Abstract
目的
多囊卵巢综合征(polycystic ovary syndrome，PCOS)是育龄妇女最常见的内分泌疾病之一，常伴有高雄激素血症、胰岛素抵抗及排卵功能障碍。孕激素受体膜组分1(progesterone receptor membrane component 1，PGRMC1)可介导孕酮抑制卵巢颗粒细胞凋亡及卵泡生长，诱导卵巢颗粒细胞葡萄糖及脂质代谢紊乱，与PCOS发生和发展密切相关。本研究旨在通过检测PGRMC1在PCOS患者及非PCOS患者血清、卵巢组织、卵泡液和卵巢颗粒细胞中的表达，分析PGRMC1对PCOS诊断及预后评估的价值，并探讨其调控卵巢颗粒细胞凋亡及糖脂代谢分子机制。
方法
纳入2021年8月至2022年3月就诊于广东省妇幼保健院(以下简称“我院”)妇产科门诊的患者123例，分为PCOS治疗前组(n=42)、PCOS治疗后组(n=36)、对照组(n=45)，采用酶联免疫吸附试验(enzyme linked immunosorbent assay，ELISA)检测其血清PGRMC1水平，并利用PGRMC1受试者操作特征(receiver operating characteristic，ROC)曲线判断PGRMC1对PCOS诊断、预后评估的临床价值。纳入2014年1月至2016年12月在我院妇产科行腹腔镜手术的患者60例，分为PCOS组和对照组(n=30)，采用免疫组织化学染色检测卵巢组织中PGRMC1蛋白质的表达及分布情况。纳入2020年12月至2021年3月就诊于我院生殖医学中心的患者22例，分为PCOS组和对照组(n=11)，采用ELISA检测卵泡液中PGRMC1水平；real-time RT-PCR检测卵巢颗粒细胞中PGRMC1 mRNA的表达水平。将人卵巢颗粒细胞系KGN细胞分为转染无干扰作用小干扰RNA(small interfering RNA，siRNA)的scrambled组和转染靶向抑制PGRMC1的特异性siRNA的siPGRMC1组，采用流式细胞术检测细胞凋亡率，real-time RT-PCR检测PGRMC1、胰岛素受体(insulin receptor，INSR)、葡萄糖转运蛋白4(glucose transporter 4，GLUT4)、极低密度脂蛋白受体(very low density lipoprotein receptor，VLDLR)、低密度脂蛋白受体(low density lipoprotein receptor，LDLR) mRNA的表达水平。
结果
PCOS治疗前组血清PGRMC1水平显著高于对照组(P<0.001)，PCOS治疗后组血清PGRMC1水平显著低于PCOS治疗前组(P<0.001)；PGRMC1用于PCOS诊断和预后评估的曲线下面积(area under curve，AUC)分别为0.932和0.893，最佳截断值分别为620.32和814.70 pg/mL。PGRMC1在卵巢颗粒细胞和卵巢间质细胞上均有表达，并在颗粒细胞上染色最深，PCOS患者卵巢组织及颗粒细胞PGRMC1平均光密度值均明显高于对照组(均P<0.05)。与对照组相比，PCOS组PGRMC1在卵巢颗粒细胞及卵泡液中表达水平显著上调(分别为P<0.001及P<0.01)。与scrambled组相比，siPGRMC1组卵巢颗粒细胞凋亡率显著增加(P<0.01)，PGRMC1和INSR mRNA的表达水平均显著下调(分别为P<0.001和P<0.05)，GLUT4、VLDLR、LDLR mRNA表达水平均显著上调(均P<0.05)。
结论
PCOS患者血清PGRMC1水平升高，并在规范治疗后下降，PGRMC1可作为PCOS诊断及预后评估的分子标志物。PGRMC1主要定位于卵巢颗粒细胞，可能在调控卵巢颗粒细胞凋亡和糖脂代谢中起关键作用。
Keywords: 多囊卵巢综合征, 孕激素受体膜组分1, 糖脂代谢, 卵巢颗粒细胞
Abstract
Objective
Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases in women with reproductive age, which is associated with hyperandrogenism, insulin resistance, and ovulatory dysfunction. Progesterone receptor membrane component 1 (PGRMC1) can mediate progesterone to inhibit the apoptosis of ovarian granulosa cells and the growth of follicles, and to induce glucolipid metabolism disorder in ovarian granulosa cells, which is closely related to the occurrence and development of PCOS. This study aims to determine the expression of PGRMC1 in serum, ovarian tissue, ovarian granulosa cells, and follicular fluid in PCOS patients and non-PCOS patients, analyze the value of PGRMC1 in diagnosis and prognosis evaluation of PCOS, and investigate its molecular mechanism on ovarian granulosa cell apoptosis and glucolipid metabolism.
