Notch-ephrinB2信号通路调节绒毛外滋养细胞在子宫螺旋动脉重铸中的作用

发布时间：2018-05-18 18:35

本文选题：EFNB2 + 绒毛外滋养细胞　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分EFNB2调节EVT功能在子宫螺旋动脉重铸中的作用目的通过研究EFNB2蛋白在妊娠早期绒毛和母体蜕膜在组织细胞水平的定位表达情况,并检测EFNB2基因在人原代EVT细胞和多个滋养细胞系细胞中的表达差异,探讨其对子宫螺旋动脉重铸过程的影响；通过基因水平调节EFNB2在人绒毛外滋养细胞系HTR-8/SVneo细胞中的表达,观察对细胞活性、凋亡、迁移、侵袭和血管形成等生物学功能的影响,探讨EFNB2在子宫螺旋动脉重铸中的作用。方法 1.免疫组织化学染色法对EFNB2蛋自在早孕绒毛组织及母体蜕膜组织的表达进行定位； 2. qRT-PCR检测人原代EVT细胞、人绒毛外滋养细胞系HTR-8/SVneo和绒癌滋养细胞系JEG-3及JAR等细胞中EFNB2mRNA水平； 3.根据转染质粒不同处理,将细胞分为空白对照组(未进行细胞转染)、抑制组(转染EFNB2抑制质粒)和过表达组(转染EFNB2过表达质粒)3组,qRT-PCR检测EFNB2mRNA表达时增加两组阴性对照组(分别转染相应质粒空白载体)； 4. qRT-PCR和Western blotting检测转染后细胞EFNB2mRNA和蛋白表达水平,了解转染效率； 5.CCK-8法测定各组细胞活性； 6. Hoechst33258染色检测转染后细胞凋亡; 7. Western blotting检测转染后细胞中抑凋亡蛋白Bcl-2及促凋亡蛋白Bax表达； 8. Trans well模型检测转染后细胞的迁移及侵袭能力； 9. Martigel胶上小管形成实验模型检测转染后细胞血管形成能力； 10.PDC共培养系统验证下调EFNB2表达对子宫螺旋动脉重铸的影响。结果 1. EFNB2广泛表达于早孕绒毛及母体蜕膜组织中,在绒毛滋养细胞层和蜕膜EVT细胞中呈强阳性表达; 2. EFNB2基因在人原代EVT细胞、人绒毛外滋养细胞系HTR-8/SVneo细胞、绒癌滋养细胞系JEG-3细胞和JAR细胞中表达量分别为：0.00067±0.00012、0.00058±0.00007、0.00042±0.00004和0.00039±0.00006,人原代EVT细胞EFNB2基因表达高于绒癌滋养细胞系(P0.05),与HTR-8/SVneo细胞表达水平无显著差异(P0.05); 3.荧光显微镜下可见转染成功的细胞强绿色荧光表达,对照组中无荧光表达,转染效率高。 4.和空白对照组相比,转染后HTR-8/SVneo中EFNB2mRNA表达水平分别为；抑制组0.14±0.09(P0.05)、抑制阴性对照组1.06±0.06(P0.05)、过表达组426.81±44.76(P0.05)、过表达阴性对照1.01±0.26(P0.05)。 5.和空白对照组相比,抑制组和过表达组中EFNB2蛋白相对表达量分别为0.25±0.04和2.19±0.07(P0.05)。 6.和空白对照组相比,抑制组细胞活性降低至0.67±0.03(P0.05),过表达组活性为1.37±0.51(P0.05)。 7.与空白对照组相比,抑制组中凋亡细胞相对数量分别为129.2±11.9%(P0.05),过表达组104.7±14.9%(P0.05)。 8.同空白对照组相比,抑制组中Bcl-2蛋白表达下降(0.35±0.02,P0.05),Bax蛋白表达增加(1.82±0.16,P0.05);过表达组中Bcl-2与Bax蛋白的相对表达量分别为：1.15±0.03、1.01±0.04(P0.05)。 9.空白对照组、抑制组及过表达组迁移细胞总数分别为：150.2±6.7、108.5±3.0和328.7.4±13.5个(P0.05)；抑制组迁移能力下降(0.69±0.03),过表达组迁移能力增强(1.95±0.08)(P0.05)。 10.空白对照组、抑制组及过表达组侵袭细胞总数分别为：140.8±3.4、42.5±1.3和265.7±14.7个(P0.05)；同空白对照组相比,抑制组侵袭能力下降(0.2±0.05),过表达组侵袭能力增强(1.80±0.21)(P0.05)。 11.与空白对照组(72.6土9.6)相比,过表达组管腔分支点数增多(100.4±20.2),抑制组数目减少(29.6±4.8)(P0.05)。 12.PDC结果表明,sh-ephrin-B2组和shNC组相比,子宫螺旋动脉重铸处于更早期阶段。结论 EFNB2可能通过介导妊娠早期EVT细胞生物学功能,参与子宫螺旋动脉重铸过程。沉默EFNB2基因表达则可以促进HTR-8/SVneo细胞凋亡,抑制细胞活性、迁移、侵袭及血管形成能力；上调EFNB2表达对细胞凋亡及活性无明显影响,但可增强上述其他各项生物学功能。第二部分Notch通路调节EFNB2对EVT功能的影响目的通过靶向基因沉默和外源性配体刺激方法,分别在基因水平和蛋白水平调节Notch1信号通路的活化情况,检测人绒毛外滋养细胞系HTR-8/SVneo细胞中EFNB2的表达水平和生物学功能的变化情况,以揭示Notch1信号通路对EVT细胞的调节作用及其可能的机制。