COX同工酶-10aa-PGIS转基因治疗神经损伤型勃起功能障碍机制的实验研究
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摘要
第一章绪论
勃起功能障碍(Erectile Dysfunction, ED)是前列腺癌根治术(Radical prostatectomy, RP)后最为常见的并发症之一,术中不可避免的损伤勃起神经造成神经失用是其最主要的发病机制,神经失用使得本世纪最伟大的发现之一,男科学最具革命性的药物-5型磷酸二脂酶抑制剂(Phosphodiesterase-5inhibitors, PDE5Is)对前列腺癌根治术后勃起功能障碍(Radical prostatectomy erectile dysfunction, RPED)的疗效疲软乏力。人们迫切希望寻找新的治疗手段。前列腺环素(Prostacyclin, PGI2)和PDE-5i均为强效的平滑肌松弛剂和血管保护剂,但是因为PGI2过短的半衰期(<2min)限制了它的临床应用。Ke-He Ruan等于2006年研发了的COX2-10aa-PGIS重组蛋白,这种蛋白同时具有COX-2和PGIS两种酶活性,并发挥3种有效的催化作用。这种特殊的结构能够把底物花生四烯酸(arachidonic acid, AA)直接高效转化成PGI2,从而使PGI2高表达。PGI2通过PGI2-IP-cAMP-PKT信号通路发挥平滑肌松弛剂和血管保护剂的作用,而这种信号通路似乎能能越过神经失用的障碍直接松弛血管平滑肌和扩张血管。这使得COX2-10aa-PGIS具有治疗RPED的潜在可能性。在第一个实验中,我们应用重组腺病毒(Adeno-virus)作为COX2-10aa-PGIS的载体,从基因层面治疗RPED,并探讨其潜在的机理。最近,Ke-He Ruan等在COX2-10aa-PGIS的基础上进一步优化结构,研发了COX1-10aa-PGIS。与前者相比,COX1-10aa-PGIS能更加稳定持久的高表达PGI2。目前的研究表明：脂质干细胞(Adipose Derived stem cells, ADSCs)具有自我更新,损伤修复,多向分化潜能和旁分泌作用。ADSCs是ED治疗的优秀候选者。由于腺病毒存在可能的生物威胁,在第二个实验中,我们进一步应用ADSCs作为COX1-10aa-PGIS基因治疗的载体,来治疗RPED,观察其疗效并探讨其可能性的分子机制。本论文正文分为两个部分陈述。
第二章Ad-COX2-10aa-PGIS基因治疗神经损伤性的勃起功能障碍及其机制的研究
目的
本实验的目的是探索Ad-COX2-10aa-PGIS基因治疗在神经损伤性勃起功能障碍中的疗效和机制
方法
我们用成年SD大鼠双侧海绵体神经损伤模型来模拟根治性前列腺癌切除术后勃起功能障碍(RPED)。32只SD大鼠随机被分为4组：组1、空白手术组(Sham surgery);组2、双侧海绵体神经挤压损伤(Bilateral cavernosal nerve crush, BCNC);组3、BCNC+空白腺病毒(Null control recombinant adenovirus, NCRA)阴茎海绵体注射；组4、BCNC+复制缺陷型重组腺病毒携带COX2-10aa-PGIS基因(Replication efficiency recombinant adenovirus carry COX2-10aa-PGIS gene, Ad-COX2-10aa-PGIS)阴茎海绵体注射。28天后,电刺激海绵体神经测海绵体内压(Intracavernosal pressure, ICP)同时记录平均动脉压(Mean arterial pressure, MAP)。所有大鼠的阴茎组织按要求处理并保存为下一步实验用。(1)免疫组化：endothelial nitric oxide synthase(eNOS), α-smooth muscle actin (ASMA) and transforming growth factor beta-1(TGF β1);(2) Western Blot for COX2-10aa-PGI;(3) Masson's Trichrome stain:平滑肌／胶原比值(smooth muscle/collagen ratios); and (4) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis.