Methods
A total of 123 patients were collected from the Department of Obstetrics and Gynecology in Guangdong Women and Children Hospital (hereinafter referred to as “our hospital”) from August 2021 to March 2022 and divided into 3 groups: a PCOS pre-treatment group (n=42), a PCOS treatment group (n=36), and a control group (n=45). The level of PGRMC1 in serum was detected by enzyme linked immunosorbent assay (ELISA). The diagnostic and prognostic value of PGRMC1 was evaluated in patients with PCOS by receiver operating characteristic (ROC) curve. Sixty patients who underwent a laparoscopic surgery from the Department of Obstetrics and Gynecology in our hospital from January 2014 to December 2016 were collected and divided into a PCOS group and a control group (n=30). The expression and distribution of PGRMC1 protein in ovarian tissues were detected by immunohistochemical staining. Twenty-two patients were collected from Reproductive Medicine Center in our hospital from December 2020 to March 2021, and they divided into a PCOS group and a control group (n=11). ELISA was used to detect the level of PGRMC1 in follicular fluid; real-time RT-PCR was used to detect the expression level of PGRMC1 mRNA in ovarian granulosa cells. Human ovarian granular cell line KGN cells were divided into a scrambled group which was transfected with small interfering RNA (siRNA) without interference and a siPGRMC1 group which was transfected with specific siRNA targeting PGRMC1. The apoptotic rate of KGN cells was detected by flow cytometry. The mRNA expression levels of PGRMC1, insulin receptor (INSR), glucose transporter 4 (GLUT4), very low density lipoprotein receptor (VLDLR), and low density lipoprotein receptor (LDLR) were determined by real-time RT-PCR.
Results
The serum level of PGRMC1 in the PCOS pre-treatment group was significantly higher than that in the control group (P<0.001), and the serum level of PGRMC1 in the PCOS treatment group was significantly lower than that in the PCOS pre-treatment group (P<0.001). The areas under curve (AUC) of PGRMC1 for the diagnosing and prognosis evaluation of PCOS were 0.923 and 0.893, respectively, and the cut-off values were 620.32 and 814.70 pg/mL, respectively. The positive staining was observed on both ovarian granulosa cells and ovarian stroma, which the staining was deepest in the ovarian granulosa cells. The average optical density of PGRMC1 in the PCOS group was significantly increased in ovarian tissue and ovarian granulosa cells than that in the control group (both P<0.05). Compared with the control group, the PGRMC1 expression levels in ovarian granulosa cells