骨髓间充质干细胞复合猪阴道脱细胞基质构建大鼠组织工程阴道的初步研究

发布时间：2018-06-08 22:00

本文选题：骨髓间充质干细胞 + 阴道脱细胞基质　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:探索应用骨髓间充质干细胞(BMSCs)复合猪阴道脱细胞基质(AVM)构建的大鼠组织工程阴道是否优于单纯应用AVM。方法:取20日龄雄性SD大鼠分离双侧股骨及胫骨,采用全骨髓贴壁法培养BMSCs。取第3代BMSCs与AVM复合培养24h,扫描电镜观察细胞在支架上的生长情况。CM.dil标记第3代细胞,荧光显微镜观察染色情况及标记效率。将第3代BMSCs以1×105/cm2的细胞密度与AVM复合培养,分别于复合后1、3、5、7天行MTT法检测BMSCs在AVM上的增殖情况。经阴道全切雌性SD大鼠阴道构建动物模型。根据构建方式不同分为三组:(1)标记的BMSCs复合AVM,即复合BMSCs组,(2)单纯AVM构建大鼠组织工程阴道,即单纯支架组,(3)正常大鼠阴道作为对照组。分别于术后1、2、3、4、8周取材行大体形态观察,HE、Masson染色行微观形态学观察,冰冻切片荧光显微镜下观察阴道组织中BMSCs向上皮细胞,平滑肌细胞、神经细胞、血管内皮细胞的分化情况。组织浴槽法对新阴道进行功能学评价,试验开始前给予124 m M的高钾溶液,若反应不良将组织条弃掉。给予频率(10~100 Hz),电压30V的电刺激,观察肌条反应情况,再分别给予不同浓度的去氧肾上腺素(10-6~2*10-4M)、酚妥拉明(10-5~2*10-4M)、卡巴胆碱(10-6~10-4M)、阿托品(10-5~10-4M)后观察肌条反应。结果:全骨髓贴壁法培养的BMSCs具有成纤维细胞样形态,90%融合时成漩涡状或放射状生长。荧光显微镜观察CM.dil标记后的BMSCs,骨髓间充质干细胞胞膜呈红色荧光,细胞核不着色,标记效率几乎可达100%。扫描电镜观察发现大体观察发现,BMSCs牢固附着于AVM上,呈长梭形。MTT法检测BMSCs与AVM复合培养后第7天细胞在支架上的细胞活性最佳。复合BMSCs组与单纯支架组均形成了新生阴道,术后8周两组新阴道均粘膜化,接近正常组织形态。HE、Masson染色后微观形态学观察发现,术后1周,复合BMSCs组可见少量上皮组织,单纯支架组仅可见未降解的AVM支架材料,两组均可见较多炎症细胞浸润。术后2周,复合BMSCs组上皮组织覆盖重建阴道全长,单纯支架组术后3周上皮组织才达重建阴道全长,术后4周两组上皮层细胞排列更加规则,更接近正常组织。复合BMSCs组纤维组织较单纯支架组排列更加规整。术后8周复合BMSCs组可见大量规整的平滑肌纤维,单纯支架组仅可见杂乱纤细的肌纤维。免疫荧光染色发现BMSCs可直接分化为平滑肌细胞、血管内皮细胞、神经细胞促进组织的再生。BMSCs也可能通过旁分泌作用、免疫调节作用促进组织再生。功能学检测发现,复合BMSCs组新阴道对药物及电刺激反应均优于单纯支架组。结论:复合BMSCs组与单纯支架组均可成功构建组织工程阴道,复合BMSCs组织工程阴道从形态学及功能学方面均优于单纯AVM支架组。BMSCs可通过直接分化作用促进大鼠组织工程阴道重建,并且可能通过旁分泌作用及免疫调节作用促进大鼠组织工程阴道重建。
[Abstract]:Objective: to explore whether the tissue engineered vagina constructed by bone marrow mesenchymal stem cell (BMSCs) combined with porcine vaginal acellular stroma AVMwas superior to AVM alone. Methods: bilateral femur and tibia were isolated from 20 day old male Sprague-Dawley rats and BMSCs were cultured by whole bone marrow adherent method. The third passage BMSCs were co-cultured with AVM for 24 hours. The growth of the cells on the scaffold was observed by scanning electron microscope. The third passage cells were labeled with CM.dil. The staining and labeling efficiency were observed by fluorescence microscope. The third generation of BMSCs were co-cultured with 1 脳 105/cm2 cell density. The proliferation of BMSCs was detected by MTT assay on the 7th day after 1 脳 105/cm2. The vaginal model of female Sprague-Dawley rats was established. According to different construction methods, BMSCs labeled with BMSCs were divided into three groups: BMSCs combined with AVM (compound BMSCs group) and simple AVM to construct rat tissue engineered vagina, that is, simple scaffold group (n = 3) normal rat vagina was used as control group. The morphology of BMSCs in vaginal tissue was observed under fluorescence microscope. The differentiation of BMSCs into epithelial cells, smooth muscle cells, nerve cells and vascular endothelial cells was observed under fluorescence microscope. The function of the new vagina was evaluated by tissue bath method. 