还原型谷胱甘肽和乌司他丁对冻融人类卵巢组织异种移植的保护作用
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摘要
背景:还原型谷胱甘肽是有效的抗氧化剂,可直接消除氧自由基,具有抑制炎症细胞因子的作用。乌司他丁能抑制氧自由基的产生,抑制多种炎症递质的产生,维持细胞膜和溶酶体膜的稳定性,还可抑制大量亢进的酶的活性,发挥了对移植器官的保护作用。目的:探讨还原型谷胱甘肽和乌司他丁在改善冻融人类卵巢组织异种移植方面的效果。方法:收集6名来源于中山大学附属第一医院年龄(31.8±5.7)岁妇女的卵巢组织,慢速程序化冻融后每一份卵巢组织随机分成4组:还原型谷胱甘肽组(谷胱甘肽组)、乌司他丁组、谷胱甘肽+乌司他丁组、空白对照组,卵巢组织培养箱中平衡15 min后移植在雄性NOD-SCID小鼠背部皮下,移植后4组小鼠分别连续5 d给药还原型谷胱甘肽、乌司他丁、还原型谷胱甘肽和乌司他丁、生理盐水。移植后第1,3,7,14,28,56和85天回收移植物,用苏木精-伊红染色法及酶消化分离荧光染色法观察卵泡组织学形态、各阶段卵泡比例及卵泡的存活率。结果与结论:(1)解冻后的卵巢组织中以始基卵泡为主(79.5±14.3)%。新鲜卵巢组织切片的卵泡存活率为97.6%,解冻后卵巢组织的卵泡存活率为(84.4±5.7)%;(2)还原型谷胱甘肽组、乌司他丁组及谷胱甘肽+乌司他丁组卵泡存活率均高于空白对照组(P=0.022),联合用药与单独用药比较无显著差异(P>0.05);移植后第3天卵泡存活率达到低值(P <0.05),移植1周后有所上升;(3)用药各组在各时间点分离卵泡后的平均卵泡存活率稍高于对照组但差异无显著性意义(P=0.114),与组织学切片部分比较,分离卵泡的存活率明显更高;(4)结果说明,使用还原型谷胱甘肽和乌司他丁可以改善人类卵巢组织异种移植后早期的卵泡存活率,联合用药暂未见优势。
BACKGROUND: Reduced glutathione, as an effective antioxidant can inhibit inflammatory cytokines. Ulinastatin can directly remove oxygen free radicals and inhibit production of oxygen free radicals and various inflammatory mediators, sustain the stability of cell membrane and lysosomal membrane, and inhibit the activity of active enzymes, thus protecting the transplanted organ. OBJECTIVE: To evaluate the effect of reduced glutathione and ulinastatin on improving the xenotransplantation efficiency of frozen-thawed human ovarian tissue. METHODS: Ovarian tissue samples from 6 women aged 31.8±5.7 years old in the First Affiliated Hospital, Sun Yat-sen University, were collected and each tissue sample was randomly divided into four groups after slow freezing: reduced glutathione, ulinastatin, glutathione + ulinastatin and blank control(normal saline) groups. Ovarian tissues were xenotransplantated under the back skin of male NOD-SCID mice after 15-minute cultivation. The mice were administered with corresponding drugs for 5 consecutive days. The grafts were collected at 1, 3, 7, 14, 28, 56, and 85 days after transplantation. Histomorphology, growth situation and viability of the follicle were observed through hematoxylin-eosin staining and enzymatic isolation method. RESULTS AND CONCLUSION: The majority of the frozen-thawed ovarian tissues were primordial follicles(79.5±14.3%). The viability of the follicle in fresh ovarian tissue was 97.6%, and(84.4±5.7)% in thawed ovarian tissue. The reduced glutathione, ulinastatin, and glutathione + ulinastatin groups showed significantl improvement in the viability of the follicle compared with the control group(P=0.022). There was no significant difference between single and combined drug groups(P > 0.05). The follicle viability reached a lowest value at 3 days after transplantation(P < 0.05), and increased after 1 week later. The mean survival rate of the follicle in the isolated follicle at different time points showed no significant difference among groups(P=0.114). Compared with the histological sections, the isolated follicle showed highest survival rate. Our results suggest that reduced glutathione and ulinastatin can increase the early viability of the follicles after xenotransplantation, and combined administration shows no extra benefit.
