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载P311微球的温敏壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的影响

张清荣 陈长友 徐娜 吕大伦 贾杰只 李文红 罗高兴 于云龙 张逸

张清荣, 陈长友, 徐娜, 等. 载P311微球的温敏壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的影响[J]. 中华烧伤与创面修复杂志, 2022, 38(10): 914-922. DOI: 10.3760/cma.j.cn501225-20220414-00135.
引用本文: 张清荣, 陈长友, 徐娜, 等. 载P311微球的温敏壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的影响[J]. 中华烧伤与创面修复杂志, 2022, 38(10): 914-922. DOI: 10.3760/cma.j.cn501225-20220414-00135.
Zhang QR,Chen CY,Xu N,et al.Effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats[J].Chin J Burns Wounds,2022,38(10):914-922.DOI: 10.3760/cma.j.cn501225-20220414-00135.
Citation: Zhang QR,Chen CY,Xu N,et al.Effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats[J].Chin J Burns Wounds,2022,38(10):914-922.DOI: 10.3760/cma.j.cn501225-20220414-00135.

载P311微球的温敏壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的影响

doi: 10.3760/cma.j.cn501225-20220414-00135
基金项目: 

国家自然科学基金国际(地区)合作与交流项目 81920108022

南通市科技项目 JC2021178, HS2020006

中国高校产学研创新基金 2021JH033

详细信息
    通讯作者:

    于云龙,Email:yuyunlong666@gmail.com

    张逸,Email:198zy@163.com

Effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats

Funds: 

Funds for International Cooperation and Exchange of the National Natural Science Foundation of China 81920108022

Nantong Science and Technology Project JC2021178, HS2020006

Industry-university-research Innovation Fund for Chinese Universities 2021JH033

More Information
  • 摘要:   目的  探讨载P311微球的温敏壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的影响。  方法  采用实验研究方法。通过油包水乳化法制备聚乙烯醇/海藻酸钠微球(单纯微球)、P311微球及异硫氰酸荧光素标记的牛血清白蛋白(FITC-BSA)微球,于光学显微镜/倒置荧光显微镜下观察形貌。制备壳聚糖溶液,将壳聚糖溶液和β-磷酸甘油二钠水合物混合制备单纯温敏水凝胶,在单纯温敏水凝胶中加入相应物质制备载单纯微球和载P311微球的温敏水凝胶,观察4种液体在37 ℃时倾斜状态下的形态变化,冷冻干燥后于扫描电子显微镜下观察微观形貌。取18只3~4周龄雄性SD大鼠,分为不进行任何处理的正常组及于背部脊柱两侧分别制作1个全层皮肤缺损创面并进行相应处理的敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组、载P311微球水凝胶组,每组3只。取5组全层皮肤缺损大鼠,于伤后0(即刻)、5、10、15 d 观察创面愈合情况,计算伤后5、10、15 d创面愈合率;取5组全层皮肤缺损大鼠伤后15 d创面和创缘组织及正常组大鼠相同部位正常皮肤组织,行苏木精-伊红染色观察组织学变化,行免疫组织化学染色观测CD31及血管内皮生长因子(VEGF)的表达,采用蛋白质印迹法检测CD31及VEGF的蛋白表达。样本数均为3。对数据行单因素方差分析、重复测量方差分析及Bonferroni校正。  结果  单纯微球呈球形,表面疏松多孔;P311微球及FITC-BSA微球表面光滑无孔隙,且FITC-BSA微球散发出均匀的绿色荧光;3种微球直径基本一致,为33.1~37.7 μm。在37 ℃时倾斜状态下,与壳聚糖溶液及单纯温敏水凝胶相比,载微球的2种水凝胶结构更稳定。载微球的2种水凝胶网状结构较壳聚糖溶液、单纯温敏水凝胶更致密,且其横断面可见直径约30 μm的微球。伤后15 d内,5组大鼠创面均不同程度愈合,其中载P311微球水凝胶组大鼠创面愈合情况最好。敷贴组、壳聚糖组大鼠伤后5、10、15 d创面愈合率分别为(26.6±2.4)%、(38.5±3.1)%、(50.9±1.5)%,(47.6±2.0)%、(58.5±3.6)%、(66.7±4.1)%,均明显低于载P311微球水凝胶组的(59.3±4.8)%、(87.6±3.2)%、(97.2±1.0)%,P<0.05或P<0.01;单纯水凝胶组大鼠伤后10、15 d及载单纯微球水凝胶组大鼠伤后15 d创面愈合率分别为(76.0±3.3)%、(84.5±3.6)%、(88.0±2.6)%,均明显低于载P311微球水凝胶组(P<0.05)。正常组大鼠正常皮肤中可见表皮、毛囊及皮脂腺,未见CD31和VEGF阳性表达;载P311微球水凝胶组大鼠伤后15 d创面已几乎完全上皮化,创面血管、毛囊、皮脂腺生成及CD31和VEGF阳性表达较其他4组全层皮肤缺损大鼠明显增加,CD31和VEGF蛋白表达均较其余5组大鼠明显增加(P<0.01)。  结论  载P311微球的温敏壳聚糖水凝胶,可以缓释包载的药物,延长药物作用时间,并通过促进创面血管生成及再上皮化,促进全层皮肤缺损大鼠创面愈合。

     

  • 1  3种聚乙烯醇/海藻酸钠微球的形貌观察。1A.单纯微球呈球形 光学显微镜×200;1B、1C.分别为单纯微球和P311微球,表面结构疏松多孔 光学显微镜×200;1D. 异硫氰酸荧光素标记的牛血清白蛋白微球表面光滑无空隙,且发出均匀绿色荧光 倒置荧光显微镜×200

