大鼠血管平滑肌增殖器官模型的机制探讨
发布时间：2018-08-16 10:23
【摘要】： 研究目的:探讨大鼠动脉在体外培养条件下其血管平滑肌细胞增殖的形成机制,为本室已建立的血管平滑肌增殖器官模型的应用提供理论依据。 实验方法: 实验分组:在无菌条件下取大鼠腹主动脉,剪成1.5cm左右小段,实验分成:20%FBS培养(其中又分为:内皮损伤、损伤+BQ123、未损伤、未损伤+ BQ123四个组)和无血清培养(其中也分为:内皮损伤、损伤+ BQ123、未损伤、未损伤+ BQ123四个组)共8个实验组。每组10个血管条,分两个培养瓶在DMEM培养基中培养10d,每隔24h半量换液一次。其中一瓶于培养第5天时加入5-Brdu(8×10-4mol/l)标记,收集后用4%多聚甲醛固定,作石蜡切片用于HE染色和免疫组织化学染色。另一瓶用于提取总RNA做RT-PCR。收集每组培养上清贮存于-80℃,用于检测ET-1。正常对照组用未培养的血管。 免疫组织化学染色:每组5个血管条各取2张切片做免疫组织化学,具体步骤如下:将切片脱蜡至水后,用含0.3%双氧水的甲醇封闭;2N盐酸37℃1h;正常羊血清封闭20 min,以上各步之间用0.1M PBS冲洗3次。5-BrDU抗体(1:250)4℃过夜;二抗(1:50)37℃30 min;SABC 37℃20 min;DAB显色3 min,以上各步之间用0.1M PBS冲洗3次。脱水,二甲苯透明,封片,显微镜下观察。每张切片都进行阳性细胞计数,每组阳性标记细胞以X±表示,用X±t检验显著性差异。 上清液中ET-1的测定:将上述收集的培养上清液,使用放射免疫试剂盒和放免检测仪,按照说明书操作测定各组样品中的内皮素含量。每一实验重复3次。结果以X±表示,组间用X±检验显著性差异。 RT-PCR检测:将上述实验收集的血管用一步法(Trizol试剂)提取总RNA,在20μl反应体系中42℃50 min,70℃15 min,逆转录mRNA为cDNA。取逆转录产物3μl加入聚合酶链反应(Polymerase Chain Reaction , PCR)体系,在50μl反应体系中进行聚合酶链反应。聚合酶链反应:94℃变性50 s,56℃退火50 s,72℃延伸1 min,共32个循环。产物经图像处理系统进行分析,同一实验重复3次。 结果: 1.血管平滑肌细胞的增殖:(1)HE染色可见,内皮损伤血管经体外培养10天后平滑肌细胞明显增生,肌纤维排列紊乱。(2)抗5-Brdu免疫组织化学染色显示,各组培养血管中膜均有标记的平滑肌增殖细胞,但内皮损伤后用20%血清培养组标记的增殖细胞明显多于无血清培养组,在培养基中加入内皮素受体阻断剂BQ123能明显抑制血管平滑肌的增殖(P〈0.05)。(3)RT-PCR检测发现,血管平滑肌增殖负调控基因Hrg-1在正常血管平滑肌中都有表达,而各组内皮损伤后体外培养10天的血管表达均明显减弱,血清培养组更为显著,加ET-1特异性阻断剂BQ123后各组表达均明显上调。这说明ET-1分泌及血清刺激对内皮损伤后体外培养的血管平滑肌细胞增殖起了重要作用。 2.血管平滑肌表型的转化:RT-PCR检测发现,血管平滑肌收缩表型标志基因SM22α在正常血管平滑肌中都有表达,但在内皮损伤后体外培养10天的血管表达明显减弱,其中血清培养组血管基本不表达,加内皮素受体阻断剂BQ123后,各组SM22α的表达均有所上调,这表明内皮素分泌和血清培养促进了血管平滑肌的表型由收缩型向分泌型的转化。 3.培养上清中ET-1的变化:内皮损伤后血管ET-1分泌增加,其中血清培养组增加更为显著(P0.01),加入ET-1受体阻断剂BQ123后,各组ET-1分泌普遍减少,这一结果说明内皮损伤和血清培养条件能促进血管ET-1分泌,而ET-1受体阻断剂BQ123能减弱上述作用。 结论:大鼠主动脉经体外培养能诱导平滑肌细胞异常增生并表型从收缩型向合成型转化,ET-1和血清作用是该模型平滑肌增殖的主要因素。本模型为研究血管平滑肌增殖性疾病的机理和防治提供了一个较好的实验平台。
[Abstract]:Objective: To investigate the mechanism of vascular smooth muscle cell proliferation in rat arteries cultured in vitro, and to provide a theoretical basis for the establishment of vascular smooth muscle proliferation organ model in our laboratory.
