小鼠胚胎发育过程中内皮和平滑肌标志物的表达变化

发布时间：2018-06-25 23:19

本文选题：血管平滑肌 + 胎肝激酶-1　；参考：《大连医科大学》2007年硕士论文

【摘要】： 研究背景和目的血管增殖失调是临床常见的病理过程,如动脉粥样硬化、同种异体移植后血管重塑、支架内再狭窄、经皮球囊血管成形术等,探讨血管平滑肌细胞(Vascular smooth muscle cells,VSMCs)的起源有助于增生性血管病的治疗。以往认为胚胎的VSMCs起源于神经嵴和胚胎中胚层;新近发现VSMCs还可能起源于成体骨髓干细胞或祖细胞、外周血的单核细胞、骨骼肌的成体干细胞和内皮细胞(Endothelium cells,ECs)等。Yamashita J等的体外研究证实,胚胎干细胞来源的胎肝激酶-1(Fetal liver kinase-1,Flk-1),即血管内皮生长因子受体-2阳性细胞在血管内皮生长因子(Vascular endothelial growth factor,VEGF)作用下可分化为血管ECs,在血小板源性生长因子-BB作用下可分化为VSMCs,因此认为Flk-1阳性细胞是血管ECs和VSMCs的共同前体,然而该理论尚缺乏在体证据支持。我们通过对胚胎血管行Flk-1、血小板内皮细胞粘附分子(Platelet endothelial cell adhesion molecule-1,PECAM-1/CD31)、Ⅷ因子相关抗原及平滑肌α-肌动蛋白(Smooth Muscleα-actin,SMα-actin)免疫组织化学染色,探讨上述标志物在血管形成初期的表达时相及空间位置关系,为VSMCs的起源提供新的在体实验依据。 Flk-1主要分布于ECs、造血细胞及其前体细胞,已有报道Flk-1最早出现于小鼠胚胎6.5 d,在ECs及造血细胞的发育中发挥重要作用;在血管新生生理过程或肿瘤等病理过程中也可观察到Flk-1呈高表达,但小鼠植入前胚胎是否表达Flk-1迄今尚未见报道。本实验拟初步探讨Flk-1在小鼠植入前胚胎发育过程中的表达规律及其在胚胎发育中的作用。实验方法 1.获取8.5～18.5 d胚胎:将母鼠分别放入公鼠笼内过夜、交配。次日清晨检查到白色阴道栓者为怀孕鼠,该天记为怀孕第0.5 d。在母鼠怀孕第8.5～18.5 d,处死母鼠,取出胚胎。 2.获取植入前胚胎:母鼠腹腔内注射孕马激素10 U,44～48 h后注射人绒毛膜促性腺激素5 U,随后将其分别放入公鼠笼内过夜、交配。12 h获取单细胞期胚胎,36 h获取2细胞期胚胎,48 h获取4细胞期胚胎,60 h获取8细胞期胚胎,84～96 h获取囊胚。从超排卵母鼠的输卵管冲出单细胞期胚胎到8细胞期胚胎,从子宫中冲出桑椹胚和囊胚。 3. 8.5～18.5 d胚胎标本置4%多聚甲醛(Paraformaldehyde,PFA)中室温固定3 h,按常规脱水后石蜡包埋,制备横断面5μm厚度连续切片,挑选胸腔部分和腹腔部分的主动脉切片染色。 4.植入前胚胎经3 g/L聚乙烯基吡咯烷酮/磷酸盐缓冲液(Polyvinylpyrrolidone/ phosphatebuffered saline,PVP/PBS)中洗涤,2.5% PFA室温固定15 min,置PVP/PBS液4℃保存,待用。 5. 8.5～18.5 d胚胎切片行苏木素-伊红染色,观察背主动脉形态学变化。 6. 8.5～18.5 d胚胎切片行Flk-1、CD31、Ⅷ因子相关抗原及SMα-actin免疫组织化学染色,观察胚胎发育过程中,背主动脉ECs及VSMCs标记物的表达变化。 7.收集植入前各期胚胎20个,采用mRNA Capture Kit试剂盒可捕获总mRNA,行Flk-1的反转录-聚合酶链(Reverse transcription polymerase chain reaction,RT-PCR)反应,观察Flk-1的表达变化规律。 8.植入前各期胚胎采用微滴操作行Flk-1全胚免疫荧光染色,激光共聚焦显微镜下观察Flk-1的表达及定位。 9.将8细胞期胚胎置于含5 mg/L Flk-1中和抗体的胚胎培养液中培养 12 h后,加入绿色荧光标记的羊抗大鼠二抗,观察透明带是否能通过抗体等大分子物质。 10.取8细胞期胚胎随机分3组,实验组加入Flk-1中和抗体5 mg/L至胚胎培养液中,空白对照组为胚胎培养液,抗体对照组加入非中和抗体5 mg/L至胚胎培养液中,培养36 h后计算囊胚形成率。采用X2检验对数据作统计学分析,P㩳0.05有统计学意义。 11. 8细胞期胚胎采用微滴操作行VEGF全胚免疫荧光染色,激光共聚焦显微镜下观察VEGF的表达及定位。实验结果 1.胚胎8.5 d在胸腔部分和腹腔部分均可见左、右背主动脉,9.5 d腹腔部分可见单一背主动脉,而胸腔部分心脏水平仍然维持左、右背主动脉的形态;8.5～9.5 d背主动脉呈现Flk-1阳性、CD31阳性、Ⅷ因子相关抗原阴性、SMα-actin阴性。 2.胚胎10.5 d左、右背主动脉在腹腔部分和胸腔下部为一条背主动脉,但仍为单层细胞围成的管腔,而在心房水平仍然可见左、右背主动脉,且呈现SMα-actin、Flk-1、CD31、Ⅷ因子相关抗原均阳性。 3.胚胎11.5 d背主动脉管壁发育为多层,内层细胞呈Flk-1阳性、SMα-actin阴性,外层细胞呈Flk-1阴性而SMα-actin阳性,血管与周围间充质无明显分界,背主动脉管壁周围可见散在SMα-actin阳性细胞。 4.