【摘要】：目的構建功能化組織工程真皮支架——負載納米銀的膠原-絲素蛋白真皮支架(collagen-fibroin scaffolds loaded with silver nanoparticles,NAg-CFS),并對其微觀結(jié)構及生物學性能進行初步評價,為納米銀在組織工程真皮中的應用尋找新的載體及可行的方式。方法通過胃蛋白酶消化與酸抽提相結(jié)合的方法,從牛跟腱中獲取Ⅰ型膠原。通過脫膠、鹽析、透析等步驟從桑蠶蠶繭中獲取絲素蛋白。將5mg/mL的膠原蛋白和5mg/mL的絲素蛋白以1:1的體積比配制成混合溶液,隨后將10mmg/mL的納米銀溶液與膠原-絲素蛋白溶液以1:1000的體積比混勻,通過冷凍-凍干法制備出負載納米銀的膠原-絲素蛋白真皮支架(NAg-CFS)和不負載納米銀的膠原-絲素蛋白真皮支架(CFS)。以NAg-CFS為實驗組,CFS為對照組,用掃描電子顯微鏡觀察上述兩種支架的微觀結(jié)構。取18只雄性SD大鼠,在每只大鼠背部脊柱中線兩側(cè)制作4個直徑為2cm的圓形全層皮膚缺損創(chuàng)面,在同一只大鼠背部創(chuàng)面交叉移植兩種支架(NAg-CFS和CFS),行同體對照,縫合后加壓包扎。在術后第7、14、28天,分別取6只SD大鼠,對創(chuàng)面進行觀察、拍照,獲取組織標本后過量麻醉處死。通過蘇木素/伊紅(HE)染色觀察創(chuàng)面炎性細胞浸潤和真皮再生情況,通過CD68免疫組織化學染色顯示創(chuàng)面的巨噬細胞,并用RT-PCR技術檢測創(chuàng)面IL-6、IL-10的mRNA相對表達水平。采用Student-t檢驗對數(shù)據(jù)進行統(tǒng)計分析。結(jié)果(1)掃描電鏡觀察結(jié)果顯示,CFS和NAg-CFS的微觀結(jié)構相似。(2)大鼠創(chuàng)面大體觀察的結(jié)果顯示,實驗組創(chuàng)面周圍炎癥反應較輕,且支架與組織結(jié)合更牢固;而對照組創(chuàng)面周圍的炎癥反應較強,支架更容易脫落。(3)蘇木素/伊紅(HE)染色結(jié)果顯示,在術后觀察的各個時相點,與對照組相比,均可見實驗組支架及創(chuàng)面內(nèi)部的炎癥細胞較少、新生組織生長速度較快。(4)CD68免疫組織化學染色及陽性細胞計數(shù)結(jié)果顯示,在術后第14和第28天,實驗組支架及創(chuàng)面內(nèi)部的巨噬細胞浸潤數(shù)量顯著少于對照組。(5)RT-PCR結(jié)果顯示,在術后第7、14、28天,在實驗組創(chuàng)面上,促炎因子IL-6的mRNA相對表達量均低于對照組,且均有顯著性差異;而實驗組創(chuàng)面抗炎因子IL-10的mRNA相對表達量在術后第14天明顯高于對照組,具有統(tǒng)計學意義(p0.05),在第7天高于對照組但無顯著性差異。結(jié)論本實驗成功構建了負載納米銀的膠原-絲素蛋白真皮支架,并初步探討該支架在大鼠移植后的變化和可能機制。結(jié)果表明該真皮支架植入創(chuàng)面后,可以緩解支架本身及其周圍創(chuàng)面組織的炎癥反應。
[Abstract]:Objective to construct a functional tissue engineering dermal scaffold (collagen-fibroin scaffolds loaded with silver nanoparticles-NAg-CFS) loaded with nano-silver, and to evaluate its microstructure and biological properties. To find a new carrier and feasible way for the application of nano-silver in tissue engineering dermis. Methods Type I collagen was obtained from bovine Achilles tendon by pepsin digestion and acid extraction. Silk fibroin was obtained from silkworm cocoon by degumming, salting out and dialysis. The collagen of 5mg/mL and the silk fibroin of 5mg/mL were mixed into a mixture solution at 1:1 volume, and then the nano-silver solution of 10mmg/mL and the collagen-fibroin solution were mixed at 1: 1000 volume. Collagen fibroin dermal scaffold (NAg-CFS) and collagen fibroin dermal scaffold (CFS).) without silver nanoparticles were prepared by freeze-drying method. The microstructures of the above two scaffolds were observed by scanning electron microscope (SEM). Eighteen male Sprague-Dawley rats were used to make 4 round full-thickness skin defect wounds with diameter of 2cm on both sides of the midline of the dorsal spine of each rat. Two kinds of scaffolds (NAg-CFS and CFS),) were cross-transplanted in the same rat back wound. Six SD rats were taken at the 14th day after operation. The wound was observed and photographed, and the tissue specimens were collected and killed under excessive anesthesia. The infiltration of inflammatory cells and dermal regeneration were observed by hematoxylin / eosin (HE) staining. The macrophages of the wound were detected by CD68 immunohistochemical staining. The relative expression of IL-6 / IL-10 was detected by RT-PCR technique. The data were analyzed by Student-t test. Results (1) the results of scanning electron microscope showed that the microstructure of NAg-CFS and CFS were similar. (2) the results of gross observation showed that the inflammatory reaction around the wound was lighter in the experimental group and the stents were more firmly bound to the tissue. In the control group, the inflammatory reaction around the wound was stronger, and the stent was more easily shed. (3) the results of hematoxylin / eosin (HE) staining showed that, compared with the control group, There were fewer inflammatory cells and faster growth rate of new tissue in the experimental group. (4) the results of CD68 immunohistochemical staining and positive cell count showed that on the 14th and 28th day after operation, the inflammatory cells in the scaffold and wound surface of the experimental group were less, and the growth rate of the new tissue was faster. The number of macrophages infiltrated in the scaffold and wound in the experimental group was significantly less than that in the control group. (5) the results of RT-PCR showed that the relative expression of mRNA of pro-inflammatory factor IL-6 on the wounds of the experimental group was lower than that of the control group on the 714 ~ (th) day after operation, and there was significant difference between the two groups. The relative expression of anti-inflammatory factor IL-10 in the experimental group was significantly higher than that in the control group on the 14th day after operation (p0.05), and on the 7th day it was higher than that in the control group, but there was no significant difference. Conclusion the collagen-fibroin dermal scaffold loaded with nano-silver was successfully constructed in this experiment, and the changes and possible mechanism of the scaffold after transplantation in rats were discussed. The results showed that the inflammatory reaction of the stent itself and its surrounding wound tissue could be alleviated after the implantation of the dermal scaffold.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R318.08;R641
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本文編號：2157036