124mm high potassium solution was given before the experiment. If the reaction was bad, the tissue strip would be discarded. The reactivity of muscle strips was observed by electrical stimulation with a frequency of 10 ~ 100 Hz and a voltage of 30 V, followed by different concentrations of noradrenaline (10 ~ (-6) ~ (-2) ~ (-4) M ~ (-1), phentoid Lemine (10 ~ (-5) ~ (2) 10 ~ (-4) M ~ (4), carbachol 10 ~ (-6) ~ (10 ~ (-4) M) and atropine (10 ~ (-5) 10 ~ (-4) M). Results: BMSCs cultured by whole bone marrow adherent method had 90% fibroblast-like morphology. The cell membrane of bone marrow mesenchymal stem cells was red fluorescent, the nucleus was not stained, and the labeling efficiency was almost 100%. Scanning electron microscopy (SEM) showed that BMSCs were firmly attached to AVM, and the cell activity on the scaffold was the best on the 7th day after co-culture of BMSCs and AVM. The new vagina was formed in both the composite BMSCs group and the simple stent group. At 8 weeks after operation, the new vagina became mucous, close to the normal tissue morphology. The microscopic morphological observation showed that a small amount of epithelial tissue was found in the composite BMSCs group at 1 week after operation. Only non-degradable AVM scaffolds were found in the pure stent group, and more inflammatory cell infiltration was observed in both groups. At 2 weeks after operation, the epithelium of composite BMSCs group was covered with the whole length of the reconstructed vagina, while that of the simple stent group reached the full length of the reconstructed vagina 3 weeks after operation, and the arrangement of epithelial cells in the two groups was more regular and closer to the normal tissue 4 weeks after operation. The arrangement of fiber tissue in composite BMSCs group was more regular than that in simple scaffold group. 8 weeks after operation, a large number of regular smooth muscle fibers were observed in the combined BMSCs group, but only in the simple scaffold group. Immunofluorescence staining showed that BMSCs could differentiate directly into smooth muscle cells, vascular endothelial cells, nerve cells, promote tissue regeneration. BMSCs may also promote tissue regeneration through paracrine and immunomodulation. The results of functional examination showed that the new vagina response to drugs and electrical stimulation in BMSCs group was better than that in stent group. Conclusion: the tissue engineered vagina can be successfully constructed in the combined BMSCs group and the simple scaffold group. The compound BMSCs tissue engineered vagina is superior to the AVM scaffold group in morphology and function. BMSCs can promote the reconstruction of rat tissue engineering vagina through direct differentiation. The paracrine and immunomodulatory effects may promote the reconstruction of tissue engineered vagina in rats.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08

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