本文選題：腭間充質細胞 + BrdU　； 參考：《大連醫(yī)科大學》2011年碩士論文

【摘要】：先天性唇腭裂是最常見的先天性發(fā)育畸形,常因為腭板短小而使手術治療難以完全修復缺損。唇腭部組織再生修復目前僅見于干細胞促進的骨組織再生,而對腭部其他組織包括骨、骨骼肌和肌腱、軟骨以及神經等未見報道。 目的:本研究利用外源性BrdU可以標記slow-cycling long term label- retaining cells(LRCs)的特性,同時利用間充質干細胞候選標志物Stro-1、P75和CD57染色,定位正常小鼠出生后腭部未分化間充質細胞,進而檢測維甲酸誘導腭裂小鼠腭部未分化間充質細胞與正常表達部位和模式的差異。 方法:選擇SPF級ICR孕鼠于E10給予維甲酸管飼做為實驗組,建立小鼠腭裂模型。利用具有分化潛能細胞的慢周期特性,在E12給孕鼠80mg/kg的BrdU腹腔注射,分別在P0對照組和實驗組獲得新生小鼠組織進行灌流固定,常規(guī)脫水、透明、浸蠟、包埋,連續(xù)4μm蠟片備用。利用免疫熒光染色法,檢測腭間充質細胞BrdU以及Stro-1、P75及CD57表達水平和分布。 結果: 1. P75和CD57陽性表達在細胞膜上。在正常對照組中,強表達在未發(fā)生骨化的腭中縫處的未分化間充質細胞,P75在鄰近上皮的間充質中也有表達。在腭上皮中CD57表達在棘細胞全層,而P75特異性地表達在鄰近基底層的棘細胞中。在維甲酸誘導腭裂組中,主要表達在血管周圍及骨化中心周圍的未分化間充質中,CD57在腭突遠中端有較強陽性表達,在上皮中均未見表達。 2. BrdU陽性表達在細胞核中,主要分為致密型強表達和散在顆粒型弱表達兩種類型。在正常對照組中,致密型強表達集中在未發(fā)生骨化的腭中縫處的未分化間充質細胞中,前后腭無明顯差異(P0.05)。在維甲酸誘導腭裂組中,致密型強表達分布于腭突正中嵴處以及血管周圍的間充質細胞中,前部腭突多于后部。維甲酸誘導腭裂組BrdU陽性表達顯著多于正常對照組(P0.01)。Stro-1與BrdU致密型強表達在血管周圍有部分重疊,但陽性表達細胞數量較少。 結論:正常小鼠在出生后腭部未分化間充質細胞數量較少,并主要局限于腭中縫處;而在出生后維甲酸誘導腭裂小鼠中,數量較多且較廣泛存在于血管周圍的腭突未分化間充質細胞為其組織工程學修復提供了可能。
[Abstract]:Congenital cleft lip and palate is the most common congenital malformation. It is often difficult to repair the defect completely because of the short palatine plate. The regenerative repair of the lip and palate tissue is only seen in the regeneration of the bone tissue promoted by stem cells, but the other tissues of the palate, including bone, skeletal muscle and tendon, cartilage and nerve, have not been reported.
Objective: This study used exogenous BrdU to mark the characteristics of slow-cycling long term label- retaining cells (LRCs), and at the same time use the candidate markers of mesenchymal stem cells Stro-1, P75 and CD57 staining, to locate the undifferentiated palatine mesenchymal cells in normal mice after birth, and then detect the undifferentiated palatine mesenchyme of palatine mice induced by retinoic acid. The difference between the cells and the normal expression sites and patterns.
Methods: the SPF grade ICR pregnant rats were given the retinoic acid tube in E10 as the experimental group, and the mouse cleft palate model was established. By using the slow cycle characteristics of the differentiated cells, the BrdU intraperitoneal injection of 80mg/kg in the pregnant mice was intraperitoneally injected into the P0 control group and the experimental group. The routine dehydration, transparency, wax impregnation, embedding, and embedding were performed in the P0 control group and the experimental group. 4 mu m wax tablet was added. The expression level and distribution of BrdU and Stro-1, P75 and CD57 in palatal mesenchymal cells were detected by immunofluorescence staining.
Result:
1. P75 and CD57 were expressed in the cell membrane. In the normal control group, undifferentiated mesenchymal cells were strongly expressed in the middle suture of the palatine without ossification. P75 was also expressed in the mesenchyme of the adjacent epithelium. In the palatine epithelium, CD57 was expressed in the whole layer of the spinous cells, and P75 was specifically expressed in the acanthosis adjacent to the basal layer. In the cleft palate group, it is mainly expressed in the undifferentiated mesenchyme around the vessels and around the ossification center. CD57 has a strong positive expression at the distal end of the palatine process, and no expression is found in the epithelium.
The positive expression of 2. BrdU in the nucleus is mainly divided into two types of dense strong expression and granular weak expression. In the normal control group, dense strong expression is concentrated in undifferentiated mesenchymal cells in the middle suture of the palatine without ossification, and there is no obvious difference between the front and back of the palate (P0.05). In the median crest of the palatine process and in the perivascular mesenchyme cells, the anterior palatine process was more than the posterior part. The positive expression of BrdU in the cleft palate group induced by retinoic acid was significantly more than that of the normal control group (P0.01) and the dense expression of.Stro-1 and BrdU was partly overlapped around the vessels, but the number of positive cells was less.
Conclusion: the number of undifferentiated mesenchyme cells in the palate is less in normal mice and is mainly limited to the middle of the palatine, but in the postnatal retinoic acid induced cleft palate mice, the number of undifferentiated palatine mesenchymal cells, which are more widespread around the vessels, provides the possibility for the repair of the tissue engineering.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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