低分子硫酸软骨素的制备及对软骨修复作用的研究

发布时间：2018-04-24 20:20

  本文选题：硫酸软骨素 + 低分子量　； 参考：《青岛大学》2014年硕士论文

【摘要】：目的 研究低分子硫酸软骨素的制备方法,并探讨其对软骨的修复作用。 方法 1、称取一定量分子质量在5万以上的硫酸软骨素(ChS),在一定温度和条件下分别用HC1、H2O2和硫酸软骨素裂解酶EAC对其进行降解,反应结束后用乙醇沉淀、脱水、干燥,产物用高效液相色谱仪测定分子量。 2、建立新西兰大白兔膝关节软骨缺失模型,36只新西兰大白兔随机分成6组,分别为对照组[蒸馏水0.4g/(kg·d)]、模型组[蒸馏水0.4g/(kg·d)]、低分子量ChS低剂量组[低分子硫酸软骨素0.2g/(kg·d)]、低分子量ChS高剂量组[低分子硫酸软骨素0.4g/(kg·d)]、高分子量ChS低剂量组[高分子硫酸软骨素0.2g/(kg·d)]和高分子量ChS高剂量组[高分子硫酸软骨素0.4g/(kg·d)],每组6只,分别进行灌胃给药。记录各组实验用兔的体重、生存时间和生活状况,对关节软骨缺损部位修复情况和病理组织学切片进行观察。应用ELISA法检测兔关节液中IL-1β、TNF-β和TGF-β的水平,并采用全自动生化分析仪及其配套的试剂盒对兔血清中钙、磷含量进行测定。 结果 1、经高效液相色谱法测定,硫酸软骨素原料的分子量为55426。用HC1降解得到的ChS,分子量为3936,颜色从白色转变成红褐色,产物粘性较大,产率较低为48.71%；而用H202在酸性条件下和用硫酸软骨素裂解酶EAC降解所得到的硫酸软骨素,降解效果较好,分子量分别为4217和4565,其外观较好,产物为乳白色,其产率分别达到64.45%和91.6%。 2、造模后伤口愈合情况较好,实验用兔生活状态和精神状态良好。造模后各组体重均有下降,但模型组与其余给予ChS组相比,体重下降明显。低分子ChS组,其软骨表面较为平整,凹陷基本已填充,较模型组与高分子ChS组效果明显。在HE染色状态下,损伤修复处均可见初期修复反应,并且给予低分子ChS组效果要优于高分子ChS组和模型组。 3、低分子ChS和高分子ChS都能够减少关节液中炎性因子的含量和增加TGF-β的含量(P0.05),且低分子ChS组与高分子ChS组相比,效果较好(P0.05)。与模型组相比,给予ChS的组别,其血清中的钙磷含量也相对较少(P0.05)。 结论 1、HC1、H2O2和硫酸软骨素裂解酶EAC都能有效地将分子量为5万以上的ChS降解为分子量在3500-5300范围内的低分子量产物。 2、低分子ChS能够更好地促进伤口愈合和软骨修复,这可能是因为低分子ChS,分子量较小,机体易于吸收,生物利用度提高,从而使得伤口愈合和软骨修复的作用较为明显。 3、高、低分子量的ChS都可以增加TGF-β的含量,降低IL-1β、TNF-α和血清中钙、磷的含量。且低分子ChS要优于高分子ChS。对于软骨修复,这可能是通过对上述因子的影响,从而产生积极的作用。
[Abstract]:Purpose To study the preparation of chondroitin sulfate and its effect on cartilage repair. Method 1. Chondroitin sulfate chondroitin sulfate (CHS) with a molecular weight of more than 50 000 was obtained and degraded by HC1H 2O 2 and chondroitin sulfate lyase EAC at certain temperature and under certain conditions. After the reaction was finished, the chondroitin sulfate was precipitated, dehydrated and dried. The molecular weight of the product was determined by high performance liquid chromatography. 2, 36 New Zealand white rabbits were randomly divided into 6 groups. Control group [distilled water 0.4g/(kg d], model group [distilled water 0.4g/(kg d], low molecular weight ChS low dose group [low molecular weight chondroitin sulfate 0.2g/(kg d], low molecular weight ChS high dose group [low molecular weight chondroitin sulfate 0.4g/(kg d], high molecular weight ChS low dose group [high molecular weight ChS low dose group] Chondroitin sulfate (0.2g/(kg d) and high molecular weight ChS (high dose group [high molecular weight chondroitin sulfate 0.4g/(kg d]), 6 rats in each group, The drugs were administered by gavage respectively. The weight, survival time and living condition of the rabbits were recorded. The repair of articular cartilage defects and histopathological sections were observed. The levels of IL-1 尾 -TNF- 尾 and TGF- 尾 in rabbit articular fluid were detected by ELISA method. The contents of calcium and phosphorus in rabbit serum were determined by automatic biochemical analyzer and kit. Result 1. The molecular weight of chondroitin sulfate was 55426 by HPLC. Chondroitin sulfate obtained by HC1 degradation, with a molecular weight of 3936, was changed from white to reddish brown, with a high viscosity and a lower yield of 48.71.The degradation of chondroitin sulfate by H202 under acidic conditions and by chondroitin sulfate lyase EAC showed better degradation effect. The molecular weight was 4217 and 4565 respectively. The product was milky white and its yield reached 64.45% and 91.6% respectively. 2. The wound healed well after modeling, and the living state and mental state of experimental rabbits were good. The weight of each group was decreased after the model was made, but the weight of the model group was significantly lower than that of the other groups treated with ChS. In the low molecular weight ChS group, the cartilage surface was flat and the hollow was basically filled, which was more effective than the model group and the high molecular weight ChS group. Under HE staining, the initial repair reaction was observed at the site of injury repair, and the effect of low molecular weight ChS group was better than that of macromolecule ChS group and model group. 3. Both low molecular weight ChS and high molecular weight ChS could decrease the content of inflammatory factor and increase the content of TGF- 尾 in articular fluid. The effect of low molecular weight ChS group was better than that of high molecular weight ChS group. Compared with the model group, the serum calcium and phosphorus contents of the ChS group were also relatively lower than that of the model group. Conclusion 1HC1H _ 2O _ 2 and chondroitin sulfate lyase EAC can effectively degrade ChS with molecular weight over 50,000 to low molecular weight products in the range of 3500-5300. 2, low molecular weight ChS can better promote wound healing and cartilage repair, which may be due to the low molecular weight, easy to be absorbed and bioavailability, which makes wound healing and cartilage repair more obvious. 3. ChS with high, low molecular weight could increase the content of TGF- 尾, decrease the content of IL-1 尾 TNF- 伪 and the contents of calcium and phosphorus in serum. And the low molecular ChS is superior to the polymer Chs. For cartilage repair, this may have a positive effect by influencing these factors.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914

【引证文献】

相关期刊论文 前1条

1 王俊;王紫薇;张金木;汪兰;乔宇;李新;扥晓艳;吴文锦;熊光权;;不同来源和纯度的硫酸软骨素理化特性的研究[J];湖北农业科学;2016年23期

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