本文選題：臍帶間充質(zhì)細胞 + 創(chuàng)面愈合　； 參考：《中國人民解放軍軍醫(yī)進修學院》2011年博士論文

【摘要】：目的探討從新生兒臍帶華通氏膠中分離、培養(yǎng)人臍帶間充質(zhì)干細胞(umbilical cord mesenchymal stem cells, UCMSCs),并對其表型鑒定以及生物學特性進行檢測。將UCMSCs與兔異體皮+自體微粒皮復合移植于兔皮膚全層缺損創(chuàng)面,觀察UCMSCs促進微粒皮修復創(chuàng)面的效果,為其臨床應用提供實驗支持。觀察UCMSCs在不同孔徑的聚碳酸酯膜上生長和遷移情況,體外快速分離、培養(yǎng)表皮干細胞(epidermal stem cells, ESCs),將UCMSCs與ESCs分別接種在聚碳酸酯膜正反兩面進行共培養(yǎng),誘導UCMSCs分化為表皮樣細胞,為以UCMSCs做為組織工程皮膚種子細胞提供實驗依據(jù)。 方法1取正常足月產(chǎn)健康新生兒臍帶,采用酶消化法及組織塊法的方法分離培養(yǎng)UCMSCs。以相差顯微鏡、HE染色及透射電鏡觀察UCMSCs形態(tài)及超微結(jié)構(gòu)；以免疫熒光及流式細胞術鑒定細胞表面標志物；通過MTT法檢測第1、3、5代細胞的增殖狀況,并繪制生長曲線；流式細胞術檢測第2、4代細胞周期；通過成脂以及成骨實驗檢測人臍帶間充質(zhì)干細胞多向分化的潛能。 2以腺病毒介導GFP基因體外轉(zhuǎn)染UCMSCs 48h,于兔耳皮下注射移植,于3d、7d、14d、21d取材冰凍切片熒光顯微鏡下觀察GFP表達情況。取成年日本大耳兔8只,隨機抽取2只分為一組,同時構(gòu)建兔背部急性皮膚全層缺損創(chuàng)面模型(每只兔2個創(chuàng)面),交換移植同等大小面積的反削斷層皮,其真皮面均勻粘附兔自體微粒皮。所有兔頭側(cè)創(chuàng)面為UCMSCs移植組(A組)、尾側(cè)為PBS空白自身對照組(B組)。于術后14d、21d觀察創(chuàng)面情況,計算21d創(chuàng)面愈合率,進行統(tǒng)計學分析。于術后21d切取創(chuàng)面愈合區(qū)域組織,對標本行HE染色觀察。 3取健康包皮環(huán)切術患者包皮皮片,采用中性蛋白酶和胰酶消化結(jié)合Ⅳ型膠原黏附法分離、培養(yǎng)hESCs,以相差顯微鏡及HE染色觀察ESCs形態(tài)及生長特點;以免疫熒光染色鑒定細胞標志CK19、P63和β1-integrin。 4UCMSCs以絲裂霉素C處理后接種于常用的0.4、3.0和8.0μm三種膜孔徑的6孔板Transwell插件中,培養(yǎng)7d,觀察、計數(shù)三種孔徑插件底部貼壁細胞,計算遷移率,并在掃描電鏡下觀察細胞在多孔膜上的生長、遷移情況。 5在聚碳酸酯膜底面預培養(yǎng)ESCs的Transwell插件內(nèi)接種UCMSCs,使UCMSCs與ESCs在聚碳酸酯膜正反兩面生長形成非直接接觸式共培養(yǎng),誘導UCMSCs向表皮細胞分化。共培養(yǎng)10d后,消化收集細胞,爬片后進行形態(tài)學觀察；并進行免疫熒光染色檢測細胞標志物CK19、P63和β1-integrin的變化；收集誘導后細胞通過Western blotting及qRT-PCR的方法,在mRNA和蛋白水平檢測CK19、P63和β1-integrin表達情況；通過流式細胞術檢測Transwell新型共培養(yǎng)與傳統(tǒng)共培養(yǎng)誘導后細胞CK19表達率。 結(jié)果1采用復合酶消化法和組織塊法培養(yǎng)均從臍帶華通氏膠中分離出成纖維樣細胞,細胞呈梭形或多角形,呈旋渦狀生長。透射電鏡觀察,細胞核大且不規(guī)則,核仁明顯,細胞漿較少,細胞器以粗面內(nèi)質(zhì)網(wǎng)和線粒體為主。MTT法繪制生長曲線顯示,各代細胞經(jīng)過1d的潛伏期后,進入對數(shù)增殖期,第7d之后開始出現(xiàn)不同的接觸抑制。細胞周期檢測80%以上的細胞處于靜止期。流式細胞儀檢測顯示分離培養(yǎng)的細胞表達CD44 (98.07%)、CD105 (95.29%)、CD73 (98.58%)、CD29 (99.50%)和HLA-Ⅰ(99.80%),不表達CD34 (0.59%)、CD31 (0.24%)、CD45 (1.91%)和HLA-DR (1.18%)。免疫熒光染色顯示,細胞CD90和CD44陽性表達, CD31和CD45陰性表達。在體外特殊誘導條件下,這些細胞能夠向脂肪細胞和成骨細胞分化,顯示它們具有多向分化潛能。 2腺病毒介導的GFP基因轉(zhuǎn)染UCMSCs后48h,熒光顯微鏡觀察顯示轉(zhuǎn)染UCMSCs可有效表達GFP。于兔耳皮下注射移植后3d、7d時取材冰凍切片觀察,UCMSCs較強表達GFP；14d、21d取材觀察仍有GFP減弱表達。術后21d創(chuàng)面愈合率A組高于B組(P0.05)。愈合后創(chuàng)面取材病理學觀察可見表皮層下大量不規(guī)則膠原纖維,成纖維細胞以及小血管,缺少皮膚附件；移植組表皮層可發(fā)現(xiàn)表皮釘突樣結(jié)構(gòu),而對照組表皮層底部平坦,與基底結(jié)合較為疏松。 3中性蛋白酶和胰酶消化結(jié)合Ⅳ型膠原黏附法分離、培養(yǎng)ESCs,形態(tài)學觀察可見hESCs呈扁圓狀多角形,融合后呈鋪路石樣生長；免疫熒光染色鑒定細胞標志CK19、P63和β1-integrin均呈陽性表達。 4培養(yǎng)7d后,0.4μm、3.0μm和8.0μm三種Transwell插件內(nèi)貼壁UCMSCs的遷移率分別為0、1.8%、8.0%,0.4μm孔徑聚碳酸酯膜膜細胞遷移率為0。