and follicular fluid in the PCOS group were significantly up-regulated (P<0.001 and P<0.01, respectively). Compared with the scrambled group, the apoptotic rate of ovarian granulosa cells was significantly increased in the siPGRMC1 group (P<0.01), the mRNA expression levels of PGRMC1 and INSR in the siPGRMC1 group were significantly down-regulated (P<0.001 and P<0.05, respectively), and the mRNA expression levels of GLUT4, VLDLR and LDLR were significantly up-regulated (all P<0.05).
Conclusion
Serum level of PGRMC1 is increased in PCOS patients, and decreased after standard treatment. PGRMC1 could be used as molecular marker for diagnosis and prognosis evaluation of PCOS. PGRMC1 mainly localizes in ovarian granulosa cells and might play a key role in regulating ovarian granulosa cell apoptosis and glycolipid metabolism.
Keywords: polycystic ovary syndrome, progesterone receptor membrane component 1, glycolipid metabolism, ovarian granulosa cell
多囊卵巢综合征(polycystic ovary syndrome，PCOS)是女性常见的生殖功能障碍与糖脂代谢异常高度关联的一种复杂内分泌代谢疾病[1]，PCOS发病率占育龄妇女的8%~13%，是引起育龄妇女无排卵性不孕的主要原因[2-3]，患者常伴有胰岛素抵抗(insulin resistance，IR)、肥胖、脂代谢异常以及代偿性高胰岛素血症等内分泌代谢紊乱的表现，影响育龄妇女身心健康和生活质量。孕激素受体膜组分1(progesterone receptor membrane component 1，PGRMC1)为孕激素膜受体，属膜相关的孕激素受体(membrane associated progesterone receptor，MAPR)家族的成员，是在生殖系统中广泛表达的孕激素结合蛋白。既往研究[4-5]报道PGRMC1在多种肿瘤组织和肿瘤细胞系中高表达，与乳腺癌、卵巢癌、子宫内膜癌的关系密切，但关于PGRMC1在卵巢颗粒细胞中的研究较少。笔者所在研究组前期的研究[6]首次发现：在PCOS患者卵巢组织中PGRMC1蛋白质高表达，是正常对照组的1.51倍。部分体内、体外实验[7-9]证实PGRMC1在调节卵巢颗粒细胞的凋亡及胰岛素抵抗方面发挥重要作用，但对其在PCOS发病机制中的研究较少。本研究拟检测PCOS患者外周血、卵巢组织、卵泡液和卵巢颗粒细胞中PGRMC1的表达情况，探讨PGRMC1用于PCOS诊断及预后评估的价值；并利用人卵巢颗粒细胞系KGN细胞，进行PGRMC1基因小干扰RNA(small interfering RNA，siRNA)转染，探讨PGRMC1参与调控卵巢颗粒细胞凋亡和糖脂代谢相关基因的分子机制，以期为将PGRMC1作为PCOS治疗的潜在生物学靶点提供实验依据。
1. 材料与方法
1.1. 试剂及仪器
人卵巢颗粒细胞系KGN细胞购自上海中乔新舟生物科技有限公司(货号：ZQ0916)，由广州中医药大学国际中医药转化医学研究所保存，睾酮(testosterone，T)、黄体生成素(luteinizing hormone，LH)、卵泡刺激素(follicle-stimulating hormone，FSH)、雌二醇(estradiol，E2)化学发光免疫试剂盒及测定仪均由德国Bayer公司提供，PGRMC1酶联免疫吸附试验(enzyme linked immunosorbent assay，ELISA)试剂盒购自武汉酶免生物科技有限公司，兔鼠通用型Streptavidin-HPR试剂盒购自北京中杉金桥生物技术公司，PGRMC1抗体购自美国Cell Signaling Technology公司，TRIzol购自美国赛默飞世尔科技公司，HiScript II Q Select RT SuperMix试剂盒、ChamQ SYBR qPCR Master Mix试剂盒均购自南京诺唯赞生物科技股份有限公司，siRNA-EndoFectin Max脂质体核酸转染试剂复合物购自上海翊圣生物科技有限公司，细胞凋亡检测试剂盒购自美国Becton，Dickinson公司，生物显微镜及多功能图像分析系统购自德国Leica公司，Varioskan LUX多功能酶标仪、ABI7500实时荧光定量PCR仪均购自美国赛默飞世尔科技公司，FACSCalibur流式细胞仪购自美国Becton，Dickinson公司。
1.2. 分组
本研究包括临床血清学样本、卵巢组织、卵泡液及颗粒细胞、人卵巢颗粒细胞系4个部分。
第一部分：根据纳入和排除标准，收集2021年8月至2022年3月就诊于广东省妇幼保健院(以下简称“我院”)妇产科的78名PCOS患者的临床资料和血清学样本，检测PCOS患者的血清PGRMC1水平。其中42名未行治疗的初诊PCOS患者为PCOS治疗前组；36名已采用短效口服避孕药干预3~6个月的PCOS患者为PCOS治疗后组；另收集同期在我院体检的45名非PCOS患者为对照组。所有患者均知情同意，此部分研究经我院医学伦理委员会批准后实施(审批号：202101174)。