BACKGROUND: Reduced glutathione, as an effective antioxidant can inhibit inflammatory cytokines. Ulinastatin can directly remove oxygen free radicals and inhibit production of oxygen free radicals and various inflammatory mediators, sustain the stability of cell membrane and lysosomal membrane, and inhibit the activity of active enzymes, thus protecting the transplanted organ. OBJECTIVE: To evaluate the effect of reduced glutathione and ulinastatin on improving the xenotransplantation efficiency of frozen-thawed human ovarian tissue. METHODS: Ovarian tissue samples from 6 women aged 31.8±5.7 years old in the First Affiliated Hospital, Sun Yat-sen University, were collected and each tissue sample was randomly divided into four groups after slow freezing: reduced glutathione, ulinastatin, glutathione + ulinastatin and blank control(normal saline) groups. Ovarian tissues were xenotransplantated under the back skin of male NOD-SCID mice after 15-minute cultivation. The mice were administered with corresponding drugs for 5 consecutive days. The grafts were collected at 1, 3, 7, 14, 28, 56, and 85 days after transplantation. Histomorphology, growth situation and viability of the follicle were observed through hematoxylin-eosin staining and enzymatic isolation method. RESULTS AND CONCLUSION: The majority of the frozen-thawed ovarian tissues were primordial follicles(79.5±14.3%). The viability of the follicle in fresh ovarian tissue was 97.6%, and(84.4±5.7)% in thawed ovarian tissue. The reduced glutathione, ulinastatin, and glutathione + ulinastatin groups showed significantl improvement in the viability of the follicle compared with the control group(P=0.022). There was no significant difference between single and combined drug groups(P > 0.05). The follicle viability reached a lowest value at 3 days after transplantation(P < 0.05), and increased after 1 week later. The mean survival rate of the follicle in the isolated follicle at different time points showed no significant difference among groups(P=0.114). Compared with the histological sections, the isolated follicle showed highest survival rate. Our results suggest that reduced glutathione and ulinastatin can increase the early viability of the follicles after xenotransplantation, and combined administration shows no extra benefit.
引文
[1]Lobo RA.Potential options for preservation of fertility in women.NEngl J Med.2005;(1):64-73.
[2]Lotz L,Maktabi A,Hoffmann I,et al.Ovarian tissue cryopreservation and retransplantation-what do patients think about it?Reproductive BioMedicine Online.2016;(4):394-400.
[3]Huser M,Crha I,Hudecek R,et al.Ovarian tissue cryopreservationnew opportunity to preserve fertility in female cancer patients.Eur JGynaecol Oncol.2007;28(4):249-55.
[4]Donnez J,Dolmans Mm Fau-Martinez-Madrid B,Martinez-Madrid B,et al.The role of cryopreservation for women prior to treatment of malignancy.Curr Opin Obstet Gynecol.2005;(4):333-338.
[5]Donnez J,Dolmans MM,Demylle D,et al.Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.Lancet.2004;364(9443):1405-1410.
[6]Meirow D,Levron J,Eldar-Geva T,et al.Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.N Engl J Med.2005;(3):318-321.
[7]Ladanyi C,Mor A,Christianson MS,et al.Recent advances in the field of ovarian tissue cryopreservation and opportunities for research.JAssist Reprod Genet.2017;(6):709-722.
[8]Li YB,Zhou CQ,Yang GF,et al.Modified vitrification method for cryopreservation of human ovarian tissues.Chin Med J(Engl).2007;120(2):110-114.
[9]Jensen AK,Macklon KT,Fedder J,et al.86 successful births and 9ongoing pregnancies worldwide in women transplanted with frozen-thawed ovarian tissue:focus on birth and perinatal outcome in40 of these children.J Assist Reprod Genet.2017;(3):325-336.
[10]Beckmann MW,Dittrich R,Lotz L,et al.Fertility protection:complications of surgery and results of removal and transplantation of ovarian tissue.Reprod Biomed Online.2018;36(2):188-196.
[11]Kim S,Lee Y,Lee S,Kim T.Ovarian tissue cryopreservation and transplantation in patients with cancer.Obstet Gynecol Science.2018;(4):431-442.
[12]Kristensen SG,Andersen CY.Cryopreservation of Ovarian Tissue:Opportunities Beyond Fertility Preservation and a Positive View Into the Future.Front Endocrinol(Lausanne).2018;9:347.
