本文選題：創(chuàng)面愈合 + 富血小板血漿��； 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：近年來,國內外學者研究發(fā)現血小板除具有止血作用外,在創(chuàng)傷修復和再生領域也發(fā)揮著重要的作用。富血小板血漿(Platelet-rich Plasma,PRP)作為血小板的常規(guī)應用形式,因其富含高濃度的復合生長因子和細胞因子等生物活性分子,廣泛用于組織工程和再生醫(yī)學領域。但是PRP的主要形式是以液體存在,單獨運用有一定局限性,通過將其負載在生物材料上,可以更好的應用在創(chuàng)面發(fā)揮止血愈創(chuàng)作用。羧甲基殼聚糖是殼聚糖經羧甲基化后的衍生物,因為其良好的水溶性,規(guī)避了殼聚糖用稀酸作為溶劑的弊端,此外,其分子中含有羥基和氨基,可與多種功能活性物質結合,充當其載體,從而使其得到廣泛的應用和深入的研究。PRP是否能與其聯合應用,國內尚未見報道。本研究將從以下幾個方面研究負載濃縮血小板紗布的制備工藝并對其進行初步功能評價。本研究中,我們以臨床上常用的可吸收性止血紗布為基礎,篩選出合適的原料為殼聚糖類,出于溶解性和降解周期的考慮,我們不用殼聚糖而選擇將羧甲基殼聚糖作為本課題制備功能性紗布的原材料,然后將其與富血小板血漿復合,設計不同梯度的濃度、溫度及混合比例等條件,對該負載濃縮血小板的可吸收性紗布的制備條件進行探究。為證明其有效性,本研究進一步從促細胞增殖、動物急性創(chuàng)傷修復和外傷止血等幾個層面對其功能進行評價。一.復合紗布制備材料的篩選以臨床上常用的可吸收性止血紗布為基礎,選取臨床上常用的幾種代表性的紗布。制備濃縮血小板,并對其進行凍干前的預處理。然后在各組紗布的表面滴加制備好的濃縮血小板,并經過預凍、凍干等程序。然后對凍干后的材料進行復溶,測量其pH值并加入相應的激活劑,觀察凝膠的的形成情況。結果發(fā)現,各組復合凍干品質地均呈現硬而脆,且只有殼聚糖類的復合凍干品能夠形成血小板凝膠,其余材料均呈現不同程度的酸性,未見凝膠形成。二.羧甲基殼聚糖/濃縮血小板復合凍干品制備條件的探究設計不同殼聚糖濃度、預凍溫度和CC/PRP共混比例等條件,制備CC/PRP復合凍干品,并對其進行肉眼觀和掃描電鏡觀察,抗壓縮性能及所負載生長因子含量等進行測試,結果發(fā)現,2-3%的殼聚糖溶液濃度,預凍溫度為-80℃,CC/PRP按1:1共混比例混合時,所制備出的復合凍干品具有較好的抗壓縮性能,且負載的生長因子含量較高。三.CC/PRP可吸收復合材料對HUVEC促增殖功能的研究將不同CC、PRP共混比例制備的新型可吸收性復合血小板功能材料用基礎培養(yǎng)基浸提48h后,用凝血酶-葡萄酸鈣溶液激活,用其浸提上清作用于HUVEC,檢測復合功能材料對HUVEC的促增值效果。研究發(fā)現,不同共混比例的復合功能材料均具有促進細胞增殖效果,且相同條件下,2%(wt/vol)濃度CC共混的復合材料,促增殖效果更好。四.CC/PRP可吸收復合材料促進小鼠皮膚全層缺損模型的研究前面的研究,我們已經發(fā)現復合止血紗布上已經成功負載了較高濃度的生長因子,并且具有促進細胞增殖的作用。為進一步證明其促進創(chuàng)面修復的能力,通過建立小鼠皮膚全層缺損模型,將48只小鼠隨機分為4組(空白組、CC/PRP組、CC組和FDP組),觀察其促創(chuàng)面修復效果,結果發(fā)現,實驗早期,CC/PRP組、CC組、FDP組均具有較高的促進創(chuàng)面愈合率,均明顯高于空白對照組。病理切片檢查提示,制備的復合材料具有縮短創(chuàng)傷炎癥期,加速傷口上皮化和創(chuàng)面愈合率、促進血管新生和膠原纖維的沉積與成熟等。由此可見,其具有明顯的促進創(chuàng)面愈合的作用。五.CC/PRP可吸收復合材料對新西蘭白兔耳動脈出血的止血作用研究32只新西蘭白兔隨機分為4組(空白組、CC/PRP組、CC組和FDP組),建立了新西蘭白兔耳動脈出血模型,觀察所制備的復合CC/PRP對外傷性動脈出血的止血作用,并與FDP、CC進行比較。結果表明,CC/PRP、CC、FDP均具有一定的止血作用,且與單獨使用CC、FDP相比,兩者聯合制備的CC/PRP具有更顯著的止血效果。綜上所述,本研究制備的CC/PRP可吸收復合功能性紗布,成功負載了生物活性分子,具有促進止血和創(chuàng)傷愈合作用,有望作為一種新型復合功能敷料應用于臨床。
[Abstract]:In recent years, scholars at home and abroad have found that platelets play an important role in the field of trauma repair and regeneration. Platelet-rich Plasma (PRP), as a conventional form of platelet, is widely used because of its high concentration of compound growth factor and cytokine, and other biological active molecules. It is in the field of tissue engineering and regenerative medicine. But the main form of PRP is the existence of liquid, which has some limitations. By loading it on biological materials, it can be better applied to the wound to play a hemostatic effect. Carboxymethyl chitosan is a derivative of chitosan after carboxymethylation, because of its good solubility in water. In addition, the chitosan with dilute acid as a solvent, in addition, its molecules contain hydroxyl and amino groups, can be combined with a variety of functional active substances and act as its carrier, so that it can be widely used and deeply study whether.PRP can be combined with its application, and it has not been reported in China. This study will study the small load concentration from the following aspects. In this study, we screened the suitable raw materials for chitosan on the basis of the commonly used absorbable hemostatic gauze. In consideration of solubility and degradation cycle, we chose carboxymethyl chitosan as a functional gauze to prepare carboxymethyl chitosan without the use of chitosan. The preparation conditions of the absorbable gauze of the concentrated platelets were investigated by combining with the platelet rich plasma, the concentration of different gradients, the temperature and the mixing ratio, etc., the preparation conditions of the absorbable gauze of the concentrated platelets were explored. Firstly, the selection of composite