【摘要】：多囊卵巢綜合征（polycystic ovarian syndrome, PCOS）是臨床最常見的婦科內(nèi)分泌疾病，亦是引起育齡期婦女不孕的最常見原因，以長期無排卵、高雄激素血癥及超聲下雙側(cè)卵巢多囊樣變?yōu)橹饕卣鳌＝陙恚琍COS被定義為一種代謝綜合征，因其常伴有代謝障礙如肥胖、胰島素抵抗、高脂血癥，且遠(yuǎn)期有發(fā)生2型糖尿病、高血壓及其他心血管疾病的風(fēng)險，嚴(yán)重影響生育年齡婦女的身心健康。至今PCOS的病因及發(fā)病機(jī)制尚未清楚。目前的研究主要集中在遺傳相關(guān)基因、環(huán)境因素及卵巢局部分子生物學(xué)改變等方面。 卵泡發(fā)育異常是PCOS主要的病理生理特征。研究顯示卵泡發(fā)育障礙可能是PCOS患者持續(xù)不排卵和臨床內(nèi)分泌改變的病理基礎(chǔ)。而卵泡的異常發(fā)育可能與顆粒細(xì)胞的凋亡相關(guān)，但具體機(jī)制尚未探明。 線粒體融合素基因2（mitofusin2, Mfn2），是嵌于線粒體外膜的一種跨膜GTP酶，介導(dǎo)線粒體融合，參與調(diào)節(jié)線粒體的形態(tài)。人體Mfn2基因缺陷或突變可致2A型腓骨肌萎縮癥等神經(jīng)退行性疾病的發(fā)生。近年來越來越多的研究顯示Mfn2除促進(jìn)線粒體融合外，還參與多種代謝綜合征如糖尿病、胰島素抵抗及肥胖的發(fā)生及細(xì)胞凋亡的調(diào)節(jié)。研究顯示小鼠Mfn1/Mfn2的缺失可致胚胎于孕中期死亡，提示Mfn2可能參與胚胎的發(fā)育。然而目前尚無Mfn2參與卵巢功能調(diào)節(jié)的研究。為明確rat mitofusin2(rMfn2)在大鼠卵泡發(fā)育中的作用，探討其是否參與PCOS的發(fā)病，我們設(shè)計(jì)此實(shí)驗(yàn)。 第一部分：rMfn2在正常大鼠及PCOS大鼠卵巢組織的表達(dá) 目的：探討rMfn2在正常大鼠及PCOS模型大鼠卵巢組織的表達(dá)差異，明確rMfn2是否參與PCOS的發(fā)病。 方法：選取6周齡SD雌性大鼠來曲唑灌胃建立PCOS模型，觀察大鼠體重及卵巢體重變化，HE染色評估卵巢形態(tài)，放射免疫法檢測血清E2、T、P、FSH及LH水平。免疫組織化學(xué)法分析rMfn2、Bcl-2及Bax在大鼠卵巢的定位表達(dá)。RT-PCR和Western blotting分別從mRNA和蛋白質(zhì)水平定量分析卵巢組織rMfn2的表達(dá)。 結(jié)果：與對照組比較，PCOS模型組大鼠體重增長明顯低于對照組，卵巢重量明顯高于對照組，差異有統(tǒng)計(jì)學(xué)意義（p0.05）；模型組血清LH、T濃度較對照組明顯增高，E2、P濃度明顯降低，差異有統(tǒng)計(jì)學(xué)意義（p0.05）；兩組血清FSH濃度無統(tǒng)計(jì)學(xué)差異（p0.05）；HE染色顯示對照組大鼠卵巢鏡下可見多個新鮮黃體和不同生長階段的各級卵泡；模型組大鼠卵巢鏡下見典型多囊改變，可見較多小卵泡及囊狀擴(kuò)張的大卵泡，卵泡內(nèi)層顆粒細(xì)胞層數(shù)明顯減少，未見黃體；免疫組織化學(xué)檢測顯示rMfn2廣泛表達(dá)于兩組大鼠顆粒細(xì)胞、卵泡膜細(xì)胞、卵泡液、黃體及卵巢間質(zhì)；rMfn2在PCOS大鼠顆粒細(xì)胞上的表達(dá)明顯弱于對照組，同時Bcl-2在PCOS大鼠顆粒細(xì)胞的表達(dá)也較對照組降低，而Bax的表達(dá)