間葉幹細胞對於睪丸扭轉後造精能力及精子品質損傷之保護機轉探討

The protective effect of mesenchymal stem cell on spermatogenesis and sperm parameter injury status post testicular torsion-detorsion

蕭志豪(Chi-Hao Hsiao)

指導教授 ： 簡銘賢

共同指導教授 ： 何慧君(Jannifer Ho)

臺北醫學大學/醫學院/臨床醫學研究所/博士(2020年)

摘要

睾丸扭轉是泌尿外科在急診常見的急症，由於缺血性損傷導致不育是常見的併發症。進行睾丸復位手術是必要的處置，但迄今為止，尚無有效的方法來進一步恢復精子生成功能。間質幹細胞（Mesenchymal stem cells, MSC）對急性組織損傷的影響已得到證實，藉由其旁分泌功能、分化和免疫調節的能力可能有益於睾丸扭轉誘發的不孕。此外，通過即時睾丸復位手術是避免不育的唯一方法，但仍然沒有有效的方法來進一步恢復精子製造效率。在一系列研究中，我們探討了局部注射MSC在睾丸扭轉導致的生殖細胞損傷中的治療功效和機制。此外，還闡述了MSC調節睾丸扭轉誘導的生殖細胞活性的分子機制。在精子生成試驗中，我們將雄性大白鼠進行720度睪丸扭轉，三小時後再進行扭轉復位，在此同時於扭轉復位前三十分鐘給予局部注射三千個眼窩脂肪幹細胞於睪丸中，七天後解剖分析。經由強森指數（Johnsen’s Score）檢驗發現眼窩脂肪幹細胞能減緩睪丸扭轉導致的造精能力下降，並減少睪丸扭轉導致缺血再灌流所誘發之氧化損傷及細胞凋亡，同時給予眼窩脂肪幹細胞能提昇血液中賀爾蒙的含量。而經由免疫組織染色發現所移植的眼窩脂肪幹細胞主要集中在睪丸間質細胞（Leydig）周圍，特別的是某些移植細胞會分化成為表現p450之細胞。而在精子功能回復試驗中，我們將雄性大白鼠進行720度睪丸扭轉，三小時後再進行扭轉復位，在此同時於扭轉復位前三十分鐘給予局部注射三千個眼窩脂肪幹細胞於睪丸中，三天後解剖分析。我們分別將睪丸、附睪丸以及精子分離出來進行分析，經由精子功能性評估，不論是在精子的數量、存活率、活動力以及ATP含量都因為眼窩脂肪幹細胞的注射而減緩睪丸扭轉再復位所導致的精子功能損傷。同時我們發現眼窩脂肪幹細胞之注射能促進phosphoglycerate kinase 2及glyceraldehyde-3-phosphate dehydrogenase的表現，活化Akt以及glycogen synthase kinase 3的路徑進而提昇醣解反應的進行而促進ATP生成。本研究證實移植間質幹細胞能有效減緩睪丸扭轉及復位時所造成之氧化損傷導致不孕以及精子功能異常，對於臨床應用能提供臨床前試驗之佐證。根據這一系列研究，來自眼窩脂肪的間葉幹細胞可以預防睾丸扭轉引起的精細胞損傷，其機制主要是通過減少氧化壓力，減少睾丸凋亡和SCF的旁分泌。 同時，由於間葉幹細胞針對Akt / GSK3機轉的活化，促進了糖解作用並增加了精子中ATP的含量，進而改善了睾丸扭轉後精子的活動力。在臨床病例中，缺血與再灌流所造成的生殖細胞損傷（如睾丸扭轉，精索靜脈曲張和缺氧性損傷）可能通過間葉幹細胞的治療得以挽救，並十分令人期待進一步的研究。

關鍵字

間葉幹細胞 ； 睪丸扭轉

並列摘要

Testicular torsion is a urological emergency and infertility is a common complication due to ischemic injury. Surgical reduction and orchiopexy is indicated, but to date there is no effective method for restoration of spermatogenesis. The effects of mesenchymal stem cells (MSC) on acute tissue injury have been demonstrated, and the abilities of paracrine support, differentiation and immune-modulation may benefit to testicular torsion-induced infertility. Besides, promptly surgical correction by orchiopexy is the only way to avoid infertility and no effective treatment for restoration of spermatogenesis. In a series of study, we investigate the therapeutic efficacy and the mechanisms of MSC in testicular torsion-induced germ cell injury when injected locally. Furthermore, the molecular mechanisms of MSC in regulating germ cell activity induced by testicular torsion-detorsion was also illustrated. In the study on spermatogenesis and infertility, Sprague–Dawley rats received surgical 720 degree torsion for 3 hours, followed by detorsion on the left testis. 20 μl of phosphate-buffered saline (PBS) without or with 3 x 104 MSC from human orbital fat stem cells (OFSEs) were given, respectively, via local injection into the left testis 30 minutes before detorsion. Histopathology with Johnsen’s score analysis, Western blot analysis for superoxide dismutase 2, Bax, Caspase-3, human insulin growth factor-1 and human stem cell factor, malondialdehyde (MDA) assay in testis and plasma, hormones level including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by ELISA Kits, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and fluorescence staining for P450, Sox-9 and VASA were performed. Animals were sacrificed and bilateral orchiectomy was performed 7 days after torsion-detorsion. Local injections of MSC prevented torsion-induced infertility judged by Johnsen's score. TUNEL assay and Western blot analysis on caspase 3 and Bax demonstrated that MSC prevented ischemic/reperfusion induced intrinsic apoptosis. MDA assay revealed that MSC significantly reduced the oxidative stress in the damaged testicular tissues. After the MSC injection, serum hormones secretion was increased, while the elevation of FSH triggered by testicular injury was balanced. MSC also produced stem cell factor in the damaged testis. Immunofluorescence staining revealed that most transplanted cells surrounded the Leydig cells. Some of transplanted cells differentiated into p450 expressing cells within 7 days. In the study on sperm function, Sprague-Dawley rats received left testis 720 degree torsion for 3 hours followed by detorsion with or without MSC. Right inguinal skin incision without testicular torsion served as control. MSC with 3 x 104 cells were locally injected into left testis 30 minutes before detorsion. Three days after the surgery, orchiotectomy was executed and the testis, epididymis and sperm were separated to each other. Functional assessments on sperm included counting sperm amount and sperm motility, staining F-actin and quantifying adenosine triphosphate (ATP) content. The hallmarks of glycogenesis and glycolysis in each tissue segment were measured by Western blot. We also found that testicular torsion-detorsion significantly decreased the amount of sperm, inhibited the motility, declined the F-actin expression, and reduced the content of ATP in sperm. Local injection of MSC improved sperm function, particularly in sperm motility. With MSC, ATP content and F-actin were preserved after testicular torsion-detorsion. MSC significantly reversed the imbalance of glycolysis in sperm and testis induced by testicular torsion-detorsion, as evidenced by increasing the expression of phosphoglycerate kinase 2 and glyceraldehyde-3-phosphate dehydrogenase-spermatogenic, activating Akt and increasing glycogen synthase kinase 3 (GSK3), which led to the increase in glycolysis cascades and ATP production. These results suggested that local injection of allogenic MSC before surgical detorsion is a simple, clinical friendly procedure to rescue torsion-induced infertility and human stem cell factor contributed the activation of Akt/GSK3 axis when sperm suffered from testicular torsion-detorsion induced germ cell injury.

並列關鍵字

Mesenchymal stem cell ； Testicular torsion

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