干细胞诱导分化为雄激素分泌细胞的研究进展

睾丸间质细胞是男性体内产生雄激素的主要细胞。干细胞可被诱导分化为具有雄激素分泌功能的睾丸间质样细胞,从而使其功能受到下丘脑与垂体调控,精确分泌维持生理功能需要的激素。故建立一套可靠高效的将干细胞诱导为雄激素分泌细胞的方法,对于治疗由于睾丸间质细胞异常引起的性腺功能低下意义重大。本文对近年来关于干细胞诱导分化为雄激素分泌细胞的研究进展予以综述。     

睾丸细胞生物学研究进展

睾丸是男性生殖腺,由生精小管和间质构成。生精小管主要由生精细胞和支持细胞组成,是精子发生场所;间质中主要是间质细胞,间质细胞合成与分泌雄激素。本文介绍睾丸3种细胞的发育分化,以及成年期睾丸细胞的结构和生物学研究进展。     

睾丸肿瘤的病因、分类及诊断

睾丸既是男性的生殖腺，又具有内分泌功能。睾丸由生精小管及其周围的间质组成。生精小管内衬生精上皮，由不同发育阶段的生殖细胞和支持细胞组成。间质中有血管、巨噬细胞、肥大细胞及间质细胞。睾丸的生精功能和产生雄激素的功能受到垂体促性腺激素的调控。促滤泡激素（FSH）促使睾丸生成精于，促黄体激素（LH）促使睾丸合成雄激素。而FSH发挥效应的靶细胞即睾丸的支持细胞，LH的靶细胞是睾丸间质中的间质细胞。睾丸生殖细胞肿瘤是源于生精小管中的一些异形细胞，其属全能的未分化细胞，可以发展为复杂的多样化的肿瘤。因此，睾丸肿瘤…     

干细胞移植治疗睾丸衰老的现状与思考

睾丸衰老的主要特点为睾酮合成能力以及生精功能逐渐下降,不仅会影响男性生育力,也与衰老相关慢性疾病关系密切。因此,延缓睾丸衰老对于改善中老年男性健康和生活质量至关重要。干细胞是具有强大的自我更新能力和多向分化潜能的细胞群体,近年来,其基础与临床应用研究不断深化,推动了细胞疗法的发展。干细胞移植目前已被用于治疗多种疾病,在衰老与再生医学领域也受到广泛关注。干细胞移植在治疗睾丸衰老中也具有巨大的应用前景。本文将回顾干细胞移植治疗睾丸衰老的相关研究历程与进展,并探讨临床转化面临的瓶颈以及疗效优化策略,以期为睾丸衰老的干细胞疗法研发及临床转化提供新思路。     

人脐带间充质干细胞在大鼠睾丸间质内向Leydig细胞分化的实验研究

目的:研究人脐带间充质干细胞(HUMSCs)在大鼠睾丸间质内向Leydig细胞分化的可行性。方法:贴壁法获得HUMSCs,分别通过流式细胞表面抗原染色与三系分化验证其纯度与多向分化能力,用CM-Dil标记HUMSCs后将其移植入大鼠睾丸间质内,并在移植后4周与8周对大鼠睾丸进行免疫荧光染色观察HUMSCs的存活与分化情况,移植后8周获得大鼠睾丸细胞悬液,通过流式细胞染色检测HUMSCs表达Leydig细胞标志物3β-HSD判断细胞分化的效率,通过流式细胞分选获得悬液内CM-Dil标记的在睾丸内分化后的HUMSCs,并对其进行培养,培养3 d后收集培养液,检测其睾酮水平。结果:Leydig细胞标志物CYP11a1在HUMSCs移植8周后有表达,而在移植4周后未见表达。3β-HSD流式染色显示其分化效率约为14.5%,流式细胞分选后细胞可存活,且其培养液内能检测出睾酮。结论:HUMSCs在大鼠睾丸间质内可向Leydig细胞分化。     

睾丸Leydig细胞干细胞研究进展

Leyd ig细胞是机体合成和分泌雄激素的主要细胞,利用Leyd ig细胞干细胞的体外定向诱导分化扩增至具有雄激素合成功能的成熟细胞再植入机体中,对雄激素缺乏的治疗具有非常广阔的临床应用前景。     