胚胎11.5 d之后,背主动脉管壁VSMCs数量增多且由不规则型转变为纺锤型,内层ECs继续呈Flk-1阳性、SMα-actin阴性,外层VSMCs Flk-1阴性、SMα-actin阳性,血管与周围间充质分界清楚,背主动脉血管周围无散在SMα-actin阳性细胞。 5.在植入前小鼠胚胎中8细胞期胚胎Flk-1表达最高,在RT-PCR反应28个循环时只有8细胞期胚胎可检测到Flk-1;循环数增加到32个时4细胞期胚胎也检测到Flk-1表达。在单细胞期、2细胞期、桑椹胚期和囊胚期胚胎均未检测到Flk-1表达。 6. 4细胞期胚胎的细胞膜上可见绿色荧光,主要定位于胚胎的外缘和细胞交界处,胞浆和胞核均未见绿色荧光;8细胞期胚胎细胞表面的绿色荧光较4细胞期胚胎增强,蛋白定位同4细胞期胚胎。单细胞期、2细胞期、桑椹胚期、囊胚期表达呈阴性。 7.取8细胞期胚胎在含Flk-1中和抗体5 mg/L的胚胎培养液中培养12 h后加入二抗,发现绿色荧光出现在胚胎的透明带内部,说明透明带可通过抗体等大分子物质。 8.取8细胞期胚胎在含Flk-1中和抗体5 mg/L的胚胎培养液中培养,各组36 h囊胚形成率如下:空白对照组80.95%(17/21),抗体对照组81.81%(18/22),实验组40.00%(10/25)。空白对照组和抗体对照组36 h囊胚形成率无统计学差异。实验组与空白对照组之间有统计学差异。但两实验组胚胎在48 h时仍可发育为囊胚。 9. 8细胞期胚胎VEGF表达呈阳性,蛋白表达定位于胚胎细胞的胞浆内。结论 1.胚胎背主动脉的VSMCs可能最早起源于Flk-1、CD31阳性细胞,后期可能来源于周围间充质细胞的募集分化。 2.在小鼠植入前胚胎发育不同阶段Flk-1表达不尽相同,Flk-1的表达可能与促进植入前胚胎发育有关。
[Abstract]:Background and purpose of research
Vascular dysregulation is a common clinical pathological process, such as atherosclerosis, vascular remodeling, stent restenosis after allograft, percutaneous balloon angioplasty, and the origin of Vascular smooth muscle cells (VSMCs), which is helpful for the treatment of angiopathy. The origin of VSMCs in the embryo was previously believed. Neural crest and embryonic mesoderm; recently, VSMCs may also be derived from adult bone marrow stem cells or progenitor cells, peripheral blood mononuclear cells, skeletal muscle adult stem cells and endothelial cells (Endothelium cells, ECs) and other.Yamashita J in vitro studies confirmed that embryonic stem cells derived from fetal liver kinase -1 (Fetal liver kinase-1, Flk-1), that is, Vascular endothelial growth factor receptor -2 positive cells can differentiate into vascular ECs under the action of vascular endothelial growth factor (Vascular endothelial growth factor, VEGF), and can differentiate into VSMCs under the action of platelet derived growth factor -BB. Therefore, the Flk-1 positive cells are the common precursors of vascular ECs and VSMCs. However, the theory is still lacking in body. Evidence is supported by immunohistochemical staining of Flk-1, platelet endothelial cell adhesion molecule (Platelet endothelial cell adhesion molecule-1, PECAM-1/CD31), factor VIII associated antigen and smooth muscle alpha actin (Smooth Muscle alpha -actin, SM alpha -actin), to explore the expression of the above markers at the early stage of angiogenesis. The relationship between the time and space position provides a new experimental basis for the origin of VSMCs.