纳入标准：1)PCOS治疗前组为符合2003年鹿特丹诊断标准[10]，近3个月未曾使用激素类及降糖调脂类药物治疗，年龄18~35岁的PCOS患者；2)PCOS治疗后组为符合2003年鹿特丹诊断标准[10]，采用生活方式管理及短效口服避孕药规范治疗3~6个月，年龄18~35岁的PCOS患者；3)对照组为无内分泌疾病史，年龄18~35岁的非PCOS患者。排除标准：1)实验室指标不完善者；2)未成年及妊娠期患者；3)高雄激素疾病如先天性肾上腺皮质增生症、库欣综合征、雄激素肿瘤患者；4)高催乳素血症、卵巢早衰、下丘脑性闭经及甲状腺功能异常患者；5)合并肿瘤、感染、其他自身免疫性疾病、肝肾功能损伤、严重心血管疾病者；6)有酗酒、药物滥用史者。所有患者均知情同意，此部分研究经我院医学伦理委员会批准后实施(审批号：202101174)。
第二部分：根据纳入和排除标准，收集2014年1月至2016年12月就诊于我院妇产科60例患者的卵巢组织，其中30名PCOS患者作为PCOS组，30名年龄和体重与PCOS组匹配的非PCOS患者为对照组。检测2组患者卵巢组织中PGRMC1的表达情况。PCOS组患者行腹腔镜下卵巢楔形切除术，取楔形切除的小部分卵巢组织进行研究；对照组患者行腹腔镜下卵巢畸胎瘤剔除术及健侧卵巢组织剖腹探查术，取用于活检的健侧卵巢组织进行研究。收集的卵巢组织样本体积应达到整个卵巢组织的十分之一，在卵巢组织表面取一块类似“金字塔样”结构的组织，该组织应包括颗粒细胞、卵泡膜细胞、卵母细胞、间质细胞等。手术由我院具有IV类腹腔镜执业资格的妇产科内分泌主任医师进行，以确保临床样本收集的准确性及代表性。所有患者均知情同意，此部分研究经我院医学伦理委员会批准后实施(审批号：201401011)。
纳入标准：1)PCOS组为符合2003年鹿特丹诊断标准[10]，接受腹腔镜治疗，年龄18~35岁的PCOS患者；2)对照组为同期就诊月经规律、临床及生化检查排除高雄激素血症，年龄18~35岁的非PCOS患者。排除标准同第一部分所述。
第三部分：根据纳入和排除标准，收集2020年12月至2021年3月在我院生殖医学中心行体外受精/卵胞质内单精子注射-胚胎移植治疗的22例不孕症患者的颗粒细胞和卵泡液，其中11例PCOS患者为PCOS组；11例单纯因男性或输卵管因素造成的不孕患者为对照组。检测2组患者颗粒细胞和卵泡液中PGRMC1的表达情况。
所有患者均接受我院生殖医学中心的常规长方案促排卵治疗。患者于月经中期皮下注射促性腺激素释放激素激动剂(gonadotrophin releasing hormone agonist，GnRH-a)曲普瑞林0.05 mg/d，进行调节治疗；于月经周期第3天起根据患者年龄、基础卵泡数和基础卵泡刺激素水平予果纳芬150~225 U/d皮下注射；当二维超声检查监测到直径16~18 mm的卵泡数>3个时，予肌肉注射人绒毛膜促性腺激素5 000~10 000 U；于注射后34~36 h，在超声引导下行穿刺取卵术，收集卵泡液5~10 mL，以3 000 g离心10 min，分别收集上层澄清的卵泡液及下层卵巢颗粒细胞。所有患者均知情同意，此部分研究经我院医学伦理委员会批准后实施(审批号：201901012)。
纳入标准：1)PCOS组为符合2003年鹿特丹诊断标准[10]，接受体外受精/卵胞质内单精子注射-胚胎移植治疗，年龄18~35岁的PCOS患者；2)对照组为单纯因输卵管或男性因素造成的不孕、月经周期及卵巢储备功能均正常，年龄18~35岁的非PCOS患者。排除标准同第一部分所述。
第四部分：将人卵巢颗粒细胞系KGN细胞分为2组，分别转染无干扰作用siRNA(scrambled组)、靶向抑制PGRMC1的特异性siRNA(siPGRMC1组)，检测敲减PGRMC1基因的表达对KGN细胞凋亡及糖脂代谢相关基因表达的影响。
所有研究在中国临床试验数据库完成注册(https:// www.chictr.org.cn，注册号：ChiCTR2100048684)。
1.3. 临床指标及多毛症、痤疮判定
采用化学发光免疫法检测血清中T、LH、FSH、E2水平。多毛症：采用改良Ferriman-Gallwey(modified Ferriman-Gallwey，mFG)评分[11]评价，≥5分诊断为多毛症。痤疮：根据《皮肤性病学》[12]中Pillsbury分类法进行诊断。
1.4. ELISA
收集患者血清或卵泡液，检测PGRMC1的水平。严格按照ELISA试剂盒说明书进行操作，设空白孔、标准孔和待测样品孔，每孔加样50 μL，在37 ℃恒温箱中孵育30 min，洗板5次，每孔加入酶标试剂 50 μL，再次在37 ℃恒温箱中孵育30 min，加入显色液避光显色，最后以空白孔调零，测量各孔在450 nm波长处的吸光度值，根据标准浓度曲线计算PGRMC1浓度。
1.5. 免疫组织化学染色
采用免疫组织化学染色检测卵巢组织中PGRMC1的表达情况。将卵巢组织用4%多聚甲醛溶液固定和石蜡包埋，并行4 μm厚连续切片。按照兔鼠通用型Streptavidin-HPR试剂盒说明书操作，将组织切片脱蜡，并以高温微波修复，滴加血清进行封闭，于室温下放置10 min。倾去血清，滴加PGRMC1一抗(1꞉500)，于4 ℃下孵育过夜。用PBS冲洗后，滴加二抗，于室温下孵育2 h。最后用二氨基联苯胺(3,3'-diaminobenzidine，DAB)避光显色，在显微镜下采集图像，阳性为黄色或棕黄色颗粒。用Image J图像分析软件对PGRMC1的表达情况进行分析，计算每张照片卵巢组织中PGRMC1阳性区域的平均光密度值。
1.6. 细胞转染
转染合成的无干扰作用siRNA(scrambled组)及靶向抑制PGRMC1的特异性siRNA(siPGRMC1组)序列见表1。转染前24 h以每孔2×105个细胞的密度将生长至对数期的KGN细胞接种于6孔板，当细胞生长至融合度达90%以上时，加入含6 μL转染试剂的 60 μL的无血清培养基，并在室温下孵育5 min，加入5 μL的转染试剂，置于室温(15~25 ℃)下孵育10 min。收集转染48 h后的细胞。
表1.
