猪骨髓间充质干细胞治疗猪全层皮损的促血管化研究
摘要
目的检测骨髓间充质干细胞治疗猪全层皮肤缺损过程中相关促血管化因子表达及血管数量变化,探讨骨髓间充质干细胞对大面积皮肤缺损治疗的临床应用价值。
方法选择2头一岁龄滇南小耳猪,多次在麻醉下使用骨髓穿刺针由髂骨处按15-20ml/次抽取骨髓,采用密度梯度离心和细胞贴壁培养法联合筛选单核细胞层。使用含15%胎牛血清和1%双抗(青霉素和链霉素)的F-12培养基,放入95%饱和湿度,37℃的CO2培养箱中培养。首次72h换液,之后每日倒置显微镜下观察细胞形态,隔日换液。达到80%融合时按1：3传代,传至第三代,留取备用。
猪麻醉后采用自身配对设计,沿脊椎两侧旁开2.5cm按24个／每头预制作3cm×3cm正方形全层皮肤缺损创面。按是否使用干细胞分为实验组(A/B/C组)和对照组(a/b/c组)。实验组按植皮方式分为三组,即微粒皮+干细胞治疗组(A组)、小皮片+干细胞治疗组(B组)、单纯干细胞治疗组(C组),按1×107/ml密度于每个创面注射干细胞1m1。术后第7天、第14天分别取新生上皮和／或肉芽组织行免疫组织化学血管平滑肌肌动蛋白染色计数微血管密度,同时采用实时荧光多聚酶链反应(RT-PCR)检测血管内皮细胞生长因子、血管细胞黏附分子、血管生成素mRNA相对表达量。
结果干细胞移植治疗实验组对创面愈合均有促进作用①第14天时创面大体观可见实验组愈合情况均优于各自对照组,实验组中B组创面已愈合优于A组的部分愈合及C组的肉芽创面。②免疫组化染色可见肉芽组织或新生表皮下血管平滑肌肌动蛋白棕染阳性的内皮细胞团簇,计数微血管密度可知第7、14天时B组血管密度显著高于其他5组(P<0.05)。③RT-PCR检测采用2.△△ct相对定量法可知血管内皮细胞生长因子mRNA在第7天时高表达,第14天表达水平显著下降。第7天时各实验组均高于对照组,实验组中C组表达最高,其次是B组。血管细胞黏附分子、血管生成素表达变化趋势与血管内皮细胞生长因子相似。
结论骨髓间充质干细胞移植可能通过促进血管新生进而促进创面愈合,其主要机制可能是通过促进VEGF、VCAM、ANG三种目的基因的表达来实现的。但是单纯干细胞治疗还不能取代治疗。
Objective:
To investigate the clinical value of BMSCs treatment in full-thickness skin defect wounds, the expression of vascular factors and microvessel density were detected in this research.
Method:
2 one-year-old pigs were employed for BMSCs isolation.15-20 ml bone marrow were harvested from ileac bone using a ileac needle and mononuclear cells were isolated using density gradient centrifugation and cultured in F-12 with 15% fetal bovine serum. After 48 hours of cell isolation medium was replaced and after then cell medium was replaced every 2 days. Culture cells were daily observed under phasecontrast microscope and passaged when cells almost 80% confluent.
24 3cm×3cm full-thickness skin wounds were made on the back of each pig after anesthesia. Every wound was divided into different group. Group A:1×107 cultured BMSCs in 1 ml PBS were administrated to each wound followed by autologous microskin grafting, and covered by acellular dermal matrix (ADM); Group B:After BMSCs administration like Group A, auto-split skin were transplanted to each wound and covered with ADM; Group C:After BMSCs administration like Group A, wounds were covered with ADM. As control groups, Group a, Group b, and Group c, all steps were similar to above but BMSCs administration was replaced by PBS injection. At 7,14 days post operations, wound tissues were harvested for histology and total RNA abstraction. Actin was detected by immunohistochemistry staining, and microvessel density (MVD) was counted under microscope. VEGF, VCAM, and ANG mRNA expression were detected by real time-PCR.
Result:
Combined with cultured BMSCs administration, full-thickness skin wounds closure were accelerated.①Among three experimental groups, wounds of Group B were complete healed at 14 days after operation, but small open wounds were still exist in Group A, and Group C. Compared with Group C, at day 14, opening wounds of Group A were smaller.②MVD analysis expressed that counting of Actin-positive cell clusters in wound beds and leading edges in Group B were higher than other groups (P< 0.05).③Relative quantitative analysis by real time PCR shows, in all groups, VEGF mRNA expression at day 7 post operation were higher than its expression at day 14. But in three experimental groups combined with BMSCs administration, VEGF mRNA were higher than control groups. Among three experimental groups, its expression in Group C was highest, and its expression in Group B was higher than Group A. VCAM, ANG mRNA expression pattern were similar to VEGF mRNA.
