维甲酸诱导腭裂小鼠出生后未分化腭间充质细胞定位研究

发布时间：2018-06-19 21:02

本文选题：腭间充质细胞 + BrdU　；参考：《大连医科大学》2011年硕士论文

【摘要】：先天性唇腭裂是最常见的先天性发育畸形,常因为腭板短小而使手术治疗难以完全修复缺损。唇腭部组织再生修复目前仅见于干细胞促进的骨组织再生,而对腭部其他组织包括骨、骨骼肌和肌腱、软骨以及神经等未见报道。目的:本研究利用外源性BrdU可以标记slow-cycling long term label- retaining cells(LRCs)的特性,同时利用间充质干细胞候选标志物Stro-1、P75和CD57染色,定位正常小鼠出生后腭部未分化间充质细胞,进而检测维甲酸诱导腭裂小鼠腭部未分化间充质细胞与正常表达部位和模式的差异。方法:选择SPF级ICR孕鼠于E10给予维甲酸管饲做为实验组,建立小鼠腭裂模型。利用具有分化潜能细胞的慢周期特性,在E12给孕鼠80mg/kg的BrdU腹腔注射,分别在P0对照组和实验组获得新生小鼠组织进行灌流固定,常规脱水、透明、浸蜡、包埋,连续4μm蜡片备用。利用免疫荧光染色法,检测腭间充质细胞BrdU以及Stro-1、P75及CD57表达水平和分布。结果: 1. P75和CD57阳性表达在细胞膜上。在正常对照组中,强表达在未发生骨化的腭中缝处的未分化间充质细胞,P75在邻近上皮的间充质中也有表达。在腭上皮中CD57表达在棘细胞全层,而P75特异性地表达在邻近基底层的棘细胞中。在维甲酸诱导腭裂组中,主要表达在血管周围及骨化中心周围的未分化间充质中,CD57在腭突远中端有较强阳性表达,在上皮中均未见表达。 2. BrdU阳性表达在细胞核中,主要分为致密型强表达和散在颗粒型弱表达两种类型。在正常对照组中,致密型强表达集中在未发生骨化的腭中缝处的未分化间充质细胞中,前后腭无明显差异(P0.05)。在维甲酸诱导腭裂组中,致密型强表达分布于腭突正中嵴处以及血管周围的间充质细胞中,前部腭突多于后部。维甲酸诱导腭裂组BrdU阳性表达显著多于正常对照组(P0.01)。Stro-1与BrdU致密型强表达在血管周围有部分重叠,但阳性表达细胞数量较少。结论:正常小鼠在出生后腭部未分化间充质细胞数量较少,并主要局限于腭中缝处;而在出生后维甲酸诱导腭裂小鼠中,数量较多且较广泛存在于血管周围的腭突未分化间充质细胞为其组织工程学修复提供了可能。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】