结果
1. COX2-10aa-PGIS基因治疗在神经损伤大鼠模型中能保存勃起功能；
2. COX2-10aa-PGIS基因治疗能增加eNOS和ASMA表达水平；
3. COX2-10aa-PGIS基因治疗能保护平滑肌／胶原比值：
4. COX2-10aa-PGIS基因治疗能降低TGF β1的表达水平；
5. COX2-10aa-PGIS基因治疗能减少细胞凋亡。
小结
我们的实验证明阴茎海绵体注射Ad-COX2-10aa-PGIS基因治疗有助于神经损伤型ED大鼠的勃起功能的康复。其机制是抗纤维化和抗细胞凋亡,从而阻止海绵体静脉漏的发生,最终保护勃起功能。
第三章脂质干细胞携带COX1-10aa-PGIS基因治疗双侧海绵体神经损伤引起的勃起功能障碍及其机制
目的
使用脂质干细胞(ADSCs)作为COX1-10aa-PGIS基因载体来治疗双侧海绵体损伤性勃起功能障碍,并探讨其潜在的机制。
方法
1.从同种异体SD大鼠生殖腺周围脂肪中提取ADSCs,在DMEM/F12中传代；
2.建立COX1-10aa-PGIS转染的ADSCs的稳定细胞系；
3. Western blot鉴定COX1-10aa-PGIS在ADSCs表达,LC-MS/MS测定其酶活性；
4.动物实验：建立成年SD大鼠双侧海绵体损伤模型来模拟前列腺癌根治术后勃起功能障碍。将SD大鼠随机被分成四组,Sham组：空白手术组；BCNC组：双侧海绵体神经挤压损伤；BCNC+ADSCs组：双侧海绵体神经挤压损伤并接受空白ADSCs阴茎海绵体注射治疗；BCNC+ADSCs with COX2-10aa-PGIS组：双侧海绵体神经挤压损伤,并接受转染COX2-10aa-PGIS基因的ADSCs阴茎海绵体注射治疗。28天后,在电刺激海绵体神经下测海绵体内压(ICP),同时监测平均动脉压(MAP)。收集所有大鼠的阴茎组织和尿液,按要求处理并保存为下一步实验用。1、免疫组化:Neural nitric oxide synthase(nNOS), α-smooth muscle actin (ASMA)和Vascular endothelial growth factor (VEGF);2、Western blot for ASMA, Transf forming growth factor β1(TGF β1)和Hypoxia-inducible factor-1α(HIF-1α);3、Liquid Chromatography-Mass Spectrometry (LC-MS/MS) for6-keto-PGFla;4、TUNEL for apoptosis.
结果
1.成功分离出ADSCs,并传代；
2.成功建立稳定表达COX1-10aa-PGIS蛋白的ADSCs with COX1-10aa-PGIS细胞系；
3. ADSCs with COX1-10aa-PGIS能在细胞中高表达PGI2；
4. ADSCs with COX1-10aa-PGIS阴茎海绵体注射后,COX1-10aa-PGIS能在海绵体组织中表达,并产生高水平的PGI2；
5. ADSCs with COX1-10aa-PGIS基因治疗组能有效改善双侧海绵体神经损伤大鼠的勃起功能；
6. ADSCs with COX1-10aa-PGIS上调阴茎海绵体中nNOS, ASMA和VEGF表达水平；
7.ADSCs with COX1-10aa-PGIS下调阴茎海绵体中TGF β1和H工F-1á表达水平；
8. ADSCs with COX1-10aa-PGIS能抑制神经损伤后阴茎海绵体内的细胞凋亡。
小结
我们的试验结果证明COX1-10aa-PGIS基因能成功的转入ADSCs,建立稳定高表达PGI2的ADSCs with COX1-10aa-PGIS的细胞系。ADSCs with COX1-10aa-PGIS应用于大鼠神经损伤性ED模型能促进勃起功能的康复。其机制为ADSCs的组织修复和旁分泌作用以及COX1-10aa-PGIS的抗缺氧、抗纤维化、抗凋亡的作用。
全文结论：
我们的研究表明：
1. Ad-COX2-10aa-PGIS基因治疗能促进神经损伤性ED的勃起功能的恢复,其机制是通过抗纤维化和抗凋亡来保护神经损伤后的勃起组织。
2. COX1-10aa-PGIS基因能成功的转染ADSCs并建立稳定高表达PGI2的细胞系,ADSCs with COX1-10aa-PGIS治疗可改善神经损伤型ED大鼠模型的勃起功能。其机制为ADSCs的损伤修复和旁分泌作用以及COX1-1Oaa-PGIS的抗缺氧、抗纤维化、抗凋亡的作用。
Chapter One:Introduction
Post radical prostatectomy erectile dysfunction is a very common complication which is almost unavoidable and without effective rescue method. Erectile function impaired immediately following RP is thought due to the damage to the cavernous nerves, which is known as neuropraxia. The most popular medication of PDE5-Is may not be effective in the early stage after RP due to the lack of cavernous nerve impulse to release nitric oxide to increase cGMP. Therefore, there is an acute need for exploration of novel rehabilitation approaches. Both PGI2and PDE-5i are well known potent smooth muscle relaxant and vasodilators. Benefiting by PGI2-IP-cAMP-PKT signal pathway, PGI2therapy may