表皮细胞悬液与微粒皮联合修复深度创面的基础研究

发布时间：2018-03-09 01:23

  本文选题：皮肤缺损　切入点：表皮细胞　出处：《中国人民解放军医学院》2014年硕士论文　论文类型：学位论文

【摘要】：目的 研究自体表皮细胞悬液与自体微粒皮联合修复深度创面的效果，为特大面积深度烧伤创面修复提供实验基础。 方法 1.大鼠表皮细胞分离培养与基因标记 （1）用1.25g/L中性蛋白酶消化大鼠背部皮肤，分离表皮，2.5g/L胰蛋白酶消化表皮，体外分离培养原代表皮细胞（epidermis cells，ECs），免疫组织化学法检测细胞的抗原表达，将原代细胞随机分为两组，改良组和传统组，改良组细胞采用低浓度(0.25g/L胰蛋白酶)消化液消化传代，传统组采用传统浓度（2.5g/L胰蛋白酶）消化液消化传代，锥虫蓝染色法计数细胞存活率，噻唑蓝比色法检测贴壁细胞数量（以吸光度值表示）。倒置相差显微镜观察细胞形态，流式细胞仪检测细胞周期，β-半乳糖苷酶染色法计数衰老细胞比率。 （2）利用含RFP基因的慢病毒，以不同感染复数（MOI值分别为0,5,20,50,100）转染ECs，荧光显微镜下观察RFP的表达情况，流式细胞仪检测细胞转染效率，MTT法评价转染慢病毒对ECs增殖的影响，确定最佳病毒感染复数。 2.动物模型建立与创面修复 （1）动物模型建立：16只成年SD大鼠随机分为2组，模型组和对照组。每组8只。将大鼠麻醉，刮毛、消毒后，在背部量取3.3cm×3cm(纵长横短)皮肤范围，切除标记范围内的全层皮肤至深筋膜层，形成全层皮肤缺损创面。模型组创面周围缝合3.3cm×3cm钢丝圈，对照组不作处理，两组大鼠创面交叉移植同种异体中厚皮，纱布打包固定。术后7d拆除敷料，并于术后7d、14d、21d使用数码相机照相，采用图像分析软件(Image J,V1.30)计算两组大鼠创面收缩率。 （2）创面修复：50只成年SD大鼠随机分成5组，微粒皮（1:10）移植组，微粒皮（1:20）+细胞悬液移植组、微粒皮（1:20）移植组、细胞悬液移植组、空白组对照组，每组10只。制作大鼠全层皮肤缺损创面抗挛缩模型，将各组移植物分别移植到相应创面，覆盖异体中厚皮（异体皮来自25只成年Wistar大鼠）。观察大张异体皮成活情况及脱落时间，用数码照相结合图像分析软件(Image J,V1.30)测量创面愈合率。于术后14d、21d取组织块以甲醛固定，制作病理切片，HE染色，免疫组织化学染色检测Ⅳ型胶原与层黏连蛋白表达。微粒皮（1:20）+细胞悬液移植组大鼠，表皮细胞荧光标记后移植，使用小动物活体成像技术检测创面荧光表达。 结果 1.大鼠表皮细胞分离培养与基因标记 （1）消化培养得到的细胞经免疫组织化学染色，表面表达CK19和CK14等表皮细胞标志抗原，证实分离出的细胞为表皮细胞。首次传代细胞改良组细胞存活率[（94.56±1.74）%]显著高于传统组[（66.22±2.57）%,t=15.815, P0.05]；传代细胞接种1h、12h、24h，改良组贴壁细胞吸光光度值（0.205±0.015,0.225±0.014,0.265±0.021）均显著高于传统组（0.176±0.015,0.196±0.011,0.221±0.019, t值分别为2.947,3.517,3.476, P值均小于0.05）。细胞经5次传代后，镜下观察改良组细胞为圆形或小的多角形，或呈鹅卵石样铺满瓶底；传统组细胞胞体变大，增殖缓慢，呈片状覆盖瓶底；改良组S+G2/M期细胞所占百分率[(30.55±2.29)%]显著高于传统组[(17.01±5.58)%, t=3.890, P0.05]；改良组衰老细胞比率[（10.87±2.01）%]显著低于传统组[（75.93±4.11）%, t=24.624, P0.05]。 （2）显微镜下观察细胞发现，转染24h后有微弱荧光，48h后荧光增强，72h后荧光更强。当以MOI=5,20,50,100转染细胞72h后，RFP阳性表达率分别为1.68%,17.15%,47.53%,69.90%；病毒转染72h后，MOI=0,5,20,50,100各组细胞吸光度值分别为1.14±0.143,1.05±0.073,1.02±0.090,1.03±0.141,0.912±0.102，组间比较差异有统计学意义（F=3.022，P0.05），两两比较MOI=0未转染组和MOI=5组细胞吸光度值高于MOI=100组（P0.05）。 2.动物模型建立与创面修复 （1）动物模型建立：术后7d、14d、21d，模型组创面收缩率分别为（1.3±0.5）%,（1.9±0.9）%,（2.6±1.3）%均显著低于（8.6±1.2）%,（37.4±2.6）%,（65.0±3.7）%（t值分别为15.911,36.581,44.847，P均小于0.05）。 （2）创面修复： ①大体观察：术后5d，异体皮颜色红润，术后14d异体皮干燥，开始脱落。术后21d异体皮基本脱落，空白组大鼠异体皮脱落时间晚于实验组。