羊膜制备工艺的研究以及改良

发布时间：2018-06-16 18:43

本文选题：羊膜 + 灭菌　；参考：《重庆医科大学》2010年硕士论文

【摘要】： 目的通过对现有羊膜制备工艺进行研究比较,分别从灭菌、保存条件以及对传统羊膜贴载物改进这几个方面入手,以期寻找出一种对羊膜损伤小,便于制备,易于保存,更加方便临床使用且易推广的方法。本实验共分为三部分: 实验一:检测常用灭菌方式对羊膜上皮性状影响实验二:探讨不同保存条件对羊膜性状的影响实验三:将无纺材料(聚丙烯无纺布,Polypropylene Non-woven Substrates)作为新型羊膜贴载物的研究方法实验一:取健康剖宫产产妇胎盘羊膜,随机分为四组,分别以抗菌素(含青霉素10,00U/ml,庆大霉素10,00U/ml、二性霉素B2.5μg/ml)、过氧乙酸/乙醇混合液(过氧乙酸12.5ml,96%乙醇125ml,蒸馏水362.5ml)、γ-射线、0.5%碘伏灭菌,行病原学检测确认达到临床运用标准后,HE染色观察羊膜上皮细胞形态变化;免疫荧光、RT-PCR检测肝细胞生长因子(HGF)、金属蛋白酶组织抑制剂-1(TIMP-1)及其mRNA的表达,并设未经任何灭菌处理的羊膜作为对照。实验二:将按常规方法制备好的羊膜随机分为三组,分别于4℃,-20℃以及-80℃保存1月,3月,6月后,分别行光镜以及电镜观察,比较不同保存组之间羊膜上皮细胞以及基质的变化,评估不同保存条件对羊膜的影响。实验三:用新型无纺布替代硝酸纤维滤膜作为羊膜贴载物,探讨羊膜与无纺布的不同贴载模式,并将羊膜分别贴载于硝酸纤维滤膜、无纺布后各置于纯甘油-80℃保存1个月,行HE染色、透射电镜检测,观察羊膜上皮细胞以及基质的形态。结果实验一:所采用的几种灭菌方法病原学培养结果均为阴性,经过氧乙酸/乙醇混合液和抗菌药处理的羊膜细胞形态完好,而经γ-射线处理的细胞形态损害较大。经灭菌处理后,羊膜细胞的HGF和TIMP-1蛋白表达均有所下降(P0.05),由高到低依次为PES组、抗菌药组、碘伏组和γ-射线组;HGF和TIMP-1的mRNA表达也有下降(P0.05),其中γ-射线处理组下降趋势更明显(P0.05),其余3个处理组间无明显差别(P0.05)。实验二:经4℃保存的羊膜随着时间的延长羊膜上皮细胞破坏加重,在保存到3个月的时候,羊膜上皮细胞丢失较为严重。经-20℃和-80℃保存的羊膜上皮细胞随保存时间延长也出现破坏,但损害程度较小,且保存3个月与保存6个月相比较无明显差异。在相同时间点上,-20℃保存的羊膜与-80℃保存的羊膜相比较,上皮细胞有差异但不显著,远优于4℃保存。另外,羊膜基质在4℃保存6个月后发生变化,胶原纤维减少,而-20℃和-80℃保存的羊膜基质无明显变化。实验三:与传统的硝酸纤维滤膜相比较,无纺布作为羊膜贴载物更具优势:其拥有良好韧性,经折叠不易变型;保存时两者可紧密贴合不分开;临床使用时与羊膜易于辨别,在生理盐水中复水较短时间即可将其两者分开;形态学观察发现与传统的硝酸纤维滤膜相比较,无纺布对羊膜上皮细胞以及基质形态无明显影响。结论 1、就现行常用的灭菌方式抗菌素浸泡、过氧乙酸/乙醇混合液浸泡、γ-射线照射、碘伏浸泡而言,过氧乙酸/乙醇混合液浸泡对羊膜的损伤最小,值得进一步研究。 2、相对于其他保存温度而言,-20℃能较好的维持羊膜的形态,且该温度是现有普通家用冰箱都达到的条件,故该法可大范围推广,特别是适用于经济不发达地区的应用。 3、无纺布作为羊膜贴载物比硝酸纤维膜更利于临床使用,简化了羊膜植片制作程序,且对羊膜形态无明显影响。将其作为羊膜载体是一种方便、有效、安全、可靠的途径。
[Abstract]:objective
Through the research and comparison of the existing amniotic membrane preparation technology, we start with the sterilization, the preservation conditions and the improvement of the traditional amniotic membrane, in order to find out a method of small damage to the amniotic membrane, easy to prepare, easy to save, more convenient for clinical use and easy to popularize.
The experiment is divided into three parts:
Experiment 1: detect the influence of common sterilization methods on the amniotic epithelium
Experiment two: To explore the effects of different preservation conditions on amniotic characteristics.
Experiment three: nonwoven material (polypropylene non-woven fabric, Polypropylene Non-woven Substrates) was used as a new amniotic membrane carrier.
Method
Experiment 1: the placental amniotic membrane of healthy cesarean parturient was randomly divided into four groups, with antibiotics (including penicillin 10,00U/ml, gentamicin 10,00U/ml, amphotericin B2.5 mu g/ml), peracetic acid / ethanol mixture (peracetic acid 12.5ml, 96% ethanol 125ml, distilled water 362.5ml), gamma ray, and 0.5% iodophor, which were confirmed to be clinical by pathogenic detection. After using the standard, the morphologic changes of amniotic epithelial cells were observed by HE staining, immunofluorescence, RT-PCR detection of hepatocyte growth factor (HGF), metalloproteinase tissue inhibitor -1 (TIMP-1) and the expression of mRNA, and the amniotic membrane without any sterilization treatment as control.
Experiment two: the amniotic membrane prepared by conventional methods was divided into three groups randomly, at 4, -20 and -80, respectively, in January, March, and June. The changes of amniotic epithelial cells and matrix between different preservation groups were compared, and the effects of different preservation conditions on amniotic membrane were evaluated.
