本文選題：子癇前期 + 胎盤(pán)灌注　； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：第一部分miRNA-126及EPCs在子癇前期發(fā)病機(jī)制中的作用 目的 探討miRNA-126在子癇前期患者臍血EPCs及胎盤(pán)組織中的表達(dá)及其與胎盤(pán)血管形成中的作用。 方法 1.分離、培養(yǎng)對(duì)照組和子癇前期組臍血EPCs,利用細(xì)胞形態(tài)學(xué)和免疫熒光染色(DiI-ac-LDL和FITC-UEA-I)檢測(cè)鑒定EPCs純度,并觀察記錄群落計(jì)數(shù)及群落直徑； 2.采用逆轉(zhuǎn)錄聚合酶鏈反應(yīng)檢測(cè)臍血EPCs及胎盤(pán)組織miRNA-126的表達(dá)； 3.采用相關(guān)性分析臍血EPCs及miRNA-126的表達(dá)； 4.采用免疫組織化學(xué)染色檢測(cè)胎盤(pán)組織血管密度。 結(jié)果 1.分離培養(yǎng)單個(gè)核細(xì)胞,培養(yǎng)7天后可見(jiàn)細(xì)胞呈集落樣分布,中心為圓形細(xì)胞,在其周?chē)鸀榧忓N形的細(xì),,此類(lèi)型集落被稱(chēng)為colony-forming units(CFUs)。熒光顯微鏡下細(xì)胞呈現(xiàn)FITC-UEA-I(綠色熒光)和DiI-ac-LDL(紅色熒光)雙染色陽(yáng)性,此時(shí)期的細(xì)胞為正在分化的早期EPC。子癇前期組臍靜脈血中EPCs數(shù)量(77±13)明顯低于正常孕婦組(136±23)；對(duì)照組臍血EPCs集落數(shù)量較子癇前期組明顯升高(8.7±2.2和4.3±1.3),且對(duì)照組CUFs群落直徑明顯高于子癇前期孕婦。 2.子癇前期臍血EPCs中miR-126表達(dá)量(0.55±0.36)明顯低于對(duì)照組(1.0±0.15)；子癇前期胎盤(pán)中miR-126表達(dá)量(0.38±0.22)明顯低于對(duì)照組(0.88±0.25)。 3.子癇前期組EPCs數(shù)量及miR-126表達(dá)水平呈現(xiàn)正相關(guān)(r=0.65,P0.05)。對(duì)照組EPCs數(shù)量及miR-126表達(dá)水平無(wú)相關(guān)性。 4.子癇前期組孕婦胎盤(pán)的微血管數(shù)量(54.8±5.72)較正常組孕婦胎盤(pán)的微血管數(shù)量(77.6±7.92)顯著減少；臍血管S/D比值子癇前期組(3.08±±0.95)明顯高于正常組(2.37±0.63)(p均0.05),見(jiàn)表1。 結(jié)論 子癇前期患者臍血中EPCs數(shù)量及群落明顯減少,EPCs中miR-126表達(dá)下降可能與胎盤(pán)血管減少,子癇前期的發(fā)生密切相關(guān)。 第二部分miR-126對(duì)EPCs功能及血管新生的調(diào)控作用目的 研究上調(diào)miR-126的表達(dá)對(duì)EPCs細(xì)胞增殖、分化、遷移和菌落形成能力的影響,了解miR-126在EPCs形成血管過(guò)程中的作用。 方法 1.分離、培養(yǎng)子癇前期患者臍血EPCs,將細(xì)胞分為實(shí)驗(yàn)組(轉(zhuǎn)染niR-126mimic)、陰性對(duì)照組(轉(zhuǎn)染mimic control)和空白對(duì)照組(不進(jìn)行細(xì)胞轉(zhuǎn)染)3組； 2.采用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)miR-126的表達(dá),了解轉(zhuǎn)染效率及過(guò)表達(dá)的效率；同時(shí),采用PCR技術(shù)檢測(cè)miR-126下游基因PIK3R2和P13K的mRNA表達(dá)水平。 3.采用免疫印跡技術(shù)檢測(cè)miR-126下游基因PIK3R2和P13K蛋白的表達(dá)； 4.采用MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)比色法測(cè)定轉(zhuǎn)染后的細(xì)胞活力： 5.采用Transwell模型遷移實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染后細(xì)胞的遷移能力； 6.采用重懸貼壁法檢測(cè)轉(zhuǎn)染后細(xì)胞的分化能力及菌落形成能力 結(jié)果 1.轉(zhuǎn)染后miR-126mimic的EPCs在熒光顯微鏡下可見(jiàn)強(qiáng)的紅色熒光,熒光陽(yáng)性細(xì)胞占總細(xì)胞數(shù)的90%以上。