events' become two individuals in each group of experiments. The number of pigs can also save on feeding costs, rearing, and the health of each group. You can take at least ten knots - two of them: high: the difference between the groups becomes smaller; ^ Side vertebral body Xiao and Guan Xi Xiao _ 'simulating intervertebral health _ environment, so the carrier outside 2 201031746 = large batch culture' as a high efficiency promotion of the plate 5 to test the effectiveness and safety of the drug (4) (4) under the dance and attached [embodiment] Figure for the treatment of the touch-residue method,
5月一併參閱圖一以及圖二A至圖二E 圖一係綠示根據本發In May, please refer to Figure 1 and Figure 2A to Figure 2E.
EE
5一具體實施例的治療劑篩檢方法之流程圖。圖：日A、S 根據圖-的、冶療綱檢方法各步狀示干， 自法包含Μ步驟：料，執行步驟 盤。清注意，方 =二並且’該等椎間盤係取自該脊椎動物的適當脊椎部位(： 其第十節胸椎⑽)至第七節腰雜7)之間取 ^ R如圖二A所示，本具體實施例所使用之脊椎動物係約二至三 之成年迷你豬隻3。當然，於實際應財，也可選用A他 物(例如’但不限於，牛、兔子、鼠、猴子等)來進行。在 王身氣麻情況下犧牲後，盡快取出脊椎30 ,並自脊椎30取出痛 =固，整椎間盤3〇〇置於培養皿4〇中(如圖二3所示）。此椎間盤 匕含部分骨、終板(end plate)、纖維環300a以及中央之髓核 組織300b。該複數個椎間盤取下後，迅速將圍繞 = 肌肉及結締軟組織剃除，保持減汙染狀況下，迅速 胞培養室之無菌操作台做後續處理。 201031746 ^由實驗豬體内取下椎間盤組織時，仍有曝露於空氣中而導 可能性’因此如何保持零污染狀況，以避免組織在培養 =程^受到汙染，顯得格外重要。為改善無g取得組織之過程， =由，内取*賴盤時，先將之以該第—無菌溶液制，其中 if了無菌溶液包含無菌生理食鹽水以及蛾酒，並且該無菌生理 食鹽水與該碘酒的比例介於丨：1至1〇〇 :丨之間。 經可能會對細胞造絲害，因此吾人先測試組織在稀 ❿ 時間’並經由環狀切面觀察破酒是否會侵入組織 證實’在以前述的第—賴溶液處理後的椎間盤之外 ΐ衣么、f、、l酉侵入呈色痕跡。因此’本發明在取出椎間盤組織 1 : ^刖述之第一無菌溶液浸潤適當時間(例如，10〜600秒）， 以3有1至50倍抗生素之無菌生理食鹽水浸潤若干時間 4拉i〇 t匕鐘）:'經實驗證實’經此步驟處理的組織於無菌培養 相中★口養，皆無受感染之情形發生。 接著，執行步驟S12 ,如圖二c所示，以鑷子42挾梏兮姑 二US般並以針頭44將一分解溶液注射至該椎間盤300 :::去 隹間盤的部分中央髓核組織现。其中該分解溶液包含木瓜5 is a flow chart of a therapeutic screening method of a specific embodiment. Fig.: Days A and S According to the figure--, the method of physical therapy is shown in the steps of the method, and the self-method includes the steps: material, and the step is executed. Note that square = two and 'the intervertebral discs taken from the appropriate vertebrae of the vertebrate (: its tenth thoracic vertebrae (10)) to the seventh section of the waist (7) are taken as shown in Figure 2A. The vertebrate animals used in this embodiment are about two to three adult mini-pigs. Of course, in the actual financial situation, you can also choose A (such as 'but not limited to, cattle, rabbits, rats, monkeys, etc.) to carry out. After the sacrificial condition of the king, the spine 30 is removed as soon as possible, and the pain is removed from the spine 30. The entire disc 3 is placed in the petri dish (as shown in Figure 2). The intervertebral disc contains a portion of bone, an end plate, an annulus 300a, and a central nucleus pulposus tissue 300b. After the multiple intervertebral discs are removed, the muscles and connective soft tissues are quickly shaved, and the aseptic processing table of the cell culture chamber is quickly processed for subsequent treatment. 201031746 ^ When the intervertebral disc tissue is removed from the experimental pigs, there is still exposure to the air and the possibility of 'how to maintain zero pollution', so as to avoid tissue contamination in the culture = process ^ is particularly important. In order to improve the process of obtaining the tissue without g, the internal solution is taken as the first sterile solution, wherein the sterile solution contains sterile physiological saline and moth wine, and the sterile physiological saline solution The ratio to the iodine is between 丨:1 to 1〇〇:丨. It may cause damage to the cells, so we first test the tissue at the time of dilute and observe whether the broken wine will invade the tissue through the circular section