[13]Estrada E,Rodríguez-Gil JE,Rivera Delálamo MM,et al.Effects of reduced glutathione on acrosin activity in frozen-thawed boar spermatozoa.Reprod Fertil Dev.2017;(2):283-293.
[14]Tao Z,Hu FQ,Li CF,et al.Effect of ulinastatin,a human urinary protease inhibitor,on heatstroke-induced apoptosis and inflammatory responses in rats.Exp Ther Med.2017;(1):335-341.
[15]Cho YS,Shin MS,Ko IG,et al.Ulinastatin inhibits cerebral ischemia-induced apoptosis in the hippocampus of gerbils.Mol Med Rep.2015;12(2):1796-802.
[16]Zhou L,Rui JA,Zhou RL,et al.Liver injury after intermittent or continuous hepatic pedicle clamping and its protection by reduced glutathione.Hepatobiliary Pancreat Dis Int.2004;3(2):209-213.
[17]Guan L,Liu H,Fu P,et al.The Protective Effects of Trypsin Inhibitor on Hepatic Ischemia-Reperfusion Injury and Liver Graft Survival.Oxid Med Cell Longev.2016;2016:1429835.
[18]Dolmans MM,Michaux N,Fau-Camboni A,et al.Evaluation of Liberase,a purified enzyme blend,for the isolation of human primordial and primary ovarian follicles.Hum Reprod.2006;(2):413-420.
[19]Martinez-Madrid B,Dolmans MM,Langendonckt AV,et al.Ficoll density gradient method for recovery of isolated human ovarian primordial follicles.Fertil Steril.2004;(6):1648-1653.
[20]Dolmans MM,Michaux N,Camboni A,et al.Evaluation of Liberase,a purified enzyme blend,for the isolation of human primordial and primary ovarian follicles.Hum Reprod.2006;21(2):413-420.
[21]Gougeon A.Dynamics of follicular growth in the human:a model from preliminary results.Hum Reprod.1986;1:81-87
[22]Candy CJ,Wood MJ,Whittingham DG.Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries.J Reprod Fertil.1997;110(1):11-19.
[23]Israely T,Dafni H,Fau-Nevo N,et al.Angiogenesis in ectopic ovarian xenotransplantation:multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI.Magn Reson Med.2004;(4):741-750.
[24]Kim SS,Yang Hw,Fau-Kang HG,et al.Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant(ascorbic acid)treatment.Fertil Steril.2004;(3):679-685.
[25]Hreinsson J,Zhang P,Fau-Swahn ML,et al.Cryopreservation of follicles in human ovarian cortical tissue.Comparison of serum and human serum albumin in the cryoprotectant solutions.Hum Reprod.2003;(11):2420-2428.
[26]焦桂萍,赵兵,袁志柳,等.还原型谷胱甘肽对大鼠肺缺血再灌注损伤的保护作用[J].中国临床保健杂志,2006,9(2):142-144.
[27]Kevelaitis E,Nyborg NC,MenaschéP.Protective effect of reduced glutathione on endothelial function of coronary arteries subjected to prolonged cold storage.Transplantation.1997;64(4):660-663.
[28]大西冶夫.Ulinastatin.Biomedical Sciences.1984,5(2):76-77.
[29]曾文红,白国强.乌司他丁研究进展[J].中华肝胆外科杂志,2001,7(1):52-54.
[30]Matsumi M,Mizobuchi S,Kaku R,et al.Changes of urinary tripsin inhibitor in blood and urine,as well as serum cytokines in living related liver transplantation.Masui.2003;52(3):251-256.
[31]马兴久,耿振宏,李德春.乌司他汀对肝脏缺血再灌注损伤的防护作用[J].医师进修杂志,2005,28(7):22-28.
[32]钱叶本,赵红川,熊奇如,等.乌司他丁在肝脏缺血再灌注损伤和肝脏再生中的作用[J].中华肝胆外科杂志,2001,11(7):701-2.
[33]于金贵,周广利,孟冬梅,等.乌司他丁对心脏直视患者体外循环期炎性细胞因子和自由基的影响[J].中华麻醉学杂志,2001,21(12):724-26.
[34]Zhang XQ,Sun JL,Chen YJ,et al.Amelioration of ischemia-reperfusion injury of transplanted small intestine by ulinastatin:effects on accumulation and adhesion of neutrophil.Transplant Proc.2005;37(10):4464-4466.