directly dilate the artery and sinusoid trabeculae, and overcome the cavernous nerve injury barrier. However, it's short time half-life (<2min) limits its clinical application. In2006, Ruan et al reported an innovative engineering of a recombinant protein COX2-10aa-PGIS with triple catalytic activities directly converting AA into PGI2. This special protein structure could induce PGI2high express. It looks COX2-10aa-PGIS could be the promising candidate for RPED treatment. We tried to use adenovirus with COX1-10aa-PGIS gene (Ad-COX2-10aa-PGIS) to BCNC ED. The effect was observed and the underlying mechanisms were explored. Recently, Ruan et al optimized the structure of the recombinant protein named as COX1-10aa-PGIS. Adenovirus showed some limitation to clinical use. ADSCs are arising as a new therapy for ED, The aim of our second study was to applying the Adipose Derived Stem Cells (ADSCs) with COX1-10aa-PGIS gene to BCNC Rat ED model and observing the effect, then exploring the underlying mechanisms. The main body of this paper is divided into two parts and narrated as following.
Chapter Two:COX2-10aa-PGIS Gene Therapy Improving Erectile Function in Rats after Cavernous Nerve Injury
Objective
The purpose of this study was to explore the effect and mechanism of COX2-10aa-PGIS gene therapy in penile rehabilitation.
Methods
Bilateral cavernous nerve crush (BCNC) in adult Sprague-Dawley(SD) rats was used to mimic radical prostatectomy induced ED. SD rats were randomly assigned into four groups:1. sham surgery;2. BCNC;3. BCNC+null control recombinant adenovirus(NCRA) intracavernous injection; and4. BCNC+Ad-COX2-10aa-PGIS intracavernous injection.28days later, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime the blood pressure was monitored. At the end of the measurement, the penis was harvested and processed for (1) immunohistochemistry analysis for:endothelial nitric oxide synthase(eNOS), a-smooth muscle actin (ASMA) and transforming growth factor beta-1(TGFβ1);(2) Western Blot for COX2-10aa-PGI;(3) Masson's Trichrome stain for smooth muscle/collagen ratios; and (4) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis.
Results
1. COX2-10aa-PGIS gene therapy preserved erectile function in the BCNC rat model;
2. COX2-10aa-PGIS gene therapy increased eNOS and ASMA expression and decreased TGF(β1;
3. COX2-10aa-PGIS gene therapy reduced cell apoptosis after cavernous nerve injury;
4. COX2-10aa-PGIS gene therapy preserved smooth muscle/collagen ratios.
Conclusion
Our data demonstrated that COX2-10aa-PGIS protected erectile function after cavernous nerve injury through anti-fibrosis and anti-apoptotic mechanism.