siRNA序列
Table 1 siRNA sequences
| Gene name | Gene ID | Transcript ID | Species | Sequences (5'-3') |
|---|---|---|---|---|
| siPGRMC1a | 10857 | NM_006667.5 | Human | Forward: GGUGUUCGAUGUGACCAAAUU |
| Reverse: UUUGGUCACAUCGAACACCUU | ||||
| siPGRMC1b | 10857 | NM_006667.5 | Human | Forward: UGAGUACGAUGACCUUUCUUU |
| Reverse: AGAAAGGUCAUCGUACUCAUU | ||||
| siPGRMC1c | 10857 | NM_006667.5 | Human | Forward: UGUGCUUUGGUCAGUUAAUUU |
| Reverse: AUUAACUGACCAAAGCACAUU | ||||
| siRNANC | ― | ― | Human | Forward: UUCUCCGAACGUGUCACGUUU |
| Reverse: ACGUGACACGUUCGGAGAAUU |
PGRMC1: Progesterone receptor membrane component 1; siRNA: Small interfering RNA; NC: Negative control.
1.7. Real-time RT-PCR
采用real-time RT-PCR检测卵巢颗粒细胞中PGRMC1及转染后各组KGN细胞中PGRMC1、胰岛素受体(insulin receptor，INSR)、葡萄糖转运蛋白4(glucose transporter 4，GLUT4)、极低密度脂蛋白受体(very low density lipoprotein receptor，VLDLR)、低密度脂蛋白受体(low density lipoprotein receptor，LDLR)基因的表达情况。采用TRIzol提取颗粒细胞总mRNA，HiScript II Q Select RT SuperMix试剂盒将RNA反转录为cDNA，ChamQ SYBR qPCR Master MiX试剂盒。PCR引物使用Premier 5.0软件设计，由生工生物工程(上海)股份有限公司合成，序列见表2。PCR反应条件如下：首先于50 ℃ 2 min，95 ℃ 5 min；随后95 ℃ 15 s，55 ℃ 30 s，72 ℃ 1 min扩增39个循环；再于72 ℃延伸10 min。将PCR扩增产物进行凝胶电泳并采集图像，以GAPDH为内参，使用2-△△Ct计算各目的基因的相对表达水平(表2)。
表2.
引物序列
Table 2 Primer sequences
| Gene | Primer sequences (5'-3') |
|---|---|
| GAPDH | Forward: ACCACCATGGAGAAGGCTGG |
| Reverse: CTCAGTGTAGCCCAGGATGC | |
| PGRMC1 | Forward: TTCGATGTGACCAAAGGCCG |
| Reverse: CAGCAGTGAGGTCAGAAAGGT | |
| INSR | Forward: CCTGAAGCCAAGGCTGATGA |
| Reverse: ACGTAGAAATAGGTGGGTTCCG | |
| GLUT4 | Forward: ACTGGCCATTGTTATCGGCA |
| Reverse: GTCAGGCGCTTCAGACTCTT | |
| VLDLR | Forward: CACCGGAACCGGGAGAAAA |
| Reverse: GGCGCCACAGCTGATTTCAT | |
| LDLR | Forward: CAGCTACCCCTCGAGACAGA |
| Reverse: CACTGTCCGAAGCCTGTTCT |
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PGRMC1: Progesterone receptor membrane component 1; INSR: Insulin receptor; GLUT4: Glucose transporter 4; LDLR: Low density lipoprotein receptor; VLDLR: Very low density lipoprotein receptor.
1.8. 流式细胞术
采用膜联蛋白V(Annexin V)-异硫氰酸荧光素(fluorescein isothiocyanate，FITC)/碘化丙啶(propidium iodide，PI)双染法检测转染后KGN细胞的凋亡情况。以每孔1×105个细胞的密度将KGN细胞接种于6孔板中，当细胞生长至融合度达80%时，改用无血清培养基培养细胞24 h，按照前述方法分组转染，转染后48 h，用胰蛋白酶消化细胞，制备成细胞悬液，离心后重悬细胞，加入Annexin V-FITC和PI各5 μL混匀，避光反应1 h后，用流式细胞仪检测细胞凋亡情况。
1.9. 统计学处理
釆用SPSS 25.0软件进行数据分析。符合正态分布的计量资料以均数±标准差( ±s)表示，用Leneve检验分析2组的方差齐性，2组方差平齐使用t检验比较组间差异，方差不齐使用t'检验。计数资料以例(%)表示，采用χ2检验分析。多组间使用单因素方差分析，后进行LSD多重检验分析2组间差异。采用受试者操作特征(receiver operating characteristic，ROC)曲线及曲线下面积(area under the curve，AUC)评价血清中PGRMC1水平对PCOS诊断与判断预后的价值。以α=0.05(双侧)作为检验水准，P<0.05为差异有统计学意义。
2. 结 果
2.1. 临床特征比较
PCOS治疗前组、PCOS治疗后组和对照组3组在年龄、体重指数(body mass index，BMI)方面的差异均无统计学意义(均P>0.05，表3)。PCOS治疗前组腰臀比(waist hip rate，WHR)、痤疮发病率、多毛症发病率、LH/FSH、血清T和E2水平均明显高于对照组(均P<0.05，表3)。PCOS治疗后组BMI、痤疮发病率、多毛症发病率、LH/FSH、血清T和E2水平均明显低于PCOS治疗前组(均P<0.05，表3)。
表3.