較對照組增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義（p0.05）；RT-PCR和Westernblotting結(jié)果顯示PCOS模型組大鼠卵巢組織rMfn2mRNA及蛋白表達(dá)均明顯低于對照組，差異有統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論：來曲唑成功誘導(dǎo)PCOS大鼠模型；PCOS大鼠顆粒細(xì)胞凋亡可能增加；rMfn2廣泛表達(dá)于大鼠顆粒細(xì)胞、卵泡膜細(xì)胞、卵泡液、黃體及卵巢間質(zhì)，PCOS大鼠顆粒細(xì)胞rMfn2的表達(dá)下調(diào)可能參與PCOS的發(fā)生。 第二部分：Lenti-GFP-rMfn2過表達(dá)載體的構(gòu)建及鑒定 目的：構(gòu)建并鑒定rMfn2過表達(dá)的慢病毒載體（lenti-GFP-rMfn2）。 方法：pLenO-GTP載體酶切線性化，與PCR擴(kuò)增的rMfn2基因連接、轉(zhuǎn)化，提取重組質(zhì)粒，通過酶切法和DNA測序?qū)χ亟M質(zhì)粒進(jìn)行鑒定。將鑒定正確的重組質(zhì)粒轉(zhuǎn)染293T細(xì)胞，收集提純病毒，同時采用流式細(xì)胞儀檢測活細(xì)胞比率測定病毒滴度。 結(jié)果：重組質(zhì)粒經(jīng)酶切和DNA測序鑒定正確。重組質(zhì)粒轉(zhuǎn)染293T細(xì)胞可見綠色熒光表達(dá)，提示質(zhì)粒重組成功。病毒滴度測定滴度為2.2×108TU/ml。 結(jié)論：Lenti-GFP-rMfn2過表達(dá)載體構(gòu)建成功。 第三部分：rMfn2對大鼠顆粒細(xì)胞的作用及信號通路的初步研究 目的：探討rMfn2對正常大鼠卵巢顆粒細(xì)胞增殖凋亡的作用及相關(guān)信號通路。 方法：體外大鼠原代顆粒細(xì)胞的培養(yǎng)及鑒定。細(xì)胞免疫熒光檢測rMfn在顆粒細(xì)胞的定位表達(dá)。Lenti-GFP-rMfn2體外轉(zhuǎn)染大鼠顆粒細(xì)胞，Western blotting鑒定轉(zhuǎn)染結(jié)果。轉(zhuǎn)染成功后MTT法繪制顆粒細(xì)胞生長曲線，流式細(xì)胞儀檢測細(xì)胞周期及凋亡率，Western blotting檢測rMfn2及相關(guān)信號通路蛋白Bcl-2、Bax、Akt、p-Akt的表達(dá)。 結(jié)果：細(xì)胞免疫熒光顯示rMfn表達(dá)于正常大鼠顆粒細(xì)胞胞漿。Westernblotting檢測顯示Lenti-GFP-rMfn2成功轉(zhuǎn)染大鼠顆粒細(xì)胞，MTT檢測顯示rMfn2過表達(dá)組（MFN組）細(xì)胞的增殖速率明顯高于其余兩組細(xì)胞（正常對照CON組和空病毒GFP組），差異有統(tǒng)計(jì)學(xué)意義（p0.05）；細(xì)胞周期檢測顯示MFN組細(xì)胞在G1期所占比例明顯下降，S及G2期所占比例明顯增加，差異有統(tǒng)計(jì)學(xué)意義（p0.05）。凋亡率檢測顯示CON組、GFP組及MFN組三組細(xì)胞凋亡率均很低，三組間差異無統(tǒng)計(jì)學(xué)意義（p0.05）。Western blotting檢測顯示MFN組顆粒細(xì)胞Bcl-2的表達(dá)明顯增加，Bax的表達(dá)減少，差異有統(tǒng)計(jì)學(xué)意義（p0.05）；MFN組p-Akt蛋白的表達(dá)增加，差異有統(tǒng)計(jì)學(xué)意義（p0.05），Akt在三組間表達(dá)無統(tǒng)計(jì)學(xué)差異（p0.05）。 