本文選題：EFNB2 + 絨毛外滋養(yǎng)細胞��； 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：第一部分EFNB2調(diào)節(jié)EVT功能在子宮螺旋動脈重鑄中的作用 目的 通過研究EFNB2蛋白在妊娠早期絨毛和母體蛻膜在組織細胞水平的定位表達情況,并檢測EFNB2基因在人原代EVT細胞和多個滋養(yǎng)細胞系細胞中的表達差異,探討其對子宮螺旋動脈重鑄過程的影響；通過基因水平調(diào)節(jié)EFNB2在人絨毛外滋養(yǎng)細胞系HTR-8/SVneo細胞中的表達,觀察對細胞活性、凋亡、遷移、侵襲和血管形成等生物學(xué)功能的影響,探討EFNB2在子宮螺旋動脈重鑄中的作用。 方法 1.免疫組織化學(xué)染色法對EFNB2蛋自在早孕絨毛組織及母體蛻膜組織的表達進行定位； 2. qRT-PCR檢測人原代EVT細胞、人絨毛外滋養(yǎng)細胞系HTR-8/SVneo和絨癌滋養(yǎng)細胞系JEG-3及JAR等細胞中EFNB2mRNA水平； 3.根據(jù)轉(zhuǎn)染質(zhì)粒不同處理,將細胞分為空白對照組(未進行細胞轉(zhuǎn)染)、抑制組(轉(zhuǎn)染EFNB2抑制質(zhì)粒)和過表達組(轉(zhuǎn)染EFNB2過表達質(zhì)粒)3組,qRT-PCR檢測EFNB2mRNA表達時增加兩組陰性對照組(分別轉(zhuǎn)染相應(yīng)質(zhì)�？瞻纵d體)； 4. qRT-PCR和Western blotting檢測轉(zhuǎn)染后細胞EFNB2mRNA和蛋白表達水平,了解轉(zhuǎn)染效率； 5.CCK-8法測定各組細胞活性； 6. Hoechst33258染色檢測轉(zhuǎn)染后細胞凋亡; 7. Western blotting檢測轉(zhuǎn)染后細胞中抑凋亡蛋白Bcl-2及促凋亡蛋白Bax表達； 8. Trans well模型檢測轉(zhuǎn)染后細胞的遷移及侵襲能力； 9. Martigel膠上小管形成實驗?zāi)Ｐ蜋z測轉(zhuǎn)染后細胞血管形成能力； 10.PDC共培養(yǎng)系統(tǒng)驗證下調(diào)EFNB2表達對子宮螺旋動脈重鑄的影響。 結(jié)果 1. EFNB2廣泛表達于早孕絨毛及母體蛻膜組織中,在絨毛滋養(yǎng)細胞層和蛻膜EVT細胞中呈強陽性表達; 2. EFNB2基因在人原代EVT細胞、人絨毛外滋養(yǎng)細胞系HTR-8/SVneo細胞、絨癌滋養(yǎng)細胞系JEG-3細胞和JAR細胞中表達量分別為：0.00067±0.00012、0.00058±0.00007、0.00042±0.00004和0.00039±0.00006,人原代EVT細胞EFNB2基因表達高于絨癌滋養(yǎng)細胞系(P0.05),與HTR-8/SVneo細胞表達水平無顯著差異(P0.05); 3.熒光顯微鏡下可見轉(zhuǎn)染成功的細胞強綠色熒光表達,對照組中無熒光表達,轉(zhuǎn)染效率高。 4.和空白對照組相比,轉(zhuǎn)染后HTR-8/SVneo中EFNB2mRNA表達水平分別為；抑制組0.14±0.09(P0.05)、抑制陰性對照組1.06±0.06(P0.05)、過表達組426.81±44.76(P0.05)、過表達陰性對照1.01±0.26(P0.05)。 5.和空白對照組相比,抑制組和過表達組中EFNB2蛋白相對表達量分別為0.25±0.04和2.19±0.07(P0.05)。 6.和空白對照組相比,抑制組細胞活性降低至0.67±0.03(P0.05),過表達組活性為1.37±0.51(P0.05)。 7.與空白對照組相比,抑制組中凋亡細胞相對數(shù)量分別為129.2±11.9%(P0.05),過表達組104.7±14.9%(P0.05)。 8.同空白對照組相比,抑制組中Bcl-2蛋白表達下降(0.35±0.02,P0.05),Bax蛋白表達增加(1.82±0.16,P0.05);過表達組中Bcl-2與Bax蛋白的相對表達量分別為：1.15±0.03、1.01±0.04(P0.05)。 9.空白對照組、抑制組及過表達組遷移細胞總數(shù)分別為：150.2±6.7、108.5±3.0和328.7.4±13.5個(P0.05)；抑制組遷移能力下降(0.69±0.03),過表達組遷移能力增強(1.95±0.08)(P0.05)。 10.空白對照組、抑制組及過表達組侵襲細胞總數(shù)分別為：140.8±3.4、42.5±1.3和265.7±14.7個(P0.05)；同空白對照組相比,抑制組侵襲能力下降(0.2±0.05),過表達組侵襲能力增強(1.80±0.21)(P0.05)。 11.與空白對照組(72.6土9.6)相比,過表達組管腔分支點數(shù)增多(100.4±20.2),抑制組數(shù)目減少(29.6±4.8)(P0.05)。 12.PDC結(jié)果表明,sh-ephrin-B2組和shNC組相比,子宮螺旋動脈重鑄處于更早期階段。 結(jié)論 EFNB2可能通過介導(dǎo)妊娠早期EVT細胞生物學(xué)功能,參與子宮螺旋動脈重鑄過程。沉默EFNB2基因表達則可以促進HTR-8/SVneo細胞凋亡,抑制細胞活性、遷移、侵襲及血管形成能力；上調(diào)EFNB2表達對細胞凋亡及活性無明顯影響,但可增強上述其他各項生物學(xué)功能。 第二部分Notch通路調(diào)節(jié)EFNB2對EVT功能的影響 目的 通過靶向基因沉默和外源性配體刺激方法,分別在基因水平和蛋白水平調(diào)節(jié)Notch1信號通路的活化情況,檢測人絨毛外滋養(yǎng)細胞系HTR-8/SVneo細胞中EFNB2的表達水平和生物學(xué)功能的變化情況,以揭示Notch1信號通路對EVT細胞的調(diào)節(jié)作用及其可能的機制。 方法 1.CCK-8法檢測不同濃度sD114對HTR-8/SVneo細胞活性的影響,確定sD114加藥濃度； 2. qRT-PCR和Western blotting檢測轉(zhuǎn)染對HTR-8/SVneo細胞中Notch1基因表達的抑制作用； 3.