對(duì)照組中無(wú)熒光表達(dá)。轉(zhuǎn)染后EPCs中miR-126的表達(dá)水平分別為實(shí)驗(yàn)組7.5±1.8,陰性對(duì)照組3.8±2.2、空白對(duì)照組miR-126(3.3±1.5)： 2.空白對(duì)照組、陰性對(duì)照組、實(shí)驗(yàn)組下游基因PIK3R2的mRNA表達(dá)水平分別為1.0±0.32,1.34±0.29和0.25±0.28。下游基因P13K的mRNA表達(dá)水平分別為0.46±0.22,0.54±0.27和0.85±0.23。下游基因的蛋白表達(dá)水平趨勢(shì)與mRNA的表達(dá)水平趨勢(shì)一致。實(shí)驗(yàn)組與陰性對(duì)照組比較,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)；空白對(duì)照組與陰性對(duì)照組相比,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 3.轉(zhuǎn)染24小時(shí)后EPCs的細(xì)胞活力,實(shí)驗(yàn)組的吸光度A值為(0.78±0.12),陰性對(duì)照組為(0.46±0.07),空白對(duì)照組為(0.53±0.09)。實(shí)驗(yàn)組的A值與陰性對(duì)照組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；空白對(duì)照組與陰性對(duì)照組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 4.轉(zhuǎn)染24小時(shí)后空白對(duì)照組、陰性對(duì)照組、實(shí)驗(yàn)組遷移入Transwell小室下室的細(xì)胞總數(shù)分別為：84.2±2.7、93.2±3.1和136.4±2.5個(gè)。實(shí)驗(yàn)組高于陰性對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；空白對(duì)照組與陰性對(duì)照組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。轉(zhuǎn)染miR-126能明顯增強(qiáng)EPCs的遷移能力。 5.轉(zhuǎn)染24小時(shí)后,計(jì)數(shù)梭形細(xì)胞數(shù)目,實(shí)驗(yàn)組為(96.1±10.3)個(gè),陰性對(duì)照組為(53.2±11.3)個(gè),空白對(duì)照組為(47.4±9.6)。各組間比較,實(shí)驗(yàn)組與陰性對(duì)照組差異有統(tǒng)計(jì)學(xué)意義(P0.05)；陰性對(duì)照組與空白對(duì)照組之間的差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 miR-126表達(dá)水平的升高可以上調(diào)EPCs的增殖、遷移和分化能力。 第三部分局部轉(zhuǎn)染niiRNA-126改善子癇前期大鼠胎盤(pán)灌注 目的 通過(guò)agomir-126局部轉(zhuǎn)染模型大鼠胎盤(pán),觀察轉(zhuǎn)染agomir-126后胎盤(pán)形態(tài)學(xué)和微血管密度等方面的變化,探討miR-126在改善孕鼠胎盤(pán)血管生長(zhǎng)的可能作用和機(jī)制。 方法 1.利用L-NAME建立高血壓孕鼠模型； 2.模型鼠分組,并采用胎盤(pán)局部注射轉(zhuǎn)染技術(shù)將agomir-126轉(zhuǎn)染入孕鼠胎盤(pán)組織； 3.轉(zhuǎn)染后的孕鼠組織行冰凍切片,熒光顯微鏡下觀察agomir-126表達(dá)以了解轉(zhuǎn)染效果； 4.采用超聲微泡技術(shù)實(shí)時(shí)測(cè)量孕20天大鼠胎盤(pán)血流灌注 5.處死大鼠,測(cè)定胎盤(pán)、胎鼠的大小、重量及外觀； 6.利用實(shí)時(shí)熒光定量PCR檢測(cè)轉(zhuǎn)染后孕鼠胎盤(pán)組織miR-126的表達(dá)水平； 7.利用CD31因子免疫組化實(shí)驗(yàn)檢測(cè)孕21天大鼠轉(zhuǎn)染后的胎盤(pán)血管密度。 結(jié)果 1.實(shí)驗(yàn)選取的21只孕鼠,麻醉及感染死亡1只,早產(chǎn)1只；其余孕鼠均存活,于妊娠第21天剖腹取胎。 2.轉(zhuǎn)染后胎盤(pán)組織的冰凍切片在熒光下觀察可見(jiàn)紅色Cy3大面積高表達(dá),體內(nèi)轉(zhuǎn)染效果理想； 3.