脂肪干细胞移植对野百合碱诱发的肺动脉高压大鼠肺动脉压的量效和时效作用
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摘要
目的探索脂肪干细胞(ADSC)移植治疗野百合碱(MCT)诱导的肺动脉高压(PAH)大鼠的适宜细胞数和干预时间。方法 (1)MCT的建模时效和量效:雄性SD大鼠48只分为正常对照组,20 mg/kg、30 mg/kg、40 mg/kg MCT组分别予腹腔注射生理盐水、MCT20 mg/kg、30 mg/kg、40 mg/kg,4和8周后,右心室插管法检测平均肺动脉压(mPAP),称重法计算右心室肥厚指数(RVHI)。(2)ADSC的治疗量效作用:雄性SD大鼠分别予腹腔注射MCT(30只)和生理盐水(30只),1周后通过颈静脉注射分别移植0.5×10~6、1.0×10~6、3.0×10~6、5.0×10~6ADSC,其他组予等量生理盐水。移植3周后检测mPAP和RVHI。(3) ADSC的治疗时效作用:雄性SD大鼠30只,分别注射40 mg/kg MCT(24只)和生理盐水(6只)。MCT腹腔注射1 d,1、2周后分别移植1.0×10~6个ADSC。MCT注射4周后检测mPAP和RVHI。多组间比较采用单因素或双因素方差分析,两两比较采用LSD检验。结果 (1)腹腔注射4周后,30 mg/kg或40 mg/kg MCT组mPAP和RVHI均升高[mPAP值(24.89±3.31) mmHg,(27.19±2.11)mmHg比(15.80±0.42)mmHg,差异有统计学意义(P均<0.05);RVHI值0.42±0.06,0.47±0.04比0.25±0.02,差异有统计学意义(P均<0.05)]。8周后,20 mg/kg或30 mg/kg MCT组mPAP和RVHI均恢复正常,而40 mg/kg MCT组大鼠全部死亡。(2) 40 mg/kg MCT诱导的PAH大鼠mPAP和RVHI均升高。移植1.0×10~6个ADSC可降低PAH大鼠的mPAP[(17.24±0.66)mmHg比(27.19±1.73)mmHg,P <0.05]。移植0.5×10~6、3.0×10~6、5.0×10~6个ADSC不能降低PAH大鼠的mPAP和RVHI。(3)MCT腹腔注射1周和2周后,移植1.0×10~6个ADSC可降低PAH大鼠的mPAP。结论 40 mg/kg MCT造模4周可建立稳定的PAH大鼠模型;造模1或2周后移植1.0×10~6个ADSC能有效降低PAH大鼠的mPAP。
Objective To investigate the effect of dose and timing of adipose-derived stem cells(ADSC) transplantation on pulmonary arterial pressure in monocrotaline(MCT)-induced pulmonary arterial hypertension(PAH) in rats. Methods(1) MCT-induced PAH in rats: 48 male SD rats were injected intraperitoneally with normal saline, 20 mg/kg, 30 mg/kg and 40 mg/kg MCT respectively. Mean pulmonary arterial pressure(mPAP) was determined by right heart catheterization and right ventricular hypertrophy index(RVHI) was detected by weighting method at 4 and 8 weeks after MCT injection.(2) Dose of ADSC: male SD rats were administrated with 40 mg/kg MCT(n = 30) and normal saline(n = 30). Normal saline or different numbers of ADSC(0.5×10~6, 1.0×10~6, 3.0×10~6, 5.0×10~6) were injected through jugular vein at 1 week after intraperitoneal injection of MCT. mPAP and RVHI were detected at 3 weeks after ADSC transplantation.(3) Timing of ADSC transplantation: 30 male SD rats were administrated with 40 mg/kg MCT(n = 30) and normal saline(n = 6). One day, 1 or 2 weeks after intraperitoneal injection of 40 mg/kg MCT, 1.0×10~6 ADSC were transplanted into the rats. mPAP and RVHI were detected at 4 weeks after MCT injection. Differences among groups were compared using one-way or two-way analysis of variance, and pairwise comparison was performed using LSD test. Results(1) 4 weeks after intraperitoneal injection of 30 or 40 mg/kg MCT, both mPAP and RVHI increased [mPAP(mm Hg): 24.89±3.31, 27.19±2.11 vs15.80±0.42, P < 0.05; RVHI: 0.42±0.06, 0.47±0.04 vs 0.25±0.02, P < 0.05)]. 8 weeks after MCT injection, mPAP and RVHI returned to normal in the 20 and 30 mg/kg MCT groups. All rats died in the 40 mg/kg MCT group.(2) Both mPAP and RVHI in the 40 mg/kg MCT group elevated. 3 weeks after transplantation of 1.0×10~6 ADSC, mPAP declined(27.19±1.73) mmHg vs(17.24±0.66) mmHg(P < 0.05). However, mPAP did not decrease in rats given 0.5×10~6, 3.0×10~6 or 5.0×10~6 ADSC.(3) 1 or 2 weeks after intraperitoneal injection of 40 mg/kg MCT,mPAP reduced in PAH rats given 1.0×10~6 ADSC. Conclusions 4 weeks after a single intraperitoneal injection of 40 mg/kg MCT, a stable PAH rat model could be established. 1 or 2 weeks after intraperitoneal injection of 40 mg/kg MCT, transplantation of 1.0×10~6 ADSC could effectively reduce mPAP in PAH rats.