Experimental methods:
The abdominal aorta of rats was cut into about 1.5 cm segments under aseptic condition. The experiment was divided into 8 experimental groups: 20% FBS culture (which was divided into four groups: endothelial injury, injury + BQ123, non-injury, non-injury + BQ123) and serum-free culture (which was also divided into four groups: endothelial injury, injury + BQ123, non-injury, non-injury + BQ123). One bottle was labeled with 5-Brdu (8 x 10-4 mol/l) on the 5th day of culture, and then fixed with 4% paraformaldehyde for HE staining and immunohistochemical staining. The other bottle was used to extract total RNA for RT-PCR. The supernatant was stored at -80 C for detecting ET-1. in normal control group with uncultured blood vessels.
Immunohistochemical staining: Each group of 5 blood vessel strips were taken 2 slices for immunohistochemistry, the specific steps are as follows: dewaxing to water, with 0.3% hydrogen peroxide methanol blocking; 2N hydrochloric acid 37 1h; normal sheep serum blocking 20 minutes, above each step with 0.1M PBS washing 3.5-BrDU antibody (1:250) 4 second antibody (1:50) 37 30 min; SABC 37 20 min; DAB color 3 min, between the above steps with 0.1M PBS washing three times. Dehydration, xylene transparent, sealed slices, microscopic observation. Each slice of positive cells were counted, each group of positive labeled cells expressed in X t, with X t test significant difference.
Determination of ET-1 in supernatant: The culture supernatant collected above was determined by radioimmunoassay kit and radioimmunoassay instrument according to the instructions. Each experiment was repeated three times. The results showed that there was significant difference between the groups by X + test.
RT-PCR Detection: Total RNA was extracted by one-step method (Trizol reagent) from the blood vessels collected from the above experiments, and the reverse transcription mRNA was expressed as cDNA at 42 50 min, 70 15 min in 20_ ml reaction system. The product was analyzed by image processing system and repeated three times in the same experiment.
Result:
1. Proliferation of vascular smooth muscle cells: (1) HE staining showed that vascular smooth muscle cells were obviously proliferated and myofibrils were disordered after 10 days of culture in vitro. (2) Anti-5-Brdu immunohistochemical staining showed that smooth muscle proliferating cells were marked in the media of cultured vessels in each group, but increased marked by 20% serum culture group after endothelial injury. The proliferation of vascular smooth muscle was significantly inhibited by the addition of endothelin receptor blocker BQ123 (P_0.05). (3) RT-PCR detection showed that Hrg-1, a negative regulator of vascular smooth muscle proliferation, was expressed in normal vascular smooth muscle, while the expression of Hrg-1 was observed in all groups 10 days after endothelial injury. The expression of ET-1 was significantly increased after the addition of ET-1 specific blocker BQ123, which indicated that the secretion of ET-1 and the stimulation of serum played an important role in the proliferation of vascular smooth muscle cells cultured in vitro after endothelial injury.
2. Phenotypic transformation of vascular smooth muscle: RT-PCR detection showed that SM22alpha, a marker of vascular smooth muscle contraction phenotype, was expressed in normal vascular smooth muscle, but the expression of SM22alpha was significantly decreased after 10 days of endothelial injury in vitro culture, and the expression of SM22alpha in serum culture group was almost non-expressed. After BQ123, SM22alpha was expressed in all groups. The expression of endothelin was up-regulated, suggesting that endothelin secretion and serum culture promoted the transformation of vascular smooth muscle phenotype from contractile to secretory.
3. Changes of ET-1 in culture supernatant: The secretion of ET-1 increased after endothelial injury, especially in serum culture group (P 0.01). After the addition of ET-1 receptor blocker BQ123, the secretion of ET-1 decreased in all groups. These results suggest that endothelial injury and serum culture conditions can promote the secretion of ET-1, but ET-1 receptor blocker BQ123 can weaken the above mentioned secretion. Effect.
CONCLUSION: Rat aorta cultured in vitro can induce abnormal proliferation of smooth muscle cells and phenotype transformation from contractile to synthetic. ET-1 and serum are the main factors of smooth muscle proliferation in this model.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332
本文编号：2185695
[Abstract]:Objective: To investigate the mechanism of vascular smooth muscle cell proliferation in rat arteries cultured in vitro, and to provide a theoretical basis for the establishment of vascular smooth muscle proliferation organ model in our laboratory.
Experimental methods:
The abdominal aorta of rats was cut into about 1.5 cm segments under aseptic condition. The experiment was divided into 8 experimental groups: 20% FBS culture (which was divided into four groups: endothelial injury, injury + BQ123, non-injury, non-injury + BQ123) and serum-free culture (which was also divided into four groups: endothelial injury, injury + BQ123, non-injury, non-injury + BQ123). One bottle was labeled with 5-Brdu (8 x 10-4 mol/l) on the 5th day of culture, and then fixed with 4% paraformaldehyde for HE staining and immunohistochemical staining. The other bottle was used to extract total RNA for RT-PCR. The supernatant was stored at -80 C for detecting ET-1. in normal control group with uncultured blood vessels.
