Mfn2在PCOS大鼠卵巢的表达及其对大鼠卵泡发育的影响与机制的初步探讨

发布时间：2018-09-01 20:38

【摘要】：多囊卵巢综合征（polycystic ovarian syndrome, PCOS）是临床最常见的妇科内分泌疾病，亦是引起育龄期妇女不孕的最常见原因，以长期无排卵、高雄激素血症及超声下双侧卵巢多囊样变为主要特征。近年来，PCOS被定义为一种代谢综合征，因其常伴有代谢障碍如肥胖、胰岛素抵抗、高脂血症，且远期有发生2型糖尿病、高血压及其他心血管疾病的风险，严重影响生育年龄妇女的身心健康。至今PCOS的病因及发病机制尚未清楚。目前的研究主要集中在遗传相关基因、环境因素及卵巢局部分子生物学改变等方面。卵泡发育异常是PCOS主要的病理生理特征。研究显示卵泡发育障碍可能是PCOS患者持续不排卵和临床内分泌改变的病理基础。而卵泡的异常发育可能与颗粒细胞的凋亡相关，但具体机制尚未探明。线粒体融合素基因2（mitofusin2, Mfn2），是嵌于线粒体外膜的一种跨膜GTP酶，介导线粒体融合，参与调节线粒体的形态。人体Mfn2基因缺陷或突变可致2A型腓骨肌萎缩症等神经退行性疾病的发生。近年来越来越多的研究显示Mfn2除促进线粒体融合外，还参与多种代谢综合征如糖尿病、胰岛素抵抗及肥胖的发生及细胞凋亡的调节。研究显示小鼠Mfn1/Mfn2的缺失可致胚胎于孕中期死亡，提示Mfn2可能参与胚胎的发育。然而目前尚无Mfn2参与卵巢功能调节的研究。为明确rat mitofusin2(rMfn2)在大鼠卵泡发育中的作用，探讨其是否参与PCOS的发病，我们设计此实验。第一部分：rMfn2在正常大鼠及PCOS大鼠卵巢组织的表达目的：探讨rMfn2在正常大鼠及PCOS模型大鼠卵巢组织的表达差异，明确rMfn2是否参与PCOS的发病。方法：选取6周龄SD雌性大鼠来曲唑灌胃建立PCOS模型，观察大鼠体重及卵巢体重变化，HE染色评估卵巢形态，放射免疫法检测血清E2、T、P、FSH及LH水平。免疫组织化学法分析rMfn2、Bcl-2及Bax在大鼠卵巢的定位表达。RT-PCR和Western blotting分别从mRNA和蛋白质水平定量分析卵巢组织rMfn2的表达。结果：与对照组比较，PCOS模型组大鼠体重增长明显低于对照组，卵巢重量明显高于对照组，差异有统计学意义（p0.05）；模型组血清LH、T浓度较对照组明显增高，E2、P浓度明显降低，差异有统计学意义（p0.05）；两组血清FSH浓度无统计学差异（p0.05）；HE染色显示对照组大鼠卵巢镜下可见多个新鲜黄体和不同生长阶段的各级卵泡；模型组大鼠卵巢镜下见典型多囊改变，可见较多小卵泡及囊状扩张的大卵泡，卵泡内层颗粒细胞层数明显减少，未见黄体；免疫组织化学检测显示rMfn2广泛表达于两组大鼠颗粒细胞、卵泡膜细胞、卵泡液、黄体及卵巢间质；rMfn2在PCOS大鼠颗粒细胞上的表达明显弱于对照组，同时Bcl-2在PCOS大鼠颗粒细胞的表达也较对照组降低，而Bax的表达较对照组增强，差异有统计学意义（p0.05）；RT-PCR和Westernblotting结果显示PCOS模型组大鼠卵巢组织rMfn2mRNA及蛋白表达均明显低于对照组，差异有统计学意义（p0.05）。结论：来曲唑成功诱导PCOS大鼠模型；PCOS大鼠颗粒细胞凋亡可能增加；rMfn2广泛表达于大鼠颗粒细胞、卵泡膜细胞、卵泡液、黄体及卵巢间质，PCOS大鼠颗粒细胞rMfn2的表达下调可能参与PCOS的发生。第二部分：Lenti-GFP-rMfn2过表达载体的构建及鉴定目的：构建并鉴定rMfn2过表达的慢病毒载体（lenti-GFP-rMfn2）。方法：pLenO-GTP载体酶切线性化，与PCR扩增的rMfn2基因连接、转化，提取重组质粒，通过酶切法和DNA测序对重组质粒进行鉴定。将鉴定正确的重组质粒转染293T细胞，收集提纯病毒，同时采用流式细胞仪检测活细胞比率测定病毒滴度。结果：重组质粒经酶切和DNA测序鉴定正确。重组质粒转染293T细胞可见绿色荧光表达，提示质粒重组成功。病毒滴度测定滴度为2.2×108TU/ml。结论：Lenti-GFP-rMfn2过表达载体构建成功。第三部分：rMfn2对大鼠颗粒细胞的作用及信号通路的初步研究目的：探讨rMfn2对正常大鼠卵巢颗粒细胞增殖凋亡的作用及相关信号通路。方法：体外大鼠原代颗粒细胞的培养及鉴定。细胞免疫荧光检测rMfn在颗粒细胞的定位表达。Lenti-GFP-rMfn2体外转染大鼠颗粒细胞，Western blotting鉴定转染结果。转染成功后MTT法绘制颗粒细胞生长曲线，流式细胞仪检测细胞周期及凋亡率，Western blotting检测rMfn2及相关信号通路蛋白Bcl-2、Bax、Akt、p-Akt的表达。结果：细胞免疫荧光显示rMfn表达于正常大鼠颗粒细胞胞浆。