孕D15子癇組和治療組血壓均高于對(duì)照組(p0.05)；D20時(shí)子癇組的血壓高于對(duì)照組和治療組,子癇組和治療組間差異無(wú)顯著性(p0.05)。 4.超聲微泡實(shí)時(shí)胎盤(pán)造影可清楚顯示胎盤(pán)的大小,直徑以及血流灌注情況。二維常規(guī)超聲下測(cè)量三組胎盤(pán)最大切面的面積,子癇組的切面面積明顯小于其它兩組,且超聲微泡造影下第8秒圖像顯示子癇組的充盈程度明顯低于其它兩組。大鼠胎盤(pán)的時(shí)間一強(qiáng)度曲線顯示子癇組的到達(dá)時(shí)間(AT)和達(dá)峰時(shí)間(TTP)均晚于對(duì)照組和治療組(P0.05),治療組曲線與對(duì)照組較為一致,但到達(dá)時(shí)間(AT)略晚于對(duì)照組。 5.體內(nèi)轉(zhuǎn)染后,三組的胎鼠重量(g)分別為：對(duì)照組5.26±0.06,子癇組4.15+0.08,治療組5.04±0.11；三組的胎盤(pán)重量(g)分別為：對(duì)照組0.60±0.03,子癇組0.43±0.02,治療組0.52±0.04；三組的胎盤(pán)直徑(cm)分別為：對(duì)照組1.43±0.14,子癇組1.25±0.18,治療組1.40±0.11。子癇組的胎盤(pán)胎鼠明顯小于其它兩組,且子癇組的胎盤(pán)外觀較其它兩組胎盤(pán)蒼白。治療組與子癇組相比較,胎鼠重量和胎盤(pán)重量差異具有統(tǒng)計(jì)學(xué)意義和胎鼠重量(p0.05)。 6.轉(zhuǎn)染后各組胎盤(pán)中miR-126的表達(dá)水平,治療組為4.2±0.31,對(duì)照組為1.0±0.27,子癇組為0.693±0.16。各組分別比較,治療組與對(duì)照組差異有統(tǒng)計(jì)學(xué)意義(P0.05)；對(duì)照組與子癇組差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 7.對(duì)照組和治療組的絨毛間質(zhì)疏松,CD34染色陽(yáng)性率高;子癇組的絨毛間質(zhì)稍密,CD34表達(dá)降低(×200)。治療組孕鼠胎盤(pán)的微血管數(shù)量(69.2±4.15)較子癇組(39.1±5.34)顯著增多；治療組與對(duì)照組(76.44±5.23)比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論 利用miR-126類(lèi)似物agomir-126在胎盤(pán)內(nèi)轉(zhuǎn)染技術(shù)可有效增加miR-126在孕鼠胎盤(pán)的表達(dá)水平,且能夠增強(qiáng)孕鼠胎盤(pán)的血管形成能力,改善子癇前期孕鼠模型的妊娠結(jié)局,提示miR-126有望成為胎盤(pán)促血管新生的新靶點(diǎn)。
[Abstract]:Part one: the role of miRNA-126 and EPCs in the pathogenesis of preeclampsia.
objective
Objective to investigate the expression of miRNA-126 in umbilical cord blood EPCs and placental tissue and its role in placental angiogenesis in preeclampsia.
Method
1. the cord blood EPCs was cultured in the control group and the preeclampsia group. The purity of EPCs was detected by cell morphology and immunofluorescence staining (DiI-ac-LDL and FITC-UEA-I), and the count of the community and the diameter of the community were observed.
2. reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of EPCs and miRNA-126 in umbilical cord blood.
3. correlation analysis was used to analyze the expression of EPCs and miRNA-126 in cord blood.
4. the blood vessel density of placenta tissue was detected by immunohistochemistry.