Conclusion:
BMSCs could accelerate full-thickness skin wound healing by improving angiogenesis, and the main mechanism may promote the expression of VEGF, VCAM, and ANG.. But only local BMSCs administration could not replace auto-split skin grafting.
方法选择2头一岁龄滇南小耳猪,多次在麻醉下使用骨髓穿刺针由髂骨处按15-20ml/次抽取骨髓,采用密度梯度离心和细胞贴壁培养法联合筛选单核细胞层。使用含15%胎牛血清和1%双抗(青霉素和链霉素)的F-12培养基,放入95%饱和湿度,37℃的CO2培养箱中培养。首次72h换液,之后每日倒置显微镜下观察细胞形态,隔日换液。达到80%融合时按1：3传代,传至第三代,留取备用。
猪麻醉后采用自身配对设计,沿脊椎两侧旁开2.5cm按24个／每头预制作3cm×3cm正方形全层皮肤缺损创面。按是否使用干细胞分为实验组(A/B/C组)和对照组(a/b/c组)。实验组按植皮方式分为三组,即微粒皮+干细胞治疗组(A组)、小皮片+干细胞治疗组(B组)、单纯干细胞治疗组(C组),按1×107/ml密度于每个创面注射干细胞1m1。术后第7天、第14天分别取新生上皮和／或肉芽组织行免疫组织化学血管平滑肌肌动蛋白染色计数微血管密度,同时采用实时荧光多聚酶链反应(RT-PCR)检测血管内皮细胞生长因子、血管细胞黏附分子、血管生成素mRNA相对表达量。
结果干细胞移植治疗实验组对创面愈合均有促进作用①第14天时创面大体观可见实验组愈合情况均优于各自对照组,实验组中B组创面已愈合优于A组的部分愈合及C组的肉芽创面。②免疫组化染色可见肉芽组织或新生表皮下血管平滑肌肌动蛋白棕染阳性的内皮细胞团簇,计数微血管密度可知第7、14天时B组血管密度显著高于其他5组(P<0.05)。③RT-PCR检测采用2.△△ct相对定量法可知血管内皮细胞生长因子mRNA在第7天时高表达,第14天表达水平显著下降。第7天时各实验组均高于对照组,实验组中C组表达最高,其次是B组。血管细胞黏附分子、血管生成素表达变化趋势与血管内皮细胞生长因子相似。
结论骨髓间充质干细胞移植可能通过促进血管新生进而促进创面愈合,其主要机制可能是通过促进VEGF、VCAM、ANG三种目的基因的表达来实现的。但是单纯干细胞治疗还不能取代治疗。
Objective:
To investigate the clinical value of BMSCs treatment in full-thickness skin defect wounds, the expression of vascular factors and microvessel density were detected in this research.
Method:
2 one-year-old pigs were employed for BMSCs isolation.15-20 ml bone marrow were harvested from ileac bone using a ileac needle and mononuclear cells were isolated using density gradient centrifugation and cultured in F-12 with 15% fetal bovine serum. After 48 hours of cell isolation medium was replaced and after then cell medium was replaced every 2 days. Culture cells were daily observed under phasecontrast microscope and passaged when cells almost 80% confluent.
24 3cm×3cm full-thickness skin wounds were made on the back of each pig after anesthesia. Every wound was divided into different group. Group A:1×107 cultured BMSCs in 1 ml PBS were administrated to each wound followed by autologous microskin grafting, and covered by acellular dermal matrix (ADM); Group B:After BMSCs administration like Group A, auto-split skin were transplanted to each wound and covered with ADM; Group C:After BMSCs administration like Group A, wounds were covered with ADM. As control groups, Group a, Group b, and Group c, all steps were similar to above but BMSCs administration was replaced by PBS injection. At 7,14 days post operations, wound tissues were harvested for histology and total RNA abstraction. Actin was detected by immunohistochemistry staining, and microvessel density (MVD) was counted under microscope. VEGF, VCAM, and ANG mRNA expression were detected by real time-PCR.
Result:
Combined with cultured BMSCs administration, full-thickness skin wounds closure were accelerated.①Among three experimental groups, wounds of Group B were complete healed at 14 days after operation, but small open wounds were still exist in Group A, and Group C. Compared with Group C, at day 14, opening wounds of Group A were smaller.②MVD analysis expressed that counting of Actin-positive cell clusters in wound beds and leading edges in Group B were higher than other groups (P< 0.05).③Relative quantitative analysis by real time PCR shows, in all groups, VEGF mRNA expression at day 7 post operation were higher than its expression at day 14. But in three experimental groups combined with BMSCs administration, VEGF mRNA were higher than control groups. Among three experimental groups, its expression in Group C was highest, and its expression in Group B was higher than Group A. VCAM, ANG mRNA expression pattern were similar to VEGF mRNA.
Conclusion:
BMSCs could accelerate full-thickness skin wound healing by improving angiogenesis, and the main mechanism may promote the expression of VEGF, VCAM, and ANG.. But only local BMSCs administration could not replace auto-split skin grafting.
引文
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