    2  壳聚糖溶液及3种温敏水凝胶在37 ℃水浴3 min后的形貌。2A.壳聚糖溶液仍为液态;2B、2C、2D.分别为单纯温敏水凝胶、载单纯微球的温敏水凝胶、载P311微球的温敏水凝胶,均变成凝胶态,且图2C、2D温敏水凝胶结构较图2B更加稳定

    注:微球为聚乙烯醇/海藻酸钠微球,温敏水凝胶由β-磷酸甘油二钠水合物与壳聚糖制成

    3  壳聚糖溶液及3种温敏水凝胶冻干后的横断面结构 扫描电子显微镜×200。3A.壳聚糖呈多孔网状结构;3B.温敏水凝胶网状结构较图3A更加致密;3C、3D.分别为载单纯微球的温敏水凝胶及载P311微球的温敏水凝胶,微球被包裹在凝胶中,且凝胶结构较图3A、3B更加致密

    注:微球为聚乙烯醇/海藻酸钠微球,温敏水凝胶由β-磷酸甘油二钠水合物与壳聚糖制成;图中箭头指示微球

    4  5组全层皮肤缺损大鼠伤后各时间点创面情况。4A、4B、4C、4D.分别为敷贴组伤后0(即刻)、5、10、15 d创面情况,伤后15 d仍有部分创面未愈合;4E、4F、4G、4H.分别为壳聚糖组伤后0、5、10、15 d创面情况,图4F创面面积较图4E明显缩小,图4G、4H创面面积相近;4I、4J、4K、4L及4M、4N、4O、4P.分别为单纯水凝胶组及载单纯微球水凝胶组伤后0、5、10、15 d创面情况,创面面积均较组内前一时间点明显缩小,但伤后15 d均有小部分创面未愈合;4Q、4R、4S、4T.分别为载P311微球水凝胶组伤后0、5、10、15 d创面情况,创面逐渐缩小,且伤后15 d创面已基本愈合

    注:右侧圆形物为参照物;创面均贴手术敷贴,微球为聚乙烯醇/海藻酸钠微球,水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶

    5  正常组大鼠正常皮肤组织及5组全层皮肤缺损大鼠伤后15 d创面组织学观察 苏木精-伊红×40。5A.正常组大鼠正常皮肤组织毛囊、皮脂腺排列整齐;5B.敷贴组创面仅有少许血管生成;5C、5D.分别为壳聚糖组和单纯水凝胶组,创面部分上皮化,有少许血管生成;5E.载单纯微球水凝胶组创面大部分上皮化,有部分皮脂腺、毛囊及血管生成;5F.载P311微球水凝胶组创面已几乎完全上皮化,有大量皮脂腺、毛囊及血管生成

    注:黑色箭头指示皮脂腺,黄色箭头指示毛囊,红色箭头指示新生血管;正常组大鼠取与其他5组大鼠相同部位正常皮肤组织;创面均贴手术敷贴,微球为聚乙烯醇/海藻酸钠微球,水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶

    6  正常组大鼠正常皮肤组织及5组全层皮肤缺损大鼠伤后15 d创面组织中CD31及VEGF阳性表达(均为棕色) 二氨基联苯胺-苏木精×400。6A.正常组未见CD31阳性表达;6B、6C、6D、6E、6F.分别为敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组、载P311微球水凝胶组,CD31阳性表达逐渐增多,图6F中CD31阳性表达明显多于图6B、6C、6D、6E;6G.正常组未见VEGF阳性表达;6H、6I、6J、6K、6L.分别为敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组及载P311微球水凝胶组,VEGF阳性表达逐渐增多,图6L中VEGF阳性表达明显多于图6H、6I、6J、6K

    注:正常组大鼠取与其他5组大鼠相同部位正常皮肤组织;创面均贴手术敷贴,微球为聚乙烯醇/海藻酸钠微球,水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶;VEGF为血管内皮生长因子

    7  蛋白质印迹法检测正常组大鼠正常皮肤组织及5组全层皮肤缺损大鼠伤后15 d创面组织中CD31、VEGF蛋白表达水平

    注:创面均贴手术敷贴,微球为聚乙烯醇/海藻酸钠微球,水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶;VEGF为血管内皮生长因子,GAPDH为3-磷酸甘油醛脱氢酶;条带上方1、2、3、4、5、6分别指示正常组、敷贴组、壳聚糖组、单纯水凝胶组、载单纯微球水凝胶组、载P311微球水凝胶组

    表1  5组全层皮肤缺损大鼠伤后各时间点创面愈合率比较(%,x¯±s

    组别鼠数(只)5 d10 d15 d
    敷贴组326.6±2.4a38.5±3.1a50.9±1.5a
    壳聚糖组347.6±2.0b58.5±3.6a66.7±4.1a
    单纯水凝胶组350.4±3.076.0±3.3b84.5±3.6b
    载单纯微球水凝胶组357.6±2.783.2±3.788.0±2.6b
    载P311微球水凝胶组359.3±4.887.6±3.297.2±1.0
    F52.34106.55129.84
    P<0.001<0.001<0.001
    注:创面均贴手术敷贴,微球为聚乙烯醇/海藻酸钠微球,水凝胶指由β-磷酸甘油二钠水合物与壳聚糖制成的温敏水凝胶;处理因素主效应,F=323.44,P<0.001;时间因素主效应,F=245.30,P<0.001;两者交互作用,F=4.24,P=0.005;与载P311微球水凝胶组相比,aP<0.01,bP<0.05
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