Westernblotting检测显示Lenti-GFP-rMfn2成功转染大鼠颗粒细胞，MTT检测显示rMfn2过表达组（MFN组）细胞的增殖速率明显高于其余两组细胞（正常对照CON组和空病毒GFP组），差异有统计学意义（p0.05）；细胞周期检测显示MFN组细胞在G1期所占比例明显下降，S及G2期所占比例明显增加，差异有统计学意义（p0.05）。凋亡率检测显示CON组、GFP组及MFN组三组细胞凋亡率均很低，三组间差异无统计学意义（p0.05）。Western blotting检测显示MFN组颗粒细胞Bcl-2的表达明显增加，Bax的表达减少，差异有统计学意义（p0.05）；MFN组p-Akt蛋白的表达增加，差异有统计学意义（p0.05），Akt在三组间表达无统计学差异（p0.05）。结论：rMfn2过表达促进正常大鼠颗粒细胞的增殖，其机制是激活PI3K/Akt信号通路，引起下游Bcl-2表达增加，Bad及Bax表达减少，从而促进颗粒细胞增殖。第四部分：rMfn2对正常大鼠及PCOS大鼠卵泡发育的影响研究目的：观察rMfn2对正常大鼠及PCOS大鼠卵泡发育的影响。方法：60只2月龄SD雌性大鼠随机分成以下四组：对照组（CON组，仅注射生理盐水），空病毒组（GFP组，注射Lenti-GFP慢病毒颗粒），目的病毒组（MFN组，注射Lenti-GFP-rMfn2慢病毒颗粒），目的病毒+来曲唑组（ML组，在Lenti-GFP-rMfn2慢病毒颗粒注射30天后开始来曲唑灌胃连续23天），来曲唑组（LT组，采用第一部分PCOS模型标本）。荧光显微镜观察卵巢组织及全身其他组织rMfn2的表达；realtime RT-PCR和Western blotting定量检测rMfn2及性激素相关受体（ER、PR、FSHR、LHR）的表达；HE染色评估卵巢形态，放射免疫法检测血清E2、T、P、FSH及LH水平。结果：慢病毒转染第30天，real time RT-PCR和Western blotting检测结果显示MFN组大鼠卵巢rMfn2呈过表达状态；荧光显微镜及Westernblotting检测显示rMfn2过表达载体转染大鼠卵巢，其过表达存在时间依赖性，，随着时间的延长表达逐渐增强，直至转染第60天仍持续稳定表达。Western blotting检测显示转染第60天MFN组ER及PR在子宫的表达高于GFP组，差异有统计学意义（p0.05）；FSHR及LHR的表达，GFP及MFN两组间比较无统计学差异（p0.05）。HE染色显示转染第60天MFN组大鼠卵巢镜下见较多黄体，始基卵泡及多个发育良好的窦状卵泡；ML组大鼠卵巢镜下仍可见囊状扩张卵泡，但较LT组明显减少，同时可见黄体组织、始基卵泡、窦状卵泡甚至排卵前卵泡，且卵泡内卵母细胞存在。放射免疫检测显示MFN组大鼠血清E2、P水平明显高于CON组、ML组及LT组，T水平远低于CON组、ML组及LT组，差异有统计学意义（p0.05）；ML组与LT组比较，血清FSH与T水平变化不大，E2、P水平明显增加，LH水平降低，差异有统计学意义（p0.05）。结论：rMfn2过表达影响大鼠生殖内分泌功能，促进正常大鼠卵泡发育；rMfn2过表达可能抑制来曲唑诱导的大鼠卵巢多囊样变，具体机制有待进一步研究。
[Abstract]:Polycystic ovarian syndrome (PCOS) is the most common gynecological endocrine disease and the most common cause of infertility in women of childbearing age. PCOS is characterized by chronic anovulation, hyperandrogenism and bilateral ovarian polycystic degeneration under ultrasound. In recent years, PCOS has been defined as a metabolic syndrome because of its frequent occurrence. Metabolic disorders such as obesity, insulin resistance, hyperlipidemia, and long-term risk of type 2 diabetes mellitus, hypertension and other cardiovascular diseases seriously affect the physical and mental health of women of reproductive age. Until now, the etiology and pathogenesis of PCOS have not been clear. Some sub biological changes.