睾丸功能的激素调节及其临床意义

睾丸是一个男性特有的腺体组织。它有二种结构形式:(1)存在睾丸间质内的间质细胞(LeydigCell);(2)存在于曲细精管内的支持细胞(SertoliCell)。具有二种特殊的功能:(1)间质细胞分泌雄激素以维持男性的性征和性功能;(2)支持细胞则产生精子以保证男性的生育力。睾丸的这些功能是在神经下丘脑-垂体-睾丸激素的调节下保持相对的稳定。若其中任一环节的损害均可能导致睾丸功能的紊乱而产生性功能和/或生育力的障碍。了解睾丸功能的神经一内分泌激素调节对临床诊治该类疾病将不无裨益,故本文摘要于以介绍。一、雄激素分泌的激素调节 (一)神经一下丘脑的激素调节: 1.神经调节:已知,各种内外环境因素的刺激均     

Leydig细胞培养与移植

Ｌｅｙｄｉｇ细胞是一种大而圆形的细胞，位于睾丸曲细精管间质内，占间质的５％～１２％，一个２０岁男子睾丸含有７×１０８个Ｌｅｙｄｉｇ细胞，其主要功能是分泌睾酮，调节精子的发生、促进性分化及青春期性成熟［１］。下丘脑垂体性男性性腺功能低下，雄性激素分泌不足，临床上主要采用人工合成的雄激素替代治疗，长期服用则有许多不良反应。国内外学者试图通过分离、培养、移植Ｌｅｙｄｉｇ细胞，提高病人血浆睾酮水平，恢复男性第二性征，改善男性的性功能［２～５］。现综述如下。１　Ｌｅｙｄｉｇ细胞功能调节Ｌｅｙｄｉｇ细胞是睾…     

邻苯二甲酸酯类化合物与睾丸源性生殖障碍综合征

流行病学研究显示妇女在怀孕期间接触邻苯二甲酸酯类化合物后,所产男婴易患隐睾、尿道下裂、成年期睾丸肿瘤及精液质量低下等症状。这类症状可统称为睾丸源性生殖障碍综合征(testicular dysgenesis syndrome,TDS)。TDS可能是由男性胎儿睾丸的发育在子宫内受到影响不能发育出具有正常功能的睾丸间质(Leydig)细胞和支持(Sertoli)细胞引起的。例如,睾丸间质细胞接触邻苯二甲酸酯类化合物后,睾丸间质细胞的两种产物———睾酮和胰岛素样生长因子3(INSL3)———受到了抑制,两者对于睾丸下降起关键作用。成年期睾丸的胎儿型间质细胞和支持细胞错位可能是精子生成减少的原因。     

小鼠睾丸雄激素生成酶活性的测定

睾丸间质细胞是体内产生雄激素的主要细胞，其合成功能依赖于细胞内一系列将胆固醇转化为雄激素的酶，主要有３β－羟类固醇脱氢酶，１７α－羟化酶和１７－甾酮还原酶，这些酶活性的大小可直接反映间质细胞的合成功能。     

小鼠胚胎睾丸Leydig细胞培养、纯化及其功能研究

目的:探讨小鼠胚胎睾丸Leyd ig细胞体外培养、鉴定、纯化的方法,并进行形态学观察、分泌睾酮能力等生物学特性检测。方法:选择胎龄16 d的胎鼠睾丸,0.03%胶原酶Ⅰ消化,3β-羟基类固醇脱氢酶(HSD)染色鉴定及纯度测定,锥虫蓝染色检测细胞活率。放免法测定Leyd ig细胞不同培养时间及密度下分泌睾酮的水平。结果:Leyd ig细胞培养前和培养72 h后纯度分别为(45.10±1.66)%和(81.17±2.32)%;培养液中可检测到睾酮,睾酮水平与Leyd ig细胞数、培养时间相关,单个Leyd ig细胞睾酮分泌能力逐日下降。结论:该法分离的胚胎Leyd ig细胞纯度较高,生长性好,保持增殖和分泌睾酮的生物学特性,可应用于相关的研究。     