[35]Nugent D,Newton H Fau-Gallivan L,Gallivan L,et al.Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts.J Reprod Fertil.1998;(2):341-346.
[36]Schnorr J,Oehninger S,Fau-Toner J,et al.Functional studies of subcutaneous ovarian transplants in non-human primates:steroidogenesis,endometrial development,ovulation,menstrual patterns and gamete morphology.Hum Reprod.2002;(3):612-619.
[37]Gadea J,Gumbao D,Fau-Matas C,et al.Supplementation of the thawing media with reduced glutathione improves function and the in vitro fertilizing ability of boar spermatozoa after cryopreservation.JAndrol.2005;(6):749-756.
[38]Luo CQ,Long LL,Li XH,et al.Human ovarian tissue xenografted in asepticallywounded mice to conserve follicles.Int J Clin Exp Med.2017;(9):13482-13490.
[39]Nisolle M,Casanas-Roux F,Fau-Qu J,et al.Histologic and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice.Fertil Steril.2000;(1):122-129.
[2]Lotz L,Maktabi A,Hoffmann I,et al.Ovarian tissue cryopreservation and retransplantation-what do patients think about it?Reproductive BioMedicine Online.2016;(4):394-400.
[3]Huser M,Crha I,Hudecek R,et al.Ovarian tissue cryopreservationnew opportunity to preserve fertility in female cancer patients.Eur JGynaecol Oncol.2007;28(4):249-55.
[4]Donnez J,Dolmans Mm Fau-Martinez-Madrid B,Martinez-Madrid B,et al.The role of cryopreservation for women prior to treatment of malignancy.Curr Opin Obstet Gynecol.2005;(4):333-338.
[5]Donnez J,Dolmans MM,Demylle D,et al.Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.Lancet.2004;364(9443):1405-1410.
[6]Meirow D,Levron J,Eldar-Geva T,et al.Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.N Engl J Med.2005;(3):318-321.
[7]Ladanyi C,Mor A,Christianson MS,et al.Recent advances in the field of ovarian tissue cryopreservation and opportunities for research.JAssist Reprod Genet.2017;(6):709-722.
[8]Li YB,Zhou CQ,Yang GF,et al.Modified vitrification method for cryopreservation of human ovarian tissues.Chin Med J(Engl).2007;120(2):110-114.
[9]Jensen AK,Macklon KT,Fedder J,et al.86 successful births and 9ongoing pregnancies worldwide in women transplanted with frozen-thawed ovarian tissue:focus on birth and perinatal outcome in40 of these children.J Assist Reprod Genet.2017;(3):325-336.
[10]Beckmann MW,Dittrich R,Lotz L,et al.Fertility protection:complications of surgery and results of removal and transplantation of ovarian tissue.Reprod Biomed Online.2018;36(2):188-196.
[11]Kim S,Lee Y,Lee S,Kim T.Ovarian tissue cryopreservation and transplantation in patients with cancer.Obstet Gynecol Science.2018;(4):431-442.
[12]Kristensen SG,Andersen CY.Cryopreservation of Ovarian Tissue:Opportunities Beyond Fertility Preservation and a Positive View Into the Future.Front Endocrinol(Lausanne).2018;9:347.
[13]Estrada E,Rodríguez-Gil JE,Rivera Delálamo MM,et al.Effects of reduced glutathione on acrosin activity in frozen-thawed boar spermatozoa.Reprod Fertil Dev.2017;(2):283-293.
[14]Tao Z,Hu FQ,Li CF,et al.Effect of ulinastatin,a human urinary protease inhibitor,on heatstroke-induced apoptosis and inflammatory responses in rats.Exp Ther Med.2017;(1):335-341.
[15]Cho YS,Shin MS,Ko IG,et al.Ulinastatin inhibits cerebral ischemia-induced apoptosis in the hippocampus of gerbils.Mol Med Rep.2015;12(2):1796-802.
[16]Zhou L,Rui JA,Zhou RL,et al.Liver injury after intermittent or continuous hepatic pedicle clamping and its protection by reduced glutathione.Hepatobiliary Pancreat Dis Int.2004;3(2):209-213.
[17]Guan L,Liu H,Fu P,et al.The Protective Effects of Trypsin Inhibitor on Hepatic Ischemia-Reperfusion Injury and Liver Graft Survival.Oxid Med Cell Longev.2016;2016:1429835.
