人脐带间充质干细胞治疗肢体火器伤的动物实验研究

发布时间：2018-02-15 21:35

本文关键词： 人脐带间充质干细胞火器伤创伤愈合　出处：《福建医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的： 1.探讨人脐带间充质干细胞（human umbilical cord-derived mesenchymalstem cells，hUCMSCs）体外分离、纯化和培养方法，并鉴定其生物学特性，为后续研究奠定基础。 2.采用不同投射物对猪后肢肌肉软组织进行致伤，评估、建立一种局部伤情稳定性高、可重复性好、实战模拟性强的火器伤动物模型，为后续研究奠定基础。 3.提出一种新的火器伤治疗方法：局部清创引流+局部注射hUCMSCs，并在猪后肢火器伤模型中评价其疗效。方法： 1.采用组织块贴壁法从健康产妇脐带中分离hUCMSCs，传代培养后光镜下进行细胞形态学观察，通过流式细胞仪检测细胞表面抗原，并检测hUCMSCs成脂、成骨、成软骨诱导分化的潜能。 2.分别采用“五六”式弹道枪发射7.62mm制式弹及“五三”式滑膛枪发射钢制球形破片射击8只随机分配的长白猪双侧后肢，通过观察伤情、伤道入出口面积、伤道最大横截面积、伤道组织病理学分区，计算伤道组织细菌计数来评价模型伤情的稳定性。 3.取长白猪20只，7.62mm制式弹致伤后随机分为2组：手术清创引流+hUCMSCs注射组（A组）、手术清创引流+无菌生理盐水注射组（B组）。通过检测伤道组织细菌量、观察组织病理学改变以及伤道愈合情况来评价各组的创伤愈合效果。结果： 1.组织块贴壁法分离的hUC-MSCs强表达CD90、CD29、CD73、CD105，弱表达CD106，不表达CD45、CD34及HLA-DR，，能够分化为脂肪细胞、骨细胞和软骨细胞。 2.两种投射物均可造成伤情稳定且具有良好可重复性的单纯软组织贯通伤，两组间伤道入出口面积无显著差异（p＞0.05），但伤道最大横截面积有显著差异（p0.05），两种方法建立的动物模型均具有典型火器伤病理学特点，细菌计数检测显示伤道组织污染程度均较重，两组间无显著差异（p＞0.05）。 3.致伤后经不同处理， A组的伤道组织细菌量显著低于B组（p＜0.05）。两组间震荡区病理学表现不同，Ａ组炎症反应轻于Ｂ组，A组伤道愈合效果优于B组。结论： 1.组织块贴壁法是一种理想的hUC-MSCs体外分离培养方法，根据这种方法培养出的hUC-MSCs纯度高、生物学特征稳定，可用于后续实验研究。 2.2种投射物致伤效果相似，相比之下，采用“五六”式弹道枪发射7.62mm制式弹更适合模拟战时真实伤情，适用于进一步的实验研究。 3. hUC-MSCs治疗肢体火器伤有效。
[Abstract]:Objective:. 1. To investigate the methods of isolation, purification and culture of human umbilical cord-derived mesenchymalstem cells (hUCMSCs) from human umbilical cord mesenchymal stem cells in vitro. 2. Using different projectiles to injure the muscle and soft tissue of pig hind limb, a kind of animal model of firearm injury with high stability, reproducibility and simulated combat was established, which laid a foundation for further study. 3. A new method for the treatment of firearm injury: local debridement and drainage, local injection of hUCMSCsand evaluation of its curative effect in the model of pig hind limb firearm injury. Methods:. 1. HUCMSCs were isolated from umbilical cord of healthy parturient by tissue mass adherent method. Cell morphology was observed under light microscope after passage culture, cell surface antigen was detected by flow cytometry, and the potential of hUCMSCs fat-forming, osteogenesis and chondrogenic differentiation was detected. 2. Using "56-type ballistic gun" to fire 7.62mm standard projectile and "53-type" shotgun firing steel spherical fragment to shoot 8 randomly allocated pig hind limbs. By observing the injury condition, the wound path entrance and exit area and the maximum cross section area of the wound path were observed. To evaluate the stability of the injury model, the bacterial count of wound tissue was calculated in order to evaluate the stability of the model. 3. Twenty Landrace pigs were randomly divided into two groups after injury with 7.62mm standard bomb: surgical debridement and drainage of hUCMSCs injection group (group A) and surgical drainage of sterile saline injection group (group B). Histopathological changes and wound healing were observed to evaluate the wound healing effect in each group. Results:. 1. HUC-MSCs isolated by tissue mass adherent method strongly expressed CD90, CD29, CD73, CD105, weakly expressed CD106, and did not express CD45, CD34 and HLA-DR.It could differentiate into adipocytes, osteocytes and chondrocytes. 2. Both projectiles can cause stable injuries and simple soft tissue penetrating injuries with good repeatability. There was no significant difference in the entrance and exit area between the two groups (p > 0.05), but there was a significant difference in the maximum cross-sectional area of the wound path between the two groups. The animal models established by the two methods had the characteristic of typical firearm wound pathology, and the bacterial count test showed that the wound tissue was polluted seriously. There was no significant difference between the two groups (P > 0.05). 3.The amount of bacteria in wound canal of group A was significantly lower than that of group B (P < 0.05), and the inflammatory reaction of group A was lighter than that of group B (P < 0.05), and the effect of wound healing in group A was better than that in group B. Conclusion:. 1. Tissue mass adherence method is an ideal method for isolation and culture of hUC-MSCs in vitro. The hUC-MSCs obtained by this method has high purity and stable biological characteristics, and can be used for further experimental study. 2.2 the effect of projectile injury is similar. By contrast, the 7.62mm projectile with "five or six" ballistic gun is more suitable for simulating real injury in combat, and suitable for further experimental study. 3. HUC-MSCs is effective in the treatment of firearm injury.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R82

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