根據(jù)處理的差異,將細胞分為空白對照組、抑制組(轉(zhuǎn)染Notch1抑制質(zhì)粒)、加藥組(0.5mg/mLsDll4)和抑制加藥組(轉(zhuǎn)染Notch1抑制質(zhì)粒后加入0.5mg/mLsD114)4組; 4.CCK-8測定各組細胞活性； 5. Western blotting檢測轉(zhuǎn)染后細胞中抑凋亡蛋白Bcl-2及促凋亡蛋白Bax表達情況； 6. Transwell模型檢測轉(zhuǎn)染后細胞的遷移及侵襲能力； 7. qRT-PCR和Western blotting檢測各組細胞中Notch1信號通路激活情況對EFNB2基因表達的影響； 8. Transwell模型檢測Notch1信號通路激活后對EFNB2抑制的HTR-8/SVneo細胞遷移及侵襲能力影響。 結(jié)果 1.與空白對照組相比,sD114終濃度為0.125、0.25、0.5和1.0μg/mL時,各組細胞活性分別為：(0.96±0.03,),(1.15±0.09),(1.16±0.10)和(1.08±0.08)(P0.05)。 2.同空白對照組相比,轉(zhuǎn)染的HTR-8/SVneo細胞中Notch1mRNA相對表達水平分別為；抑制組0.60±0.14(P0.05)、陰性對照組1.01±0.04(P0.05)；抑制組中Notch1蛋白相對表達水平為0.25±0.04(P0.05)。 3.和空白對照組相比,抑制組細胞活性為0.65±0.13(P0.05),加藥組細胞活性為1.07±0.05(P0.05),抑制后加藥組細胞活性為0.56±0.14(P0.05),與抑制組無顯著差異(P0.05)； 4.同空白對照組相比,抑制組中Bcl-2蛋白表達下降(0.47±0.02,P0.05),Bax蛋白表達增加(2.144±0.05,P0.05)；加藥組中Bcl-2與Bax蛋白的相對表達量分別為1.01±0.09和0.93±0.04(P0.05)。 5.同空白對照組相比,抑制組、加藥組和抑制后加藥組遷移指數(shù)分別為0.30±±0.03、2.49±0.25和2.84±0.14(P0.05)； 6.同空白對照組相比,抑制組、加藥組和抑制后加藥組侵襲指數(shù)分別為0.15±0.02,、2.84±0.12和2.46±0.18(P0.05)； 7.與空白對照組相比,抑制組和加藥組細胞EFNB2基因mRNA相對表達量分別為0.24±0.08和3.41±0.17(P0.05)。 8.與空白對照組相比,抑制組和加藥組細胞EFNB2基因蛋白水平相對表達量分別為0.43±0.17和1.79±0.25(P0.05)。 9. EFNB2抑制加藥組遷移與侵襲能力分別為EFNB2抑制組的7.52±3.12和7.48±3.67(P0.05) 結(jié)論 Notchl信號通路可以通過調(diào)節(jié)EFNB2的表達,影響EVT細胞的生物學(xué)功能,參與子宮螺旋動脈重鑄的過程,調(diào)控胎盤血管網(wǎng)絡(luò)發(fā)育。 第三部分Notch信號通路調(diào)節(jié)dNK對EVT協(xié)同作用的研究 目的 sDll4激活體外培養(yǎng)ddNK細胞中Notch信號通路,檢測dNK細胞活性、凋亡、IFN-y的分泌、EFNB2基因表達及其對HTR-8/SVneo細胞遷移能力的影響,探討Notch/EFNB2途徑是否可以通過調(diào)節(jié)ddNK生物學(xué)功能協(xié)助EVT參與子宮螺旋動脈重鑄過程。 方法 1.流式細胞學(xué)檢測體外分離培養(yǎng)的dNK細胞純度； 2.CCK-8法檢測細胞活性； 3. Caspase-3分光光度法檢測細胞凋亡情況； 4. qRT-PCR和ELISA檢測dNK細胞中IFN-y mRNA水平和分泌情況； 5. Transwell共培養(yǎng)模型檢測HTR-8/SVneo細胞遷移能力。 結(jié)果 1.流式細胞儀分析檢測所提取細胞CD56+CD3"的細胞純度90%。 2.與空白對照組相比,實驗組dNK細胞活性為1.01±0.05(P0.05)。 3.同空白組相比,實驗組細胞caspase-3相對表達量為0.79±0.14(P0.05)。 4.同空白對照組相比,加藥組中IFN-y mRNA水平為6.22±0.24(P0.05)；空白對照組和實驗組分泌量分別為3175.56±141.55pg/mL和4226.28±158.09pg/mL (P0.05). 5.同空白對照組相比,實驗組細胞EFNB2mRNA相對表達量為2.53±0.75(P0.05)。 6.與空白對照組相比,加藥組、共培養(yǎng)組和共培養(yǎng)加藥組HTR-8/SVneo細胞遷移指數(shù)分別為：0.71±0.24(P0.05)、0.85±0.22(P0.05)和2.28±0.33(P0.05)。 結(jié)論 D114/Notch信號通路可能通過調(diào)節(jié)EFNB2影響dNK細胞生物學(xué)功能,對EVT細胞在子宮螺旋動脈重鑄過程中的生物學(xué)效應(yīng)發(fā)揮直接和間接的協(xié)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 鹵 0.0000012 , 0.0000058 鹵 0.00007 , 0.0000042 鹵 0.0000004 and 0.0000039 鹵 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 鹵 0.09 ( P0.05 ) was inhibited , 1.06 鹵 0.06 in the negative control group ( P0.05 ) , 426.81 鹵 44.76 in the overexpression group ( P0.05 ) , and 1.01 鹵 0.26 in the negative control group ( P0.05 ) .