对照组、PCOS治疗前组和PCOS治疗后组人群的临床特征
Table 3 Clininal characteristics of the subjects in the control, PCOS pre-treatment and PCOS treatment groups
| Groups | n | Age/year | BMI/(kg·m-2) | WHR | Acne/[No.(%)] |
|---|---|---|---|---|---|
| Control | 45 | 25.80±3.88 | 21.33±3.42 | 0.75±0.07 | 7(15.6) |
| PCOS pre-treatment | 42 | 24.62±4.98 | 22.20±3.00 | 0.83±0.06** | 31(73.8)* |
| PCOS treatment | 36 | 23.81±3.65 | 20.70±2.88† | 0.83±0.05 | 22(61.1)† |
| Groups | Hypertrichosis/[No.(%)] | LH/FSH | T/(nmol·L-1) | E2/(pmol·L-1) |
|---|---|---|---|---|
| Control | 3(6.7) | 0.94±0.41 | 0.81±0.32 | 152.69±152.81 |
| PCOS pre-treatment | 30(71.4)* | 1.87±0.98*** | 1.73±0.64*** | 260.93±239.09* |
| PCOS treatment | 13(36.1)† | 1.57±1.06† | 1.34±0.44†† | 154.19±145.68† |
Measurement data are represented as ±s; count data are expressed as No.(%). *P<0.05, **P<0.01, ***P<0.001 vs the control group; †P<0.05, ††P<0.01 vs the PCOS pre-treatment group. PCOS: Polycystic ovary syndrome; BMI: Body mass index; WHR: Waist-to-hip ratio; LH/FSH: Luteinizing hormone/follicle-stimulating hormone; T: Testosterone; E2: Estradiol.
2.2. PCOS患者血清中PGRMC1水平
PCOS治疗前组血清PGRMC1水平显著高于对照组[(928.7±254.8) pg/mL vs (509.6±197.4) pg/mL，P<0.001，图1A)]，而PCOS治疗后组患者血清PGRMC1水平显著低于PCOS治疗前组[(606.6±153.0) pg/mL vs (928.7±254.8) pg/mL，P<0.001，图1A]。采用ROC曲线评估PGRMC1对PCOS诊断及预后评估的价值，AUC分别为0.932(95% CI 0.881~0.982，P<0.001)及0.893(95% CI 0.823~0.962，P<0.001)，表明PGRMC1具有较好的辅助诊断及预后评估价值。区分PCOS治疗前组和对照组的最佳截断值为620.32 pg/mL，对应的敏感性为97.60%，特异性为75.60%；区分PCOS治疗前组和PCOS治疗后组的最佳截断值为814.70 pg/mL，对应的敏感性为69.00%，特异性为97.20%(图1B、1C)。
图1.
血清PGRMC1水平及ROC曲线分析
Figure 1 Serum level of PGRMC1 and ROC curve analysis
A: Serum level of PGRMC1 in the subjects of the control, PCOS pre-treatment, and PCOS treatment groups determined by ELISA; B: Diagnostic value of PGRMC1 by ROC curve; C: Therapeutic value of PGRMC1 by ROC curve. ***P<0.001. PGRMC1: Progesterone receptor membrane component 1; ROC: Receiver operating characteristic; PCOS: Polycystic ovary syndrome; ELISA: Enzyme linked immunosorbent assay; AUC: Area under the curve; CI: Confidence interval.
2.3. PGRMC1在PCOS患者卵巢组织中的表达升高
免疫组织化学染色结果显示：PGRMC1蛋白质主要定位在卵巢颗粒细胞，少部分在卵巢间质细胞中，并且在颗粒细胞中染色最深(图2)。PCOS组卵巢组织和颗粒细胞PGRMC1的平均光密度值均明显高于对照组[(0.21±0.12) vs (0.13±0.07)，(0.22±0.10) vs (0.12±0.07)，均P<0.05]。
图2.
免疫组织化学染色检测PGRMC1在对照组(A)和PCOS组(B)患者卵巢组织中的表达情况
Figure 2 Expression of PGRMC1 in ovarian tissues of patients in the control group (A) and the PCOS group (B) determined by immunohistochemistry staining
The black arrow shows the granulosa cells. PGRMC1: Progesterone receptor membrane component 1; PCOS: Polycystic ovary syndrome.
2.4. PGRMC1在PCOS患者卵巢颗粒细胞及卵泡液中的表达上调
与对照组相比，PCOS组卵巢颗粒细胞(图3A)及卵泡液(图3B)中PGRMC1表达水平均显著上调(分别为P<0.001及P<0.01)。
图3.
