臺灣博碩士論文加值系統

本研究以廢棄豬眼球作為原料，分為現場採樣及冷凍解凍處理組，現場採樣處理組於豬隻屠宰後立即取出眼球內玻璃體並萃取其玻尿酸，分別以A、B及C三種純化方法進行萃取純化，經冷凍乾燥後成玻尿酸萃取物(hyaluronic acid, HA)粉末，進行成分分析。冷凍解凍處理組將眼球摘下後急速冷凍，分別以室溫解凍(room temperature, RT)、冷藏解凍 (refrigeration, 4℃)及遠紅外線解凍(far infrared, FIR)三種方法進行解凍，再以現場採樣處理組中最佳萃取方法進行冷凍解凍處理組HA之萃取純化並分析其成分；根據成分分析結果選出豬眼球玻璃體HA最佳萃取方法之成品進行抗氧化能力分析及細胞試驗，細胞試驗中成品與人類軟骨肉瘤細胞(SW1353)共培養，探討其對於促進細胞增生、Interleukin-1β (IL-1β)誘導下細胞存活率、蛋白聚醣(Proteoglycan, PG)分泌情形及退化性關節炎相關細胞因子基因表現量之影響，藉此以體外模式初步評估HA延緩退化性關節炎病程之能力。
結果顯示：每顆豬眼球中約有3.63克之玻璃體及0.48克之水晶體。現場採樣處理組玻璃體呈透明無色、黏稠狀，水晶體呈完整之橢圓形，而冷凍解凍處理組之水晶體被分解難與玻璃體做分離，且玻璃體混濁為黑色液體狀，與血液混合。現場採樣處理組每100克玻璃體用A、B及C三種萃取法，分別可萃取出2.3588 mg、1.2027 mg及2.1728 mg之純HA，其分子量800 kDa以上分別有20.797%、9.093%及45.430%，而冷凍解凍處理組用RT、4℃及FIR三種方法解凍再以C法萃取HA，分別可萃取出0.8957 mg、0.9262 mg及1.1589 mg之純HA，但HA被分解，分子量皆小於50 kDa。因此選擇現場採樣C處理組之冷凍乾燥成品進行後續試驗，HA成品製備成濃度為0.1、0.3、0.5、1.0及10 mg/mL之溶液，另有僅添加IL-1β誘發發炎之negative control (NC)組，無添加IL-1β及HA之blank組當作對照。體外抗氧化能力試驗中，豬玻璃體HA具有良好之螯合亞鐵離子能力，濃度高之處理組效果最佳，其於10 mg/mL之螯合率可達53.99%；DPPH自由基清除能力及還原力之抗氧化效果較差，且不同濃度之HA間無顯著差異；超氧陰離子清除能力方面，隨著HA濃度下降，超氧陰離子清除能力越低。HA於細胞增生試驗中，共培養1天後，各濃度處理組細胞增生量低於blank組，培養第3天各濃度處理組細胞增生量顯有高於blank組的趨勢，第5天後各濃度處理組細胞增生量皆低於blank組(p < 0.05)。在IL-1β誘發發炎下細胞增生率試驗中共培養5天後，與blank組相比NC組有下降之趨勢，而各濃度處理組中以HA 1.0 mg/mL組之細胞增生量顯著高於NC組(p < 0.05)，且隨濃度降低，增生量逐漸降低。在IL-1β誘發發炎下HA對SW1353蛋白聚醣分泌量之影響，NC組之蛋白聚醣分泌量顯著低於blank組，而各濃度HA處理組之蛋白聚醣分泌量顯著高於NC組(p < 0.05)，且隨著濃度增加，蛋白聚醣分泌量增加。HA在IL-1β誘發發炎下SW1353中促發炎細胞因子基因表現量之結果方面，MMP-3、MMP-13、TNF-α及IL-8之表現量，NC組顯著高於blank組，而各濃度HA處理組濃度高者抑制能力越佳，HA 1.0組顯著低於NC組(p < 0.05)；而iNOS之表現量NC組低於blank組，但無顯著差異，HA處理組iNOS表現量有較低之趨勢，但無顯著差異。綜觀上述，本試驗從豬眼球萃取玻尿酸最佳的方法為現場採樣C處理組，其可萃取較多之玻尿酸，且分子量大於800kDa之比例較多；於體外試驗中能有減緩退化性關節炎病程，未來可進一步於體內試驗中探討其對於退化性關節炎之影響，以提升豬隻屠宰副產物之附加價值，增加業界收益並減少屠宰廢棄物之汙染。

Porcine eyeballs are seldom to eat and be treated as livestock waste. However, hyaluronic acid in vitreous has a very high value in use. Some studies show that hyaluronic acid can improve osteoarthritis (OA) symptoms and increase viscosity and lubrication of joint fluid. The purpose of this study, porcine eyeballs as a raw material were divided into sampling immediately and sampling freezing and thawing treatment groups after slaughtered of pigs. Sampling immediately treatment group cut the eyeballs, removed and collected the vitreous, and the vitreous were extracted hyaluronic acid by A, B and C three purification methods respectively and freeze-dried into hyaluronic acid extraction powder (HA) and analyzed HA composition. In sampling freezing and thawing treatment group, porcine eyeballs were frozen immediately after slaughter of pigs. The frozen porcine eyeballs were thawed at room temperature (RT), refrigeration (4℃) and far infrared (FIR), respectively, and then using the method which was the best method in sampling immediately to extract HA. HA from sampling freeze and thawing treatment group was analyzed for its composition. According to the results of HA composition analysis, we selected the best extraction method of HA to analyze for antioxidant activity and cell experiments. In cell experiments, chondrosarcoma cells (SW1353) were studied the effects on promoting cell proliferation, cell viability after interleukin-1β (IL-1β) induced inflammation, proteoglycans (PG) secretion and the expression level of OA proinflammatory cytokines including metallopreoteinases-3 (MMP-3), metallopreoteinases-13 (MMP-13), tumor necrosis factor -α (TNF-α), inducible nitric oxide synthase (iNOS) and interleukin-8 (IL-8).