Flk-1 is mainly distributed in ECs, hematopoietic cells and progenitor cells. It has been reported that Flk-1 was first appeared in mouse embryo 6.5 D and plays an important role in the development of ECs and hematopoietic cells. The high expression of Flk-1 can be observed during the physiological process of angiogenesis or in the pathological process of tumor, but the expression of Flk-1 in the preimplantation embryo of mice has not yet been found yet. This report intends to preliminarily explore the expression pattern of Flk-1 in mouse preimplantation embryo development and its role in embryonic development.
Experimental method
1. to get 8.5 to 18.5 D embryos: the female rats were put into the cage for the night and mating. The next morning, the white vaginal suppositories were checked as pregnant rats. The day was recorded as the 0.5 D. pregnancy of the pregnant mouse, and the pregnant mouse was killed and the embryo was removed.
2. to obtain preimplantation embryos: intraperitoneal injection of gestational hormone 10 U intraperitoneally, 44~48 h after injection of human chorionic gonadotropin 5 U, then put them into the male rat cage overnight, mating.12 h to obtain single cell stage embryos, 36 h to obtain 2 cell stage embryos, 48 h to obtain 4 cell stage embryos, 60 h to obtain 8 cell stage embryos, 84~96 h acquisition blastocysts. From super Oviductal ovulation of ovulation female rats rushed out of single cell stage embryos to 8 cell stage embryos, and blastocysts and blastocysts were released from the womb.
3. 8.5 ~ 18.5 D embryos were placed in 4% paraformaldehyde (Paraformaldehyde, PFA) for 3 h at room temperature. The thickness of 5 mu section of the transverse section was prepared by paraffin embedded after routine dehydration. The section of the aorta of the thoracic cavity and the abdominal part were selected and stained with the section of the aorta.
4. the preimplantation embryos were washed in 3 g/L polyvinylpyrrolidone / phosphate buffer (Polyvinylpyrrolidone/ phosphatebuffered saline, PVP/PBS), 2.5% PFA was fixed at room temperature 15 min, and PVP/PBS solution was stored at 4 C for use.
5. 8.5 to 18.5 D embryos were stained with hematoxylin eosin to observe the morphological changes of the dorsal aorta.
6. 8.5 ~ 18.5 D embryos sections were stained with Flk-1, CD31, factor VIII related antigen and SM alpha -actin immunohistochemical staining. The expression of ECs and VSMCs markers in the dorsal aorta during the development of the embryo was observed.
7. a total of 20 preimplantation embryos were collected, and the mRNA Capture Kit kit was used to capture the total mRNA, and the reaction of Flk-1 reverse transcription polymerase chain (Reverse transcription polymerase chain reaction, RT-PCR) was used to observe the regularity of the expression of Flk-1.
8. pre implantation embryos were immunized with Flk-1 whole embryo immunofluorescence staining and laser scanning confocal microscopy was used to observe the expression and localization of Flk-1.
9. the 8 cell stage embryos were cultured in the embryo culture medium containing 5 mg/L Flk-1 neutralizing antibody.
After 12 h, the green fluorescent labeled Goat anti rat two antibody was added to observe whether the zona pellucida could pass macromolecules such as antibody.