睾丸Leydig干细胞分离、纯化及鉴定的体外研究

目的 改进Leydig干细胞的分离及纯化方法,观察其生物学特性并进行鉴定.方法　通过胶原酶消化、差速贴壁法及双抗体免疫磁珠分选法,从大鼠睾丸内获得Leydig干细胞.以条件培养液进行培养,采用CCK法测定增殖能力.通过PDGFRα、LIFR及3β-HSD免疫组织化学染色鉴定Leydig干细胞.经定向诱导分化后,检测Leydig细胞的睾酮分泌能力.结果　每个睾丸约可获得83 000个干细胞,经免疫组织化学染色分析,PDGFRα阳性率为(98.0±0.8)％.在条件培养液中培养的SLCs增殖明显,在1个月内能稳定维持其干性而未向Leydig细胞系分化.免疫组织化学染色结果表明,PDGFRα、LIFR阳性表达,3β-HSD为阴性表达.Leydig干细胞在含有T3和HCG的培养液中培养后,第5天即可测到睾酮分泌,分化后的细胞3β-HSD+.结论　 由差速贴壁法及双抗体免疫磁珠分选法能获得纯化的PDGFRα+/LHRˉ细胞,这些细胞符合SLCs所必须具有的特征.采用该方法获取SLCs,操作简便,细胞损伤小并且获取纯度高.     

Steroidogenic and Spermatogenic Potentials of the Male Rat after Acute Treatment with Aflatoxin B1

150 microgram of aflatoxin B1 were injected i.p]. to male rats on three occasions at intervals 48 h. Sperm production and plasma and testicular testosterone concentrations were determined in rats killed 24 h after the last dose of aflatoxin with or without stimulation with human chorionic gonadotropin (HCG). Neither sperm production nor testosterone secreting capacity was significantly depressed by the acute aflatoxin treatment. The Leydig cell function was however impaired as indicated by the extremely reduced responsiveness of the Leydig cells of the aflatoxin-pretreated rats to HCG stimulation. It may be concluded that the disruption of spermatogenesis by chronic aflatoxin treatment is preceded and caused by the impairment of the Leydig cell function and the resultant drop in testosterone in the testis.     

环境污染物三丁基氯化锡和氯化三苯锡对大鼠睾丸Leydig细胞的影响

目的:探讨环境污染物三丁基氯化锡(TBT)和氯化三苯锡(TPT)对大鼠睾丸Leyd ig细胞的影响。方法:①用0~80 nmol/L浓度的TBT和TPT处理大鼠睾丸Leyd ig(LC-540)细胞24~96 h,用四唑蓝(MTT)法确定细胞的存活率;②用DNA片段法确定是否存在细胞凋亡;③观察细胞内Ca2+螯合剂氨基苯乙烷四乙酸(BAPTA)和蛋白激酶A(PKA)抑制剂H-89、蛋白激酶C(PKC)抑制剂GF109203X、酪氨酸蛋白激酶(TPK)抑制剂Gen iste in是否可以阻断TBT所致的细胞凋亡;④检测TBT处理的原代大鼠睾丸Leyd ig细胞分泌睾酮的变化。结果:①TBT和TPT影响Leyd ig细胞存活率的强度基本相同,在20~80 nmol/L浓度时细胞存活率呈剂量和时间依赖性降低;②DNA片段法研究证实TBT和TPT可引起细胞凋亡;③BAPTA可阻断20 nmol/L的TBT所致的细胞凋亡,而PKA、PKC和TPK抑制剂对细胞存活率没有影响;④TBT可减少Leyd ig细胞分泌睾酮,并使其对人绒毛膜促性腺激素(hCG)刺激的反应性降低。结论:环境污染物TBT和TPT能直接导致睾丸Leyd ig细胞凋亡,并抑制睾酮分泌,这种致凋亡作用可能与细胞内Ca2+浓度增加有关。     

Developmental changes in testicular interstitial cell populations, LH receptors and in the response to hCG in the rat

