【作者】 刘镘利；

【导师】 王宗仁； 马静；

【作者基本信息】 第四军医大学 ， 中西医结合临床， 2013， 博士

【摘要】 目的转化生长因子-β1（Transforming growth factor-β1, TGF-β1）是一种以多肽形式存在的细胞因子，属于TGF-β超家族成员。现已证实TGF-β1在睾丸的不同发育阶段均有表达，通过自分泌或旁分泌的方式调节睾丸发育和精子发生。尽管已有研究证实TGF-β1可以下调睾丸间质细胞（Leydig）睾酮（testosterone, T）分泌、抑制该细胞增殖并影响其分化进程，但对其合成的另一重要类固醇激素雌二醇（estradiol, E2）的分泌调控以及对间质细胞间以连接蛋白为基础的缝隙连接通讯功能的影响仍是空白，因此本实验旨在探讨TGF-β1对Leydig细胞上述生理功能的作用及机制。同时，鉴于中药淫羊藿有效成分之一的淫羊藿苷（Icariin）在保护生殖细胞损伤中的作用，故拟进一步探讨该成分对TGF-β1作用后的睾丸Leydig细胞是否发挥积极保护作用。方法1.采用Percoll连续密度梯度离心法提取、纯化并原代培养成年大鼠（3月龄）睾丸Leydig细胞，探讨不同剂量TGF-β1对Leydig细胞性激素合成的影响。2.利用免疫细胞化学法检测TGF-β1作用后，Leydig细胞中芳香化酶表达；采用氚水释放实验检测芳香化酶活性；Westernblot和Real-time PCR方法检测芳香化酶编码基因CYP19表达情况，并结合图像分析技术对其表达进行半定量分析。3.利用间接免疫荧光技术检测TGF-β1作用后，缝隙连接蛋白43（connexin43,Cx43）在Leydig细胞中的分布情况；采用Western blot对Cx43蛋白磷酸化亚型含量进行检测；采用荧光漂白后恢复实验（fluore scence redistributionafter photobleaching,FRAP）对Cx43介导的GJIC进行检测。4.利用MTT法筛选Icariin作用细胞的最适浓度，随后用放射免疫法和氚水释放实验检测Icariin对TGF-β1作用的Leydig细胞进行干预后，E2含量的影响以及Icariin对芳香化酶活性的影响；采用FRAP实验检测Icariin对TGF-β1刺激后细胞间GJIC的影响。结果1. Percoll连续密度梯度离心法提取成年大鼠睾丸Leydig细胞，经3β-HSD酶细胞化学染色鉴定后，纯度符合细胞学实验要求。原代培养的Leydig细胞经不同剂量TGF-β1处理后，检测细胞上清中E2和T含量皆低于正常对照组，并呈剂量依赖趋势。同正常对照组相比，TGF-β1低剂量（1ng/ml）时即可显著抑制Leydig细胞基础水平下以及人绒毛膜促性腺激素（human chorionic gonadotrophin, hCG）刺激下E2和T的分泌量（P＜0.01）。虽然高剂量的TGF-β1（10ng/ml）对上述两种状态下T分泌量的抑制作用并未随剂量增加而增强，但仍明显低于对照组。2.免疫细胞化学染色检测到芳香化酶阳性染色呈棕黄色颗粒，分布于Leydig细胞胞浆中，随着TGF-β1处理细胞时间的延长和剂量的增加，其阳性表达强度呈逐渐减弱的趋势。利用氚水释放实验检测芳香化酶活性发现，与正常对照组53±0.71fmol mg protein^-1h^-1相比，TGF-β1浓度为5ng/ml时，其活性水平降至最低为21.43±1.07fmol mg protein^-1h^-1。作用时间延长至30h时，对其活性抑制率达到50%。利用Western blot和Real-time PCR方法对芳香化酶的编码基因CYP19表达检测发现TGF-β1作用后，CYP19在翻译和转录水平上的表达均受到不同程度的抑制，且具有时间依赖性。3.间接免疫荧光技术发现，正常情况下Leydig细胞中的Cx43呈斑点状主要散在分布于细胞膜区域，胞浆中仅少量分布。经TGF-β1处理后，Cx43表达则主要定位于胞浆中。用Westernblot对Cx43磷酸化和非磷酸化两种状态下蛋白含量的检测发现与正常对照组相比，各TGF-β1处理组中Cx43非磷酸化蛋白定量无显著差异；而经TGF-β1作用后，随着剂量增大及时间延长，磷酸化（P2+P1）Cx43蛋白定量明显增多。采用FRAP方法检测细胞间Cx43介导的GJIC发现，正常对照组中由激光淬灭所致细胞内荧光强度的减弱可随着时间的推移又逐渐恢复，并最终趋于稳定，其荧光恢复率为（81.25±1.25）%。而经TGF-β1以5ng/ml浓度作用20h后细胞内荧光强度持续减弱，且其最终平均荧光恢复率仅为（43.58±1.87）%，两者相比具有显著性差异（P＜0.01）。而经外源性雌激素干预可明显逆转TGF-β1对GJIC的下调作用。4. MTT法筛选出Icariin作用于Leydig细胞的最适浓度为10μg/ml，以该浓度干预由TGF-β1作用的Leydig细胞可显著提高E2的生成量（由0.22±0.04pg/ml上升至1.48±0.12pg/ml），同时明显上调芳香化酶活性。此外，Icariin干预后，Leydig细胞间的荧光恢复率由（24.71±1.87）%上升至（55.88±2.45）%。结论1.成功分离、纯化了大鼠睾丸Leydig细胞，经鉴定可用于原代培养下细胞学实验。外源性TGF-β1可显著抑制Leydig细胞中E2和T的分泌，提示TGF-β1对Leydig细胞的内分泌功能具有调节作用2. TGF-β1可显著抑制芳香化酶在Leydig细胞中的表达并降低其转化合成雌激素的活性，这可能通过在转录和翻译水平上下调芳香化酶编码基因CYP19的表达而实现。提示TGF-β1对雌激素的抑制作用通过下调芳香化酶的活性及抑制其编码基因的表达来实现。3. TGF-β1可显著下调Leydig细胞间的GJIC，这种抑制作用可能通过使其连接蛋白Cx43表达易位，提高其磷酸化水平来实现。同时，雌激素可有效逆转TGF-β1对GJIC的损伤，提示雌激素对GJIC具有一定保护作用。4.中药淫羊藿的有效成分Icariin可有效阻止TGF-β1对Leydig细胞中E2合成的抑制作用，这种影响可能部分通过上调雌激素合成的关键酶芳香化酶活性实现。同时Icariin可能通过增加E2合成达到保护Leydig细胞间GJIC的作用。更多还原

