低氧預(yù)處理人臍帶間充質(zhì)干細胞治療急性心肌梗死的實驗研究
發(fā)布時間：2019-05-27 06:57
【摘要】：目的： 本課題在體外收集臍帶,分離、培養(yǎng)、擴增、鑒定人臍帶間充質(zhì)干細胞(]human umbilical cord mesenchymal stem cells, hUC-MSCs),將hUC-MSCs進行不同濃度低氧預(yù)處理,觀察抗凋亡能力和分泌細胞因子能力的變化。選取中華小型豬建立急性心肌梗死模型,將低氧預(yù)處理hUC-MSCs和未處理hUC-MSCs經(jīng)心外膜移植到心肌梗死組織周圍,比較兩者在心肌組織修復(fù)中作用有無差別,并進一步探討可能存在的機制。 方法： 無菌條件下采集足月剖腹產(chǎn)胎兒臍帶,經(jīng)體外分離、培養(yǎng)、擴增hUC-MSCs至第六代,進行鑒定。按照預(yù)處理氧濃度不同分為1%02組,3%O2組,5%02組,10%02組和對照組(21%02組),各組hUC-MSCs在設(shè)定氧濃度條件下預(yù)處理24小時。通過實時定量PCR、ELISA、Annexin V-FITC/PI等方法比較各組細胞抗凋亡能力和細胞因子分泌能力不同,選取結(jié)果最佳的組進行動物實驗。選取中華小型豬,全麻下經(jīng)正中切口入胸,結(jié)扎冠狀動脈左前降支遠端1/3處,制作急性心梗模型。將實驗動物隨機分3組(6只／組)：對照組(Control)在心梗局部注射生理鹽水,正常培養(yǎng)hUC-MSCs組(Nor)注射未處理hUC-MSCs,低氧預(yù)處理組(Hyp)注射低氧預(yù)處理hUC-MSCs。細胞注射量為2.5x107/只,分10到15個點注射。術(shù)后即刻、術(shù)后6周行超聲心動圖,靜息核素心肌灌注顯像檢查。6周后人道處死動物,采集心臟標本,用Masson三染色、免疫熒光染色和TUNEL染色觀察移植干細胞在體內(nèi)定植、生存、血管新生及細胞凋亡情況。 結(jié)果： (1)hUC-MSCs表面分子表型CD73、D90、CD105陽性表達,CD11b、CD34、 CD45、CD19、HLA-DR陰性表達。實時定量PCR示促凋亡基因Bax在1%02組、3%02組表達量均顯著低于對照組(21%02組)和10%02組(p0.01),其余各組間比較無統(tǒng)計學(xué)差異�？沟蛲龌駼cl-2在所有低氧預(yù)處理組表達量均顯著高于對照組(p0.01)；在1%02組表達量顯著低于3%02組(p0.01),在1%02組表達量低于5%02組,有統(tǒng)計學(xué)意義(p0.05),其余各組間比較無統(tǒng)計學(xué)差異。凋亡誘導(dǎo)實驗后,AnnexinV/PI檢測示所有低氧預(yù)處理組hUC-MSCs與陽性對照組(21%02組)相比,早期、晚期及總凋亡數(shù)量均顯著減少(p0.01)。1%02組和3%02組hUC-MSCs早期凋亡低于10%02組,有統(tǒng)計學(xué)意義(p0.05),其余各組之間無統(tǒng)計學(xué)差異。 (2)實時定量PCR示HGF基因在1%02組、3%02組和5%02組表達量均顯著高于對照組(21%02組)(p0.01)；在1%02組和3%02組表達量顯著高于10%02組(p0.01)；VEGF在3%02組表達量較高,但與對照組比較無統(tǒng)計學(xué)差異；KGF在3%O2組和5%02組表達量均顯著高于對照組、10%02組以及1%02組(p0.01)；NGF在3%02組和5%02組表達量均顯著高于對照組(p0.01),在3%02組表達量高于10%02組和1%02組,有統(tǒng)計學(xué)差異(P0.05),在5%02組表達量著高于10%02組(p0.01)。ELISA檢測示：HGF分泌量在1%02、3%02組均顯著高于對照組和10%02組(p0.01)。VEGF分泌量在3%02組高于對照組和10%02組,有統(tǒng)計學(xué)意義(p0.05)。KGF分泌量在3%02組和5%02組均顯著高于1%02組和對照組(p0.01)。NGF分泌量在3%02組和5%02組均高于對照組,有統(tǒng)計學(xué)意義(p0.05)。 (3)干細胞移植6周后,超聲心動圖示低氧預(yù)處理組LVEDV低于對照組P0.05,各組術(shù)后6周與術(shù)后即刻LVEF的差值比較,低氧預(yù)處理組高于對照組,P0.05。術(shù)后6周,低氧預(yù)處理組和正常氧干細胞移植組△WT%均顯著高于對照組(P0.01),低氧預(yù)處理組高于正常氧干細胞移植組,有統(tǒng)計學(xué)意義(P0.05)。靜息放射核素顯像示灌注質(zhì)量缺損百分率(]mass defect percent, MDP)的差值即△MDP比較,兩干細胞治療組顯著低于對照組(P0.01)。各組術(shù)后6周與術(shù)后即刻LVEF的差值比較,低氧預(yù)處理組高于對照組,有統(tǒng)計學(xué)意義(P0.05)。Masson三染色示低氧預(yù)處理組纖維組織面積小于正常氧細胞組,后者小于對照組,有統(tǒng)計學(xué)意義(Hyp：37.14±1.78%, Nor:41.41±0.58%, Control:47.20±3.07%, P0.05)。低氧預(yù)處理組與對照組相比纖維組織面積顯著減少(P0.01)。 (4)免疫熒光測定顯示：在低氧預(yù)處理組存活的hUC-MSCs即人p2微球蛋白陽性細胞(Hyp:39.0±7.6cells/hpf, Nor:18.7±5.7cells/hpf,)明顯多于正常氧培養(yǎng)組,有統(tǒng)計學(xué)意義(P0.05)。TUNEL染色法示兩干細胞治療組凋亡細胞均顯著少于對照組(Hyp:10.0±2.6cells/hpf, Nor:49.0±6.6cells/hpf, Control:87.0±9.0cells/hpf, P0.01),低氧預(yù)處理組凋亡細胞顯著少于正常氧培養(yǎng)組(P0.01)。低氧預(yù)處理組Isolactin B4陽性累計光密度值(integrated optical density,IOD)顯著多于正常氧培養(yǎng)組和對照組(Hyp:517856.7±43670.5pixels/hpf, Nor:238294.7±66840.0pixels/hpf, Control:137114.0±15168.7pixels/hpf, P0.01),正常氧培養(yǎng)組Isolactin B4陽性IOD多于對照組,有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論： (1)3%低氧預(yù)處理能提高hUC-MSCs抗凋亡能力和分泌細胞因子的能力。 (2)移植低氧預(yù)處理hUC-MSCs能在一定程度上改善心肌梗死后心臟功能的恢復(fù)。 (3)低氧預(yù)處理hUC-MSCs在移植局部有更強的生存能力,能減少心梗局部瘢痕面積,減少纖維化。能通過旁分泌作用促進血管新生,減少心肌細胞凋亡。
[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鹵1.78%, Nor:41.41鹵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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R542.22
，
本文編號：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鹵1.78%, Nor:41.41鹵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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R542.22
，
本文編號：2485971
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