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

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

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

8 . Compared with blank control group , the expression of Bcl - 2 protein decreased ( 0.35 鹵 0.02 , P < 0.05 ) , Bax protein expression increased ( 1 . 82 鹵 0.16 , P < 0 . 05 ) , and the relative expression of Bcl - 2 and Bax protein in the overexpression group was 1.15 鹵 0.03 , 1.01 鹵 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 鹵 6.7 , 108.5 鹵 3.0 and 322.8 . 7.4 鹵 13.5 ( P0.05 ) .
The migration ability of the inhibition group decreased ( 0.69 鹵 0.03 ) , and the migration ability of the overexpression group was enhanced ( 1.95 鹵 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 鹵 3.4 , 42.5 鹵 1.3 and 265.7 鹵 14.7 ( P0.05 ) .
Compared with the blank control group , the invasion ability of the inhibition group was decreased ( 0.2 鹵 0.05 ) , and the invasion ability of the overexpression group was enhanced ( 1 . 80 鹵 0.21 ) ( P0.05 ) .

11 . Compared with the blank control group ( 72.6 鹵 9.6 ) , the number of branch points increased ( 100.4 鹵 20.2 ) in the over - expression group , and the number of inhibition groups decreased ( 29.6 鹵 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 鹵 0.03 , ) , ( 1.15 鹵 0.09 ) , ( 1.16 鹵 0.10 ) and ( 1.08 鹵 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 鹵 0.14 ( P0.05 ) , and the negative control group was 1.01 鹵 0.04 ( P0.05 ) .
The relative expression level of Notch1 protein in the inhibition group was 0.25 鹵 0.04 ( P0.05 ) .

3 . Compared with the blank control group , the activity of the inhibition group was 0.65 鹵 0.13 ( P0.05 ) , the activity of the treated group was 1.07 鹵 0.05 ( P0.05 ) , the cell activity of the treated group was 0.56 鹵 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 鹵 0.02 , P0.05 ) , Bax protein expression increased ( 2.144 鹵 0.05 , P0.05 ) .
The relative expression of Bcl - 2 and Bax protein in the dosing group was 1.01 鹵 0.09 and 0.93 鹵 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 鹵 0.03 , 2.49 鹵 0.25 and 2.84 鹵 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 鹵 0.02 , 2.84 鹵 0.12 and 2.46 鹵 0.18 ( P0.05 ) .


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

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

9 . EFNB2 inhibited 7.52 鹵 3.12 and 7.48 鹵 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 鹵 0.05 ( P0.05 ) .

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

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

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

6 . Compared with the blank control group , the migration indexes of H8 / SVneo cells were 0.71 鹵 0.24 ( P0.05 ) , 0.85 鹵 0.22 ( P0.05 ) and 2.28 鹵 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 .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R714

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