Experiment three: a new type of nonwoven fabric was used instead of nitric acid fiber membrane as a amniotic membrane. The different mode of amniotic membrane and nonwoven fabric was studied. The amniotic membrane was attached to the membrane of nitric acid fiber, and the nonwoven fabric was stored in pure glycerol at -80 for 1 months. The form of amniotic epithelial cells and matrix were observed by HE staining and transmission electron microscopy.
Result
Experiment 1: the results of several sterilization methods were all negative. The morphology of amniotic cells treated with oxy acetic acid / ethanol mixture and antibacterials was intact, and the cell morphology of the cells treated by gamma ray was more damaged. After sterilization, the expression of HGF and TIMP-1 protein in amniotic cells decreased from high to low (P0.05). For group PES, antibacterials, iodophor group and gamma ray group, the expression of mRNA in HGF and TIMP-1 decreased (P0.05), and the decreasing trend of gamma ray treatment group was more obvious (P0.05), and there was no significant difference between the other 3 treatment groups (P0.05).
Experiment two: the amniotic membrane epithelial cell destruction increased with time at 4 C, and the amniotic epithelial cells were lost more seriously at the time of preservation to 3 months. The amniotic epithelial cells preserved at -20 and -80 C were also damaged with the prolonged preservation time, but the damage degree was less, and the preservation of the amniotic epithelial cells was less than that of 3 months, and was less than the preservation of 6 months. At the same time point, the amniotic membrane preserved at -20 C and the amniotic membrane preserved at -80 C showed that the epithelial cells were different but not significant, far superior to 4 C. In addition, the amniotic membrane matrix was changed after 6 months of preservation at 4 C, and the collagen fibers decreased, but the amniotic membrane matrix preserved at -20 and -80 C had no obvious change.
Experiment three: compared with the traditional nitrate filter membrane, nonwoven fabric is more advantageous as amniotic membrane: it has good toughness and can not be easily folded through folding. The two can be closely fitted when preserved; it is easy to distinguish between the amniotic membrane and the amniotic membrane when used in clinical use. Compared with traditional nitrocellulose filter membrane, non-woven fabrics had no significant effect on amniotic epithelial cells and matrix morphology.
conclusion
1, it is worth further study on the minimal damage of the amniotic membrane by immersion in antiseptic methods, peroxy acetic acid / ethanol mixture, gamma ray irradiation and iodophor immersion.
2, compared with other preservation temperatures, -20 C can maintain the form of amniotic membrane better, and the temperature is the condition of existing ordinary household refrigerators. Therefore, this method can be extended in a wide range, especially for the application of economically underdeveloped areas.
3, nonwoven fabric as amniotic membrane is more beneficial to clinical use than nitric acid fiber membrane. It simplifies the procedure of making amniotic membrane and has no obvious effect on amniotic membrane. It is a convenient, effective, safe and reliable way to use it as amniotic carrier.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R77;R318.08

【共引文献】