PGRMC1在对照组和PCOS组患者卵巢颗粒细胞和卵泡液中的表达情况(n=11， ±s)
Figure 3 Expression of PGRMC1 in the ovarian granulosa cells and follicular fluid of patients in the control group and the PCOS group (n=11, ±s)
A: Expression of PGRMC1 mRNA in the ovaria granulosa cells determined by real-time RT-PCR; B: Expression of PGRMC1 in follicular fluid determined by ELISA. **P<0.01, ***P<0.001 vs the control group. PGRMC1: Progesterone receptor membrane component 1; PCOS: Polycystic ovary syndrome; ELISA: Enzyme linked immunosorbent assay.
2.5. 敲减 PGRMC1 基因表达促进卵巢颗粒细胞凋亡
采用转染靶向抑制PGRMC1的特异性siRNA敲减PGRMC1的表达，结果发现：转染siPGRMC1a的KGN细胞PGRMC1 mRNA的表达水平被抑制的最显著(P<0.001，图4A)，故以下实验siPGRMC1组均转染siPGRMC1a。与scrambled组相比，siPGRMC1组KGN细胞凋亡率明显增加(13.9% vs 20.0%，P<0.01，图4B)。
图4.
敲减 PGRMC1 基因表达对KGN细胞凋亡的影响
Figure 4 Effects of knocking down PGRMC1 gene expression on apoptosis in KGN cells
A: Relative mRNA expression levels of PGRMC1 in KGN cells determined by real-time RT-PCR; B: Apoptotic rate of KGN cells determined by flow cytometry. **P<0.01, ***P<0.001 vs the scrambled group. PGRMC1: Progesterone receptor membrane component 1; KGN: Ovarian granulosa cells of human.
2.6. 敲减 PGRMC1 基因表达对糖脂代谢相关基因 表达的影响
与scrambled组相比，siPGRMC1组INSR基因的表达下调(P<0.05)，GLUT4、VLDLR、LDLR基因的表达均上调(均P<0.05，图5)。
图5.
敲减 PGRMC1 基因表达对KGN细胞糖脂代谢相关基因表达的影响
Figure 5 Effects of knocking down PGRMC1 gene on the expression of genes related to glucolipid metabolism in KGN cells
Relative mRNA expression levels of PGRMC1 in KGN cells determined by real-time RT-PCR. *P<0.05 vs the scrambled group. INSR: Insulin receptor; GLUT4: Glucose transporter 4; LDLR: Low density lipoprotein receptor; VLDLR: Very low density lipoprotein receptor.
3. 讨 论
本研究发现：PGRMC1在PCOS治疗前组血清中表达水平较高，经短效口服避孕药治疗及生活方式干预后，血清PGRMC1水平显著降低。血清PGRMC1表达与PCOS发展相关。ROC曲线分析结果显示：PGRMC1可能有良好的辅助PCOS诊断及预后评估价值(最佳截断值分别为620.32、814.70 pg/mL；敏感性分别为97.60%、69.00%；特异性分别为75.60%、97.20%)。通过比较PGRMC1在PCOS患者及非PCOS患者卵巢组织、卵泡液、卵巢颗粒细胞中的表达情况发现，PGRMC1在PCOS患者中高表达。以上结果均提示PGRMC1可以成为PCOS辅助诊断和预后评估的新生物学标志物。
本研究对PCOS患者及对照组人群进行了卵巢组织PGRMC1的免疫组织化学染色，结果显示：PGRMC1主要定位在卵巢颗粒细胞中，少部分定位在卵巢间质细胞，在PCOS患者卵巢组织及卵巢颗粒细胞中高表达。Real-time RT-PCR对2组卵巢颗粒细胞中PGRMC1 mRNA表达水平进行定量分析发现：PCOS组卵巢颗粒细胞中PGRMC1表达上调。这表明PGRMC1可能在卵巢颗粒细胞中发挥其生物学功能。
卵巢颗粒细胞异常可能造成卵母细胞的成熟延迟，引起卵泡闭锁，排卵功能障碍，月经异常，其在PCOS发病中起至关重要的作用[13-14]。本研究采用人卵巢颗粒细胞系KGN细胞对PGRMC1的生物学功能进行验证发现：下调PGRMC1可促进KGN细胞凋亡。这与Zheng等[15]的研究一致。Peluso等[16]在患者卵巢颗粒/黄体细胞中发现破坏PGRMC1和PGRMC2的相互作用，可通过NF-κB/p65通路诱发卵巢颗粒细胞有丝分裂和凋亡。也有研究[8]提出PGRMC1与纤溶酶原激活物抑制剂1相互作用形成复合物，作为孕酮的膜受体结合蛋白，发挥孕酮介导的抗卵巢颗粒细胞凋亡作用。Yuan等[7]发现PGRMC1抑制剂AG205和PGRMC1 siRNA均能逆转孕酮诱导的细胞外信号，调节蛋白激酶ERK1/2信号通路，抑制卵巢颗粒细胞分化。因此，本研究推测PGRMC1作为孕酮膜的成分之一，与其他孕酮膜受体蛋白组成复合体，通过诱导炎症通路及ERK1/2信号通路，调节卵巢颗粒细胞抗凋亡、分化作用，从而影响卵母细胞减速分裂及卵泡成熟，在PCOS的发病机制中起重要调节作用。
