分步诱导人源脐带间充质干细胞为睾丸间质细胞的机制研究

Androgen deficiency results from a variety of diseases and is commenly treated with exogenous hormones. However, long term use of hormone replacement therapy would cause a lot of complications and the appropriate dosage is difficult to determine, especially for boys. Leydig cells produce the largest portion of androgen and therefore cell transplantation therapy is an effective treatment for androgen deficiency. But due to the lack of capability of mitosis in mature Leydig cells, the long term efficacy of cell transplantation still needs further investigation to be confirmed. In this study, human umbilical mesenchymal stem cells will be induced into Leidig cells and the proliferation ability will be maintained for better efficacy of cell transplantation therapy. Together with the development characteristics of Leydig cells and our previous experience in experiments, miltiple step induction strategy will be employed by adding exogenous factors to mimic the natural differentiation process. The mechanisms involved in the differentiation will also be addressed. The changes in the expression of luteinizing hormone receptors and testosterone synthetases will be investigated by in vitro experiments. The induced Leydig cells will be embedded by matrigel to mimic the morphology in testicles and implanted into the scrotum of prepubertal castrated nude mice. The changes in serum levels of testosterone and luteinizing hormones will be determined. The final aim of the study is to explain the mechanism of Leydig cells transformation. And through re-establishment of the capability of testosterone production by cell transplantation, the negative feedback regulation between intragenous androgen and luteinizing hormones will be re-constructed to induce physiological hormon levels. This study may provide the animal study results for further clinical investigations.

多种疾病造成雄激素分泌不足，目前主要采取的治疗方法是外源性激素替代。但长期应用激素造成多种并发症，且剂量难以调控。体内雄激素主要由睾丸间质细胞合成，细胞移植具有治疗效果。但成熟的睾丸间质细胞缺乏有丝分裂能力，细胞移植的长期效果有待观察。本课题诱导人源脐带间充质干细胞为睾丸间质细胞，保留细胞增殖能力，提高细胞移植疗效。结合睾丸间质细胞的发育特点和前期实验基础，拟采取分步诱导技术，分阶段添加外源性诱导因子，模拟细胞自然分化过程，阐明细胞转化机制。体外实验重点观察细胞黄体生成素受体和睾酮合成酶系的变化。诱导后的细胞以基质胶包裹，模拟睾丸形态后移植入青春期去势裸鼠阴囊。观察受体动物血清睾酮和黄体生成素含量变化。通过实验,探索睾丸间质细胞转化机制。结合细胞移植，重建机体合成睾酮的能力。产生内源性雄激素与垂体分泌的黄体生成素形成负反馈调节，激素水平符合生理所需，为进一步临床治疗提供实验基础。

本课题设计的目的是为了利用分步诱导分化技术将人脐带间充质干细胞分步诱导为有功能的睾丸间质细胞， 并移植进入睾丸功能不良的动物体内，达到提高受体性激素水平的目的。经过几年的实验，我们通过贴壁法从人脐带中获得了纯度高，分化潜能好的人脐带间充质干细胞。随后，课题组按照Leydig细胞发育成熟的四个阶段（Leydig干细胞、Leydig祖细胞、不成熟Leydig细胞和成熟Leydig细胞），使用睾丸间质细胞条件培养基（CM）及睾丸间质细胞诱导培养基 (DIM)对干细胞进行分步诱导，并选择不同的时间节点进行鉴定。发现，在干细胞诱导的第3天，细胞形态仍为纺锤状，且经PCR鉴定，Leydig细胞特异性标志酶相关基因尚未表达。干细胞诱导7天后，干细胞形态逐渐变为多角形，细胞质中细胞器数量逐渐丰富，LHR基因表达为阳性，Leydig细胞相关标志酶有弱表达。诱导10天后，诱导细胞出现Leydig细胞特异性标志酶的阳性表达。2周后，细胞形态为类圆形，胞质染色证明Leydig细胞标志酶的阳性表达。由此，根据Leydig细胞特异性标志酶出现的时间点，课题组将干细胞诱导分化过程分为4个阶段，即类Leydig干细胞、类Leydig祖细胞、不成熟类Leydig细胞、和成熟Leydig细胞。随后，我们尝试对干细胞进行体内诱导。先使用红色荧光染料CM-DIL预染干细胞，随后将其注射入大鼠睾丸，分别于2周，3周，4周后收取实验组大鼠睾丸进行CYP11a1荧光染色。发现诱导3周后的干细胞可阳性表达CYP11a1。随后通过流式细胞分选技术获得了睾丸细胞悬液中的红色荧光人源细胞，并进行离体培养，发现其在体外仍可定植并分裂，流式鉴定结果显示诱导4周后，约24%干细胞可阳性表达3β-HSD。课题组使用二甲磺酸乙烷（EDS）腹腔注射8周龄大鼠制作性腺功能低下大鼠模型。雄性大鼠性腺功能低下模型建立后，本研究将CM-Dil标记的人脐带间充质干细胞移植入实验组模型鼠睾丸内，对照组模型注射等体积PBS，分别于细胞移植后7、14、21天抽取模型鼠血液检测血清睾酮值变化情况，统计结果发现移植细胞约三周后，实验组睾酮数值明显高于对照组（p＜0.05）。移植入大鼠睾丸内的HUMSCs可向Leydig细胞方向分化，并通过分化为Leydig细胞促进了EDS性腺功能低下大鼠模型的睾酮恢复。