方法 1.CCK-8法检测不同浓度sD114对HTR-8/SVneo细胞活性的影响,确定sD114加药浓度； 2. qRT-PCR和Western blotting检测转染对HTR-8/SVneo细胞中Notch1基因表达的抑制作用； 3.根据处理的差异,将细胞分为空白对照组、抑制组(转染Notch1抑制质粒)、加药组(0.5mg/mLsDll4)和抑制加药组(转染Notch1抑制质粒后加入0.5mg/mLsD114)4组; 4.CCK-8测定各组细胞活性； 5. Western blotting检测转染后细胞中抑凋亡蛋白Bcl-2及促凋亡蛋白Bax表达情况； 6. Transwell模型检测转染后细胞的迁移及侵袭能力； 7. qRT-PCR和Western blotting检测各组细胞中Notch1信号通路激活情况对EFNB2基因表达的影响； 8. Transwell模型检测Notch1信号通路激活后对EFNB2抑制的HTR-8/SVneo细胞迁移及侵袭能力影响。结果 1.与空白对照组相比,sD114终浓度为0.125、0.25、0.5和1.0μg/mL时,各组细胞活性分别为：(0.96±0.03,),(1.15±0.09),(1.16±0.10)和(1.08±0.08)(P0.05)。 2.同空白对照组相比,转染的HTR-8/SVneo细胞中Notch1mRNA相对表达水平分别为；抑制组0.60±0.14(P0.05)、阴性对照组1.01±0.04(P0.05)；抑制组中Notch1蛋白相对表达水平为0.25±0.04(P0.05)。 3.和空白对照组相比,抑制组细胞活性为0.65±0.13(P0.05),加药组细胞活性为1.07±0.05(P0.05),抑制后加药组细胞活性为0.56±0.14(P0.05),与抑制组无显著差异(P0.05)； 4.同空白对照组相比,抑制组中Bcl-2蛋白表达下降(0.47±0.02,P0.05),Bax蛋白表达增加(2.144±0.05,P0.05)；加药组中Bcl-2与Bax蛋白的相对表达量分别为1.01±0.09和0.93±0.04(P0.05)。 5.同空白对照组相比,抑制组、加药组和抑制后加药组迁移指数分别为0.30±±0.03、2.49±0.25和2.84±0.14(P0.05)； 6.同空白对照组相比,抑制组、加药组和抑制后加药组侵袭指数分别为0.15±0.02,、2.84±0.12和2.46±0.18(P0.05)； 7.与空白对照组相比,抑制组和加药组细胞EFNB2基因mRNA相对表达量分别为0.24±0.08和3.41±0.17(P0.05)。 8.与空白对照组相比,抑制组和加药组细胞EFNB2基因蛋白水平相对表达量分别为0.43±0.17和1.79±0.25(P0.05)。 9. EFNB2抑制加药组迁移与侵袭能力分别为EFNB2抑制组的7.52±3.12和7.48±3.67(P0.05) 结论 Notchl信号通路可以通过调节EFNB2的表达,影响EVT细胞的生物学功能,参与子宫螺旋动脉重铸的过程,调控胎盘血管网络发育。第三部分Notch信号通路调节dNK对EVT协同作用的研究目的 sDll4激活体外培养ddNK细胞中Notch信号通路,检测dNK细胞活性、凋亡、IFN-y的分泌、EFNB2基因表达及其对HTR-8/SVneo细胞迁移能力的影响,探讨Notch/EFNB2途径是否可以通过调节ddNK生物学功能协助EVT参与子宫螺旋动脉重铸过程。方法 1.流式细胞学检测体外分离培养的dNK细胞纯度； 2.CCK-8法检测细胞活性； 3. Caspase-3分光光度法检测细胞凋亡情况； 4. qRT-PCR和ELISA检测dNK细胞中IFN-y mRNA水平和分泌情况； 5. Transwell共培养模型检测HTR-8/SVneo细胞迁移能力。结果 1.流式细胞仪分析检测所提取细胞CD56+CD3"的细胞纯度90%。 2.与空白对照组相比,实验组dNK细胞活性为1.01±0.05(P0.05)。 3.同空白组相比,实验组细胞caspase-3相对表达量为0.79±0.14(P0.05)。 4.同空白对照组相比,加药组中IFN-y mRNA水平为6.22±0.24(P0.05)；空白对照组和实验组分泌量分别为3175.56±141.55pg/mL和4226.28±158.09pg/mL (P0.05). 5.同空白对照组相比,实验组细胞EFNB2mRNA相对表达量为2.53±0.75(P0.05)。 6.与空白对照组相比,加药组、共培养组和共培养加药组HTR-8/SVneo细胞迁移指数分别为：0.71±0.24(P0.05)、0.85±0.22(P0.05)和2.28±0.33(P0.05)。结论 D114/Notch信号通路可能通过调节EFNB2影响dNK细胞生物学功能,对EVT细胞在子宫螺旋动脉重铸过程中的生物学效应发挥直接和间接的协同作用。
[Abstract]:The role of the first part of EFNB2 in the reconstruction of uterine spiral artery