The results showed: sampling immediately treatment group, there was about 3.63 g vitreous and 0.48 g lens in every porcine eyeball. Vitreous was colorless and viscous, and lens was completely oval. But in sampling freeze and thawing treatment group, vitreous and lens were difficult to separate and became black liquid mixed with blood. Sampling immediately treatment group were extracted HA by A, B and C method, and it could be extracted puried HA 2.3588 mg, 1.2027 mg and 2.1728 mg respectively. Molecular weight distributions more than 800 kDa were 20.797%, 9.093% and 45.430% respectively. Sampling freezing and thawing treatment groups used RT, 4℃ and FIR to thaw porcine eyeballs then extracted HA by C method, and it could be extracted puried HA 0.8957 mg, 0.9262 mg and 1.1589 mg respectively. However, HA from sampling freezing and thawing were decomposed, and molecular weight were less than 50 kDa. Therefore sampling immediately treatment group and extracted HA by C method was the best purification method and used it for follow-up test. HA solution was prepared at concentrations of 10, 1.0, 0.5, 0.3 and 0.1 mg/mL, negative control (NC) group was added only IL-1β-induced inflammation, and blank group didn’t add IL-1β and HA. Antioxidant activity, the effects of ferrous ion chelating ability decreased significantly with the decrease of HA concentration (p < 0.05). HA at 10 mg/mL had the highest chelation activity (53.99%). The effects of DPPH scavenging activity and reduction power had worse antioxidant activity, and there were no significantly differences in different HA concentrations. The effect of superoxide anion scavenging activity decreased with the decrease of HA concentration (p < 0.05). In cell proliferation assay, culturing for 1 day, various concentrations of HA were lower than blank. At the third day various concentrations of HA were higher than blank, but there were no significantly differences. At the fifth day various concentrations of HA were lower than blank (p < 0.05). In cell proliferation assay after IL-1β-induced inflammatory, NC was lower than blank, but there were no significantly differences. Cell proliferation of HA was significantly higher than NC when HA concentrations was in 1.0 mg/mL (p < 0.05) and decreased with decrease of HA concentration. The effect of SW1353 PG secretion that NC was significantly lower than blank (p < 0.05), HA treated group significantly higher than NC (p < 0.05), and PG decreased with HA concentration decreased. The gene expression of MMP-3, MMP-13, TNF-α and IL-8, NC were significantly higher than blank (p < 0.05). The gene expression increased with decrease of HA concentration (p < 0.05), and HA concentration at 1 mg/mL had better suppression effect. The gene expression of iNOS, NC was lower than blank but no significantly differences. Gene expression of iNOS increased with decrease of HA concentration but no significantly differences. In Summary, the better extract process on HA from pig eyeballs is C. It can extract more HA and more molecular weight distribution more than 800 kDa. The extraction HA of porcine eyeballs can slow down the progression of OA. In the future, we expect to investigate in vivo mechanisms and evaluate the feasibility of HA to improve OA to enhance the added value of porcine by-products and reduce environmental pollution.