掃描電鏡下觀察到細胞在3.0μm和8.0gm孔徑聚碳酸酯膜底面生長以及穿越微孔的現(xiàn)象,而在0.4μm孔徑膜則未發(fā)現(xiàn)。 5UCMSCs與ESCs在聚碳酸酯膜正反兩面非直接接觸式共培養(yǎng)10d后,誘導組UCMSCs形態(tài)呈扁圓狀多角形,而對照組細胞形態(tài)未變化；免疫熒光染色、Western blotting檢測誘導組細胞CK19、P63陽性表達,對照組則為陰性；實時定量PCR檢測CK19、P63 mRNA表達,誘導組表達水平顯著高于對照組,差異具有統(tǒng)計學意義(P0.01)。而β1-integrin的各種表達誘導組細胞與對照組相比沒有明顯減弱。流式細胞術檢測CK19陽性率,新型共培養(yǎng)誘導組(54.3%)與傳統(tǒng)Transwell共培養(yǎng)誘導組(11.4%)相比,差異具有統(tǒng)計學意義(P0.01)。 結(jié)論通過兩種方法能夠從臍帶分離、培養(yǎng)出MSCs, UCMSCs增殖能力強,經(jīng)細胞表面標志物、生物學特性以及多向分化能力檢測,符合MSCs評價標準。UCMSCs可促進創(chuàng)面微粒皮生長,縮短創(chuàng)面愈合時間。Ⅳ型膠原黏附法能夠快速、有效分離表皮干細胞；而0.4μm孔徑的聚碳酸酯膜能夠阻止細胞遷移通過,在該孔徑聚碳酸酯膜正反兩面近距離共培養(yǎng)UCMSCs和ESCs,能夠較為有效地誘導UCMSCs向表皮樣細胞分化。UCMSCs能夠成為組織工程皮膚所需理想的種子細胞。
[Abstract]:Objective to study the isolation of umbilical cord mesenchymal stem cells (UCMSCs) from Huatong's gum in newborn umbilical cord, and to detect the phenotypic identification and biological characteristics of human umbilical cord mesenchymal stem cells (UCMSCs). The transplantation of UCMSCs with rabbit skin and autologous particle skin to the full-thickness skin defect of rabbit skin was carried out, and UCMSCs promoted the particle skin. To repair the effect of the wound, we provide experimental support for its clinical application. Observe the growth and migration of UCMSCs on polycarbonate membranes with different pore sizes, rapid separation in vitro, culture of epidermal stem cells (epidermal stem cells, ESCs), and co culture UCMSCs and ESCs in the positive and negative sides of polycarbonate membrane, and induce UCMSCs to differentiate into epidermis. The like cells provide an experimental basis for using UCMSCs as a seed cell for tissue-engineered skin.
Methods 1 healthy newborn umbilical cord was obtained from normal term, and UCMSCs. was isolated and cultured by enzyme digestion method and tissue block method. The morphology and ultrastructure of UCMSCs were observed by phase contrast microscope, HE staining and transmission electron microscopy. The cell surface markers were identified by immunofluorescence and flow cytometry, and the proliferation of 1,3,5 cells was detected by MTT method. The growth curve was plotted and the cell cycle of the 2,4 generation was detected by flow cytometry; the multidirectional differentiation potential of human umbilical cord mesenchymal stem cells was detected by lipid formation and osteogenesis.