Chapter Three:Adipose Derived Stem Cells with Coxl-10aa-PGIS Gene Improve Erectile Function in Rats with Bilateral Cavernous Nerve Crush
Objective:Use Adipose Derived Stem Cells as the carrier of COX1-10aa-PGIS gene to improve the penile rehabilitation after bilateral cavernous nerve crush (BCNC) in the rat ED model.
Methods
Adipose Derived Stem Cells (ADSCs) were isolated from perigonadal fat of Sprague-Dawley (SD) rats and cultured in DMEM/F12medium. The COX1-10aa-PGIS gene was transferred into ADSCs. ADSCs with COX1-10aa-PGIS stable cell line was filtered by G418and identified by Western Blot and LC-MSMS. BCNC in adult SD rats was used to replicate radical prostatectomy induced ED. SD rats were randomly assigned into four groups:1. sham surgery;2. BCNC;3. BCNC+ADSCs intracavernous injection;4、BCNC+ADSCs with COX1-10aa-PGIS gene intracavernous injection;28days later, intracavernous pressure (ICP) was recorded under cavernous nerve stimulation; meanwhile the blood pressure was monitored. At the end of the measurement, the penis and urine was harvested and processed as required for1、Immunohistochemistry for Neural nitric oxide synthase(nNOS),a-smooth muscle actin (ASMA) and Vascular endothelial growth factor (VEGF) expression level;2、Western blot for ASMA, Transf forming growth factor β1(TGFβ1) and Hypoxia-inducible factor-1a(HIF-1a);3、Liquid Chromatography-Mass Spectrometry (LC-MS/MS) for6-keto-PGF1a;4、TUNEL for apoptosis.
Results
1. The ADSCs was successfully isolated and passaged.
2. ADSCs with COX1-10aa-PGIS gene stable cell line were successfully established.
3. The ADSCs stable cell line with COX1-10aa-PGIS gene which can high express PGI2was identified by LC-MS/MS, G418and Western blot.
4. ADSCs with COX1-10aa-PGIS gene therapy improved erectile function in rats after BCNC injury.
5. ADSCs with COX1-10aa-PGIS up-regulated nNOS, ASMA and VEGF expression level in Corpus cavernosum
6. ADSCs with COX1-10aa-PGIS down-regulated TGFβ1and HIF-1a expression level in Corpus cavernosum
7. ADSCs with COX1-10aa-PGIS inhibit cell apoptosis after cavernous nerve injury
Conclusion
Our data also suggested that COX1-10aa-PGIS gene can be successfully transferred into ADSCs for gene therapy for the treatment of ED after cavernous nerve injury in an animal model. ADSC with COX1-10aa-PGIS gene might be a potential candidate in post radical prostatectomy penile rehabilitation. The underlying mechanisms of ADSCs with COX1-10aa-PGIS protected erectile function after cavernous nerve injury may through anti-hypoxia, anti-fibrosis and anti-apoptotic, and paracrine secretion and wound healing by ADSCs.
Conclusion of the whole paper
1. Our data demonstrated that COX2-10AA-PGIS protected erectile function after cavernous nerve injury through anti-fibrosis and anti-apoptotic mechanism.
2. Our data also suggested that COX1-10aa-PGIS gene can be successfully transferred into ADSCs for gene therapy for the treatment of ED after cavernous nerve injury in an animal model. ADSC with COX1-10aa-PGIS gene might be a potential candidate in post radical prostatectomy penile rehabilitation. The underlying mechanisms of ADSCs with COX1-10aa-PGIS protected erectile function after cavernous nerve injury may through anti-hypoxia, anti-fibrosis and anti-apoptotic, and paracrine secretion and wound healing by ADSCs.