Objective To investigate the effect of dose and timing of adipose-derived stem cells(ADSC) transplantation on pulmonary arterial pressure in monocrotaline(MCT)-induced pulmonary arterial hypertension(PAH) in rats. Methods(1) MCT-induced PAH in rats: 48 male SD rats were injected intraperitoneally with normal saline, 20 mg/kg, 30 mg/kg and 40 mg/kg MCT respectively. Mean pulmonary arterial pressure(mPAP) was determined by right heart catheterization and right ventricular hypertrophy index(RVHI) was detected by weighting method at 4 and 8 weeks after MCT injection.(2) Dose of ADSC: male SD rats were administrated with 40 mg/kg MCT(n = 30) and normal saline(n = 30). Normal saline or different numbers of ADSC(0.5×10~6, 1.0×10~6, 3.0×10~6, 5.0×10~6) were injected through jugular vein at 1 week after intraperitoneal injection of MCT. mPAP and RVHI were detected at 3 weeks after ADSC transplantation.(3) Timing of ADSC transplantation: 30 male SD rats were administrated with 40 mg/kg MCT(n = 30) and normal saline(n = 6). One day, 1 or 2 weeks after intraperitoneal injection of 40 mg/kg MCT, 1.0×10~6 ADSC were transplanted into the rats. mPAP and RVHI were detected at 4 weeks after MCT injection. Differences among groups were compared using one-way or two-way analysis of variance, and pairwise comparison was performed using LSD test. Results(1) 4 weeks after intraperitoneal injection of 30 or 40 mg/kg MCT, both mPAP and RVHI increased [mPAP(mm Hg): 24.89±3.31, 27.19±2.11 vs15.80±0.42, P < 0.05; RVHI: 0.42±0.06, 0.47±0.04 vs 0.25±0.02, P < 0.05)]. 8 weeks after MCT injection, mPAP and RVHI returned to normal in the 20 and 30 mg/kg MCT groups. All rats died in the 40 mg/kg MCT group.(2) Both mPAP and RVHI in the 40 mg/kg MCT group elevated. 3 weeks after transplantation of 1.0×10~6 ADSC, mPAP declined(27.19±1.73) mmHg vs(17.24±0.66) mmHg(P < 0.05). However, mPAP did not decrease in rats given 0.5×10~6, 3.0×10~6 or 5.0×10~6 ADSC.(3) 1 or 2 weeks after intraperitoneal injection of 40 mg/kg MCT,mPAP reduced in PAH rats given 1.0×10~6 ADSC. Conclusions 4 weeks after a single intraperitoneal injection of 40 mg/kg MCT, a stable PAH rat model could be established. 1 or 2 weeks after intraperitoneal injection of 40 mg/kg MCT, transplantation of 1.0×10~6 ADSC could effectively reduce mPAP in PAH rats.
引文
1 Lai YC,Potoka KC,Champion HC,et al.Pulmonary arterial hypertension the clinical syndrome[J].Circ Res,2014,115(1):115-130.