to confirm 'in the case of the intervertebral disc treated with the aforementioned first-lying solution. , f,, l酉 invade the traces of color. Therefore, the present invention in the removal of the intervertebral disc tissue 1: the first sterile solution infiltrated for a suitable time (for example, 10 to 600 seconds), infiltrated with 3 times 1 to 50 times the sterile physiological saline of the antibiotic for some time 4 〇i〇 t匕钟): 'It has been confirmed by experiments that the tissue treated by this step is in the sterile culture phase. ★ The mouth is no infection. Next, step S12 is performed, as shown in FIG. 2c, and a decomposition solution is injected into the intervertebral disc 300 with a needle 44 as follows: a part of the central nucleus pulposus tissue of the intercondylar disc is present. . Wherein the decomposition solution comprises papaya
之F1C ，並且該木瓜酵素之濃度介於lou至1，000U 行^述^ 應用中’也可選用其他適#的分解溶液來進 :現阳：二=軟= 們在模擬體内組織培養時，先分別以不同濃产 木Ν酵素作測試’發現一周後，lou至1000U濃声 二 核_ ’且能餘留些許之髓^織，作為 ' 夺之模板’因此往後的試驗設計將以此為依據。 如圖一 D所不，注射木瓜酵素後，將椎間盤3〇〇置於試管 201031746 R -中卞又時,’等待木瓜酵素清除中央髓核組'織300b。如 300 用t在將待測治療劑注射至該椎間盤獅之中央 二、dr 1可先以滅菌生理*鹽水沖洗該中央空腔3000，以 去除被桃之t央髓她織3_及多制木瓜酵素。F1C, and the concentration of the papaya enzyme is between lou and 1,000 U. In the application, 'you can also use other decomposition solutions for the solution: Yang: two = soft = when we simulate tissue culture in vivo First, test with different concentrated hibiscus enzymes. 'After a week, lou to 1000U thick sound two core _ 'and can leave a little more marrow, as a template for 'winning', so the future design will be This is the basis. As shown in Figure 1, D, after injecting papaya enzyme, the intervertebral disc 3〇〇 was placed in a test tube. 201031746 R - Lieutenant was again, 'waiting for papaya enzyme to clear the central nucleus pulpus group' woven 300b. For example, if 300 is used to inject the therapeutic agent to be tested into the center of the intervertebral disc lion, dr 1 may first rinse the central cavity 3000 with sterile physiological saline solution to remove the meditation of the peach. Papaya enzymes.
城ίί ^彳!^S14 ’如圖二E所示，注職制治療劑至 的中央空腔中。於本具體實施例中，吾人係以2〇G 泳作為注射該待測物質的媒介。因此，我們預先測試， 中央髓核體積約為〇.01〜lml，而脊髓注射針體積約為 •〜m ’因此所注射之該待測物質總體積需取約g〇〇imi，才 生在注射時有外漏或是注射體積不足之情形發生。該待測 /口療劑包含虽含血小板血漿(platelet_rich咖腹，pRp)衍生之生 長因子複合物及/或間葉幹細胞(mesenchymal stem cells, MSCs)及/ 或胚胎幹細胞(embryonic stem eelk，耶⑶。 參 ，後，，行步驟S16，將注射該待測物質後之該椎間盤置於 一無菌培養環境中，並持續培養一預設時間(如圖二D所示）。在 實際應用中，該無菌培養環境存在於一 DMEM/F12培養基中， 並且該預設時間係一至二個月。 於一具體實施例中，將該椎間盤經過無菌過程處理後，置於 含有培養基DMEM/F12(w/ 10%FBS)之50ml試管中，並移至細胞 培養箱培養。經過四周(W4)後將之取出，萃取中央髓核組織之 抓八’測定其主要軟骨分化基因表現，並以體内組織(in vivo IVD tissue，W0)作為對照。 最後’執行步驟S18 ’評估該待測治療劑對該椎間盤的影 響。我們以外型巨觀、基因檢測、蛋白質表現、組織染色等方 式，來判定缺損組織再生情形： 201031746 i. 巨觀觀察(macromorphology) 組織取下後’以手術刀(20G)延冠狀切面切下，並以高解析度 鏡頭(UVP Bioimaging system)照相定量，可直接觀察中央腾核組 之外觀，正常髓核會表現組織飽滿度，並呈透明狀態，代表其保 水功能正常，能維持組織内膨壓，抵抗外來應力，並維持生物體 脊椎高度。 11. 基因表現(RT-PCR) 觀察完巨觀外型後，小心取下髓核組織，萃取其totalRNA， 再經過RT-PCR ’檢測軟骨特異性分化基因，主要包含π collagen以及蛋白酷類aggrecan，證明再生物質之初步效果。 iii. 矩陣基因分化路徑探討(Real_time pCR_based町吵assay) 孝人骨細胞受到在生物質誘導後，會經由特定路徑進行軟骨分 化過程。我們將其應用至矩陣基因分析套組(SuperajTaykit^，可 在一批次實驗後，大量分析並篩選其表現之路徑相關基因。 iv. 組織免疫及特異性染色 々當軟骨細胞分化成熟後，會大量表現軟骨基質是其最大特 徵，我們以組織免疫染色及蛋白醣類特異染色來證實其組織分化 程度為了證實組織是否會過度分化而導致骨組織產生，我們亦 iLP以及職—a疏來特異性觀察其骨基質於組織中 >儿降情形。 =明之另-_在於提供—種椎間盤培養方法，用以模擬 體内退化性椎間盤之組織培養。 般拉^，圖二。圖二係繪示根據本發明之—具體實施例的椎間 。法之流程圖。根據一具體實施例，本發明之椎間盤培養 201031746 Ϊ ii 自一脊椎動物獲得複數娜間 椎間ί，容液分別處理該複數個 無菌溶液包含無菌生理食鹽水3填酒， 間。該…、鹵生理艮鹽水與該碘酒的比例介於i:丨至ι〇〇 ··工之 ❹ 待測===:=;?養:法的可行性，並且對照 Ϊ醉ί:木瓜酵素(chymopa二:該 树明出制触齡行讀，並與經由 骨特!