低氧预处理人脐带间充质干细胞治疗急性心肌梗死的实验研究
发布时间：2019-05-27 06:57
【摘要】：目的： 本课题在体外收集脐带,分离、培养、扩增、鉴定人脐带间充质干细胞(]human umbilical cord mesenchymal stem cells, hUC-MSCs),将hUC-MSCs进行不同浓度低氧预处理,观察抗凋亡能力和分泌细胞因子能力的变化。选取中华小型猪建立急性心肌梗死模型,将低氧预处理hUC-MSCs和未处理hUC-MSCs经心外膜移植到心肌梗死组织周围,比较两者在心肌组织修复中作用有无差别,并进一步探讨可能存在的机制。 方法： 无菌条件下采集足月剖腹产胎儿脐带,经体外分离、培养、扩增hUC-MSCs至第六代,进行鉴定。按照预处理氧浓度不同分为1%02组,3%O2组,5%02组,10%02组和对照组(21%02组),各组hUC-MSCs在设定氧浓度条件下预处理24小时。通过实时定量PCR、ELISA、Annexin V-FITC/PI等方法比较各组细胞抗凋亡能力和细胞因子分泌能力不同,选取结果最佳的组进行动物实验。选取中华小型猪,全麻下经正中切口入胸,结扎冠状动脉左前降支远端1/3处,制作急性心梗模型。将实验动物随机分3组(6只／组)：对照组(Control)在心梗局部注射生理盐水,正常培养hUC-MSCs组(Nor)注射未处理hUC-MSCs,低氧预处理组(Hyp)注射低氧预处理hUC-MSCs。细胞注射量为2.5x107/只,分10到15个点注射。术后即刻、术后6周行超声心动图,静息核素心肌灌注显像检查。6周后人道处死动物,采集心脏标本,用Masson三染色、免疫荧光染色和TUNEL染色观察移植干细胞在体内定植、生存、血管新生及细胞凋亡情况。 结果： (1)hUC-MSCs表面分子表型CD73、D90、CD105阳性表达,CD11b、CD34、 CD45、CD19、HLA-DR阴性表达。实时定量PCR示促凋亡基因Bax在1%02组、3%02组表达量均显著低于对照组(21%02组)和10%02组(p0.01),其余各组间比较无统计学差异。抗凋亡基因Bcl-2在所有低氧预处理组表达量均显著高于对照组(p0.01)；在1%02组表达量显著低于3%02组(p0.01),在1%02组表达量低于5%02组,有统计学意义(p0.05),其余各组间比较无统计学差异。凋亡诱导实验后,AnnexinV/PI检测示所有低氧预处理组hUC-MSCs与阳性对照组(21%02组)相比,早期、晚期及总凋亡数量均显著减少(p0.01)。1%02组和3%02组hUC-MSCs早期凋亡低于10%02组,有统计学意义(p0.05),其余各组之间无统计学差异。 (2)实时定量PCR示HGF基因在1%02组、3%02组和5%02组表达量均显著高于对照组(21%02组)(p0.01)；在1%02组和3%02组表达量显著高于10%02组(p0.01)；VEGF在3%02组表达量较高,但与对照组比较无统计学差异；KGF在3%O2组和5%02组表达量均显著高于对照组、10%02组以及1%02组(p0.01)；NGF在3%02组和5%02组表达量均显著高于对照组(p0.01),在3%02组表达量高于10%02组和1%02组,有统计学差异(P0.05),在5%02组表达量著高于10%02组(p0.01)。ELISA检测示：HGF分泌量在1%02、3%02组均显著高于对照组和10%02组(p0.01)。VEGF分泌量在3%02组高于对照组和10%02组,有统计学意义(p0.05)。KGF分泌量在3%02组和5%02组均显著高于1%02组和对照组(p0.01)。NGF分泌量在3%02组和5%02组均高于对照组,有统计学意义(p0.05)。 (3)干细胞移植6周后,超声心动图示低氧预处理组LVEDV低于对照组P0.05,各组术后6周与术后即刻LVEF的差值比较,低氧预处理组高于对照组,P0.05。术后6周,低氧预处理组和正常氧干细胞移植组△WT%均显著高于对照组(P0.01),低氧预处理组高于正常氧干细胞移植组,有统计学意义(P0.05)。静息放射核素显像示灌注质量缺损百分率(]mass defect percent, MDP)的差值即△MDP比较,两干细胞治疗组显著低于对照组(P0.01)。各组术后6周与术后即刻LVEF的差值比较,低氧预处理组高于对照组,有统计学意义(P0.05)。Masson三染色示低氧预处理组纤维组织面积小于正常氧细胞组,后者小于对照组,有统计学意义(Hyp：37.14±1.78%, Nor:41.41±0.58%, Control:47.20±3.07%, P0.05)。低氧预处理组与对照组相比纤维组织面积显著减少(P0.01)。 (4)免疫荧光测定显示：在低氧预处理组存活的hUC-MSCs即人p2微球蛋白阳性细胞(Hyp:39.0±7.6cells/hpf, Nor:18.7±5.7cells/hpf,)明显多于正常氧培养组,有统计学意义(P0.05)。TUNEL染色法示两干细胞治疗组凋亡细胞均显著少于对照组(Hyp:10.0±2.6cells/hpf, Nor:49.0±6.6cells/hpf, Control:87.0±9.0cells/hpf, P0.01),低氧预处理组凋亡细胞显著少于正常氧培养组(P0.01)。低氧预处理组Isolactin B4阳性累计光密度值(integrated optical density,IOD)显著多于正常氧培养组和对照组(Hyp:517856.7±43670.5pixels/hpf, Nor:238294.7±66840.0pixels/hpf, Control:137114.0±15168.7pixels/hpf, P0.01),正常氧培养组Isolactin B4阳性IOD多于对照组,有统计学意义(P0.05)。 结论： (1)3%低氧预处理能提高hUC-MSCs抗凋亡能力和分泌细胞因子的能力。 (2)移植低氧预处理hUC-MSCs能在一定程度上改善心肌梗死后心脏功能的恢复。 (3)低氧预处理hUC-MSCs在移植局部有更强的生存能力,能减少心梗局部瘢痕面积,减少纤维化。能通过旁分泌作用促进血管新生,减少心肌细胞凋亡。
[Abstract]:Purpose: In this paper, the umbilical cord, the separation, the culture, the amplification, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were collected in vitro, and the hUC-MSCs were pre-treated with different concentrations of hypoxia, and the anti-apoptotic ability and the ability to secrete cytokines were observed. The model of acute myocardial infarction (AMI) was established by the selection of the model of acute myocardial infarction (AMI), and the treatment of hUC-MSCs and the untreated hUC-MSCs from the epicardium to the surrounding tissues of the myocardial infarction were compared. System. Methods: The umbilical cord of the fetus with full-term caesarean section was collected under the condition of sterility. The umbilical cord of the fetus was isolated and cultured in vitro, and the hUC-MSCs were amplified to the sixth generation. The pre-treated oxygen concentration was divided into 1%02 groups,3% O2 group,5%02 group,10%02 group and control group (21%02 group), each group of hUC-MSCs were pre-treated under the condition of setting oxygen concentration. By