2 Strem BM,Hicok KC,Zhu M,et al.Multipotential differentiation of adipose tissue-derived stem cells[J].Keio J Med,2005,54(3):132-141.
3 Konno M,Hamabe A,Hasegawa S,et al.Adipose-derived mesenchymal stem cells and regenerative medicine[J].Dev Growth Differ,2013,55(3):309-318.
4 Eguchi M,Ikeda S,Kusumoto S,et al.Adipose-derived regenerative cell therapy inhibits the progression of monocrotaline-induced pulmonary hypertension in rats[J].Life Sci,2014,118(2):306-312.
5梁敏烈,谢良地,李宏亮,等.脂肪间充质干细胞早期干预对野百合碱诱发的肺动脉高压大鼠肺小动脉功能的影响[J].中国病理生理杂志,2013,29(10):1729-1735.
6郑苏梨,谢良地,李宏亮,等.脂肪间充质干细胞对野百合碱诱发的肺动脉高压大鼠肺动脉钙离子通道的影响[J].中国病理生理杂志,2013,29(6):961-968.
7梁敏烈,谢良地,李宏亮,等.脂肪间充质干细胞延迟干预对野百合碱诱发的肺动脉高压大鼠肺小动脉功能的影响[J].中华高血压杂志,2013,29(9):731-737.
8 Liang M,Li H,Zheng S,et al.Comparison of early and delayed transplantation of adipose tissue-derived mesenchymal stem cells on pulmonary arterial function in monocrotaline-induced pulmonary arterial hypertensive rats[J].European Heart Journal Supplements,2015,17(suppl_F):F4-F12.
9 Luo L,Lin T,Zheng S,et al.Adipose-derived stem cells attenuate pulmonary arterial hypertension and ameliorate pulmonary arterial remodeling in monocrotaline-induced pulmonary hypertensive rats[J].Clin Exp Hypertens,37(3):241-248.
10 Luo L,Zheng W,Lian G,et al.Combination treatment of adiposederived stem cells and adiponectin attenuates pulmonary arterial hypertension in rats by inhibiting pulmonary arterial smooth muscle cell proliferation and regulating the AMPK/BMP/Smad pathway[J].Int J Mol Med,41(1):51-60.
11 Tofovic SP,Salah EM,Mady HH,et al.Estradiol metabolites attenuate monocrotaline-induced pulmonary hypertension in rats[J].J Cardiovasc Pharmacol,2005,46(4):430-437.
12 Rosenberg HC,Rabinovitch M.Endothelial injury and vascular reactivity in monocrotaline pulmonary hypertension[J].Am J Physiol,1988,255(6 Pt 2):H1484-1491.
13 Lalich JJ,Merkow L.Pulmonary arteritis produced in rat by feeding crotalaria spectabilis[J].Lab Invest,1961,10:744-750.
14 Kay JM,Harris P,Heath D.Pulmonary hypertension produced in rats by ingestion of crotalaria spectabilis seeds[J].Thorax,1967,22(2):176-179.
15 Bourin P,Bunnell BA,Casteilla L,et al.Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells:A joint statement of the international federation for adipose therapeutics and science(ifats)and the international society for cellular therapy(isct)[J].Cytotherapy,2013,15(6):641-648.
16 Vaillancourt M,Ruffenach G,Meloche J,et al.Adaptation and remodelling of the pulmonary circulation in pulmonary hypertension[J].Can J Cardiol,2015,31(4):407-415.
17 Jungmann PM,Mehlhorn AT,Schmal H,et al.Nanomechanics of human adipose-derived stem cells:Small gtpases impact chondrogenic differentiation[J].Tissue Eng Part A,2012,18(9-10):1035-1044.
18 Bartunek J,Wijns W,Heyndrickx GR,et al.Timing of intracoronary bone-marrow-derived stem cell transplantation after st-elevation myocardial infarction[J].Nat Clin Pract Cardiovasc Med,2006,3Suppl 1:S52-56.
19 Gomez-Arroyo JG,Farkas L,Alhussaini AA,et al.The monocrotaline model of pulmonary hypertension in perspective[J].Am J Physiol Lung Cell Mol Physiol,2012,302(4):L363-369.(收稿日期:2018-03-06)
2 Strem BM,Hicok KC,Zhu M,et al.Multipotential differentiation of adipose tissue-derived stem cells[J].Keio J Med,2005,54(3):132-141.