Purpose

To investigate the expression of EFNB2 protein in human primary and maternal decidual cells at the early stage of pregnancy , and to examine the difference of EFNB2 gene expression in human primary and multiple feeder cell lines , and to investigate the effect of EFNB2 gene on the process of uterine spiral artery re - casting ;
The effects of EFNB2 on cell activity , apoptosis , migration , invasion and angiogenesis were observed by gene - level regulation of EFNB2 in human chorionic trophoblasts .

method

1 . Immunohistochemical staining was used to locate the expression of EFNB2 eggs in early pregnant chorionic villi and maternal decidual tissue ;

2.qRT - PCR was used to detect the level of EFNB2mRNA in human primary cell , human chorionic trophoblasts , human chorionic trophoblasts , human chorionic trophoblasts , human chorionic trophoblasts , JEG - 3 and JAR cells .

3 . According to different treatment of transfection plasmid , the cells were divided into blank control group ( without cell transfection ) , inhibition group ( transfected EFNB2 inhibitory plasmid ) and overexpression group ( transfected EFNB2 overexpression plasmid ) 3 groups , and the expression of EFNB2mRNA was detected by qRT - PCR .

4.qRT - PCR and Western blotting were used to detect the expression of EFNB2mRNA and protein in transfected cells .

5 . CCK - 8 method was used to determine the activity of each group .

6 . Apoptosis of transfected cells was detected by hoechst 33258 staining .

7 . Western blotting was used to detect the expression of anti - apoptotic protein Bcl - 2 and pro - apoptotic protein Bax in transfected cells .

8 . Transwell model was used to detect the migration and invasion ability of transfected cells .

9.Martigel ' s small tube formation experiment model to detect the formation ability of transfected cells ;

10 . The effect of down - regulation EFNB2 expression on the re - casting of uterine spiral artery was verified by PDC co - culture system .

Results

1 . EFNB2 is widely expressed in the villi of early pregnancy and decidual tissue of maternal decidual membrane , which is strongly expressed in trophoblasts and decidual cells .

2 . The expression of EFNB2 gene was 0.0000067 卤 0.0000012 , 0.0000058 卤 0.00007 , 0.0000042 卤 0.0000004 and 0.0000039 卤 0.0000006 , respectively , and the expression level of EFNB2 gene was higher than that of choriocarcinoma trophoblasts ( P0.05 ) .

3 . The fluorescence microscope showed that the transfected cells had strong green fluorescence and no fluorescence in the control group , and the transfection efficiency was high .