means of real-time quantitative PCR, ELISA, Annexin V-FITC/ PI and other methods, the anti-apoptotic ability of each group and the secretion ability of the cytokines were compared. The animal experiment was performed. The small-sized pigs were selected. The central incision was cut into the chest under general anesthesia, and the left anterior descending branch of the coronary artery was ligated to 1/3. The experimental animals were randomly divided into 3 groups (6/ group): the control group (control) was injected with normal saline in the stem, the hUC-MSCs were not treated with the normal culture of the hUC-MSCs, and the hypoxic preconditioning group (Hyp) was injected into the hypoxic preconditioning group (Hyp) to pre-treat the hUC. -MSCs. The injection volume of the cells is 2.5x107/ min, and it is 10 to 1. Five points were injected. At the end of the operation, an echocardiogram and a resting nuclide myocardial perfusion imaging were performed at 6 weeks after operation. After 6 weeks, the animals were sacrificed, the heart specimens were collected, and the transplanted stem cells were observed with Masson three staining, immunofluorescent staining and TUNEL staining to observe the colonization, survival, angiogenesis and fine of the transplanted stem cells in vivo. cell apoptosis Results: (1) The surface molecular phenotype of hUC-MSCs was CD73, D90, CD105 positive expression, CD11b, CD34, CD45, CD19, H. The expression of Bax in the 1%02 group and 3%02 group was significantly lower than that in the control group (21%02 group) and 10%02 (p0.01). The expression of Bcl-2 in all hypoxic preconditioning groups was significantly higher than that in the control group (p0.01). The expression of Bcl-2 in the 1%02 group was significantly lower than that of the control group (p0.01). The expression of Bcl-2 in the 1%02 group was lower than that of the control group (p0.01). The expression of Bcl-2 in the 1%02 group was lower than that of the control group (p0.01). The expression of Bcl-2 in the 1%02 group was lower than that of the control group (p0.01). The expression of Bcl-2 in the 1%02 group was lower than that of the control group (p0.01), and In the early, late and total number of apoptotic cells, the early, late and total number of apoptotic cells decreased significantly (p0.01) compared with the positive control group (21%02) after the induction of apoptosis. The early apoptosis of hUC-MSCs in the 1%02 group and the 3%02 group was lower than that of the 10%02 group. There was no statistical difference between groups. (2) The expression of HGF gene in the 1%02 group,3%02 group and 5%02 group was significantly higher than that in the control group (p0.01). The expression of VEGF in the 1%02 and 3%02 groups was significantly higher than that of the control group (p0.01); the expression of VEGF in the 3%02 group was higher, but it was similar to that of the control group (p0.01). The expression of KGF in the 3% O2 group and the 5%02 group was significantly higher than that in the control group (p0.01). The expression of NGF in the 3%02 group and the 5%02 group was significantly higher than that of the control group (p0.01), and the expression in the 3%02 group was higher than that of the control group (p0.01), and the expression level in the 3%02 group was higher than that of the control group (p0.01), and the expression level in the 3%02 group was higher than that of the control group (p0.01), and the expression level in the 3%02 group was higher than that of the control group (p0.01), and the expression level in the 3%02 group was higher than that The expression of HGF in the 5%02 group was higher than that of the control group and the control group (p0.01). The