异体皮脱落后，微粒皮（1:10）移植组，微粒皮（1:20）+细胞悬液移植组、微粒皮（1:20）移植组、细胞悬液移植组创面均可见成片的上皮。大鼠创面愈合率：微粒皮（1:10）移植组创面愈合率为（84.3±11.9）%，微粒皮（1:20）+细胞悬液移植组创面愈合率为（74.2±8.0）%，微粒皮（1:20）移植组创面愈合率为（59.2±10.8）%，细胞悬液移植组创面愈合率为（53.8±11.5）%，空白组创面愈合率为（22.7±5.5）%，组间比较差异有统计学意义（F=34.446，P0.05），两两比较，微粒皮（1:10）移植组与微粒皮（1:20）+细胞悬液移植组两组间差异无统计学意义（P0.05），均显著高于微粒皮（1:20）移植组（P0.05），细胞悬液移植组创面也可愈合但上皮薄，易液化，空白组创面愈合率均显著低于各实验组（P0.05），残余大部分肉芽创面。 ②组织学观察：HE染色，镜下观察，术后14d，微粒皮（1:10）移植组，微粒皮（1:20）+细胞悬液移植组、微粒皮（1:20）移植组、细胞悬液移植组新生上皮排挤异体皮，使其与创面部分分离，术后21d，异体皮脱落，微粒皮（1:10）移植组，微粒皮（1:20）+细胞悬液移植组、微粒皮（1:20）移植组表皮分层明显，与正常大鼠皮肤表皮相比，表皮层明显增厚，基底层呈柱状排列，细胞悬液移植组表皮层含有较多空泡，空白组术后14d异体皮下无上皮细胞层，术后21d，异体皮脱落，可见肉芽组织。微粒皮（1:10）移植组，微粒皮（1:20）+细胞悬液移植组、微粒皮（1:20）移植组表皮-真皮连接层Ⅳ型胶原与层黏连蛋白有表达，细胞悬液移植组、空白组无表达，仅在真皮层新生血管处有部分表达。 ③小动物活体成像观察：荧光标记细胞移植，创面有荧光成像。 结论 1.低浓度胰蛋白酶消化法能减轻细胞损伤，，提高细胞活性、增加传代次数，为实验研究创造良好条件。 2.慢病毒介导的RFP基因能够表达于大鼠表皮细胞中，转染效率与MOI值具有量效关系， MOI值为50可作为转染表皮细胞的合适MOI值。 3.大鼠全层皮肤缺损创面创缘缝制钢丝圈可有效减轻创面挛缩。 4.微粒皮与表皮细胞悬液联用，可提高创面愈合率，减少裸露创面，该方法可显著提高自体皮的利用效率，为大面积深度烧伤创面修复带来新思路。
[Abstract]:objective
The effect of autologous epidermal cell suspension and autologous particle skin on the repair of deep wound surface was studied to provide the experimental basis for the repair of large area deep burn wounds.
Method
Isolation and culture of epidermal cells and gene labeling of 1. rats
(1) the separation of epidermis with 1.25g/L neutral protease digestion of rat skin, 2.5g/L, trypsin digestion of epidermal, isolated and cultured in vitro primary skin cells (epidermis, cells, ECs) expression was detected by immunohistochemistry. The antigen, the primary cells were randomly divided into two groups, improved group and traditional group and improvement group cells with low concentration (0.25g/L trypsin) digestion passage, the traditional group with traditional concentration (2.5g/L trypsin) digestion passage, trypan blue staining method to count the cell survival rate, MTT colorimetric assay the number of adherent cells (as absorbance value). Cell morphology was observed under inverted microscope, cells flow cytometry cell cycle, beta galactosidase staining was used to count the cell senescence rate.