【Abstract】 ObjectiveTransforming growth factor-β1（TGF-β1）, one of members belongs to TGF-β superfamily. In the testis, TGF-β1has been shown to be present at different stages ofdevelopment. Furthermore, TGF-β1can regulate testis development and spermatogenesisthrough autocrine and paracrine pathways. Previous studies had shown that TGF-β1down-regulated testosterone secretion of rat Leydig cells, inhibited Leydig cell sproliferation and affected the process of its differentiation. However, few studies havebeen done about the regulation of estradiol secretion of this cell as well asconnexin43-based gap junction intercellular communication （GJIC） between adjacent Leydig cells induced by TGF-β1. More and more researchers reported that Icarrin, as oneof the effective constituents of epimedium, a traditional C hinese herbal medicine, hasdefinite benififial effects on male reproductive system. The mechanisms may be related toimproving the secretion of testosterone or estradiol. But wehter it has protective effects onLeydig cell s function is not clear yet. Therefore, based on above evidences the aim of thisstudy is to figure out wether there is relationship between the alteration of estradiolsecretion and abnormal Cx43-based GJIC in primary cultured rat Leydig cells induced byTGF-β1. And furtherly to assay wether Icariin can protect Leydig cell from damaging byTGF-β1.Methods1．Isolation and purification of Leydig cells from3-month-old SD rats throughcontinuous Percoll density gradient centrifugation. After administration of differentconcentrations of TGF-β1to primarycultured Leydigcells, the effect of TGF-β1onbasaland hCG stimulated estradiol and testostorone secretions in vitro were evaluated.2．After administration of TGF-β1, immunocytochemical method was used to observethe expression of aromatase in Leydig cells; tritium release assay was used to investigatearomatase activity; Western blot and Real-time PCR were used to detect expression ofgene CYP19and data were measured by image analysis software.3．Observation of the location of connexin43in TGF-β1-treated Leydig cells byindirect immunofluorescence; Furthermore, the total protein and phosphorylated informswere detected by Western blotting. The function of Cx43-based GJIC between Leydigcells were investigated by FRAP techniques.4．Determined the optimal concentration of Icariin administrated in primary culturedLeydig cells by MTT. Observation of effect of Icariin on secretion of estradiol andP450arom activity induced by TGF-β1. Furthermore, FRAP experiment was used todetect the effect of Icariin on GJIC between Leydig cells treated by TGF-β1. Results1. When the purified Leydig cells were treated with different doses of TGF-β1invitro, the basal and hCG-stimulated estradiol and testosterone secretion were affected ina dose-dependent manner. Compared with control group, low concentration of TGF-β1（1ng/ml） inhibited basal and hCG-stimulated estradiol and testosterone secretionobviously（P＜0.01）. Although the inhibition of relatively high dose of TGF-β1（10ng/ml） didn t enhance with the dose increased, the result was still lower thancontrol group.2. Immunocytochemistry revealed that aromatase was localized in cytoplasm andpresented an attenuated trend in both dose and time-course TGF-β1treatment groups. Intritium