secretion of VEGF was higher than that in the control group and the 10%02 group (p0.01). The secretion of VEGF was higher in the 3%02 group. In the control group and the 10%02 group, there was a statistical significance (p0.05). The secretion of KGF was significantly higher in the 3%02 group and in the 5%02 group than in the control group (p0.01). The amount of NGF secretion was higher in the 3%02 group and the 5%02 group than in the control group. (3) After 6 weeks of stem cell transplantation, the LVEDV of the hypoxic preconditioning group was lower than that of the control group (P0.05). The control group was higher than that in the control group (P 0.05). The pre-treatment group and the normal oxygen-stem cell transplantation group were significantly higher than those in the control group (P0.01), and the hypoxic preconditioning group was higher than that of the normal oxygen stem cell transplantation group. There was a statistical significance (P0.05). The difference of the percentage of perfusion mass defect (MDP) and the difference of the mass defect (MDP) of the rest radiation nuclide imaging (MDP) were compared, and the treatment group of the two stem cells showed significant difference. Compared with the control group (P0.01), the difference of LVEF between the two groups was higher than that of the control group, and the hypoxic preconditioning group was higher than that of the control group (P <0.05). The tissue area of the hypoxic preconditioning group was lower than that of the normal oxygen cell group, and the latter was lower than that of the control group (Hyp:37). .14卤1.78%, Nor:41.41卤0.58%, Control:47.20 (3.07%, P0.05). Compared with the control group, the hypoxic preconditioning group compared with the control group. The area was significantly reduced (P0.01). (4) Immunofluorescence assay showed that human p2 microglobulin positive cells (Hyp: 39.0, 7.6 cells/ hpf, Nor: 18.7-5.7 cells/ Compared with the control group (Hyp: 10.0, 2.6cells/ hpf, Nor: 49.0, 6.6cells/ hpf, Control: 87.0-9.0 cells/ hpf, P0.01), the apoptotic cells of the hypoxic preconditioning group were significantly higher than those of the control group (Hyp: 10.6cells/ hpf, Control: 87.0-9.6cells/ hpf, P0.01). The positive cumulative optical density (IOD) of Iolactin B4 in the hypoxic preconditioning group was significantly higher than that of the normal oxygen culture group and the control group (Hyp: 517856.7, 43670.5 pels/ hpf, Nor: 238294.7, 66840.0 pels/ hpf, Control: 137114.0, 15168.7 pixels/ hpf, P0.01), and the positive IOD of Iolactin B4 in the normal oxygen culture group was more than that of the control group. Group. Yes. Conclusion: (1)3% hypoxic preconditioning can improve hUC- The ability of MSCs to anti-apoptosis and the secretion of cytokines. (2) Transplantation of hypoxic preconditioning hUC-MSCs Can improve the recovery of cardiac function after myocardial infarction to a certain extent. The viability of the stem can be reduced, the local area of the myocardial infarction can be reduced, and the fibrosis can be reduced.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R542.22
，
本文编号：2485971
[Abstract]:Purpose: In this paper, the umbilical cord, the separation, the culture, the amplification, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were collected in vitro, and the hUC-MSCs were pre-treated with different concentrations of hypoxia, and the anti-apoptotic ability and the ability to secrete cytokines were observed. The model of acute myocardial infarction (AMI) was established by the selection of the model of acute myocardial infarction (AMI), and the treatment of hUC-MSCs and the untreated hUC-MSCs from the epicardium to the surrounding tissues of