Abnormal follicular development is the main pathophysiological feature of PCOS. Studies have shown that follicular dysplasia may be the pathological basis of persistent anovulation and clinical endocrine changes in PCOS patients.
Mitochondrial fusion factor 2 (Mfn2), a transmembrane GTP enzyme embedded in the mitochondrial outer membrane, mediates mitochondrial fusion and participates in the regulation of mitochondrial morphology. Defects or mutations in the human Mfn2 gene can cause neurodegenerative diseases such as type 2A peroneal muscular atrophy. In addition, it is also involved in the regulation of various metabolic syndrome such as diabetes, insulin resistance, obesity and apoptosis. Studies have shown that the loss of Mfn1/Mfn2 in mice can cause embryo death in the second trimester of pregnancy, suggesting that Mfn2 may participate in embryo development. However, there is no study on the regulation of ovarian function by Mfn2. We designed this experiment to investigate the role of follicular development in the pathogenesis of PCOS.
Part one: the expression of rMfn2 in normal rat and PCOS rat ovary tissues.
Objective: To investigate the expression of rMfn2 in ovarian tissues of normal rats and PCOS rats, and to determine whether rMfn2 is involved in the pathogenesis of PCOS.
METHODS: PCOS model was established by intragastric administration of letrozole in 6-week-old female SD rats. The changes of body weight and ovarian weight were observed. The ovarian morphology was evaluated by HE staining. Serum levels of E2, T, P, FSH and LH were detected by radioimmunoassay. Immunohistochemistry was used to analyze the localization of rMfn2, Bcl-2 and Bax in the ovaries of rats. The expression of rMfn2 in ovarian tissue was analyzed quantitatively at protein level.
Results: Compared with the control group, the weight gain of the PCOS model group was significantly lower than that of the control group, and the weight of the ovary was significantly higher than that of the control group (p0.05); the concentration of LH, T, E2, P in the model group was significantly higher than that of the control group, and the concentration of serum FSH was significantly lower (p0.05); there was no significant difference between the two groups (p0.05). HE staining showed that there were many fresh corpus luteum and follicles at different growth stages in the control group under the microscope; typical polycystic changes were observed in the ovary of the model group, with many small follicles and large cystic expanded follicles, and the number of granulosa cells in the inner layer of follicles was significantly reduced, but no luteum was found in the model group. N2 was widely expressed in granulosa cells, follicular membrane cells, follicular fluid, corpus luteum and ovarian stroma of the two groups; rMfn2 expression in granulosa cells of PCOS rats was significantly weaker than that of the control group, and the expression of Bcl-2 in granulosa cells of PCOS rats was also lower than that of the control group, while the expression of Bax was significantly higher than that of the control group (p0.05). And Western blotting results showed that the expression of rMfn2 mRNA and protein in ovarian tissue of PCOS model group was significantly lower than that of control group (p0.05).
CONCLUSION: Letrozole can induce PCOS rat model successfully; apoptosis of PCOS rat granulosa cells may increase; rMfn2 is widely expressed in rat granulosa cells, follicular membrane cells, follicular fluid, corpus luteum and ovarian stroma; down-regulation of rMfn2 expression in PCOS rat granulosa cells may be involved in the development of PCOS.
The second part: Construction and identification of Lenti-GFP-rMfn2 overexpression vector.
Objective: to construct and identify lentivirus vector (lenti-GFP-rMfn2) over expressed by rMfn2.
Methods: pLenO-GTP vector was linearized by enzyme digestion, linked with PCR-amplified rMfn2 gene, transformed, extracted and identified by enzyme digestion and DNA sequencing.