勃起功能障碍(Erectile Dysfunction, ED)是前列腺癌根治术(Radical prostatectomy, RP)后最为常见的并发症之一,术中不可避免的损伤勃起神经造成神经失用是其最主要的发病机制,神经失用使得本世纪最伟大的发现之一,男科学最具革命性的药物-5型磷酸二脂酶抑制剂(Phosphodiesterase-5inhibitors, PDE5Is)对前列腺癌根治术后勃起功能障碍(Radical prostatectomy erectile dysfunction, RPED)的疗效疲软乏力。人们迫切希望寻找新的治疗手段。前列腺环素(Prostacyclin, PGI2)和PDE-5i均为强效的平滑肌松弛剂和血管保护剂,但是因为PGI2过短的半衰期(<2min)限制了它的临床应用。Ke-He Ruan等于2006年研发了的COX2-10aa-PGIS重组蛋白,这种蛋白同时具有COX-2和PGIS两种酶活性,并发挥3种有效的催化作用。这种特殊的结构能够把底物花生四烯酸(arachidonic acid, AA)直接高效转化成PGI2,从而使PGI2高表达。PGI2通过PGI2-IP-cAMP-PKT信号通路发挥平滑肌松弛剂和血管保护剂的作用,而这种信号通路似乎能能越过神经失用的障碍直接松弛血管平滑肌和扩张血管。这使得COX2-10aa-PGIS具有治疗RPED的潜在可能性。在第一个实验中,我们应用重组腺病毒(Adeno-virus)作为COX2-10aa-PGIS的载体,从基因层面治疗RPED,并探讨其潜在的机理。最近,Ke-He Ruan等在COX2-10aa-PGIS的基础上进一步优化结构,研发了COX1-10aa-PGIS。与前者相比,COX1-10aa-PGIS能更加稳定持久的高表达PGI2。目前的研究表明：脂质干细胞(Adipose Derived stem cells, ADSCs)具有自我更新,损伤修复,多向分化潜能和旁分泌作用。ADSCs是ED治疗的优秀候选者。由于腺病毒存在可能的生物威胁,在第二个实验中,我们进一步应用ADSCs作为COX1-10aa-PGIS基因治疗的载体,来治疗RPED,观察其疗效并探讨其可能性的分子机制。本论文正文分为两个部分陈述。
第二章Ad-COX2-10aa-PGIS基因治疗神经损伤性的勃起功能障碍及其机制的研究
目的
本实验的目的是探索Ad-COX2-10aa-PGIS基因治疗在神经损伤性勃起功能障碍中的疗效和机制
方法
我们用成年SD大鼠双侧海绵体神经损伤模型来模拟根治性前列腺癌切除术后勃起功能障碍(RPED)。32只SD大鼠随机被分为4组：组1、空白手术组(Sham surgery);组2、双侧海绵体神经挤压损伤(Bilateral cavernosal nerve crush, BCNC);组3、BCNC+空白腺病毒(Null control recombinant adenovirus, NCRA)阴茎海绵体注射；组4、BCNC+复制缺陷型重组腺病毒携带COX2-10aa-PGIS基因(Replication efficiency recombinant adenovirus carry COX2-10aa-PGIS gene, Ad-COX2-10aa-PGIS)阴茎海绵体注射。28天后,电刺激海绵体神经测海绵体内压(Intracavernosal pressure, ICP)同时记录平均动脉压(Mean arterial pressure, MAP)。所有大鼠的阴茎组织按要求处理并保存为下一步实验用。(1)免疫组化：endothelial nitric oxide synthase(eNOS), α-smooth muscle actin (ASMA) and transforming growth factor beta-1(TGF β1);(2) Western Blot for COX2-10aa-PGI;(3) Masson's Trichrome stain:平滑肌／胶原比值(smooth muscle/collagen ratios); and (4) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis.
