汪富林?夏凯?冯鑫?庄锦涛?周明宽?廖武源?雷振明?项鹏?邓春华?涂响安

【摘要】目的 探討黄体生成素受体（LHCGR）对睾丸间质干细胞（SLC）增殖及分化能力的影响。方法 采用实时荧光定量PCR（qRT-PCR）及免疫荧光染色比较LHCGR-/-小鼠与LHCGR+/+小鼠睾丸内SLC标志物的表达情况。通过流式细胞术分选出SLC，进行5-乙炔基-2'-脱氧尿苷（EdU）及CCK-8增殖实验。同时诱导SLC向睾丸间质细胞（LC）分化，14 d后检测上清液睾酮水平。qRT-PCR及免疫荧光检测诱导分化后LC标志物表达水平。结果 LHCGR-/-小鼠睾丸内存在SLC，应用CD51标记并通过流式分选可获得SLC。LHCGR-/-小鼠SLC EdU+细胞比例和CCK-8增殖曲线与LHCGR+/+小鼠比较差异均无统计学意义（P均> 0.05）。LHCGR-/-小鼠SLC诱导分化后培养基上清液睾酮平均水平仅为0.01 ng/ml，低于LHCGR+/+对照组的2.71 ng/ml

（P < 0.001），其诱导分化后细胞LC标记物的表达也低于LHCGR+/+小鼠（P均< 0.001）。结论 敲除LHCGR基因后，睾丸内存在SLC且能维持正常的增殖活性，但是分化为LC受阻。

【关键词】黄体生成素受体;睾丸间质干细胞;增殖分化;睾丸间质细胞发育不良

Effect of luteinizing hormone/chorionic gonadotropin receptor on the proliferation and differentiation of stem Leydig cells Wang Fulin， Xia Kai， Feng Xin， Zhuang Jintao， Zhou Mingkuan， Liao Wuyuan， Lei

Zhenming， Xiang Peng， Deng Chunhua， Tu Xiangan. Department of Urology and Andrology， the First Affiliated Hospital of Sun Yat-sen University， Guangzhou 510080， China

Corresponding author， Tu Xiangan， E-mail： tuxiangan@ mail. sysu. edu. cn

【Abstract】Objective To evaluate the effect of luteinizing hormone/chorionic gonadotropin receptor （LHCGR） on the proliferation and differentiation of stem Leydig cells （SLCs）. Methods The expression levels of SLC markers in the testes between the LHCGR-/- and LHCGR+/+ mice were statistically compared by quantitative RT-PCR （qRT-PCR） and immunofluorescent staining. The CD51+ SLCs were sorted by flow cytometry and then subject to 5-ethynyl-2-deoxyuridine （EdU） and CCK8 assay. The SLCs were induced to differentiate into the testicular Leydig cells （LCs）. The testosterone level in the supernatant was detected after 14 d. The expression levels of LC markers after induced differentiation were quantitatively measured by qRT-PCR and immunofluorescent staining. Results SLCs were found in the testis of LHCGR-/- mice and these cells could be sorted by CD51 labeling and flow cytometry. The proportion of EdU+ cells and CCK-8 proliferation curve did not significantly differ between the LHCGR-/- and LHCGR+/+ SLCs （both P > 0.05）. After the induced differentiation of LHCGR-/- SLCs， the average testosterone concentration in the culture supernatant was 0.01 ng/ml， significantly lower than 2.71 ng/ml in the LHCGR+/+ mice （P < 0.001）. The expression levels of LC markers in the LHCGR-/- mice were significantly lower compared with those in the LHCGR+/+ mice （all P < 0.001）. Conclusion After LHCGR knockout， SLCs exist in the testis of LHCGR-/- mice and maintain normal proliferative activity. Nevertheless， the differentiation from SLCs into LCs is suppressed.

【Key words】Luteinizing hormone/chorionic gonadotropin receptor;Stem Leydig cell;

Proliferation and differentiation;Leydig cell hypoplasia

黄体生成素受体（LHCGR）失活型突变是一种罕见的常染色体隐性遗传性疾病。男性患者可表现为小阴茎，尿道下裂甚至外生殖器完全女性化，严重影响男性性征发育、损害男性生育功能，给患者及家庭带来严重的心理负担[1]。其主要的发病机制是LHCGR失活引起睾丸间质细胞（LC）发育不良，导致睾酮合成不足，继而引起生精阻滞[2]。目前临床上睾酮替代疗法对生育力的改善极其有限，而且长期激素替代会抑制精子产生[3-4]。针对该类患者，迫切需要新疗法以恢复睾酮水平及重建生育力。

睾丸间质干细胞（SLC）是LC的前体细胞，能够通过增殖及分化过程转变为LC[5]。笔者团队所进行的前期研究显示，自体SLC移植能够恢复老年食蟹猴的睾酮水平、促进精子发生，是睾酮缺乏相关疾病的理想治疗方法[6]。另一……