結(jié)論：rMfn2過表達(dá)促進(jìn)正常大鼠顆粒細(xì)胞的增殖，其機(jī)制是激活PI3K/Akt信號通路，引起下游Bcl-2表達(dá)增加，Bad及Bax表達(dá)減少，從而促進(jìn)顆粒細(xì)胞增殖。 第四部分：rMfn2對正常大鼠及PCOS大鼠卵泡發(fā)育的影響研究 目的：觀察rMfn2對正常大鼠及PCOS大鼠卵泡發(fā)育的影響。 方法：60只2月齡SD雌性大鼠隨機(jī)分成以下四組：對照組（CON組，僅注射生理鹽水），空病毒組（GFP組，注射Lenti-GFP慢病毒顆粒），目的病毒組（MFN組，注射Lenti-GFP-rMfn2慢病毒顆粒），目的病毒+來曲唑組（ML組，在Lenti-GFP-rMfn2慢病毒顆粒注射30天后開始來曲唑灌胃連續(xù)23天），來曲唑組（LT組，采用第一部分PCOS模型標(biāo)本）。熒光顯微鏡觀察卵巢組織及全身其他組織rMfn2的表達(dá)；realtime RT-PCR和Western blotting定量檢測rMfn2及性激素相關(guān)受體（ER、PR、FSHR、LHR）的表達(dá)；HE染色評估卵巢形態(tài)，放射免疫法檢測血清E2、T、P、FSH及LH水平。 結(jié)果：慢病毒轉(zhuǎn)染第30天，real time RT-PCR和Western blotting檢測結(jié)果顯示MFN組大鼠卵巢rMfn2呈過表達(dá)狀態(tài)；熒光顯微鏡及Westernblotting檢測顯示rMfn2過表達(dá)載體轉(zhuǎn)染大鼠卵巢，其過表達(dá)存在時間依賴性，，隨著時間的延長表達(dá)逐漸增強(qiáng)，直至轉(zhuǎn)染第60天仍持續(xù)穩(wěn)定表達(dá)。Western blotting檢測顯示轉(zhuǎn)染第60天MFN組ER及PR在子宮的表達(dá)高于GFP組，差異有統(tǒng)計(jì)學(xué)意義（p0.05）；FSHR及LHR的表達(dá)，GFP及MFN兩組間比較無統(tǒng)計(jì)學(xué)差異（p0.05）。HE染色顯示轉(zhuǎn)染第60天MFN組大鼠卵巢鏡下見較多黃體，始基卵泡及多個發(fā)育良好的竇狀卵泡；ML組大鼠卵巢鏡下仍可見囊狀擴(kuò)張卵泡，但較LT組明顯減少，同時可見黃體組織、始基卵泡、竇狀卵泡甚至排卵前卵泡，且卵泡內(nèi)卵母細(xì)胞存在。放射免疫檢測顯示MFN組大鼠血清E2、P水平明顯高于CON組、ML組及LT組，T水平遠(yuǎn)低于CON組、ML組及LT組，差異有統(tǒng)計(jì)學(xué)意義（p0.05）；ML組與LT組比較，血清FSH與T水平變化不大，E2、P水平明顯增加，LH水平降低，差異有統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論：rMfn2過表達(dá)影響大鼠生殖內(nèi)分泌功能，促進(jìn)正常大鼠卵泡發(fā)育；rMfn2過表達(dá)可能抑制來曲唑誘導(dǎo)的大鼠卵巢多囊樣變，具體機(jī)制有待進(jìn)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R711.75

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