Result
1. the mononuclear cells were isolated and cultured. The cells were found to be colony like distribution after 7 days. The center was round cells, and the spindle shaped thin cells around it. This type of colony was called colony-forming units (CFUs). The cells showed FITC-UEA-I (green fluorescence) and DiI-ac-LDL (red fluorescence) double staining positive under fluorescence microscope. The number of EPCs (77 + 13) in the umbilical vein blood of the early EPC. preeclampsia group was significantly lower than that of the normal pregnant group (136 + 23), and the number of EPCs colony in the umbilical cord blood in the control group was significantly higher than that in the preeclampsia group (8.7 + 2.2 and 4.3 + 1.3), and the diameter of the CUFs community in the control group was significantly higher than that of the preeclampsia.
The expression of miR-126 in EPCs (0.55 + 0.36) of EPCs in preeclampsia was significantly lower than that of the control group (1 + 0.15), and the expression of miR-126 in preeclampsia (0.38 + 0.22) was significantly lower than that of the control group (0.88 + 0.25).
3. there was a positive correlation between the number of EPCs and miR-126 expression in preeclampsia group (r=0.65, P0.05). There was no correlation between the number of EPCs and the expression level of miR-126 in the control group.
The number of microvessels in placenta of preeclampsia group (54.8 + 5.72) in preeclampsia group was significantly lower than that of normal pregnant women (77.6 + 7.92), and the S/D ratio of umbilical blood vessels (3.08 + 0.95) in preeclampsia group was significantly higher than that of normal group (2.37 + 0.63) (2.37 + 0.63) (0.05 P), see table 1..
conclusion
The number and number of EPCs in cord blood of preeclampsia patients were significantly reduced. The decrease of miR-126 expression in EPCs may be closely related to the decrease of placental blood vessels and the occurrence of preeclampsia.
The second part of miR-126 regulates EPCs function and angiogenesis.
To study the effect of up regulation of miR-126 on proliferation, differentiation, migration and colony forming ability of EPCs cells, and to understand the role of miR-126 in EPCs formation.
Method
1. the umbilical cord blood EPCs was isolated from preeclampsia, and the cells were divided into experimental group (transfected niR-126mimic), negative control group (transfected mimic control) and blank control group (no cell transfection).
2. the expression of miR-126 was detected by real time fluorescence quantitative PCR, and the efficiency of transfection efficiency and overexpression were detected. At the same time, the expression level of mRNA in the downstream miR-126 gene PIK3R2 and P13K was detected by PCR technique.
3. Western blotting was used to detect the expression of PIK3R2 and P13K genes in miR-126 downstream genes.
4. the cell viability of transfected cells was determined by MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) colorimetry.
5. Transwell model migration assay was used to detect the migration ability of transfected cells.
6. suspension attachment assay was used to detect the differentiation ability and colony forming ability of the cells after transfection.
Result
After 1. transfection, the EPCs of miR-126mimic showed strong red fluorescence under the fluorescence microscope. The fluorescent positive cells accounted for more than 90% of the total number of cells. There was no fluorescent expression in the control group. The level of miR-126 expression in EPCs after transfection was 7.5 + 1.8 in the experimental group, 3.8 in the negative control group, and miR-126 in the blank group (3.3 + 1.5).
2. blank control group, negative control group, the mRNA expression level of downstream gene PIK3R2 in the experimental group was 1 + 0.32,1.34 + 0.29 and 0.25 + 0.28. downstream genes P13K mRNA expression level was 0.46 + 0.22,0.54 + 0.27 and 0.85 + 0.23. downstream genes, respectively, and the trend of protein expression level was consistent with the expression level of mRNA. The difference was statistically significant (P0.05), while there was no significant difference between the blank control group and the negative control group (P0.05).
3. the cell viability of EPCs after 24 hours transfection, the absorbance A value of the experimental group was (0.78 + 0.12), the negative control group was (0.46 + 0.07), and the blank control group was (0.53 + 0.09). The A value of the experimental group was statistically significant compared with the negative control group (P0.05). There was no significant difference between the blank control group and the negative control group (P0.05).
4. after 24 hours transfection, the blank control group, negative control group, the total number of cells migrated into the Transwell compartment were 84.2 + 2.7,93.2 + 3.1 and 136.4 + 2.5 respectively. The experimental group was higher than the negative control group, the difference was statistically significant (P0.05); there was no significant difference between the blank control group and the negative group (P0.05). MiR transfected to miR -126 can significantly enhance the mobility of EPCs.