the myocardial infarction were compared. System. Methods: The umbilical cord of the fetus with full-term caesarean section was collected under the condition of sterility. The umbilical cord of the fetus was isolated and cultured in vitro, and the hUC-MSCs were amplified to the sixth generation. The pre-treated oxygen concentration was divided into 1%02 groups,3% O2 group,5%02 group,10%02 group and control group (21%02 group), each group of hUC-MSCs were pre-treated under the condition of setting oxygen concentration. By means of real-time quantitative PCR, ELISA, Annexin V-FITC/ PI and other methods, the anti-apoptotic ability of each group and the secretion ability of the cytokines were compared. The animal experiment was performed. The small-sized pigs were selected. The central incision was cut into the chest under general anesthesia, and the left anterior descending branch of the coronary artery was ligated to 1/3. The experimental animals were randomly divided into 3 groups (6/ group): the control group (control) was injected with normal saline in the stem, the hUC-MSCs were not treated with the normal culture of the hUC-MSCs, and the hypoxic preconditioning group (Hyp) was injected into the hypoxic preconditioning group (Hyp) to pre-treat the hUC. -MSCs. The injection volume of the cells is 2.5x107/ min, and it is 10 to 1. Five points were injected. At the end of the operation, an echocardiogram and a resting nuclide myocardial perfusion imaging were performed at 6 weeks after operation. After 6 weeks, the animals were sacrificed, the heart specimens were collected, and the transplanted stem cells were observed with Masson three staining, immunofluorescent staining and TUNEL staining to observe the colonization, survival, angiogenesis and fine of the transplanted stem cells in vivo. cell apoptosis Results: (1) The surface molecular phenotype of hUC-MSCs was CD73, D90, CD105 positive expression, CD11b, CD34, CD45, CD19, H. The expression of Bax in the 1%02 group and 3%02 group was significantly lower than that in the control group (21%02 group) and 10%02 (p0.01). The expression of Bcl-2 in all hypoxic preconditioning groups was significantly higher than that in the control group (p0.01). The expression of Bcl-2 in the 1%02 group was significantly lower than that of the control group (p0.01). The expression of Bcl-2 in the 1%02 group was lower than that of the control group (p0.01). The expression of Bcl-2 in the 1%02 group was lower than that of the control group (p0.01). The expression of Bcl-2 in the 1%02 group was lower than that of the control group (p0.01). The expression of Bcl-2 in the 1%02 group was lower than that of the control group (p0.01), and In the early, late and total number of apoptotic cells, the early, late and total number of apoptotic cells decreased significantly (p0.01) compared with the positive control group (21%02) after the induction of apoptosis. The early apoptosis of hUC-MSCs in the 1%02 group and the 3%02 group was lower than that of the 10%02 group. There was no statistical difference between groups. (2) The expression of HGF gene in the 1%02 group,3%02 group and 5%02 group was significantly higher than that in the control group (p0.01). The expression of VEGF in the 1%02 and 3%02 groups was significantly higher than that of the control group (p0.01); the expression of VEGF in the 3%02 group was higher, but it was similar to that of the control group (p0.01). The expression of KGF in the 3% O2 group and the 5%02 group was significantly higher than that in the control group (p0.01). The expression of NGF in the 3%02 group and the 5%02 group was significantly higher than that of the control group (p0.01), and the expression in the 