發(fā)布時間：2018-01-29 13:47

  本文關鍵詞： 人臍帶間充質(zhì)干細胞 燒傷 創(chuàng)面 燒傷血清 Notch信號　出處：《中國人民解放軍醫(yī)學院》2014年博士論文　論文類型：學位論文

【摘要】：目的燒傷是平時、戰(zhàn)時的常見外傷，突發(fā)性成批燒傷的救治效果與維護社會穩(wěn)定密切相關。挽救嚴重燒傷患者生命的關鍵環(huán)節(jié)是盡早封閉創(chuàng)面。近些年的研究發(fā)現(xiàn)間充質(zhì)干細胞（Mesenchymal Stem Cells，MSCs）能夠有效修復損傷組織器官的結(jié)構(gòu)和功能�；贛SCs有效修復重建損傷組織器官的功能，本研究擬探討MSCs對嚴重燒傷創(chuàng)面的修復潛能。因此，將分離培養(yǎng)的人臍帶間充質(zhì)干細胞（humanUmbilical Cord Mesenchymal Stem Cells，hUCMSCs）移植入嚴重燒傷大鼠體內(nèi)，探討hUCMSCs對嚴重燒傷大鼠創(chuàng)面愈合的調(diào)控作用及機制，從而為臨床嚴重燒傷創(chuàng)面的治療提供新的思路和實驗依據(jù)。 方法(1)采用3種酶消化法（單純膠原酶Ⅱ消化法、膠原酶Ⅱ+胰蛋白酶消化法、膠原酶Ⅱ+透明質(zhì)酸酶消化法）及組織塊貼壁法分離培養(yǎng)hUCMSCs。免疫熒光染色法/流式鑒定細胞的表面標志物CD105、CD90、CD44、CD73、HLA-I、CD45、HLA-DR、CD31和vWF；油紅O染色、阿利新藍染色和Von Kossa鈣沉積法鑒定細胞成脂、成軟骨和成骨的分化潛能；倒置相差顯微鏡和透射電鏡觀察細胞的形態(tài)及超微結(jié)構(gòu)；MTT法檢測不同代細胞的增殖情況以及hUCMSCs對T細胞的調(diào)節(jié)作用；碘化丙啶（PI）染色和流式檢測不同代hUCMSCs的細胞周期。(2)收集特重度燒傷患者血清和正常對照血清。使用10%胎牛血清（10%FBS）、10%正常對照血清（10%HCS）和10%燒傷3d患者血清（10%BPS3d）培養(yǎng)體系培養(yǎng)hUCMSCs。AO-EB染色法/流式檢測三組細胞的凋亡情況；倒置相差顯微鏡觀察三組細胞的生長密度；MTT法檢測三組細胞的增殖情況；碘化丙啶（PI）染色和流式檢測三組的細胞周期；β-gal染色檢測三組細胞的衰老情況。(3)取成年雄性Wistar大鼠126只，隨機分成假傷組，燒傷組和燒傷+hUCMSCs移植組。將GFP標記的hUCMSCs或PBS通過尾靜脈注入對應組大鼠體內(nèi)。Image Pro Plus軟件評估大鼠創(chuàng)面的愈合率；小動物活體熒光成像系統(tǒng)（BLI）檢測GFP-hUCMSCs在大鼠體內(nèi)的遷移情況；PCR技術檢測創(chuàng)面是否表達人類特異性DNA；免疫組化染色檢測創(chuàng)面炎癥細胞浸潤程度、新生微血管數(shù)量以及I、III型膠原表達；激光多普勒血流儀評估創(chuàng)面的血流；ELISA法檢測創(chuàng)面促炎因子、抗炎因子、VEGF、I型膠原、III型膠原的含量。(4)實驗分為3組：10%正常對照血清(10%HCS)、10%燒傷3d患者血清(10%BPS3d)和10%燒傷3d患者血清+Notch信號特異性抑制劑DAPT/GSI(DAPT/GSI+BPS)或者分為5組：10%HCS、10%BPS3d、10%HCS+20ng/mL VEGF、10%HCS+20ng/mL bFGF和10%HCS+20ng/mLVEGF+20ng/mL bFGF培養(yǎng)hUCMSCs。MTT法和臺盼藍染色計數(shù)檢測hUCMSCs的存活和增殖情況； PI染色和流式檢測細胞的增殖周期；Western Blot檢測周期蛋白D(Cyclin D)的表達情況；ELISA法檢測血清中VEGF、bFGF等細胞因子的含量；免疫熒光染色法檢測VEGFR1和bFGFR2的表達情況；Western Blot和qRT-PCR檢測Notch信號關鍵分子Notch-1和Hes-1的表達情況。 結(jié)果(1)四種方法均可獲得hUCMSCs，但膠原酶Ⅱ+透明質(zhì)酸酶消化法獲得細胞數(shù)量較多，且獲得細胞的增殖速率較快。因此，膠原酶Ⅱ+透明質(zhì)酸酶消化法是一種高效提取hUCMSCs的方法。獲得的細胞陽性表達間質(zhì)系表面標志物，但不表達內(nèi)皮系和造血系表面標志物。傳代到3代時，細胞的形態(tài)為長梭形，呈旋渦狀生長。透射電鏡示該細胞的核大且不規(guī)則，核仁明顯，胞漿較少。使用特定誘導液誘導，其可向脂肪細胞、成軟骨和成骨細胞分化。細胞周期的結(jié)果顯示，80%以上的細胞處于靜止期(G0～G1期)，符合較原始干細胞的特征。綜合以上結(jié)果確定分離得到的細胞是hUCMSCs。MTT的結(jié)果示，，各代的hUCMSCs經(jīng)1d潛伏期，2d-6d是對數(shù)增殖期，于第7d時開始出現(xiàn)不同程度的接觸抑制而進入平臺期。此外，hUCMSCs能顯著抑制由植物血凝素(PHA)誘導同種異體T細胞的增殖。(2)10%FBS、10%HCS和10%BPS3d培養(yǎng)體系培養(yǎng)的hUCMSCs形態(tài)和結(jié)構(gòu)均正常，未見細胞核著橘紅色熒光的凋亡細胞或死亡細胞；流式的結(jié)果也驗證了此結(jié)果。MTT的結(jié)果示，從第2d至第6d，10%BPS3d組細胞的增殖速度和增殖細胞量顯著快于和多于其它兩組；倒置相差顯微鏡觀察細胞增殖生長的融合結(jié)果同MTT。