5. after 24 hours transfection, the number of spindle cells was counted, the experimental group was (96.1 + 10.3), the negative control group was (53.2 + 11.3), and the blank control group was (47.4 + 9.6). The difference between the experimental group and the negative control group was statistically significant (P0.05), and the difference between the negative control group and the blank control group was not statistically significant (P0.05).
conclusion
The increased expression level of miR-126 can upregulate the proliferation, migration and differentiation of EPCs.
The third part is local transfection of niiRNA-126 to improve placental perfusion in rats with preeclampsia.
objective
Through the agomir-126 local transfection model of rat placenta, the changes of placental morphology and microvascular density after transfection of agomir-126 were observed, and the possible role and mechanism of miR-126 in improving the growth of placental blood vessels in pregnant rats were discussed.
Method
1. the model of hypertensive pregnant rats was established by L-NAME.
2. model mice were grouped, and agomir-126 was transfected into placenta of pregnant rats by local injection of placenta.
3. after transfection, the frozen sections of the pregnant rats were observed, and the expression of agomir-126 was observed under fluorescence microscope to understand the transfection effect.
4. ultrasound microbubble technique was used to measure placental blood perfusion in 20 days pregnant rats.
5. the rats were sacrificed to determine the size, weight and appearance of the placenta.
6. real-time fluorescent quantitative PCR was used to detect the expression level of miR-126 in placenta of pregnant rats.
7. using CD31 factor immunohistochemical assay to detect placental vessel density after 21 days of gestation.
Result
1. in the 21 pregnant rats selected, 1 rats were anesthetized and infected, 1 were preterm birth, the other pregnant rats survived, and the fetus was harvested on the twenty-first day of gestation.
2. after transfection, the frozen sections of placenta tissue were observed under fluorescence. The large expression of red Cy3 could be seen, and the transfection effect was ideal in vivo.
The blood pressure of the 3. D15 eclampsia group and the treatment group was higher than that of the control group (P0.05), while the blood pressure of the eclampsia group was higher than that of the control group and the treatment group at D20, and there was no significant difference between the eclampsia group and the treatment group (P0.05).
The size, diameter and blood perfusion of the placenta could be clearly displayed by 4. ultrasonic microbubble real-time placentography. The area of the largest section of the three groups of placenta was measured under two-dimensional conventional ultrasound. The area of the section of the eclampsia group was significantly smaller than that of the other two groups, and the filling degree of the eclampsia group was significantly lower than the other two groups in the 8 second image of the ultrasound microbubbles. The time intensity curve of rat placenta showed that the time of arrival (AT) and peak time (TTP) of the eclampsia group were later than that of the control group and the treatment group (P0.05). The curve of the treatment group was more consistent with the control group, but the time of arrival (AT) was slightly later than that of the control group.
5. after transfection in the body, the weight (g) of the three groups was 5.26 + 0.06, the eclampsia group was 4.15+0.08 and the treatment group was 5.04 + 0.11, and the placental weight (g) of the three groups was 0.60 + 0.03, the eclampsia group 0.43 + 0.43, and the 5.26 of the treatment group was 0.52 + 0.04. The placental fetal rats in 1.40 + 0.11. eclampsia group were obviously smaller than the other two groups, and the placental appearance of the eclampsia group was more pale than that of the other two groups. Compared with the eclampsia group, the difference of weight and placental weight of fetal rats was statistically significant and the weight of fetal rats (P0.05).
The expression level of miR-126 in the placenta of each group after 6. transfection was 4.2 + 0.31, the control group was 1 + 0.27 and the eclampsia group was 0.693 + 0.16. respectively. The difference between the treatment group and the control group was statistically significant (P0.05), and the difference between the control group and the eclampsia group was statistically significant (P0.05).
In the 7. control group and the treatment group, the villous stroma was loose, the positive rate of CD34 staining was high, the villous stroma of the eclampsia group was slightly dense and the expression of CD34 decreased (69.2 + 4.15) in the treatment group (69.2 + 4.15) was significantly higher than that of the eclampsia group (39.1 + 5.34), and the difference was not statistically significant (P0.05) in the treatment group and the control group (76.44 + 5.23).
conclusion
The use of miR-126 analogs agomir-126 in placenta transfection can effectively increase the expression level of miR-126 in placenta of pregnant mice, and can enhance the angiogenesis ability of placenta of pregnant mice and improve the pregnancy outcome of pregnant rat model in preeclampsia, suggesting that miR-126 is expected to be a new target for new placental angiogenesis.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R714.244

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