本文選題：羊膜 + 滅菌　； 參考：《重慶醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的 通過對現(xiàn)有羊膜制備工藝進(jìn)行研究比較,分別從滅菌、保存條件以及對傳統(tǒng)羊膜貼載物改進(jìn)這幾個(gè)方面入手,以期尋找出一種對羊膜損傷小,便于制備,易于保存,更加方便臨床使用且易推廣的方法。 本實(shí)驗(yàn)共分為三部分: 實(shí)驗(yàn)一:檢測常用滅菌方式對羊膜上皮性狀影響 實(shí)驗(yàn)二:探討不同保存條件對羊膜性狀的影響 實(shí)驗(yàn)三:將無紡材料(聚丙烯無紡布,Polypropylene Non-woven Substrates)作為新型羊膜貼載物的研究 方法 實(shí)驗(yàn)一:取健康剖宮產(chǎn)產(chǎn)婦胎盤羊膜,隨機(jī)分為四組,分別以抗菌素(含青霉素10,00U/ml,慶大霉素10,00U/ml、二性霉素B2.5μg/ml)、過氧乙酸/乙醇混合液(過氧乙酸12.5ml,96%乙醇125ml,蒸餾水362.5ml)、γ-射線、0.5%碘伏滅菌,行病原學(xué)檢測確認(rèn)達(dá)到臨床運(yùn)用標(biāo)準(zhǔn)后,HE染色觀察羊膜上皮細(xì)胞形態(tài)變化;免疫熒光、RT-PCR檢測肝細(xì)胞生長因子(HGF)、金屬蛋白酶組織抑制劑-1(TIMP-1)及其mRNA的表達(dá),并設(shè)未經(jīng)任何滅菌處理的羊膜作為對照。 實(shí)驗(yàn)二:將按常規(guī)方法制備好的羊膜隨機(jī)分為三組,分別于4℃,-20℃以及-80℃保存1月,3月,6月后,分別行光鏡以及電鏡觀察,比較不同保存組之間羊膜上皮細(xì)胞以及基質(zhì)的變化,評估不同保存條件對羊膜的影響。 實(shí)驗(yàn)三:用新型無紡布替代硝酸纖維濾膜作為羊膜貼載物,探討羊膜與無紡布的不同貼載模式,并將羊膜分別貼載于硝酸纖維濾膜、無紡布后各置于純甘油-80℃保存1個(gè)月,行HE染色、透射電鏡檢測,觀察羊膜上皮細(xì)胞以及基質(zhì)的形態(tài)。 結(jié)果 實(shí)驗(yàn)一:所采用的幾種滅菌方法病原學(xué)培養(yǎng)結(jié)果均為陰性,經(jīng)過氧乙酸/乙醇混合液和抗菌藥處理的羊膜細(xì)胞形態(tài)完好,而經(jīng)γ-射線處理的細(xì)胞形態(tài)損害較大。經(jīng)滅菌處理后,羊膜細(xì)胞的HGF和TIMP-1蛋白表達(dá)均有所下降(P0.05),由高到低依次為PES組、抗菌藥組、碘伏組和γ-射線組;HGF和TIMP-1的mRNA表達(dá)也有下降(P0.05),其中γ-射線處理組下降趨勢更明顯(P0.05),其余3個(gè)處理組間無明顯差別(P0.05)。 實(shí)驗(yàn)二:經(jīng)4℃保存的羊膜隨著時(shí)間的延長羊膜上皮細(xì)胞破壞加重,在保存到3個(gè)月的時(shí)候,羊膜上皮細(xì)胞丟失較為嚴(yán)重。經(jīng)-20℃和-80℃保存的羊膜上皮細(xì)胞隨保存時(shí)間延長也出現(xiàn)破壞,但損害程度較小,且保存3個(gè)月與保存6個(gè)月相比較無明顯差異。在相同時(shí)間點(diǎn)上,-20℃保存的羊膜與-80℃保存的羊膜相比較,上皮細(xì)胞有差異但不顯著,遠(yuǎn)優(yōu)于4℃保存。另外,羊膜基質(zhì)在4℃保存6個(gè)月后發(fā)生變化,膠原纖維減少,而-20℃和-80℃保存的羊膜基質(zhì)無明顯變化。 實(shí)驗(yàn)三:與傳統(tǒng)的硝酸纖維濾膜相比較,無紡布作為羊膜貼載物更具優(yōu)勢:其擁有良好韌性,經(jīng)折疊不易變型;保存時(shí)兩者可緊密貼合不分開;臨床使用時(shí)與羊膜易于辨別,在生理鹽水中復(fù)水較短時(shí)間即可將其兩者分開;形態(tài)學(xué)觀察發(fā)現(xiàn)與傳統(tǒng)的硝酸纖維濾膜相比較,無紡布對羊膜上皮細(xì)胞以及基質(zhì)形態(tài)無明顯影響。 結(jié)論 1、就現(xiàn)行常用的滅菌方式抗菌素浸泡、過氧乙酸/乙醇混合液浸泡、γ-射線照射、碘伏浸泡而言,過氧乙酸/乙醇混合液浸泡對羊膜的損傷最小,值得進(jìn)一步研究。 2、相對于其他保存溫度而言,-20℃能較好的維持羊膜的形態(tài),且該溫度是現(xiàn)有普通家用冰箱都達(dá)到的條件,故該法可大范圍推廣,特別是適用于經(jīng)濟(jì)不發(fā)達(dá)地區(qū)的應(yīng)用。 3、無紡布作為羊膜貼載物比硝酸纖維膜更利于臨床使用,簡化了羊膜植片制作程序,且對羊膜形態(tài)無明顯影響。將其作為羊膜載體是一種方便、有效、安全、可靠的途徑。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R77;R318.08

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