4 . Compared with blank control group , the expression level of EFNB2mRNA in the transfected TR8 / SVneo was respectively .
In the control group , 0.14 卤 0.09 ( P0.05 ) was inhibited , 1.06 卤 0.06 in the negative control group ( P0.05 ) , 426.81 卤 44.76 in the overexpression group ( P0.05 ) , and 1.01 卤 0.26 in the negative control group ( P0.05 ) .

5 . Compared with blank control group , the relative expression of EFNB2 protein was 0.25 卤 0.04 and 2.19 卤 0.07 ( P0.05 ) .

6 . Compared with the blank control group , the activity of the inhibition group decreased to 0.67 卤 0.03 ( P0.05 ) , and the activity of the overexpression group was 1.37 卤 0.51 ( P0.05 ) .

7 . Compared with the blank control group , the relative number of apoptotic cells in the inhibition group was 129.2 卤 11.9 % ( P0.05 ) , and the overexpression group was 104.7 卤 14.9 % ( P0.05 ) .

8 . Compared with blank control group , the expression of Bcl - 2 protein decreased ( 0.35 卤 0.02 , P < 0.05 ) , Bax protein expression increased ( 1 . 82 卤 0.16 , P < 0 . 05 ) , and the relative expression of Bcl - 2 and Bax protein in the overexpression group was 1.15 卤 0.03 , 1.01 卤 0.04 ( P0.05 ) .

9 . The total number of migrated cells in the blank control group , the inhibition group and the over - expression group was 150.2 卤 6.7 , 108.5 卤 3.0 and 322.8 . 7.4 卤 13.5 ( P0.05 ) .
The migration ability of the inhibition group decreased ( 0.69 卤 0.03 ) , and the migration ability of the overexpression group was enhanced ( 1.95 卤 0.08 ) ( P0.05 ) .

10 . The total number of invasive cells in the control group , the inhibition group and the overexpression group were 140.8 卤 3.4 , 42.5 卤 1.3 and 265.7 卤 14.7 ( P0.05 ) .
Compared with the blank control group , the invasion ability of the inhibition group was decreased ( 0.2 卤 0.05 ) , and the invasion ability of the overexpression group was enhanced ( 1 . 80 卤 0.21 ) ( P0.05 ) .

11 . Compared with the blank control group ( 72.6 卤 9.6 ) , the number of branch points increased ( 100.4 卤 20.2 ) in the over - expression group , and the number of inhibition groups decreased ( 29.6 卤 4.8 ) ( P0.05 ) .

12 . PDC results showed that the re - casting of uterine spiral arteries was in a much earlier stage compared with the shNC group .

Conclusion

EFNB2 may be involved in the process of uterine spiral artery re - casting by mediating the biological function of the early evocells of pregnancy . The expression of silent EFNB2 gene can promote the apoptosis of the cells , inhibit the cell activity , migration , invasion and angiogenesis .
The up - regulation of EFNB2 expression had no significant effect on cell apoptosis and activity , but could enhance the other biological functions .

Effect of the second partial Notch pathway on the function of EFNB2

Purpose

By targeting gene silencing and exogenous ligand stimulation methods , the expression level and the biological function of EFNB2 in human chorionic trophoblasts of human chorionic trophoblasts ( TR8 / SVneo cells ) were measured at gene level and protein level respectively .

method

1 . CCK - 8 method was used to detect the effect of sD114 on the activity of TR8 / SVneo cells , and the concentration of sD114 was determined .

2.qRT - PCR and Western blotting were used to investigate the inhibitory effect of transfection on Notch1 gene expression in the cells of HSVneo cells .

3 . According to the difference of treatment , the cells were divided into the blank control group , the inhibition group ( transfected Notch1 inhibitory plasmid ) , the dosing group ( 0.5 mg / mLsDll4 ) and the inhibitor group ( 0.5 mg / mLsD114 ) 4 group after transfection of Notch1 inhibition plasmid ;

4 . CCK - 8 determined the activity of each group .

5 . Western blotting was used to detect the expression of anti - apoptotic protein Bcl - 2 and pro - apoptotic protein Bax in transfected cells .

6 . Transwell model can detect the migration and invasion ability of transfected cells .

7.qRT - PCR and Western blotting were used to detect the effect of Notch1signal pathway activation on the expression of EFNB2 gene in each group .

8 . Transwell model was used to detect the migration and invasion ability of EFNB2 in the inhibition of EFNB2 .