3%02 group was higher than that of the control group (p0.01), and the expression level in the 3%02 group was higher than that of the control group (p0.01), and the expression level in the 3%02 group was higher than that of the control group (p0.01), and the expression level in the 3%02 group was higher than that of the control group (p0.01), and the expression level in the 3%02 group was higher than that The expression of HGF in the 5%02 group was higher than that of the control group and the control group (p0.01). The secretion of VEGF was higher than that in the control group and the 10%02 group (p0.01). The secretion of VEGF was higher in the 3%02 group. In the control group and the 10%02 group, there was a statistical significance (p0.05). The secretion of KGF was significantly higher in the 3%02 group and in the 5%02 group than in the control group (p0.01). The amount of NGF secretion was higher in the 3%02 group and the 5%02 group than in the control group. (3) After 6 weeks of stem cell transplantation, the LVEDV of the hypoxic preconditioning group was lower than that of the control group (P0.05). The control group was higher than that in the control group (P 0.05). The pre-treatment group and the normal oxygen-stem cell transplantation group were significantly higher than those in the control group (P0.01), and the hypoxic preconditioning group was higher than that of the normal oxygen stem cell transplantation group. There was a statistical significance (P0.05). The difference of the percentage of perfusion mass defect (MDP) and the difference of the mass defect (MDP) of the rest radiation nuclide imaging (MDP) were compared, and the treatment group of the two stem cells showed significant difference. Compared with the control group (P0.01), the difference of LVEF between the two groups was higher than that of the control group, and the hypoxic preconditioning group was higher than that of the control group (P <0.05). The tissue area of the hypoxic preconditioning group was lower than that of the normal oxygen cell group, and the latter was lower than that of the control group (Hyp:37). .14卤1.78%, Nor:41.41卤0.58%, Control:47.20 (3.07%, P0.05). Compared with the control group, the hypoxic preconditioning group compared with the control group. The area was significantly reduced (P0.01). (4) Immunofluorescence assay showed that human p2 microglobulin positive cells (Hyp: 39.0, 7.6 cells/ hpf, Nor: 18.7-5.7 cells/ Compared with the control group (Hyp: 10.0, 2.6cells/ hpf, Nor: 49.0, 6.6cells/ hpf, Control: 87.0-9.0 cells/ hpf, P0.01), the apoptotic cells of the hypoxic preconditioning group were significantly higher than those of the control group (Hyp: 10.6cells/ hpf, Control: 87.0-9.6cells/ hpf, P0.01). The positive cumulative optical density (IOD) of Iolactin B4 in the hypoxic preconditioning group was significantly higher than that of the normal oxygen culture group and the control group (Hyp: 517856.7, 43670.5 pels/ hpf, Nor: 238294.7, 66840.0 pels/ hpf, Control: 137114.0, 15168.7 pixels/ hpf, P0.01), and the positive IOD of Iolactin B4 in the normal oxygen culture group was more than that of the control group. Group. Yes. Conclusion: (1)3% hypoxic preconditioning can improve hUC- The ability of MSCs to anti-apoptosis and the secretion of cytokines. (2) Transplantation of hypoxic preconditioning hUC-MSCs Can improve the recovery of cardiac function after myocardial infarction to a certain extent. The viability of the stem can be reduced, the local area of the myocardial infarction can be reduced, and the fibrosis can be reduced.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R542.22
，
本文编号：2485971
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