MicroRNA-126调控内皮祖细胞功能在改善子痫前期胎盘血流灌注中的作用

发布时间：2018-05-24 10:57

本文选题：子痫前期 + 胎盘灌注　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分miRNA-126及EPCs在子痫前期发病机制中的作用目的探讨miRNA-126在子痫前期患者脐血EPCs及胎盘组织中的表达及其与胎盘血管形成中的作用。方法 1.分离、培养对照组和子痫前期组脐血EPCs,利用细胞形态学和免疫荧光染色(DiI-ac-LDL和FITC-UEA-I)检测鉴定EPCs纯度,并观察记录群落计数及群落直径； 2.采用逆转录聚合酶链反应检测脐血EPCs及胎盘组织miRNA-126的表达； 3.采用相关性分析脐血EPCs及miRNA-126的表达； 4.采用免疫组织化学染色检测胎盘组织血管密度。结果 1.分离培养单个核细胞,培养7天后可见细胞呈集落样分布,中心为圆形细胞,在其周围为纺锤形的细,,此类型集落被称为colony-forming units(CFUs)。荧光显微镜下细胞呈现FITC-UEA-I(绿色荧光)和DiI-ac-LDL(红色荧光)双染色阳性,此时期的细胞为正在分化的早期EPC。子痫前期组脐静脉血中EPCs数量(77±13)明显低于正常孕妇组(136±23)；对照组脐血EPCs集落数量较子痫前期组明显升高(8.7±2.2和4.3±1.3),且对照组CUFs群落直径明显高于子痫前期孕妇。 2.子痫前期脐血EPCs中miR-126表达量(0.55±0.36)明显低于对照组(1.0±0.15)；子痫前期胎盘中miR-126表达量(0.38±0.22)明显低于对照组(0.88±0.25)。 3.子痫前期组EPCs数量及miR-126表达水平呈现正相关(r=0.65,P0.05)。对照组EPCs数量及miR-126表达水平无相关性。 4.子痫前期组孕妇胎盘的微血管数量(54.8±5.72)较正常组孕妇胎盘的微血管数量(77.6±7.92)显著减少；脐血管S/D比值子痫前期组(3.08±±0.95)明显高于正常组(2.37±0.63)(p均0.05),见表1。结论子痫前期患者脐血中EPCs数量及群落明显减少,EPCs中miR-126表达下降可能与胎盘血管减少,子痫前期的发生密切相关。第二部分miR-126对EPCs功能及血管新生的调控作用目的研究上调miR-126的表达对EPCs细胞增殖、分化、迁移和菌落形成能力的影响,了解miR-126在EPCs形成血管过程中的作用。方法 1.分离、培养子痫前期患者脐血EPCs,将细胞分为实验组(转染niR-126mimic)、阴性对照组(转染mimic control)和空白对照组(不进行细胞转染)3组； 2.采用实时荧光定量PCR技术检测miR-126的表达,了解转染效率及过表达的效率；同时,采用PCR技术检测miR-126下游基因PIK3R2和P13K的mRNA表达水平。 3.采用免疫印迹技术检测miR-126下游基因PIK3R2和P13K蛋白的表达； 4.采用MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)比色法测定转染后的细胞活力： 5.采用Transwell模型迁移实验检测转染后细胞的迁移能力； 6.采用重悬贴壁法检测转染后细胞的分化能力及菌落形成能力结果 1.转染后miR-126mimic的EPCs在荧光显微镜下可见强的红色荧光,荧光阳性细胞占总细胞数的90%以上。对照组中无荧光表达。转染后EPCs中miR-126的表达水平分别为实验组7.5±1.8,阴性对照组3.8±2.2、空白对照组miR-126(3.3±1.5)： 2.空白对照组、阴性对照组、实验组下游基因PIK3R2的mRNA表达水平分别为1.0±0.32,1.34±0.29和0.25±0.28。下游基因P13K的mRNA表达水平分别为0.46±0.22,0.54±0.27和0.85±0.23。下游基因的蛋白表达水平趋势与mRNA的表达水平趋势一致。实验组与阴性对照组比较,差异均有统计学意义(P0.05)；空白对照组与阴性对照组相比,差异均无统计学意义(P0.05)。 3.