10%BPS3d組細胞增殖期（S）的比例顯著高于其它兩組，而衰老細胞的百分率則顯著低于其他兩組。綜上所述，嚴重燒傷血清中含有一些保護細胞且促進細胞增殖的細胞因子，因此將hUCMSCs移植治療嚴重燒傷動物模型是可行的。(3)將GFP-hUCMSCs靜脈移植入嚴重燒傷大鼠體內(nèi)，其可隨著血液循環(huán)遷移到達燒傷創(chuàng)面，顯著降低創(chuàng)面炎癥細胞浸潤程度、顯著降低創(chuàng)面促炎因子IL-1，IL-6，TNF-α水平和顯著增加抗炎因子IL-10和TNF-α的刺激基因-6（TSG-6）水平。此外，hUCMSCs還可顯著增加創(chuàng)面新生微血管的數(shù)量和VEGF水平以及顯著上調(diào)創(chuàng)面中I型膠原、III型膠原比例，進而加速了嚴重燒傷創(chuàng)面的愈合過程。(4)與10%HCS相比，10%BPS3d可顯著促進hUCMSCs快速增殖和顯著促進hUCMSCs的細胞周期從靜止期進入了增殖期，也可顯著升高Cyclin D的表達水平以及可顯著升高Notch信號關鍵分子Notch-1和Hes-1mRNA和蛋白的表達水平，而給予Notch信號特異性抑制劑DAPT/GSI后，則可顯著降低10%BPS3d誘導的hUCMSCs增殖和顯著降低Notch-1和Hes-1的表達水平。BPS中VEGF和bFGF的含量顯著高于其它細胞因子，且hUCMSCs表達了VEGF和bFGF的受體。分別應用以及聯(lián)合應用VEGF和bFGF的抗體，發(fā)現(xiàn)聯(lián)合應用二者的抗體可以顯著降低由10%BPS3d誘導hUCMSCs的增殖，相反添加外源性聯(lián)bFGF和VEGF可顯著誘導hUCMSCs的增殖和顯著上調(diào)Notch-1和Hes-1mRNA和蛋白的表達水平。說明嚴重燒傷血清中的bFGF和VEGF是促進hUCMSCs的關鍵因子。 結(jié)論hUCMSCs能顯著加速嚴重燒傷大鼠創(chuàng)面的愈合。其次是嚴重燒傷患者血清中bFGF和VEGF激活了Notch信號通路進而促進hUCMSCs的存活和增殖，這也可能是hUCMSCs在嚴重燒傷大鼠創(chuàng)面微環(huán)境中存活、增殖和發(fā)揮促愈合功能的機制之一。
[Abstract]:To burn is common in peacetime, wartime trauma, the treatment effect of sudden mass burn is closely related to the maintenance of social stability. The key is to save lives in patients with severe burn wound closure as soon as possible. In recent years, the study found that mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) can effectively repair the structure and function of tissues and organs. MSCs effective repair and reconstruction of tissue and organ function damage based on, this study intends to explore the potential of MSCs for severe burn wound repair. Therefore, the isolation and culture of human umbilical cord mesenchymal stem cells (humanUmbilical Cord Mesenchymal Stem Cells, hUCMSCs) transplanted in severely burned rats, to investigate the regulatory effect and mechanism of hUCMSCs on wound healing in severe burn in rats, so as to provide new ideas and experimental basis for the clinical treatment of severe burn wounds.
Methods (1) using 3 enzyme digestion method (simple collagenase digestion method, collagenase and trypsin, collagenase and hyaluronidase digestion) were isolated and cultured hUCMSCs. immunofluorescence staining by flow cytometry / surface markers of CD105 cells, and tissue explant method, CD90, CD44, CD73 HLA-I, CD45, HLA-DR, CD31, and vWF; oil red O staining, alcian blue staining and Von Kossa calcium deposition identification of adipogenic, chondrogenic and osteogenic differentiation potential; inverted phase contrast microscope and transmission electron microscope to observe the morphology and ultrastructure of cells; MTT method to detect cell proliferation and hUCMSCs generation regulation of T cells; propidium iodide (PI) staining and flow cytometry to detect the cell cycle of different generations of hUCMSCs. (2) collected in severe burn patients serum and normal serum. 