=椎核:後，利用_r比較軟 白多酿Ααα括第；婦原蛋白咖11 （C〇1 Π)及/或蛋 象，其美體外培養並無降低其軟骨特異基因之現 i可=現她，因錄龍外培獅__可替代體内 椎間建立體外椎間盤組織培養系統，以為未來 ί^ίί 2師選建立一筛選平台’並以幹細胞及卿衍生之 實實其應用性，並驗證於動物試财。結果皆證 分化，並^kH般^長因子複合物能在體外被證實能促進軟骨 並在椎間盤組織内增進細胞外間質之沉積。 者自t治療都是目前最朗的研究棚，運用患 性椎間盤組堪優勢。而本發明中所建立的體外退化 么，延續拄亦可作為促進椎間盤再生之藥物筛選平 口 l續在後之軟骨再生藥物開發。 201031746 相較於先别技術，本發明之治療 、 行，能將實驗規模從複雜的大型動物實 體外進 能進行的組織培養試驗，大大縮驗室即 邊椎體骨及關節軟骨組織，模擬^間^ 組織完整保留周 =大批次培養’作為高效率之促進椎間^物 術’以更縮短研發治療椎間盤退化性_ 干台技 進行藥物有效性與安全性之測試。/、纟夺程’亦可同時 發明:具例之詳述，係希望能更加清楚描迷本 _目等性的安排於本侧”請之City ίί ^彳!^S14 ’ As shown in Figure 2E, the therapeutic agent is injected into the central cavity. In this embodiment, we use 2〇G swimming as a medium for injecting the test substance. Therefore, we pre-tested that the volume of the central nucleus pulposus is about 〇.01~lml, and the volume of the spinal cord injection needle is about ~~m'. Therefore, the total volume of the substance to be tested injected should be about g〇〇imi. There is an external leak during injection or an insufficient injection volume. The test/oral therapy agent comprises growth factor complexes and/or mesenchymal stem cells (MSCs) derived from platelet plasma (platelet_rich barley, pRp) and/or embryonic stem cells (embryonic stem eelk, yeah (3) After the step S16, the intervertebral disc after the injection of the test substance is placed in a sterile culture environment, and is continuously cultured for a preset time (as shown in FIG. 2D). In practical applications, the The sterile culture environment is present in a DMEM/F12 medium, and the predetermined time is one to two months. In one embodiment, the intervertebral disc is subjected to a sterile process and then placed in a medium containing DMEM/F12 (w/10). In a 50 ml tube of %FBS), transfer it to a cell culture incubator. After four weeks (W4), remove it and extract the central nucleus pulposus tissue to measure its major cartilage differentiation gene expression and organize it in vivo (in Vivo IVD tissue, W0) as a control. Finally, 'execute step S18' to evaluate the effect of the therapeutic agent to be tested on the intervertebral disc. We have extravagant macroscopic, genetic testing, protein expression, tissue staining, etc. Defective tissue regeneration: 201031746 i. Macromorphology After the tissue is removed, it is cut with a scalpel (20G) crown-shaped section and photographed by a high-resolution lens (UVP Bioimaging system). The appearance of the nucleus group, the normal nucleus pulpus will show tissue fullness, and it is transparent, which means that its water retention function is normal, it can maintain swell pressure within the tissue, resist external stress, and maintain the height of the spine of the organism. -PCR) After observing the macroscopic appearance, carefully remove the nucleus pulposus tissue, extract its total RNA, and then detect the cartilage-specific differentiation gene by RT-PCR, which mainly contains π collagen and protein aggrecan, which proves the initial of the regenerative substance. Effect iii. Matrix gene differentiation pathway (Real_time pCR_based 吵 assa assay) 孝 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨 骨After a batch of experiments, a large number of path-related genes were analyzed and screened. iv. Tissue immunity and specific staining When the chondrocytes differentiate and mature, the cartilage matrix is the most characteristic. We use tissue immunostaining and protein saccharide-specific staining to confirm the degree of tissue differentiation. In order to confirm whether the tissue will over-differentiate and cause bone tissue, we also iLP and occupation-a sparsely