壹、 中文摘要 I
貳、 英文摘要 IV
參、 前言 1
肆、 文獻探討 3
一、 眼球玻璃體之構造 3
二、 玻尿酸簡介 5
(一) 玻尿酸之由來 5
(二) 玻尿酸之結構 5
(三) 玻尿酸之黏彈性質 9
(四) 玻尿酸於生物體內之分布 10
(五) 玻尿酸合成與降解 12
三、 玻尿酸之萃取來源 14
(一) 傳統動物組織萃取 14
(二) 微生物發酵 15
四、 玻尿酸之應用 16
(一) 醫藥方面之應用 17
(二) 化妝品方面之應用 18
(三) 保健食品方面之應用 19
五、 關節軟骨之簡介 20
(一) 關節軟骨之構造 20
(二) 關節軟骨之生化組成 23
1. 組織液 23
2. 膠原蛋白 24
3. 蛋白聚醣 24
4. 非膠原蛋白 26
六、 退化性關節炎之簡介 (Osteoarthritis, OA) 27
(一) 退化性關節炎之定義 27
(二) 退化性關節炎之分類 28
1. 原發性退化性關節炎(Primary or idiopathic OA) 28
2. 續發性退化性關節炎(Secondary OA) 29
(三) 退化性關節炎之治療方法 31
1. 運動與物理治療 31
2. 藥物治療 32
3. 保健食品 34
4. 手術治療 36
七、 退化性關節炎之發生 37
(一) 細胞因子 37
(二) 基質金屬蛋白酶(Matrix metalloproteinases, MMPs) 40
(三) 自由基 41
八、 玻尿酸與退化性關節炎之關聯 42
九、 研究目的 44
伍、 材料與方法 45
一、 試驗材料 46
二、 採樣 46
三、 樣品製備 46
(一) 現場採樣處理組 46
(二) 冷凍解凍處理組 46
四、 成品特性分析 47
(一) 製成率計算 47
(二) 玻尿酸含量分析 47
(三) 蛋白質含量分析 48
(四) 氨基葡萄糖含量分析 50
(五) 分子量分布分析 51
五、 抗氧化能力分析 52
(一) DPPH (1,1-Diphenyl-2-picryl-hydrazyl, C18H12N6O5)自由基清除能力(DPPH scavenging activity) 52
(二) 超氧陰離子清除能力(Superoxide anion scavenging activity ) 52
(三) 還原力(Reduction power) 53
(四) 亞鐵離子螯合能力(Ferrous ion chelating capacity) 54
六、 細胞試驗 55
(一) 軟骨肉瘤細胞SW1353之培養 55
(二) 玻尿酸萃取物促進細胞增生能力試驗 57
(三) IL-1β誘發發炎，玻尿酸萃取物促進細胞增生能力試驗 58
(四) 不同濃度樣品玻尿酸萃取物之蛋白聚醣含量測定 58
(五) 細胞激素表現量分析 59
1. SW1353萃取mRNA 59
2. cDNA之製備 59
2. Real-time polymerase chain reaction (QPCR, Real-time PCR) 60
七、 統計分析 62
陸、 結果與討論 63
一、 豬眼球玻璃體之重量 63
二、 豬眼球玻璃體內水晶體之重量 64
三、 現場採樣處理組玻尿酸萃取物特色分析 65
(一) 現場採樣處理組玻尿酸萃取物之產率與外觀 65
(二) 現場採樣玻尿酸萃取物成分分析 67
1. 現場採樣玻尿酸萃取物蛋白質濃度 67
2. 現場採樣玻尿酸萃取物醣類濃度分析 67
(三) 現場採樣玻尿酸萃取物分子量分布之分析 70
四、 冷凍解凍處理組玻尿酸萃取物特色分析 73
(一) 冷凍解凍處理組玻尿酸萃取物之外觀與產率 73
(二) 冷凍解凍玻尿酸萃取物成分分析 75
1. 冷凍解凍玻尿酸萃取物蛋白質濃度 75
2. 冷凍解凍玻尿酸萃取物糖類濃度分析 75
(三) 冷凍解凍玻尿酸萃取物分子量分布之分析 78
五、 抗氧化能力分析 82
(一) DPPH自由基清除能力 82
(二) 超氧陰離子清除能力 84
(三) 還原力 86
(四) 亞鐵離子螯合能力 88
六、 細胞試驗 90
(一) 軟骨肉瘤細胞SW1353之外觀 90
(二) 玻尿酸萃取物對促進細胞增生試驗 92
(三) IL-1β誘發發炎對促進細胞增生試驗 93
(四) 玻尿酸萃取物對IL-1β誘發發炎下細胞蛋白聚醣分泌量之影響 97
(五) 細胞激素表現量分析 99
柒、 結論 108
捌、 參考文獻 110

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