10% fetal bovine serum (10%FBS) and 10% normal control serum (10%HCS) and 10% burn The serum of patients with 3D injury (10%BPS3d) in vitro culture system hUCMSCs.AO-EB staining flow cytometry / apoptosis cells in three groups; inverted growth density was observed in the three groups cells microscope; the proliferation of MTT was detected in three groups of cells; propidium iodide (PI) staining and flow cytometry cell cycle in three groups; aging detection of three groups of beta cells -gal staining. (3) adult male Wistar 126 rats were randomly divided into sham injury group, burns group and +hUCMSCs group. GFP labeled hUCMSCs or PBS by intravenous injection of corresponding group rats.Image Pro Plus software to evaluate the healing rate of rats was small; animal in vivo fluorescence imaging system (BLI) for detection of GFP-hUCMSCs in the migration of rats; detection of wound whether PCR expression of human specific DNA; immunohistochemical staining was used to detect the wound inflammation cells infiltration, angiogenesis The number and I expression of type III collagen; laser Doppler flowmetry evaluation of wound blood flow; proinflammatory cytokines, detection of wound ELISA anti-inflammatory factor, VEGF, collagen type I, collagen type III. (4) were divided into 3 groups: 10% normal control serum (10%HCS), 3D (10% in serum of patients with burn 10%BPS3d) and 10% 3D patients with burn serum +Notch signal specific inhibitor DAPT/GSI (DAPT/GSI+BPS) or divided into 5 groups: 10%HCS, 10%BPS3d, 10%HCS+20ng/mL VEGF, 10%HCS+20ng/mL bFGF and 10%HCS+20ng/mLVEGF+20ng/mL bFGF in cultured hUCMSCs.MTT assay and trypan blue staining counting detection hUCMSCs survival and proliferation; PI staining and flow cytometry to detect the cell cycle; Western Blot detection of cyclin D (Cyclin D) expression; VEGF in serum were measured by ELISA, the content of bFGF and other cytokines; immunofluorescence staining was used to detect VEGFR1 expression of bFGFR2 and W; The expression of the key molecules of Notch signals, Notch-1 and Hes-1, was detected by estern Blot and qRT-PCR.