release assay, we found that aromatase activity was decreased in TGF-β1-treatedcells at concentration of5ng/ml (2121.43±1.07foml mg protein^-1h^-1) compared withcontrol cells (53±0.71fmol mg protein^-1h^-1). When time-course was up to30h, TGF-β1induced a50%inhibition of aromatase activity. Besides, TGF-β1caused significantdown-regulation of both total protein and mRNA levels of CYP19in Leydig cells in atime-dependent manner.3. Indirect immunofluorescence results indicated that after TGF-β1-treated,expression of Cx43translocated from cell membrane to cytoplasm. Besides, there s noevident difference between control and TGF-β1-treated Leydig cells in total protein ofCx43. However, after administration of TGF-β1, phosphorylated Cx43（P2+P1）increased but there was no significant alteration in non-phosphorylated Cx43（P0）. Theeffect of TGF-β1on Leydig cells was observed byFRAP techniques. The results showedthat in the control groups, the fluorescence intensity was gradually recovered at differenttimes after bleaching. The mean fluorescence recovery rate of it was （81.25±1.25）%. Incontrast to the control groups, the fluorescence recovery of the bleached cells in theTGF-β1groups was not obvious in the same time. The mean fluorescence recovery ratesof them was （43.58±1.87）%. However, administration of estradiol can reverse thedown-regulation of GJIC by TGF-β1. 4. The proper concentration of Icariin is10μg/ml. After administration of Icariin, theamount of estradiol inhibited by TGF-β1was increased from0.22±0.04pg/ml to1.48±0.12pg/ml, and this trend was accompanied by up-regulated P450arom activity.Furthermore, Icariin could reversed the mean fluorescence recovery rates of Leydig cellsinduced by TGF-β1, from （24.71±1.87）%to （55.88±2.45）%.Conclusions1. We succeeded in isolation and purification Leydig cells of adult male rat. And aftertreatment with TGF-β1, the synthesis of estradiol and testosterone were significantlydecreased. TGF-β1might participate in the regulation of endocrine secretion of Leydigcells.2. TGF-β1could suppress expression of P450arom as well as down-regulate itsactivity in Leydig cells. This effect may depend on attenuated expression of its encodegene CYP19both in translation and transcription levels. The results above indicate thatthe reduction of estradiol by TGF-β1can be attributed to attenuation of aromataseactivity and expression of its encode gene CYP19.3. TGF-β1could significantly down-regulate gap junction channels accompanied byCx43hyperphosphorylation and translocation of Cx43from the gap junctions into thecytoplasm. Besides, estradiol could attenuate inhibitory effect of TGF-β1on GJIC.Therefore, estradiol may be involved in protecting of GJIC between Leydig cells.4. Icariin could interrupt the inhibition of TGF-β1onsecretionof estradiolof Leydigcells. This effect can be largely attributed to enhancing the P450arom activity. Besides,Icariin could reverse the effect of down-regulation of TGF-β1on GJIC between Leydigcells.更多还原