脂肪间充质干细胞与细胞培养液原位注射对兔耳静脉淤血皮瓣成活和皮瓣VEGF表达的影响
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摘要
目的分析脂肪间充质干细胞与细胞培养液原位注射对兔耳静脉淤血皮瓣成活和皮瓣血管内皮生长因子(VEGF)表达的影响。方法将兔耳静脉淤血皮瓣作为实验模型,分为两组:对照组于皮瓣原位注射细胞培养液,观察组于皮瓣原位注入等量的脂肪间充质干细胞。在术后1周对两组皮瓣组织进行切取,观察细胞生长情况,比较两组皮瓣成活情况,并采用免疫组织化学法对两组皮瓣组织中的VEGF表达水平进行检测。结果原代分离的脂肪间充质干细胞在接种2 d后多数可贴壁,并且以多角形或长梭形为主。第一次传代后,细胞生长速度明显加快,于72 h后可取得75%融合。并且,传至第3代的脂肪间充质干细胞生长为显著的长梭形同时以放射状至四周分布。术后1周,观察组坏死范围显著减少,而对照组皮瓣组织肿胀明显且淤血坏死;观察组皮瓣成活率[(47. 98±9. 42)%]较对照组明显升高[(12. 08±4. 96)%](P <0. 01);免疫组织化学检测结果发现,两组均存在VEGF抗原抗体复合物,均为深棕色,并且积聚于血管腔周围。此外,在同等观察条件下,观察组细胞胞浆着色程度显著高于对照组。结论相比细胞培养液,采取脂肪间充质干细胞原位注射可有效促进皮瓣组织中VEGF的分泌,有助于生成新血管,并且能够减轻静脉淤血与坏死的程度,使得皮瓣的成活率明显提高。
Objective To investigate the effects of situ injection of adipose derived mesenchymal stem cells and cell culture fluid on the survival of skin flaps with venous congestion and VEGF expression. Methods The skin flaps with venous congestion in the rabbit ear was used as experimental model and divided into the control group receiving cell culture fluid and the observation group receiving situ injection of adipose derived mesenchymal stem cells. 1 week after operation,the flap tissue of the two groups were cut and the growth of cells was observed. The survival rate of flaps in the two groups were compared,and the expression of VEGF of flaps in the two groups was detected by immunohistochemistry. Results Most of the isolated adipose derived mesenchymal stem cells could adhere to the wall after 2 days,and they were mainly polygonal or spindle shaped. After the first passage,the cell growth rate was accelerated,and the 75% fusion could be achieved after 72 hours. Moreover,the adipose mesenchymal stem cells,which were transmitted to the third generation,grew remarkably spindle shaped and distributed radially. After 1 week,the necrosis area in the observation group was significantly reduced,while the flap swelling and necrosis of skin flap was showed in the control group; the survival rate of flaps in the observation group( 47. 98 ± 9. 42) % was significantly higher than that of the control group( 12. 08 ± 4. 96) %( P < 0. 01). There were VEGF antigen antibody complexes in the two group,with the dark brown,and accumulated in the lumen of blood vessel. The degree of cytoplasm staining in the observation group was significantly higher than that in the control group. Conclusion Compared with the cell culture fluid,taking the adipose derived mesenchymal stem cells in situ injection can effectively promote the secretion of VEGF in skin flaps,and contribute to the formation of new blood vessels,which can reduce venous congestion and necrosis,so the flap survival rate can increase significantly.
Objective To investigate the effects of situ injection of adipose derived mesenchymal stem cells and cell culture fluid on the survival of skin flaps with venous congestion and VEGF expression. Methods The skin flaps with venous congestion in the rabbit ear was used as experimental model and divided into the control group receiving cell culture fluid and the observation group receiving situ injection of adipose derived mesenchymal stem cells. 1 week after operation,the flap tissue of the two groups were cut and the growth of cells was observed. The survival rate of flaps in the two groups were compared,and the expression of VEGF of flaps in the two groups was detected by immunohistochemistry. Results Most of the isolated adipose derived mesenchymal stem cells could adhere to the wall after 2 days,and they were mainly polygonal or spindle shaped. After the first passage,the cell growth rate was accelerated,and the 75% fusion could be achieved after 72 hours. Moreover,the adipose mesenchymal stem cells,which were transmitted to the third generation,grew remarkably spindle shaped and distributed radially. After 1 week,the necrosis area in the observation group was significantly reduced,while the flap swelling and necrosis of skin flap was showed in the control group; the survival rate of flaps in the observation group( 47. 98 ± 9. 42) % was significantly higher than that of the control group( 12. 08 ± 4. 96) %( P < 0. 01). There were VEGF antigen antibody complexes in the two group,with the dark brown,and accumulated in the lumen of blood vessel. The degree of cytoplasm staining in the observation group was significantly higher than that in the control group. Conclusion Compared with the cell culture fluid,taking the adipose derived mesenchymal stem cells in situ injection can effectively promote the secretion of VEGF in skin flaps,and contribute to the formation of new blood vessels,which can reduce venous congestion and necrosis,so the flap survival rate can increase significantly.