2 transfection of GFP gene with adenovirus mediated UCMSCs 48h in vitro and subcutaneous injection in rabbit ear. The expression of GFP in 3D, 7d, 14d, 21d was observed under frozen section fluorescence microscope. 8 adult Japanese big ear rabbits were randomly selected and 2 were randomly divided into one group. At the same time, the rabbit model of the full layer defect on the skin of the rabbit back (2 wounds of each rabbit) was constructed, and the exchange shift was carried out. The true skin surface adhered to the rabbit autogenous skin with the same size and area. All the rabbit head side wounds were UCMSCs transplantation group (group A), and the tail side was PBS blank control group (group B). After 14d, 21d, the wound healing rate was observed after 14d and 21d, and statistical analysis was made to calculate the healing rate of the 21d wound. After 21d, the wound healing regional tissue was cut by 21d, right after operation. The mark was observed by HE staining.
3 the patients with circumcision of healthy circumcision were separated by neutral protease and trypsin digestion combined with type IV collagen adhesion, and hESCs was cultured. The morphology and growth characteristics of ESCs were observed by phase contrast microscope and HE staining. Immunofluorescence staining was used to identify cell markers CK19, P63 and beta 1-integrin..
4UCMSCs was treated with mitomycin C and inoculated into 6 pore Transwell plug-ins with three kinds of pore diameter of 0.4,3.0 and 8 mu m, and cultured 7d. Observe and count the wall cells at the bottom of three kinds of aperture plug-ins, calculate the mobility, and observe the growth and migration of the cells on the porous membrane under scanning electron microscope.
5 inoculating UCMSCs in the Transwell plug-in of pre culture ESCs on the bottom of polycarbonate membrane, which makes UCMSCs and ESCs grow in the positive and negative sides of the polycarbonate membrane to form a non direct contact co culture and induce the differentiation of UCMSCs into the epidermal cells. After co culture 10d, the cells are collected and the cells are collected to observe the morphologic observation after climbing the tablet, and the immunofluorescence staining is used to detect the cells. The changes in the markers CK19, P63 and beta 1-integrin; the cells were collected and induced by Western blotting and qRT-PCR to detect CK19, P63 and beta 1-integrin expressions at mRNA and protein levels, and the cell CK19 expression rate was detected by the flow cytometry for the Transwell new co culture and the traditional co culture.