10. the 8 cell stage embryos were randomly divided into 3 groups, the experimental group added Flk-1 neutralization antibody 5 mg/L to the embryo culture solution, the blank control group was the embryo culture solution, the antibody control group added non neutralizing antibody in 5 mg/L to the embryo culture solution, and then cultured 36 h to calculate the blastocyst formation rate. The data were statistically analyzed by X2 test, and P 0.05 had statistical significance.
11.8 cell stage embryos were immunized with VEGF whole embryo immunofluorescence staining by microdroplet operation. The expression and localization of VEGF were observed under confocal laser scanning microscope.
experimental result
1. the 8.5 D of the embryo had left and right dorsal aorta in the thoracic cavity and the abdominal part, and the single dorsal aorta in the abdominal cavity of 9.5 D, while the level of the thoracic cavity still maintained the left and right dorsal aorta, and the 8.5 to 9.5 D dorsal aorta showed Flk-1 positive, CD31 positive, and negative of the child associated antigen, and SM alpha -actin negative.
2. the 2. embryo was 10.5 d left, the right dorsal aorta was a dorsal aorta in the part of the abdominal cavity and the lower part of the thoracic cavity, but it was still a lumen enclosed by the monolayer cells, while the left and right dorsal aorta remained at the level of the atrium, and the SM alpha -actin, Flk-1, CD31, and factor VIII related antigens were all positive.
3. the wall of the dorsal aorta of 11.5 D of the embryo was multilayered, the inner layer cells were Flk-1 positive, SM alpha -actin was negative, the outer cells were Flk-1 negative and SM alpha -actin positive. There was no clear demarcation between the blood vessels and the surrounding mesenchyme, and the SM a -actin positive cells were scattered around the wall of the dorsal aorta.
4. after 11.5 D of the embryo, the number of VSMCs in the wall of the dorsal aorta increased and changed from irregular type to spindle type. The inner ECs continued to be Flk-1 positive, SM alpha -actin negative, the outer VSMCs Flk-1 negative, the SM alpha -actin positive, the blood vessels and the surrounding mesenchyme clear, and the circumference of the dorsal aorta did not scatter in SM alpha -actin positive cells.
5. the expression of Flk-1 was highest in the 8 cell stage embryo of the preimplantation mouse embryo. Only 8 cell stage embryos could detect Flk-1 in the 28 cycles of RT-PCR reaction; the expression of Flk-1 was also detected in 4 cell stage embryos when the number of cycles increased to 32. The Flk-1 expression was not detected at the single cell stage, 2 cell stage, morula and blastocyst stage embryos.
The green fluorescence was found on the membrane of the 6.4 cell stage embryo, mainly located at the outer edge of the embryo and the junction of the cell, the cytoplasm and the nucleus were not green fluorescence, the green fluorescence of the 8 cell stage embryo cells was stronger than the 4 cell stage embryo, the protein localization was with the 4 cell stage embryo, the single cell stage, the 2 cell stage, the morula stage, and the blastocyst expression were negative. Sex.
7. the 8 cell stage embryos were cultured 12 h in the embryo culture medium containing Flk-1 neutralization antibody 5 mg/L and added two resistance. It was found that the green fluorescence appeared in the zona pellucid zone of the embryo, indicating that the zona pellucida could pass through the antibody and other macromolecules.
8. the 8 cell stage embryos were cultured in the embryo culture medium containing Flk-1 neutralization antibody 5 mg/L. The formation rate of 36 h blastocysts in each group was as follows: the blank control group was 80.95% (17/21), the antibody control group was 81.81% (18/22), and the experimental group was 40% (10/25). There was no statistical difference between the blank control group and the antibody control group in the 36 h blastocyst formation rate. Between the experimental group and the blank control group, there was a significant difference between the experimental group and the blank control group. Statistical difference. But two of embryos in the experimental group could still develop into blastocysts at 48 h.
9.8 the expression of VEGF was positive in cell stage embryos, and the protein expression was localized in the cytoplasm of embryonic cells.
conclusion
1. the VSMCs of embryonic dorsal aorta probably originated from Flk-1 and CD31 positive cells. Later, it may originate from recruitment and differentiation of peripheral mesenchymal cells.
2. the expression of Flk-1 is different in different stages of mouse preimplantation embryo development, and the expression of Flk-1 may be related to the development of preimplantation embryos.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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