转染24小时后EPCs的细胞活力,实验组的吸光度A值为(0.78±0.12),阴性对照组为(0.46±0.07),空白对照组为(0.53±0.09)。实验组的A值与阴性对照组相比,差异有统计学意义(P0.05)；空白对照组与阴性对照组相比,差异无统计学意义(P0.05)。 4.转染24小时后空白对照组、阴性对照组、实验组迁移入Transwell小室下室的细胞总数分别为：84.2±2.7、93.2±3.1和136.4±2.5个。实验组高于阴性对照组,差异有统计学意义(P0.05)；空白对照组与阴性对照组相比,差异无统计学意义(P0.05)。转染miR-126能明显增强EPCs的迁移能力。 5.转染24小时后,计数梭形细胞数目,实验组为(96.1±10.3)个,阴性对照组为(53.2±11.3)个,空白对照组为(47.4±9.6)。各组间比较,实验组与阴性对照组差异有统计学意义(P0.05)；阴性对照组与空白对照组之间的差异无统计学意义(P0.05)。结论 miR-126表达水平的升高可以上调EPCs的增殖、迁移和分化能力。第三部分局部转染niiRNA-126改善子痫前期大鼠胎盘灌注目的通过agomir-126局部转染模型大鼠胎盘,观察转染agomir-126后胎盘形态学和微血管密度等方面的变化,探讨miR-126在改善孕鼠胎盘血管生长的可能作用和机制。方法 1.利用L-NAME建立高血压孕鼠模型； 2.模型鼠分组,并采用胎盘局部注射转染技术将agomir-126转染入孕鼠胎盘组织； 3.转染后的孕鼠组织行冰冻切片,荧光显微镜下观察agomir-126表达以了解转染效果； 4.采用超声微泡技术实时测量孕20天大鼠胎盘血流灌注 5.处死大鼠,测定胎盘、胎鼠的大小、重量及外观； 6.利用实时荧光定量PCR检测转染后孕鼠胎盘组织miR-126的表达水平； 7.利用CD31因子免疫组化实验检测孕21天大鼠转染后的胎盘血管密度。结果 1.实验选取的21只孕鼠,麻醉及感染死亡1只,早产1只；其余孕鼠均存活,于妊娠第21天剖腹取胎。 2.转染后胎盘组织的冰冻切片在荧光下观察可见红色Cy3大面积高表达,体内转染效果理想； 3.孕D15子痫组和治疗组血压均高于对照组(p0.05)；D20时子痫组的血压高于对照组和治疗组,子痫组和治疗组间差异无显著性(p0.05)。 4.超声微泡实时胎盘造影可清楚显示胎盘的大小,直径以及血流灌注情况。二维常规超声下测量三组胎盘最大切面的面积,子痫组的切面面积明显小于其它两组,且超声微泡造影下第8秒图像显示子痫组的充盈程度明显低于其它两组。大鼠胎盘的时间一强度曲线显示子痫组的到达时间(AT)和达峰时间(TTP)均晚于对照组和治疗组(P0.05),治疗组曲线与对照组较为一致,但到达时间(AT)略晚于对照组。 5.体内转染后,三组的胎鼠重量(g)分别为：对照组5.26±0.06,子痫组4.15+0.08,治疗组5.04±0.11；三组的胎盘重量(g)分别为：对照组0.60±0.03,子痫组0.43±0.02,治疗组0.52±0.04；三组的胎盘直径(cm)分别为：对照组1.43±0.14,子痫组1.25±0.18,治疗组1.40±0.11。子痫组的胎盘胎鼠明显小于其它两组,且子痫组的胎盘外观较其它两组胎盘苍白。治疗组与子痫组相比较,胎鼠重量和胎盘重量差异具有统计学意义和胎鼠重量(p0.05)。 6.转染后各组胎盘中miR-126的表达水平,治疗组为4.2±0.31,对照组为1.0±0.27,子痫组为0.693±0.16。各组分别比较,治疗组与对照组差异有统计学意义(P0.05)；对照组与子痫组差异有统计学意义(P0.05)。 7.对照组和治疗组的绒毛间质疏松,CD34染色阳性率高;子痫组的绒毛间质稍密,CD34表达降低(×200)。治疗组孕鼠胎盘的微血管数量(69.2±4.15)较子痫组(39.1±5.34)显著增多；治疗组与对照组(76.44±5.23)比较,差异无统计学意义(P0.05)。结论利用miR-126类似物agomir-126在胎盘内转染技术可有效增加miR-126在孕鼠胎盘的表达水平,且能够增强孕鼠胎盘的血管形成能力,改善子痫前期孕鼠模型的妊娠结局,提示miR-126有望成为胎盘促血管新生的新靶点。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.244

【参考文献】