gauze preparation materials is based on the commonly used absorbable hemostat as a basis to select several typical representative gauze used clinically. To prepare concentrated platelets and pretreat them before freeze drying. After the procedure of prefreezing and freeze drying, the material was redissolved, the pH value was measured and the corresponding activator was added to observe the formation of the gel. The results showed that the quality of the freeze-dried compound was hard and crisp, and only the composite lyophilized freeze-dried products of chitosan could form the platelets gel, and the rest of the materials showed different degrees. Two. The preparation conditions of carboxymethyl chitosan / concentrated platelets were prepared by the preparation of different chitosan concentration, prefreezing temperature and CC/PRP blending ratio, and the preparation of CC/PRP composite freeze dried products, and its naked eye view and scanning electron microscope observation, anti compression performance and the load growth factor content and so on. The results showed that the concentration of chitosan solution in 2-3%, the prefreezing temperature of -80 C, and the mixture of CC/PRP in 1:1 blending ratio, the prepared freeze-dried products had better compression resistance, and the load growth factor content was higher. Three.CC/PRP absorbable composite materials for HUVEC proliferation promoting function will be different CC, PRP blending ratio system The new absorbable composite platelet functional material was extracted with 48h, activated by thrombin - calcium acid calcium solution, and used to extract the supernatant on HUVEC. The effect of compound functional material on HUVEC was detected. Under conditions, 2% (wt/vol) concentration of CC blend composites, the effect of promoting proliferation is better. Four.CC/PRP absorbable composites promote the study of the model of full layer skin defect in mice. We have found that the composite hemostatic gauze has successfully loaded the higher concentration of growth factor and has the effect of promoting cell proliferation. The 48 mice were randomly divided into 4 groups (blank group, CC/PRP group, CC group and FDP group) and the effect of wound healing was observed. The results showed that early experiment, group CC/PRP, CC group and FDP group had higher healing rate of wound healing, which were significantly higher than that of blank control. Pathological section examination suggested that the prepared composite could shorten the period of wound inflammation, accelerate wound epithelialization and wound healing, promote the deposition and maturation of angiogenesis and collagen fibers. This shows that it has a significant effect on promoting wound healing. Five.CC/PRP absorbable composite material for New Zealand white rabbit ear bleeding 32 New Zealand white rabbits were randomly divided into 4 groups (blank group, group CC/PRP, group CC and group FDP), and a New Zealand white rabbit ear artery hemorrhage model was established. The hemostatic effect of the compound CC/PRP was observed and compared with FDP and CC. The results showed that CC/PRP, CC and FDP all had a certain hemostatic effect, and with the single one. Compared with CC alone, the combination of FDP and CC/PRP has more significant hemostatic effect. To sum up, the CC/PRP can absorb the compound functional gauze, which has successfully loaded the bioactive molecules, and has the effect of promoting hemostasis and wound healing. It is expected to be used as a new type of compound functional dressing for clinical application.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R318.08

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