多项研究[17-19]显示：IR和高胰岛素血症是PCOS发病中重要的病理及生理基础，PCOS患者的卵巢局部存在IR，卵巢内胰岛素信号通路的异常可能与其生殖功能障碍密切相关，胰岛素在调节机体能量代谢及生长中发挥着重要作用，在卵巢中，胰岛素与排卵以及卵子质量相关[20-21]。PGRMC1属于膜相关孕激素受体蛋白家族，是一种参与多种细胞进程的多功能蛋白质，广泛存在于真核生物中，能够结合并调节细胞色素P450酶，参与调节细胞周期、孕酮4信号通路和类固醇的应答、甘油三酯代谢及葡萄糖代谢[22-23]。本研究发现通过敲减PGRMC1基因表达会导致INSR的表达水平下调，GLUT4、LDLR和VLDLR表达水平上调。研究[9]发现PGRMC1可与INSR相互作用并直接调节INSR，在胰岛刺激下加速与INSR结合，增加了细胞质膜INSR水平，但是抑制下游信号传递，而采用PGRMC1抑制剂AG502反而促进Akt磷酸化，激活胰岛素信号通路，因此推测PGRMC1与INSR结合后可能延迟了质膜上INSR的增加，抑制其作为细胞内复合体的活性，从而抑制胰岛素受体通路。GLUT4是葡萄糖跨膜转运的载体，也是机体的外周组织摄取葡萄糖的关键蛋白质，在PI3K/Akt通路激活后由细胞内转移至细胞膜，GLUT4表达量的下降使得外周组织对葡萄糖的摄取及利用能力降低，引起IR[24]。Furuhata等[25]在小鼠脂肪细胞中发现，当胰岛素信号刺激脂肪细胞时，转录激活因子/环磷酸腺苷反应元件结合蛋白和过氧化物酶体增殖物激活受体激活PGRMC1基因表达，PGRMC1与LDLR、VLDLR或GLUT4相互作用并促进它们易位至质膜，调节脂质摄取及脂肪酸从头合成，促进脂质积累。结合本次实验结果初步推测：PGRMC1可能在卵巢颗粒细胞凋亡中起关键作用；PGRMC1可能在卵巢颗粒细胞中与细胞表面INSR结合，下调INSR在复合体内活性，抑制胰岛素信号通路，从而下调GLUT4，导致葡萄糖升高；同时PGRMC1在质膜水平降低LDLR和VLDLR表达，促进脂肪酸合成及脂质摄取，导致脂质积累(图6)。本研究存在的不足之处是只初步探讨了PGRMC1敲减后的调节机制，有待进一步开展PGRMC1质粒过表达细胞实验及动物实验探索PGRMC1具体作用靶点和相关分子通路。
图6.
PGRMC1在颗粒细胞中调节糖脂代谢机制图
Figure 6 Mechanism of glucolipid metabolism mediated by PGRMC1 in granulosa cells
PGRMC1: Progesterone receptor membrane component 1; INSR: Insulin receptor; IRS: Insulin receptor substrate; PI3K: Phosphatidylinositol 3-kinase; PIP3: Phosphatidylinositol 3,4,5-trisphosphate; PDK1: Phosphoinositide-dependent kinase 1; Akt: Protein kinase B; GLUT4: Glucose transporter member 4; VLDL: Very low density lipoprotein; LDL: Low density lipoprotein; LDLR/VLDLR: Low density lipoprotein receptor/very low density lipoprotein receptor; P: Phosphorylation. GCs: Granulosa cells.
综上所述，PCOS患者血清、卵泡液、卵巢颗粒细胞中均检测到PGRMC1高表达。敲减PGRMC1基因促进卵巢颗粒细胞凋亡，PGRMC1可能参与卵巢颗粒细胞葡萄糖代谢和脂质累积。
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《中南大学学报(医学版)》编辑部
基金资助
广东省自然科学基金(2021A1515010763)；澳门特别行政区科学技术发展基金(0048/2021/A1)；广州市科技计划(201904010458)。
This work was supported by the Natural Science Foundation of Guangdong Province (2021A1515010763), Macao Special Administrative Region Science and Technology Development Fund (0048/2021/A1), and the Science and Technology Program of Guangzhou (201904010458), China.
利益冲突声明
作者声称无任何利益冲突。
作者贡献
周嘉禾 数据统计分析、论文撰写和修订；陈志静 临床和细胞实验的实施；李洁明 数据采集；邓群娣、彭秀红 病例收集；李荔 实验设计、论文修改。所有作者阅读并同意最终的文本。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202304538.pdf
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