引文
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[13] Semyari H,Rajipour M,Sabetkish S,et al. Evaluating the bone regeneration in calvarial defect using osteoblasts differentiated from adiposederived mesenchymal stem cells on three different scaffolds:an animal study[J]. Cell Tissue Bank,2016,17(1):69-83.
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[15]孔霞,郑飞,郭凌郧,等. VEGF通过细胞外信号调节激酶途径促进骨髓源间充质干细胞的增殖[J].中国实验血液学杂志,2010,18(5):1292-1296.
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[3] Quimby JM,Webb TL,Randall E,et al. Assessment of intravenous adipose-derived allogeneic mesenchymal stem cells for the treatment of feline chronic kidney disease:a randomized,placebo-controlled clinical trial in eight cats[J]. J Feline Med Surg,2016,18(2):165-171.
[4] Marconi S,Castiglione G,Turano E,et al. Human adipose-derived mesenchymal stem cells systemically injected promote peripheral nerve regeneration in the mouse model of sciatic crush[J]. Tissue Eng Part A,2012,18(11-12):1264-1272.
[5]张浩,廖伟雄,李冀,等.兔骨髓栓来源的间充质干细胞的分离培养及其生物学特性的研究[J].中国实验血液学杂志,2015,23(2):500-505.
[6] Panés J,García-Olmo D,Van Assche G,et al. Expanded allogeneic adipose-derived mesenchymal stem cells(Cx601)for complex perianal fistulas in Crohn's disease:a phase 3 randomised,double-blind controlled trial[J]. Lancet,2016,388(10051):1281-1290.
[7]黄凤华,郑新民,姚惟琦,等.自体脂肪间充质干细胞对大鼠肾局部缺血再灌注损伤的保护作用[J].中华实验外科杂志,2012,29(12):2419-2421,封3.
[8] Vishnubalaji R,Al-Nbaheen M,Kadalmani B,et al. Comparative investigation of the differentiation capability of bone-marrow-and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis[J]. Cell Tissue Res,2012,347(2):419-427.
[9]伞光,宋佳.血小板衍生内皮细胞生长因子转染脂肪间充质干细胞促进移植脂肪血管化[J].中国组织工程研究,2015,19(41):6600-6605.
[10] Koh YG,Kwon OR,Kim YS,et al. Adipose-derived mesenchymal stem cells with microfracture versus microfracture alone:2-year follow-up of a prospective randomized trial[J]. Arthroscopy,2016,32(1):97-109.
[11] Han D,Huang W,Li X,et al. Melatonin facilitates adipose-derived mesenchymal stem cells to repair the murine infarcted heart via the SIRT1 signaling pathway[J]. J Pineal Res,2016,60(2):178-192.
[12]毕晓娟,郭晨明,李亮,等.三维培养诱导人脂肪间充质干细胞微球的成软骨分化能力[J].中国组织工程研究,2015,19(1):24-29.
[13] Semyari H,Rajipour M,Sabetkish S,et al. Evaluating the bone regeneration in calvarial defect using osteoblasts differentiated from adiposederived mesenchymal stem cells on three different scaffolds:an animal study[J]. Cell Tissue Bank,2016,17(1):69-83.
[14] Squillaro T,Peluso G,Galderisi U. Clinical trials with mesenchymal stem cells:an update[J]. Cell Transplant,2016,25(5):829-848.
[15]孔霞,郑飞,郭凌郧,等. VEGF通过细胞外信号调节激酶途径促进骨髓源间充质干细胞的增殖[J].中国实验血液学杂志,2010,18(5):1292-1296.