Results: The recombinant plasmid was identified correctly by enzyme digestion and DNA sequencing. The green fluorescent expression was observed in 293T cells transfected with the recombinant plasmid, suggesting that the recombinant plasmid was successfully recombined.
Conclusion: the Lenti-GFP-rMfn2 overexpression vector was successfully constructed.
The third part: the effect of rMfn2 on rat granulosa cells and the signal transduction pathway.
Objective: To investigate the effect of rMfn2 on the proliferation and apoptosis of granulosa cells in normal rats and the related signaling pathways.
Methods: The primary rat granulosa cells were cultured and identified in vitro. The localized expression of rMfn in granulosa cells was detected by immunofluorescence. Lenti-GFP-rMfn2 was transfected into rat granulosa cells in vitro and the results were identified by Western blotting. Tern blotting was used to detect the expression of rMfn2 and related signaling pathway proteins Bcl-2, Bax, Akt and p-Akt.
Results: Immunofluorescence showed that rMfn was expressed in the cytoplasm of normal rat granulosa cells. Western blotting assay showed that Lenti-GFP-rMfn2 was successfully transfected into rat granulosa cells. MTT assay showed that the proliferation rate of rMfn2 overexpression group (MFN group) was significantly higher than that of the other two groups (normal control group CON and empty virus GFP group). Significance (p0.05); Cell cycle test showed that MFN group cells in G1 phase decreased significantly, S and G2 phase increased significantly, the difference was statistically significant (p0.05). The expression of Bcl-2 and Bax in granulosa cells of MFN group increased significantly (p0.05), but the expression of Bax in granulosa cells of MFN group decreased significantly (p0.05).
CONCLUSION: Overexpression of rMfn2 promotes the proliferation of normal rat granulosa cells by activating PI3K/Akt signaling pathway, increasing the expression of Bcl-2 downstream, decreasing the expression of Bad and Bax, thus promoting the proliferation of granulosa cells.
The fourth part: the effect of rMfn2 on follicular development in normal rats and PCOS rats.
Objective: To observe the effect of rMfn2 on follicular development in normal rats and PCOS rats.
Methods: Sixty two-month-old female SD rats were randomly divided into four groups: control group (CON group, saline only), empty virus group (GFP group, Lenti-GFP lentiviral particles), target virus group (MFN group, Lenti-GFP-rMfn2 lentiviral particles) and target virus + letrozole group (ML group, 30 days after Lenti-GFP-rMfn2 lentiviral particles injection). The expression of rMfn2 in ovarian tissues and other tissues of the whole body was observed by fluorescence microscope; the expression of rMfn2 and sex hormone-related receptors (ER, PR, FSHR, LHR) was quantitatively detected by realtime RT-PCR and Western blotting; the ovarian morphology was evaluated by HE staining; and the expression of rMfn2 and sex hormone-related receptors (ER, PR, FSHR, LHR) was detected by radioimmunoassay. Methods serum levels of E2, T, P, FSH and LH were measured.
Results: On the 30th day of lentiviral transfection, real-time RT-PCR and Western blotting showed that rMfn2 was overexpressed in the ovaries of rats in MFN group, while fluorescence microscopy and Western blotting showed that the overexpression of rMfn2 in the ovaries of rats transfected with lentiviral vectors was time-dependent and increased gradually with time until the ovaries were transfected. Western blotting showed that the expression of ER and PR in the uterus of MFN group was higher than that of GFP group on the 60th day (p0.05). There was no significant difference in the expression of FSHR and LHR, GFP and MFN between the two groups (p0.05). HE staining showed that there were more corpus luteum and primordium in the ovary of MFN Group on the 60th day. Follicles and many well-developed sinusoidal follicles were found in ML group, but cystic dilated follicles were still found in ML group, but significantly less than those in LT group, and luteal tissue, primordial follicles, sinusoidal follicles and even pre-ovulatory follicles were also found. Radioimmunoassay showed that serum E2 and P levels in MFN group were significantly higher than those in CON group and ML group. And LT group, T levels were significantly lower than CON group, ML group and LT group, the difference was statistically significant (p0.05); ML group and LT group, serum FSH and T levels changed little, E2, P levels increased significantly, LH levels decreased, the difference was statistically significant (p0.05).
CONCLUSION: Overexpression of rMfn2 affects the reproductive endocrine function and promotes follicular development in normal rats. Overexpression of rMfn2 may inhibit letrozole-induced ovarian polycystic degeneration in rats, and the mechanism needs further study.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.75

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