[18]Dolmans MM,Michaux N,Fau-Camboni A,et al.Evaluation of Liberase,a purified enzyme blend,for the isolation of human primordial and primary ovarian follicles.Hum Reprod.2006;(2):413-420.
[19]Martinez-Madrid B,Dolmans MM,Langendonckt AV,et al.Ficoll density gradient method for recovery of isolated human ovarian primordial follicles.Fertil Steril.2004;(6):1648-1653.
[20]Dolmans MM,Michaux N,Camboni A,et al.Evaluation of Liberase,a purified enzyme blend,for the isolation of human primordial and primary ovarian follicles.Hum Reprod.2006;21(2):413-420.
[21]Gougeon A.Dynamics of follicular growth in the human:a model from preliminary results.Hum Reprod.1986;1:81-87
[22]Candy CJ,Wood MJ,Whittingham DG.Effect of cryoprotectants on the survival of follicles in frozen mouse ovaries.J Reprod Fertil.1997;110(1):11-19.
[23]Israely T,Dafni H,Fau-Nevo N,et al.Angiogenesis in ectopic ovarian xenotransplantation:multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI.Magn Reson Med.2004;(4):741-750.
[24]Kim SS,Yang Hw,Fau-Kang HG,et al.Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant(ascorbic acid)treatment.Fertil Steril.2004;(3):679-685.
[25]Hreinsson J,Zhang P,Fau-Swahn ML,et al.Cryopreservation of follicles in human ovarian cortical tissue.Comparison of serum and human serum albumin in the cryoprotectant solutions.Hum Reprod.2003;(11):2420-2428.
[26]焦桂萍,赵兵,袁志柳,等.还原型谷胱甘肽对大鼠肺缺血再灌注损伤的保护作用[J].中国临床保健杂志,2006,9(2):142-144.
[27]Kevelaitis E,Nyborg NC,MenaschéP.Protective effect of reduced glutathione on endothelial function of coronary arteries subjected to prolonged cold storage.Transplantation.1997;64(4):660-663.
[28]大西冶夫.Ulinastatin.Biomedical Sciences.1984,5(2):76-77.
[29]曾文红,白国强.乌司他丁研究进展[J].中华肝胆外科杂志,2001,7(1):52-54.
[30]Matsumi M,Mizobuchi S,Kaku R,et al.Changes of urinary tripsin inhibitor in blood and urine,as well as serum cytokines in living related liver transplantation.Masui.2003;52(3):251-256.
[31]马兴久,耿振宏,李德春.乌司他汀对肝脏缺血再灌注损伤的防护作用[J].医师进修杂志,2005,28(7):22-28.
[32]钱叶本,赵红川,熊奇如,等.乌司他丁在肝脏缺血再灌注损伤和肝脏再生中的作用[J].中华肝胆外科杂志,2001,11(7):701-2.
[33]于金贵,周广利,孟冬梅,等.乌司他丁对心脏直视患者体外循环期炎性细胞因子和自由基的影响[J].中华麻醉学杂志,2001,21(12):724-26.
[34]Zhang XQ,Sun JL,Chen YJ,et al.Amelioration of ischemia-reperfusion injury of transplanted small intestine by ulinastatin:effects on accumulation and adhesion of neutrophil.Transplant Proc.2005;37(10):4464-4466.
[35]Nugent D,Newton H Fau-Gallivan L,Gallivan L,et al.Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts.J Reprod Fertil.1998;(2):341-346.
[36]Schnorr J,Oehninger S,Fau-Toner J,et al.Functional studies of subcutaneous ovarian transplants in non-human primates:steroidogenesis,endometrial development,ovulation,menstrual patterns and gamete morphology.Hum Reprod.2002;(3):612-619.
[37]Gadea J,Gumbao D,Fau-Matas C,et al.Supplementation of the thawing media with reduced glutathione improves function and the in vitro fertilizing ability of boar spermatozoa after cryopreservation.JAndrol.2005;(6):749-756.
[38]Luo CQ,Long LL,Li XH,et al.Human ovarian tissue xenografted in asepticallywounded mice to conserve follicles.Int J Clin Exp Med.2017;(9):13482-13490.
[39]Nisolle M,Casanas-Roux F,Fau-Qu J,et al.Histologic and ultrastructural evaluation of fresh and frozen-thawed human ovarian xenografts in nude mice.Fertil Steril.2000;(1):122-129.
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