Results

1 . When the final concentration of sD114 was 0.125 , 0.25 , 0.5 and 1.0 渭g / mL compared with blank control group , the activity of each group was ( 0.96 卤 0.03 , ) , ( 1.15 卤 0.09 ) , ( 1.16 卤 0.10 ) and ( 1.08 卤 0.08 ) ( P0.05 ) .

2 . The relative expression level of Notch1mRNA was expressed in transfected TR8 / SVneo cells compared with blank control group .
The inhibition group was 0.60 卤 0.14 ( P0.05 ) , and the negative control group was 1.01 卤 0.04 ( P0.05 ) .
The relative expression level of Notch1 protein in the inhibition group was 0.25 卤 0.04 ( P0.05 ) .

3 . Compared with the blank control group , the activity of the inhibition group was 0.65 卤 0.13 ( P0.05 ) , the activity of the treated group was 1.07 卤 0.05 ( P0.05 ) , the cell activity of the treated group was 0.56 卤 0.14 ( P0.05 ) , there was no significant difference with the inhibition group ( P0.05 ) ;

4 . Compared with blank control group , the expression of Bcl - 2 protein decreased ( 0.47 卤 0.02 , P0.05 ) , Bax protein expression increased ( 2.144 卤 0.05 , P0.05 ) .
The relative expression of Bcl - 2 and Bax protein in the dosing group was 1.01 卤 0.09 and 0.93 卤 0.04 respectively ( P0.05 ) .

5 . Compared with the blank control group , the migration index of the inhibition group , the dosing group and the post - inhibition group was 0.30 卤 0.03 , 2.49 卤 0.25 and 2.84 卤 0.14 ( P0.05 ) .

6 . Compared with the blank control group , the invasion index of the inhibition group , the dosing group and the inhibition group was 0.15 卤 0.02 , 2.84 卤 0.12 and 2.46 卤 0.18 ( P0.05 ) .

7 . Compared with the blank control group , the relative expression of EFNB2 mRNA was 0.24 卤 0.08 and 3.41 卤 0.17 ( P0.05 ) .

8 . Compared with the control group , the relative expression of EFNB2 gene was 0.43 卤 0.17 and 1.79 卤 0.25 respectively ( P0.05 ) .

9 . EFNB2 inhibited 7.52 卤 3.12 and 7.48 卤 3.67 of EFNB2 ( P0.05 ) .

Conclusion

Notchl signal pathway can regulate EFNB2 expression , affect the biological function , participate in the process of uterine spiral artery re - casting , regulate the development of placental vascular network .

Study on the synergistic effect of the third partial Notch signaling pathway with dNK

Purpose

sDll4 activated the Notch signaling pathway in ddNK cells in vitro to detect the effects of dNK cell activity , apoptosis , IFN - y secretion , EFNB2 gene expression and its effects on the migration ability of TR8 / SVneo cells .

method

1 . Flow cytometry detects the purity of dNK cells cultured in vitro ;

2 . CCK - 8 method was used to detect cell activity .

3 . Caspase - 3 spectrophotometry was used to detect the apoptosis of cells .

4.qRT - PCR and ELISA were used to detect the levels and secretion of IFN - y mRNA in dNK cells .

5 . Transwell co - culture model was used to detect the migration ability of HSVneo cells .

Results

1 . Flow cytometry analysis detected 90 % of the cell purity of the extracted cells CD56 + CD3 " .

2 . Compared with blank control group , the activity of dNK cells in experimental group was 1.01 卤 0.05 ( P0.05 ) .

3 . Compared with blank group , the relative expression of caspase - 3 in experimental group was 0.79 卤 0.14 ( P0.05 ) .

4 . Compared with blank control group , the mRNA level of IFN - y mRNA was 6.22 卤 0.24 ( P0.05 ) .
The secretion of the blank control group and the experimental group were 317.56 卤 145.55pg / mL and 4226 . 28 卤 158.09pg / mL , respectively ( P0.05 ) .

5 . Compared with blank control group , the relative expression of EFNB2mRNA in experimental group was 2.53 卤 0.75 ( P0.05 ) .

6 . Compared with the blank control group , the migration indexes of H8 / SVneo cells were 0.71 卤 0.24 ( P0.05 ) , 0.85 卤 0.22 ( P0.05 ) and 2.28 卤 0.33 ( P0.05 ) .

Conclusion

The D114 / Notch signaling pathway may affect the biological function of dNK cells by regulating EFNB2 , and play a direct and indirect synergistic effect on the biological effects of EVTs in the process of re - casting of uterine spiral artery .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R714

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