结果
1. COX2-10aa-PGIS基因治疗在神经损伤大鼠模型中能保存勃起功能；
2. COX2-10aa-PGIS基因治疗能增加eNOS和ASMA表达水平；
3. COX2-10aa-PGIS基因治疗能保护平滑肌／胶原比值：
4. COX2-10aa-PGIS基因治疗能降低TGF β1的表达水平；
5. COX2-10aa-PGIS基因治疗能减少细胞凋亡。
小结
我们的实验证明阴茎海绵体注射Ad-COX2-10aa-PGIS基因治疗有助于神经损伤型ED大鼠的勃起功能的康复。其机制是抗纤维化和抗细胞凋亡,从而阻止海绵体静脉漏的发生,最终保护勃起功能。
第三章脂质干细胞携带COX1-10aa-PGIS基因治疗双侧海绵体神经损伤引起的勃起功能障碍及其机制
目的
使用脂质干细胞(ADSCs)作为COX1-10aa-PGIS基因载体来治疗双侧海绵体损伤性勃起功能障碍,并探讨其潜在的机制。
方法
1.从同种异体SD大鼠生殖腺周围脂肪中提取ADSCs,在DMEM/F12中传代；
2.建立COX1-10aa-PGIS转染的ADSCs的稳定细胞系；
3. Western blot鉴定COX1-10aa-PGIS在ADSCs表达,LC-MS/MS测定其酶活性；
4.动物实验：建立成年SD大鼠双侧海绵体损伤模型来模拟前列腺癌根治术后勃起功能障碍。将SD大鼠随机被分成四组,Sham组：空白手术组；BCNC组：双侧海绵体神经挤压损伤；BCNC+ADSCs组：双侧海绵体神经挤压损伤并接受空白ADSCs阴茎海绵体注射治疗；BCNC+ADSCs with COX2-10aa-PGIS组：双侧海绵体神经挤压损伤,并接受转染COX2-10aa-PGIS基因的ADSCs阴茎海绵体注射治疗。28天后,在电刺激海绵体神经下测海绵体内压(ICP),同时监测平均动脉压(MAP)。收集所有大鼠的阴茎组织和尿液,按要求处理并保存为下一步实验用。1、免疫组化:Neural nitric oxide synthase(nNOS), α-smooth muscle actin (ASMA)和Vascular endothelial growth factor (VEGF);2、Western blot for ASMA, Transf forming growth factor β1(TGF β1)和Hypoxia-inducible factor-1α(HIF-1α);3、Liquid Chromatography-Mass Spectrometry (LC-MS/MS) for6-keto-PGFla;4、TUNEL for apoptosis.
结果
1.成功分离出ADSCs,并传代；
2.成功建立稳定表达COX1-10aa-PGIS蛋白的ADSCs with COX1-10aa-PGIS细胞系；
3. ADSCs with COX1-10aa-PGIS能在细胞中高表达PGI2；
4. ADSCs with COX1-10aa-PGIS阴茎海绵体注射后,COX1-10aa-PGIS能在海绵体组织中表达,并产生高水平的PGI2；
5. ADSCs with COX1-10aa-PGIS基因治疗组能有效改善双侧海绵体神经损伤大鼠的勃起功能；
6. ADSCs with COX1-10aa-PGIS上调阴茎海绵体中nNOS, ASMA和VEGF表达水平；
7.ADSCs with COX1-10aa-PGIS下调阴茎海绵体中TGF β1和H工F-1á表达水平；
8. ADSCs with COX1-10aa-PGIS能抑制神经损伤后阴茎海绵体内的细胞凋亡。
小结
我们的试验结果证明COX1-10aa-PGIS基因能成功的转入ADSCs,建立稳定高表达PGI2的ADSCs with COX1-10aa-PGIS的细胞系。ADSCs with COX1-10aa-PGIS应用于大鼠神经损伤性ED模型能促进勃起功能的康复。其机制为ADSCs的组织修复和旁分泌作用以及COX1-10aa-PGIS的抗缺氧、抗纤维化、抗凋亡的作用。
全文结论：
我们的研究表明：
1. Ad-COX2-10aa-PGIS基因治疗能促进神经损伤性ED的勃起功能的恢复,其机制是通过抗纤维化和抗凋亡来保护神经损伤后的勃起组织。
2. COX1-10aa-PGIS基因能成功的转染ADSCs并建立稳定高表达PGI2的细胞系,ADSCs with COX1-10aa-PGIS治疗可改善神经损伤型ED大鼠模型的勃起功能。其机制为ADSCs的损伤修复和旁分泌作用以及COX1-1Oaa-PGIS的抗缺氧、抗纤维化、抗凋亡的作用。
Chapter One:Introduction
Post radical prostatectomy erectile dysfunction is a very common complication which is almost unavoidable and without effective rescue method. Erectile function impaired immediately following RP is thought due to the damage to the cavernous nerves, which is known as neuropraxia. The most popular medication of PDE5-Is may not be effective in the early stage after RP due to the lack of cavernous nerve impulse to release nitric oxide to increase cGMP. Therefore, there is an acute need for exploration of novel rehabilitation approaches. Both PGI2and PDE-5i are well known potent smooth muscle relaxant and vasodilators. Benefiting by PGI2-IP-cAMP-PKT signal pathway, PGI2therapy may