Results 1 the fibroblast like cells were isolated from the Huatong's gum with compound enzyme digestion method and tissue block method. The cells were spindle shaped or polygonal, and the cells were vortexed. The cell nuclei were large and irregular, the nucleolus was obvious, and the cytoplasm was less. The growth curves were plotted by the.MTT method of rough surface endoplasmic reticulum and mitochondria. After the incubation period of 1D, each cell entered the logarithmic proliferation period. After 7d, different contact inhibition began to appear. Cell cycle detection more than 80% cells were at rest. Flow cytometry showed that the cultured cells expressed CD44 (98.07%), CD105 (95.29%), CD73 (98.58%), CD29 (99.50%) and HLA- I (99.80%). CD34 (0.59%), CD31 (0.24%), CD45 (1.91%) and HLA-DR (1.18%). Immunofluorescence staining showed that the positive expression of CD90 and CD44 and negative expression of CD31 and CD45 were expressed. Under special induction conditions, these cells could differentiate into adipocytes and osteoblasts, showing their multipotential differentiation potential.
2 adenovirus mediated GFP gene was transfected to 48h after UCMSCs, and the fluorescence microscope showed that transfection of UCMSCs could effectively express GFP. in 3D after subcutaneous injection of rabbit ear, and the frozen section was observed in 7d, and UCMSCs strongly expressed GFP; 14d, 21d observation still had the attenuation of GFP. Pathological observation showed a large number of irregular collagen fibers under the epidermis, fibroblasts and small vessels and lack of skin appendages. The epidermis of the transplantation group could find the peg like structure of epidermis, while the epidermis of the control group was flat and loosely combined with the base.
3 neutral protease and trypsin digestion combined with type IV collagen adhesion method were isolated and cultured for ESCs. Morphological observation showed that hESCs showed flat polygonal polygon. After fusion, it was paved stone like growth. Immunofluorescence staining was used to identify cell markers CK19, P63 and beta 1-integrin were all positive.
4 after culture of 7D, the migration rates of UCMSCs in the three Transwell plug-ins of 0.4, 3, and 8 m were 0,1.8%, 8%, and 0.4 mu m membrane cell membrane cell migration rate was 0. scanning electron microscopy to observe the growth of the cells at the bottom of the 3 and 8.0gm aperture polycarbonate membrane and through the micropores, while the 0.4 mu m aperture membrane was not found.
After the 5UCMSCs and ESCs were co cultured on the positive and negative two sides of the polycarbonate membrane, the UCMSCs morphology of the induced group was flat round polygon, but the cell morphology of the control group was not changed, and the immunofluorescence staining, Western blotting detected the cells CK19, P63 positive and negative in the control group, and the real-time quantitative PCR detection CK19, P63 mRNA expression, The expression level of the induced group was significantly higher than that in the control group (P0.01), but the cells in the induction group of beta 1-integrin were not significantly decreased compared with the control group. The positive rate of CK19 in the flow cytometry, the new co culture induction group (54.3%) and the traditional Transwell Co culture induction group (11.4%) were statistically significant. Meaning (P0.01).
Conclusion by two methods, MSCs can be isolated from the umbilical cord, and the proliferation ability of UCMSCs is strong. The cell surface markers, biological characteristics and multidirectional differentiation ability are detected. The MSCs evaluation standard.UCMSCs can promote the growth of the wound particle skin and shorten the healing time of the wound. The polycarbonate membrane with 0.4 micron m aperture can prevent cell migration from passing through, and co culture UCMSCs and ESCs in the near and opposite sides of the porous polycarbonate membrane. It can effectively induce UCMSCs to differentiate into epidermal like cells and become ideal seed cells for tissue engineering skin.

【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R329

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