Results (1) the four methods can get hUCMSCs, but collagenase and hyaluronic acid from enzyme digestion and cell number, cell proliferation rate. Therefore, collagenase II + hyaluronic acid enzyme digestion method is an efficient method for hUCMSCs extraction. The positive expression of cell surface markers of mesenchymal lineage get, but not the expression of endothelial and hematopoietic markers. The passage to the 3 generation, the morphology of the cells were fusiform, showing the vortex like growth. The transmission electron microscope showed that the cells with large nuclei and irregular nucleoli, less cytoplasm. Use specific induction medium induced, the fat to cells, chondrogenic and osteogenic differentiation. The cell cycle showed that more than 80% of the cells in the stationary phase (G0 ~ G1), with the more primitive stem cell characteristics. Based on the above results to determine the isolated cells is the result of hUCMSCs.MTT shows, the generation of hUCMSCs by 1D potential Ummer, 2d-6d is the logarithmic growth phase, began to appear different degree of contact inhibition and into the platform on the 7d. In addition, hUCMSCs significantly inhibited by phytohemagglutinin (PHA) induced by allogeneic T cell proliferation. (2) 10%FBS, 10%HCS and 10%BPS3d culture hUCMSCs the morphology and structure of the culture system were normal. Neclei with orange red fluorescence in apoptotic cells or dead cells; flow cytometry results also verified the results of.MTT showed that from the 2D to the 6D, the proliferation rate and cell proliferation capacity of 10%BPS3d cells were significantly higher than the group and more than the other two groups; observation of fusion cell proliferation and growth of the cells in MTT.10%BPS3d group the proliferative phase inverted microscope (S) were significantly higher than those of the other two groups, while the percentage of senescent cells is significantly lower than the other two groups. To sum up, the serious burn serum contains some protective cells and promote cell Cell proliferation factor, therefore the hUCMSCs transplantation in the treatment of severe burn animal model is feasible. (3) the GFP-hUCMSCs transplanted into rats with severe burns, which can migrate to the blood circulation of burn wounds, significantly reduced inflammatory cell infiltration of the wound, wound significantly reduced proinflammatory cytokines IL-1, IL-6, TNF- and alpha level increased stimulation of anti-inflammatory factor IL-10 and TNF- alpha gene -6 (TSG-6) level. In addition, hUCMSCs also significantly increased the wound microvascular number and VEGF levels and significantly increased the wound in type I collagen, type III collagen ratio, and then accelerate the healing process of severe burn wounds. (4) compared with 10%HCS. 10%BPS3d can significantly promote the hUCMSCs rapid proliferation and significantly promote cell cycle hUCMSCs in proliferative stage from quiescence, the expression level can be significantly increased Cyclin and D significantly increased Notch signal key The expression level of Notch-1 and Hes-1mRNA and protein molecules, and give Notch a specific inhibitor of DAPT/GSI signal, can significantly reduce 10%BPS3d induced hUCMSCs proliferation and significantly reduce the content of VEGF and bFGF Notch-1 and the expression level of Hes-1 in.BPS was significantly higher than that of other cytokines and the expression of hUCMSCs, VEGF and bFGF were used and the receptor. The combined application of VEGF antibody and bFGF antibody, found that the combination of the two can be significantly reduced by 10%BPS3d induced hUCMSCs proliferation, instead of adding exogenous bFGF and VEGF could significantly induce hUCMSCs proliferation and significantly up-regulated expression of Notch-1 and Hes-1mRNA and protein. Severe burn serum levels of bFGF and VEGF is a key factor the promotion of hUCMSCs.
Conclusion hUCMSCs can obviously accelerate wound healing of rats with severe burns. Followed by bFGF and VEGF in serum of patients with severe burns in the activation of Notch signaling pathway and promote the survival and proliferation of hUCMSCs, which may also be hUCMSCs survival in severe burn rats wound microenvironment, proliferation and promote healing mechanism of function.

【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R644

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