Immunohistochemical staining: Each group of 5 blood vessel strips were taken 2 slices for immunohistochemistry, the specific steps are as follows: dewaxing to water, with 0.3% hydrogen peroxide methanol blocking; 2N hydrochloric acid 37 1h; normal sheep serum blocking 20 minutes, above each step with 0.1M PBS washing 3.5-BrDU antibody (1:250) 4 second antibody (1:50) 37 30 min; SABC 37 20 min; DAB color 3 min, between the above steps with 0.1M PBS washing three times. Dehydration, xylene transparent, sealed slices, microscopic observation. Each slice of positive cells were counted, each group of positive labeled cells expressed in X t, with X t test significant difference.
Determination of ET-1 in supernatant: The culture supernatant collected above was determined by radioimmunoassay kit and radioimmunoassay instrument according to the instructions. Each experiment was repeated three times. The results showed that there was significant difference between the groups by X + test.
RT-PCR Detection: Total RNA was extracted by one-step method (Trizol reagent) from the blood vessels collected from the above experiments, and the reverse transcription mRNA was expressed as cDNA at 42 50 min, 70 15 min in 20_ ml reaction system. The product was analyzed by image processing system and repeated three times in the same experiment.
Result:
1. Proliferation of vascular smooth muscle cells: (1) HE staining showed that vascular smooth muscle cells were obviously proliferated and myofibrils were disordered after 10 days of culture in vitro. (2) Anti-5-Brdu immunohistochemical staining showed that smooth muscle proliferating cells were marked in the media of cultured vessels in each group, but increased marked by 20% serum culture group after endothelial injury. The proliferation of vascular smooth muscle was significantly inhibited by the addition of endothelin receptor blocker BQ123 (P_0.05). (3) RT-PCR detection showed that Hrg-1, a negative regulator of vascular smooth muscle proliferation, was expressed in normal vascular smooth muscle, while the expression of Hrg-1 was observed in all groups 10 days after endothelial injury. The expression of ET-1 was significantly increased after the addition of ET-1 specific blocker BQ123, which indicated that the secretion of ET-1 and the stimulation of serum played an important role in the proliferation of vascular smooth muscle cells cultured in vitro after endothelial injury.
2. Phenotypic transformation of vascular smooth muscle: RT-PCR detection showed that SM22alpha, a marker of vascular smooth muscle contraction phenotype, was expressed in normal vascular smooth muscle, but the expression of SM22alpha was significantly decreased after 10 days of endothelial injury in vitro culture, and the expression of SM22alpha in serum culture group was almost non-expressed. After BQ123, SM22alpha was expressed in all groups. The expression of endothelin was up-regulated, suggesting that endothelin secretion and serum culture promoted the transformation of vascular smooth muscle phenotype from contractile to secretory.
3. Changes of ET-1 in culture supernatant: The secretion of ET-1 increased after endothelial injury, especially in serum culture group (P 0.01). After the addition of ET-1 receptor blocker BQ123, the secretion of ET-1 decreased in all groups. These results suggest that endothelial injury and serum culture conditions can promote the secretion of ET-1, but ET-1 receptor blocker BQ123 can weaken the above mentioned secretion. Effect.
CONCLUSION: Rat aorta cultured in vitro can induce abnormal proliferation of smooth muscle cells and phenotype transformation from contractile to synthetic. ET-1 and serum are the main factors of smooth muscle proliferation in this model.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332
【相似文献】
相关重要报纸文章 前2条
1 张骁；束梅英;HCY是冠心病发病的独立危险因素[N];中国医药报;2004年
2 记者 吴仲国;日用万能细胞生成血管[N];科技日报;2000年
相关博士学位论文 前2条
1 张延武;滋肾宁心方对去势雌兔合并动脉粥样硬化模型的实验研究[D];湖南中医药大学;2009年
2 武晓静;MSCs修复血管内膜及对平滑肌部分生物学行为的影响[D];第三军医大学;2004年
相关硕士学位论文 前9条
1 杨芹;降钙素基因相关肽对大鼠血管平滑肌增殖及表型转化的影响[D];苏州大学;2006年
2 张艳林;大鼠血管平滑肌增殖器官模型的机制探讨[D];苏州大学;2007年
3 马颖;咪达普利、缬沙坦抑制动脉粥样硬化的作用及其机制[D];青岛大学;2008年
4 周纪星;咪达普利、缬沙坦抑制动脉粥样硬化的作用及其可能机理[D];青岛大学;2007年
5 王彩英;大鼠血管平滑肌增殖器官和细胞模型的建立[D];苏州大学;2006年
6 伍新中;生物器官模型数字化与纹理映射技术研究[D];西北工业大学;2007年
7 李毅;氟伐他汀抑制气道平滑肌增殖及对哮喘气道重建治疗作用的研究[D];第四军医大学;2001年
8 马绍刚;氯沙坦对糖尿病大鼠大脑中动脉病变影响[D];广西医科大学;2002年
9 许莹;冠心病患者血浆总同型半胱氨酸水平的研究[D];中国医科大学;2004年
，本文编号：2185695
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2185695.html
最近更新
教材专著
