本文選題：皮膚缺損　切入點(diǎn)：表皮細(xì)胞　出處：《中國人民解放軍醫(yī)學(xué)院》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 研究自體表皮細(xì)胞懸液與自體微粒皮聯(lián)合修復(fù)深度創(chuàng)面的效果，為特大面積深度燒傷創(chuàng)面修復(fù)提供實(shí)驗(yàn)基礎(chǔ)。 方法 1.大鼠表皮細(xì)胞分離培養(yǎng)與基因標(biāo)記 （1）用1.25g/L中性蛋白酶消化大鼠背部皮膚，分離表皮，2.5g/L胰蛋白酶消化表皮，體外分離培養(yǎng)原代表皮細(xì)胞（epidermis cells，ECs），免疫組織化學(xué)法檢測細(xì)胞的抗原表達(dá)，將原代細(xì)胞隨機(jī)分為兩組，改良組和傳統(tǒng)組，改良組細(xì)胞采用低濃度(0.25g/L胰蛋白酶)消化液消化傳代，傳統(tǒng)組采用傳統(tǒng)濃度（2.5g/L胰蛋白酶）消化液消化傳代，錐蟲藍(lán)染色法計(jì)數(shù)細(xì)胞存活率，噻唑藍(lán)比色法檢測貼壁細(xì)胞數(shù)量（以吸光度值表示）。倒置相差顯微鏡觀察細(xì)胞形態(tài)，流式細(xì)胞儀檢測細(xì)胞周期，β-半乳糖苷酶染色法計(jì)數(shù)衰老細(xì)胞比率。 （2）利用含RFP基因的慢病毒，以不同感染復(fù)數(shù)（MOI值分別為0,5,20,50,100）轉(zhuǎn)染ECs，熒光顯微鏡下觀察RFP的表達(dá)情況，流式細(xì)胞儀檢測細(xì)胞轉(zhuǎn)染效率，MTT法評價(jià)轉(zhuǎn)染慢病毒對ECs增殖的影響，確定最佳病毒感染復(fù)數(shù)。 2.動物模型建立與創(chuàng)面修復(fù) （1）動物模型建立：16只成年SD大鼠隨機(jī)分為2組，模型組和對照組。每組8只。將大鼠麻醉，刮毛、消毒后，在背部量取3.3cm×3cm(縱長橫短)皮膚范圍，切除標(biāo)記范圍內(nèi)的全層皮膚至深筋膜層，形成全層皮膚缺損創(chuàng)面。模型組創(chuàng)面周圍縫合3.3cm×3cm鋼絲圈，對照組不作處理，兩組大鼠創(chuàng)面交叉移植同種異體中厚皮，紗布打包固定。術(shù)后7d拆除敷料，并于術(shù)后7d、14d、21d使用數(shù)碼相機(jī)照相，采用圖像分析軟件(Image J,V1.30)計(jì)算兩組大鼠創(chuàng)面收縮率。 （2）創(chuàng)面修復(fù)：50只成年SD大鼠隨機(jī)分成5組，微粒皮（1:10）移植組，微粒皮（1:20）+細(xì)胞懸液移植組、微粒皮（1:20）移植組、細(xì)胞懸液移植組、空白組對照組，每組10只。制作大鼠全層皮膚缺損創(chuàng)面抗攣縮模型，將各組移植物分別移植到相應(yīng)創(chuàng)面，覆蓋異體中厚皮（異體皮來自25只成年Wistar大鼠）。觀察大張異體皮成活情況及脫落時間，用數(shù)碼照相結(jié)合圖像分析軟件(Image J,V1.30)測量創(chuàng)面愈合率。于術(shù)后14d、21d取組織塊以甲醛固定，制作病理切片，HE染色，免疫組織化學(xué)染色檢測Ⅳ型膠原與層黏連蛋白表達(dá)。微粒皮（1:20）+細(xì)胞懸液移植組大鼠，表皮細(xì)胞熒光標(biāo)記后移植，使用小動物活體成像技術(shù)檢測創(chuàng)面熒光表達(dá)。 結(jié)果 1.大鼠表皮細(xì)胞分離培養(yǎng)與基因標(biāo)記 （1）消化培養(yǎng)得到的細(xì)胞經(jīng)免疫組織化學(xué)染色，表面表達(dá)CK19和CK14等表皮細(xì)胞標(biāo)志抗原，證實(shí)分離出的細(xì)胞為表皮細(xì)胞。首次傳代細(xì)胞改良組細(xì)胞存活率[（94.56±1.74）%]顯著高于傳統(tǒng)組[（66.22±2.57）%,t=15.815, P0.05]；傳代細(xì)胞接種1h、12h、24h，改良組貼壁細(xì)胞吸光光度值（0.205±0.015,0.225±0.014,0.265±0.021）均顯著高于傳統(tǒng)組（0.176±0.015,0.196±0.011,0.221±0.019, t值分別為2.947,3.517,3.476, P值均小于0.05）。細(xì)胞經(jīng)5次傳代后，鏡下觀察改良組細(xì)胞為圓形或小的多角形，或呈鵝卵石樣鋪滿瓶底；傳統(tǒng)組細(xì)胞胞體變大，增殖緩慢，呈片狀覆蓋瓶底；改良組S+G2/M期細(xì)胞所占百分率[(30.55±2.29)%]顯著高于傳統(tǒng)組[(17.01±5.58)%, t=3.890, P0.05]；改良組衰老細(xì)胞比率[（10.87±2.01）%]顯著低于傳統(tǒng)組[（75.93±4.11）%, t=24.624, P0.05]。 （2）顯微鏡下觀察細(xì)胞發(fā)現(xiàn)，轉(zhuǎn)染24h后有微弱熒光，48h后熒光增強(qiáng)，72h后熒光更強(qiáng)。當(dāng)以MOI=5,20,50,100轉(zhuǎn)染細(xì)胞72h后，RFP陽性表達(dá)率分別為1.68%,17.15%,47.53%,69.90%；病毒轉(zhuǎn)染72h后，MOI=0,5,20,50,100各組細(xì)胞吸光度值分別為1.14±0.143,1.05±0.073,1.02±0.090,1.03±0.141,0.912±0.102，組間比較差異有統(tǒng)計(jì)學(xué)意義（F=3.022，P0.05），兩兩比較MOI=0未轉(zhuǎn)染組和MOI=5組細(xì)胞吸光度值高于MOI=100組（P0.05）。 2.動物模型建立與創(chuàng)面修復(fù) （1）動物模型建立：術(shù)后7d、14d、21d，模型組創(chuàng)面收縮率分別為（1.3±0.5）%,（1.9±0.9）%,（2.6±1.3）%均顯著低于（8.6±1.2）%,（37.4±2.6）%,（65.0±3.7）%（t值分別為15.911,36.581,44.847，P均小于0.05）。 （2）創(chuàng)面修復(fù)： ①大體觀察：術(shù)后5d，異體皮顏色紅潤，術(shù)后14d異體皮干燥，開始脫落。術(shù)后21d異體皮基本脫落，空白組大鼠異體皮脫落時間晚于實(shí)驗(yàn)組。異體皮脫落后，微粒皮（1:10）移植組，微粒皮（1:20）+細(xì)胞懸液移植組、微粒皮（1:20）移植組、細(xì)胞懸液移植組創(chuàng)面均可見成片的上皮。大鼠創(chuàng)面愈合率：微粒皮（1:10）移植組創(chuàng)面愈合率為（84.3±11.9）%，微粒皮（1:20）+細(xì)胞懸液移植組創(chuàng)面愈合率為（74.2±8.0）%，微粒皮（1:20）移植組創(chuàng)面愈合率為（59.2±10.8）%，細(xì)胞懸液移植組創(chuàng)面愈合率為（53.8±11.5）%，空白組創(chuàng)面愈合率為（22.7±5.5）%，組間比較差異有統(tǒng)計(jì)學(xué)意義（F=34.446，P0.05），兩兩比較，微粒皮（1:10）移植組與微粒皮（1:20）+細(xì)胞懸液移植組兩組間差異無統(tǒng)計(jì)學(xué)意義（P0.05），均顯著高于微粒皮（1:20）移植組（P0.05），細(xì)胞懸液移植組創(chuàng)面也可愈合但上皮薄，易液化，空白組創(chuàng)面愈合率均顯著低于各實(shí)驗(yàn)組（P0.05），殘余大部分肉芽創(chuàng)面。 ②組織學(xué)觀察：HE染色，鏡下觀察，術(shù)后14d，微粒皮（1:10）移植組，微粒皮（1:20）+細(xì)胞懸液移植組、微粒皮（1:20）移植組、細(xì)胞懸液移植組新生上皮排擠異體皮，使其與創(chuàng)面部分分離，術(shù)后21d，異體皮脫落，微粒皮（1:10）移植組，微粒皮（1:20）+細(xì)胞懸液移植組、微粒皮（1:20）移植組表皮分層明顯，與正常大鼠皮膚表皮相比，表皮層明顯增厚，基底層呈柱狀排列，細(xì)胞懸液移植組表皮層含有較多空泡，空白組術(shù)后14d異體皮下無上皮細(xì)胞層，術(shù)后21d，異體皮脫落，可見肉芽組織。微粒皮（1:10）移植組，微粒皮（1:20）+細(xì)胞懸液移植組、微粒皮（1:20）移植組表皮-真皮連接層Ⅳ型膠原與層黏連蛋白有表達(dá)，細(xì)胞懸液移植組、空白組無表達(dá)，僅在真皮層新生血管處有部分表達(dá)。 ③小動物活體成像觀察：熒光標(biāo)記細(xì)胞移植，創(chuàng)面有熒光成像。 結(jié)論 1.低濃度胰蛋白酶消化法能減輕細(xì)胞損傷，，提高細(xì)胞活性、增加傳代次數(shù)，為實(shí)驗(yàn)研究創(chuàng)造良好條件。 2.慢病毒介導(dǎo)的RFP基因能夠表達(dá)于大鼠表皮細(xì)胞中，轉(zhuǎn)染效率與MOI值具有量效關(guān)系， MOI值為50可作為轉(zhuǎn)染表皮細(xì)胞的合適MOI值。 3.大鼠全層皮膚缺損創(chuàng)面創(chuàng)緣縫制鋼絲圈可有效減輕創(chuàng)面攣縮。 4.微粒皮與表皮細(xì)胞懸液聯(lián)用，可提高創(chuàng)面愈合率，減少裸露創(chuàng)面，該方法可顯著提高自體皮的利用效率，為大面積深度燒傷創(chuàng)面修復(fù)帶來新思路。
[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:
鈶犲ぇ浣撹瀵燂細(xì)鏈悗5d,寮備綋鐨鑹茬孩娑

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