directly dilate the artery and sinusoid trabeculae, and overcome the cavernous nerve injury barrier. However, it's short time half-life (<2min) limits its clinical application. In2006, Ruan et al reported an innovative engineering of a recombinant protein COX2-10aa-PGIS with triple catalytic activities directly converting AA into PGI2. This special protein structure could induce PGI2high express. It looks COX2-10aa-PGIS could be the promising candidate for RPED treatment. We tried to use adenovirus with COX1-10aa-PGIS gene (Ad-COX2-10aa-PGIS) to BCNC ED. The effect was observed and the underlying mechanisms were explored. Recently, Ruan et al optimized the structure of the recombinant protein named as COX1-10aa-PGIS. Adenovirus showed some limitation to clinical use. ADSCs are arising as a new therapy for ED, The aim of our second study was to applying the Adipose Derived Stem Cells (ADSCs) with COX1-10aa-PGIS gene to BCNC Rat ED model and observing the effect, then exploring the underlying mechanisms. The main body of this paper is divided into two parts and narrated as following.
Chapter Two:COX2-10aa-PGIS Gene Therapy Improving Erectile Function in Rats after Cavernous Nerve Injury
Objective
The purpose of this study was to explore the effect and mechanism of COX2-10aa-PGIS gene therapy in penile rehabilitation.
Methods
Bilateral cavernous nerve crush (BCNC) in adult Sprague-Dawley(SD) rats was used to mimic radical prostatectomy induced ED. SD rats were randomly assigned into four groups:1. sham surgery;2. BCNC;3. BCNC+null control recombinant adenovirus(NCRA) intracavernous injection; and4. BCNC+Ad-COX2-10aa-PGIS intracavernous injection.28days later, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime the blood pressure was monitored. At the end of the measurement, the penis was harvested and processed for (1) immunohistochemistry analysis for:endothelial nitric oxide synthase(eNOS), a-smooth muscle actin (ASMA) and transforming growth factor beta-1(TGFβ1);(2) Western Blot for COX2-10aa-PGI;(3) Masson's Trichrome stain for smooth muscle/collagen ratios; and (4) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis.
Results
1. COX2-10aa-PGIS gene therapy preserved erectile function in the BCNC rat model;
2. COX2-10aa-PGIS gene therapy increased eNOS and ASMA expression and decreased TGF(β1;
3. COX2-10aa-PGIS gene therapy reduced cell apoptosis after cavernous nerve injury;
4. COX2-10aa-PGIS gene therapy preserved smooth muscle/collagen ratios.
Conclusion
Our data demonstrated that COX2-10aa-PGIS protected erectile function after cavernous nerve injury through anti-fibrosis and anti-apoptotic mechanism.