High doses of hCG were administered to immature rats of different ages and the animals killed 48 h later. Serum testosterone increased 2 to 4-fold over control values 48 h after hCG. In-vitro androgen production showed different patterns according to age. Animals younger than 35 days, when treated with hCG, retained the ability to respond to in-vitro gonadotrophic stimulation. This ability was lost in testes from rats aged 45 days. The number of free LH-receptors 48 h after hCG diminished with increasing age to become non-detectable at 35 and 45 days. In control animals the proportion of differentiated Leydig cells in relation to their mesenchymal precursors increased progressively with age to reach highest values at 45 days. hCG administration induced a shift of the cellular composition of the interstitium toward the more mature cell types. hCG has a predominantly trophic action on mesenchymal precursors in young rats, promoting their differentiation. These effects are minimal in the differentiated Leydig cells in older animals. It is proposed that the observed biochemical responses are the result of the balance between the increase in LH receptors and steroidogenic enzymes in the developing new generation of young Leydig cells and the down-regulation of receptors and enzymatic lesions in fully differentiated Leydig cells.     

Feeding hydroalcoholic extract powder of Lepidium meyenii (maca) increases serum testosterone concentration and enhances steroidogenic ability of Leydig cells in male rats

Although Lepidium meyenii (maca), a plant growing in Peru's central Andes, has been traditionally used for enhancing fertility and reproductive performance in domestic animals and human beings, effects of maca on reproductive organs are still unclear. This study examined whether feeding the hydroalcoholic extract powder of maca for 6 weeks affects weight of the reproductive organs, serum concentrations of testosterone and luteinising hormone (LH), number and cytoplasmic area of immunohistochemically stained Leydig cells, and steroidogenesis of cultured Leydig cells in 8‐week‐old male rats. Feeding the extract powder increased weight of seminal vesicles, serum testosterone level and cytoplasmic area of Leydig cells when compared with controls. Weight of prostate gland, serum LH concentration and number of Leydig cells were not affected by the maca treatment. The testosterone production by Leydig cells significantly increased when cultured with 22R‐hydroxycholesterol or pregnenolone and tended to increase when cultured with hCG by feeding the extract powder. The results show that feeding the hydroalcoholic extract powder of maca for 6 weeks increases serum testosterone concentration associated with seminal vesicle stimulation in male rats, and this increase in testosterone level may be related to the enhanced ability of testosterone production by Leydig cells especially in the metabolic process following cholesterol.     

Apoptosis related gene products in differentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate

Androgen secreting Leydig cells in the adult are differentiated with a very low turnover, however, Leydig cell tumours can arise spontaneously or after treatment with toxins. This study in the rat investigated whether changes in components of programmed cell death could be involved. In contrast to their absence in differentiated Leydig cells, antiapoptotic Bcl-2 and proapoptotic Bax were expressed in tumours. Bak and Bcl-xl were found in both tumour and normal Leydig cells. Apoptosis was induced in subcutaneous implants of Leydig cell tumour by ethane dimethanesulphonate (EDS) which is known to kill differentiated Leydig cells. The marked regression of the tumour following EDS treatment was transient and re-growth occurred between 6 and 14 days later. Tumour regression and growth was associated with a similar weight pattern in the seminal vesicles caused by changes in serum testosterone. During tumour regression, clusterin and Bax proteins were elevated but Bak, Bcl-xl and Bcl-2 were unchanged. Fas-R, Fas-L and Bax were upregulated after tumour regression had taken place. These data show that Leydig cell tumours possess many of the apoptosis related gene products and can die by apoptosis, however, regulation is clearly different in differentiated and mitotic Leydig cells.     

大鼠睾丸间质细胞移植的实验研究

目的:观察大鼠同种异体睾丸间质细胞移植后的血清睾酮变化水平。方法:采用Percoll方法分离提取SD大鼠两侧睾丸间质细胞,异体移植后每月测定受体血清睾酮1次,连续3次。结果:睾丸间质细胞移植后,受体动物血清睾酮含量逐渐升高。移植术后3个月,血清睾酮水平明显上升,与出生后2个月内的动物比较,差异具有显著性。结论:异体睾丸间质细胞移植治疗男性原发性性腺功能低下症可能具有良好的应用前景。     

Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells

Late-onset hypogonadism (LOH) is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone (LH)-stimulated steroidogenic acute regulatory (StAR) protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species (ROS) levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H(2)O(2), resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.