(2) with RFP gene by lentivirus, with different multiplicities of infection (MOI = 0,5,20,50100) transfected by ECs, fluorescence microscope to observe the expression of RFP, cell transfection efficiency was detected by flow cytometry. The effect of MTT method to evaluate the transfection of Lentivirus on the proliferation of ECs, to determine the best virus complex.
2. animal model establishment and wound repair
(1) the establishment of animal model: 16 adult SD rats were randomly divided into 2 groups, model group and control group. Each group had 8 rats. The rats were anesthetized, shave, after disinfection, take 3.3cm * 3cm in the back (the amount of longitudinal and transverse short range) skin, resection of markers within the skin to the deep fascia layer, form perfect layer skin defect. The model group wound suture around 3.3cm * 3cm steel wire ring, the control group was not treated, two groups of rats wound cross transplantation of allogeneic skin, gauze packing fixation. Postoperative 7d dressing was removed, and after 7d, 14d, 21d using a digital camera mining, using image analysis software (Image J, V1.30) to calculate the two groups of rats wound contraction rate.
(2) wound repair: 50 adult SD rats were randomly divided into 5 groups, microskin (1:10) transplantation group, microskin (1:20) + cell suspension transplantation group, microskin transplantation group (1:20), cell suspension transplantation group, blank control group, 10 rats in each group. All rats cutaneous wound anti contracture model, each group grafts were transplanted into the corresponding wound coverage, allogeneic skin (skin allograft from 25 adult Wistar rats). Observation of large skin allograft survival and shedding time, using digital camera with image analysis software (Image J, V1.30) to measure the wound healing rate. After 14d, 21d off the tissue to formaldehyde fixation, making pathological section, HE staining, immunohistochemical staining of type IV collagen and laminin expression. Microskin (1:20) + cell suspension transplantation rats, epidermal cell labeling after transplantation, using in vivo fluorescence imaging detection technology of small animal wounds Expression.
Result
Isolation and culture of epidermal cells and gene labeling of 1. rats
(1) digestion of cultured cells by immunohistochemical staining, the expression of CK19 and CK14 surface antigen markers of epidermal cells, showed that the isolated cells into epidermal cells. The first cell group improved the survival rate of cells [(94.56 + 1.74) when it was significantly higher than that of traditional group [(66.22 + 2.57)%, t=15.815, P0.05] inoculation; 12h, 1H cell, 24h, modified group of adherent cells absorbance (0.205 + 0.015,0.225 + 0.014,0.265 + 0.021) was significantly higher than that of the traditional group (0.176 + 0.015,0.196 + 0.011,0.221 + 0.019, t = 2.947,3.517,3.476, P < 0.05). Cells after 5 generations, under the microscope the observation group improved cells were round or polygonal or a small, cobblestone covered the bottom of the bottle; the traditional group of cells became large, slow proliferation, patchy coverage of the bottom of the bottle; the modified group S+G2/M phase cell percentage of [(30.55 + 2.29) when it was significantly higher than that of traditional group [(17.01 + 5.58 %, t=3.890, P0.05]); improved ratio of senescent cells group (10.87 + 2.01) when it was significantly lower than that of traditional group [(75.93 + 4.11)%, t=24.624, P0.05].
(2) the microscope was found after transfection of 24h weak fluorescence, 48h fluorescence enhancement, 72h fluorescence is stronger. When the MOI=5,20,50100 72h transfected cells, the positive expression rates of RFP were 1.68%, 17.15%, 47.53%, 69.90%; the virus after transfection of 72h, MOI=0,5,20,50100 of each group cell absorbance values were 1.14 + 0.143,1.05 + 0.073,1.02 + 0.090,1.03 + 0.141,0.912 + 0.102, there were significant differences between the groups (F=3.022, P0.05), 22 MOI=0 group and MOI=5 group without transfection cell absorbance is higher than that of MOI=100 group (P0.05).
2. animal model establishment and wound repair
(1) establishment of animal models: 7d, 14d and 21d after operation. The wound contraction rates of model group were (1.3 + 0.5)%, (1.9 + 0.9)%, (2.6 + 1.3)% respectively, which were significantly lower than (8.6 + 1.2)%, (37.4, 2.6%), (2.6%), t value was 15.911,36.581,44.847, P was less than that of precipitation.
(2) wound repair:
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