3 Konno M,Hamabe A,Hasegawa S,et al.Adipose-derived mesenchymal stem cells and regenerative medicine[J].Dev Growth Differ,2013,55(3):309-318.
4 Eguchi M,Ikeda S,Kusumoto S,et al.Adipose-derived regenerative cell therapy inhibits the progression of monocrotaline-induced pulmonary hypertension in rats[J].Life Sci,2014,118(2):306-312.
5梁敏烈,谢良地,李宏亮,等.脂肪间充质干细胞早期干预对野百合碱诱发的肺动脉高压大鼠肺小动脉功能的影响[J].中国病理生理杂志,2013,29(10):1729-1735.
6郑苏梨,谢良地,李宏亮,等.脂肪间充质干细胞对野百合碱诱发的肺动脉高压大鼠肺动脉钙离子通道的影响[J].中国病理生理杂志,2013,29(6):961-968.
7梁敏烈,谢良地,李宏亮,等.脂肪间充质干细胞延迟干预对野百合碱诱发的肺动脉高压大鼠肺小动脉功能的影响[J].中华高血压杂志,2013,29(9):731-737.
8 Liang M,Li H,Zheng S,et al.Comparison of early and delayed transplantation of adipose tissue-derived mesenchymal stem cells on pulmonary arterial function in monocrotaline-induced pulmonary arterial hypertensive rats[J].European Heart Journal Supplements,2015,17(suppl_F):F4-F12.
9 Luo L,Lin T,Zheng S,et al.Adipose-derived stem cells attenuate pulmonary arterial hypertension and ameliorate pulmonary arterial remodeling in monocrotaline-induced pulmonary hypertensive rats[J].Clin Exp Hypertens,37(3):241-248.
10 Luo L,Zheng W,Lian G,et al.Combination treatment of adiposederived stem cells and adiponectin attenuates pulmonary arterial hypertension in rats by inhibiting pulmonary arterial smooth muscle cell proliferation and regulating the AMPK/BMP/Smad pathway[J].Int J Mol Med,41(1):51-60.
11 Tofovic SP,Salah EM,Mady HH,et al.Estradiol metabolites attenuate monocrotaline-induced pulmonary hypertension in rats[J].J Cardiovasc Pharmacol,2005,46(4):430-437.
12 Rosenberg HC,Rabinovitch M.Endothelial injury and vascular reactivity in monocrotaline pulmonary hypertension[J].Am J Physiol,1988,255(6 Pt 2):H1484-1491.
13 Lalich JJ,Merkow L.Pulmonary arteritis produced in rat by feeding crotalaria spectabilis[J].Lab Invest,1961,10:744-750.
14 Kay JM,Harris P,Heath D.Pulmonary hypertension produced in rats by ingestion of crotalaria spectabilis seeds[J].Thorax,1967,22(2):176-179.
15 Bourin P,Bunnell BA,Casteilla L,et al.Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells:A joint statement of the international federation for adipose therapeutics and science(ifats)and the international society for cellular therapy(isct)[J].Cytotherapy,2013,15(6):641-648.
16 Vaillancourt M,Ruffenach G,Meloche J,et al.Adaptation and remodelling of the pulmonary circulation in pulmonary hypertension[J].Can J Cardiol,2015,31(4):407-415.
17 Jungmann PM,Mehlhorn AT,Schmal H,et al.Nanomechanics of human adipose-derived stem cells:Small gtpases impact chondrogenic differentiation[J].Tissue Eng Part A,2012,18(9-10):1035-1044.
18 Bartunek J,Wijns W,Heyndrickx GR,et al.Timing of intracoronary bone-marrow-derived stem cell transplantation after st-elevation myocardial infarction[J].Nat Clin Pract Cardiovasc Med,2006,3Suppl 1:S52-56.
19 Gomez-Arroyo JG,Farkas L,Alhussaini AA,et al.The monocrotaline model of pulmonary hypertension in perspective[J].Am J Physiol Lung Cell Mol Physiol,2012,302(4):L363-369.(收稿日期:2018-03-06)