Chapter Three:Adipose Derived Stem Cells with Coxl-10aa-PGIS Gene Improve Erectile Function in Rats with Bilateral Cavernous Nerve Crush
Objective:Use Adipose Derived Stem Cells as the carrier of COX1-10aa-PGIS gene to improve the penile rehabilitation after bilateral cavernous nerve crush (BCNC) in the rat ED model.
Methods
Adipose Derived Stem Cells (ADSCs) were isolated from perigonadal fat of Sprague-Dawley (SD) rats and cultured in DMEM/F12medium. The COX1-10aa-PGIS gene was transferred into ADSCs. ADSCs with COX1-10aa-PGIS stable cell line was filtered by G418and identified by Western Blot and LC-MSMS. BCNC in adult SD rats was used to replicate radical prostatectomy induced ED. SD rats were randomly assigned into four groups:1. sham surgery;2. BCNC;3. BCNC+ADSCs intracavernous injection;4、BCNC+ADSCs with COX1-10aa-PGIS gene intracavernous injection;28days later, intracavernous pressure (ICP) was recorded under cavernous nerve stimulation; meanwhile the blood pressure was monitored. At the end of the measurement, the penis and urine was harvested and processed as required for1、Immunohistochemistry for Neural nitric oxide synthase(nNOS),a-smooth muscle actin (ASMA) and Vascular endothelial growth factor (VEGF) expression level;2、Western blot for ASMA, Transf forming growth factor β1(TGFβ1) and Hypoxia-inducible factor-1a(HIF-1a);3、Liquid Chromatography-Mass Spectrometry (LC-MS/MS) for6-keto-PGF1a;4、TUNEL for apoptosis.
Results
1. The ADSCs was successfully isolated and passaged.
2. ADSCs with COX1-10aa-PGIS gene stable cell line were successfully established.
3. The ADSCs stable cell line with COX1-10aa-PGIS gene which can high express PGI2was identified by LC-MS/MS, G418and Western blot.
4. ADSCs with COX1-10aa-PGIS gene therapy improved erectile function in rats after BCNC injury.
5. ADSCs with COX1-10aa-PGIS up-regulated nNOS, ASMA and VEGF expression level in Corpus cavernosum
6. ADSCs with COX1-10aa-PGIS down-regulated TGFβ1and HIF-1a expression level in Corpus cavernosum
7. ADSCs with COX1-10aa-PGIS inhibit cell apoptosis after cavernous nerve injury
Conclusion
Our data also suggested that COX1-10aa-PGIS gene can be successfully transferred into ADSCs for gene therapy for the treatment of ED after cavernous nerve injury in an animal model. ADSC with COX1-10aa-PGIS gene might be a potential candidate in post radical prostatectomy penile rehabilitation. The underlying mechanisms of ADSCs with COX1-10aa-PGIS protected erectile function after cavernous nerve injury may through anti-hypoxia, anti-fibrosis and anti-apoptotic, and paracrine secretion and wound healing by ADSCs.
Conclusion of the whole paper
1. Our data demonstrated that COX2-10AA-PGIS protected erectile function after cavernous nerve injury through anti-fibrosis and anti-apoptotic mechanism.
2. Our data also suggested that COX1-10aa-PGIS gene can be successfully transferred into ADSCs for gene therapy for the treatment of ED after cavernous nerve injury in an animal model. ADSC with COX1-10aa-PGIS gene might be a potential candidate in post radical prostatectomy penile rehabilitation. The underlying mechanisms of ADSCs with COX1-10aa-PGIS protected erectile function after cavernous nerve injury may through anti-hypoxia, anti-fibrosis and anti-apoptotic, and paracrine secretion and wound healing by ADSCs.
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