observe the bone matrix in the tissue> child drop. =Mingzhi-_ is to provide a disc culture method to simulate tissue culture of degenerative intervertebral disc in vivo. General pull ^, Figure 2. Figure 2 illustrates an intervertebral space in accordance with an embodiment of the present invention. Flow chart of the law. According to a specific embodiment, the intervertebral disc culture of the present invention 201031746 Ϊ ii obtains a plurality of intervertebral discs from a vertebrate, and the liquid solution separately treats the plurality of sterile solutions containing sterile physiological saline 3 filling. The ratio of the brine to the iodine is between i: 丨 to ι〇〇··工之❹ to be tested ===:=; 养: the feasibility of the law, and the control is drunk ί: papaya Enzyme (chymopa II: The tree is clearly read by the age of the tree, and with the bones through the bones! = vertebral nucleus: after the use of _r compared with soft white and more oysters αα; the original protein coffee 11 (C〇1 Π) and / or egg, its beauty in vitro culture does not reduce its cartilage-specific genes can now = now, because the recording of the dragon outside the lion __ can replace the internal intervertebral disc to establish an extracorporeal disc tissue culture system, thinking of the future ί^ίί 2 divisions to establish a screening platform 'and the practical application of stem cells and Qing, and verified the animal test. The results are all differentiated, and ^kH-like long-term factor complex can be confirmed in vitro to promote Cartilage and the deposition of extracellular interstitial space in the intervertebral disc tissue. The treatment of t is the most research shed at present, and it is advantageous to use the intervertebral disc group. However, the in vitro degradation established in the present invention can be continued. As a drug to promote the regeneration of intervertebral discs, the development of cartilage regenerative drugs continued. Compared with the prior art, the treatment and treatment of the present invention can carry out the tissue culture test of the scale of the experiment from the complex large animal body, and greatly reduce the room and the vertebral body bone and the articular cartilage tissue, and simulate ^^ Tissue complete retention week = large batch culture 'as a high-efficiency promotion of intervertebral body surgery' to further shorten the development of treatment of disc degeneration _ dry Taiwan technology for drug efficacy and safety testing. /, 纟 程 ' ' Simultaneous invention: a detailed description of the case, it is hoped that the arrangement of this article can be more clearly described on the side.
11 201031746 【圖式簡單說明】 圖一係繪示根據本發明之一具體實施例的治療劑篩檢方法 流程圖。 圖二A至圖二E則係繪示根據圖一的治療劑篩檢方法各步驟 之不意圖。 m 圖三係繪示根據本發明之-具體實施例的椎間盤培養方法之 流程圖。 【主要元件符號說明】 S10〜S18、S50〜S54 :流程步驟 鬌 3 :豬隻 300 :椎間盤 300b :聽核組織 40 :培養皿 44 :針頭 30 :脊椎 300a :纖維環 3000 :中央空腔 42 .録子 46 :試管 1211 201031746 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing a method of screening a therapeutic agent according to an embodiment of the present invention. Figures 2A through 2E illustrate the various steps of the therapeutic screening method according to Figure 1. m Figure 3 is a flow chart showing a method of culturing a disc according to a specific embodiment of the present invention. [Description of main component symbols] S10~S18, S50~S54: Flow procedure 鬌3: Pig 300: Intervertebral disc 300b: Auditory nuclear tissue 40: Petri dish 44: Needle 30: Spine 300a: Fiber ring 3000: Central cavity 42. Record 46: Test tube 12