大鼠血管平滑肌增殖器官模型的機制探討
發(fā)布時間：2018-08-16 10:23
【摘要】： 研究目的:探討大鼠動脈在體外培養(yǎng)條件下其血管平滑肌細胞增殖的形成機制,為本室已建立的血管平滑肌增殖器官模型的應(yīng)用提供理論依據(jù)。 實驗方法: 實驗分組:在無菌條件下取大鼠腹主動脈,剪成1.5cm左右小段,實驗分成:20%FBS培養(yǎng)(其中又分為:內(nèi)皮損傷、損傷+BQ123、未損傷、未損傷+ BQ123四個組)和無血清培養(yǎng)(其中也分為:內(nèi)皮損傷、損傷+ BQ123、未損傷、未損傷+ BQ123四個組)共8個實驗組。每組10個血管條,分兩個培養(yǎng)瓶在DMEM培養(yǎng)基中培養(yǎng)10d,每隔24h半量換液一次。其中一瓶于培養(yǎng)第5天時加入5-Brdu(8×10-4mol/l)標記,收集后用4%多聚甲醛固定,作石蠟切片用于HE染色和免疫組織化學(xué)染色。另一瓶用于提取總RNA做RT-PCR。收集每組培養(yǎng)上清貯存于-80℃,用于檢測ET-1。正常對照組用未培養(yǎng)的血管。 免疫組織化學(xué)染色:每組5個血管條各取2張切片做免疫組織化學(xué),具體步驟如下:將切片脫蠟至水后,用含0.3%雙氧水的甲醇封閉;2N鹽酸37℃1h;正常羊血清封閉20 min,以上各步之間用0.1M PBS沖洗3次。5-BrDU抗體(1:250)4℃過夜;二抗(1:50)37℃30 min;SABC 37℃20 min;DAB顯色3 min,以上各步之間用0.1M PBS沖洗3次。脫水,二甲苯透明,封片,顯微鏡下觀察。每張切片都進行陽性細胞計數(shù),每組陽性標記細胞以X±表示,用X±t檢驗顯著性差異。 上清液中ET-1的測定:將上述收集的培養(yǎng)上清液,使用放射免疫試劑盒和放免檢測儀,按照說明書操作測定各組樣品中的內(nèi)皮素含量。每一實驗重復(fù)3次。結(jié)果以X±表示,組間用X±檢驗顯著性差異。 RT-PCR檢測:將上述實驗收集的血管用一步法(Trizol試劑)提取總RNA,在20μl反應(yīng)體系中42℃50 min,70℃15 min,逆轉(zhuǎn)錄mRNA為cDNA。取逆轉(zhuǎn)錄產(chǎn)物3μl加入聚合酶鏈反應(yīng)(Polymerase Chain Reaction , PCR)體系,在50μl反應(yīng)體系中進行聚合酶鏈反應(yīng)。聚合酶鏈反應(yīng):94℃變性50 s,56℃退火50 s,72℃延伸1 min,共32個循環(huán)。產(chǎn)物經(jīng)圖像處理系統(tǒng)進行分析,同一實驗重復(fù)3次。 結(jié)果: 1.血管平滑肌細胞的增殖:(1)HE染色可見,內(nèi)皮損傷血管經(jīng)體外培養(yǎng)10天后平滑肌細胞明顯增生,肌纖維排列紊亂。(2)抗5-Brdu免疫組織化學(xué)染色顯示,各組培養(yǎng)血管中膜均有標記的平滑肌增殖細胞,但內(nèi)皮損傷后用20%血清培養(yǎng)組標記的增殖細胞明顯多于無血清培養(yǎng)組,在培養(yǎng)基中加入內(nèi)皮素受體阻斷劑BQ123能明顯抑制血管平滑肌的增殖(P〈0.05)。(3)RT-PCR檢測發(fā)現(xiàn),血管平滑肌增殖負調(diào)控基因Hrg-1在正常血管平滑肌中都有表達,而各組內(nèi)皮損傷后體外培養(yǎng)10天的血管表達均明顯減弱,血清培養(yǎng)組更為顯著,加ET-1特異性阻斷劑BQ123后各組表達均明顯上調(diào)。這說明ET-1分泌及血清刺激對內(nèi)皮損傷后體外培養(yǎng)的血管平滑肌細胞增殖起了重要作用。 2.血管平滑肌表型的轉(zhuǎn)化:RT-PCR檢測發(fā)現(xiàn),血管平滑肌收縮表型標志基因SM22α在正常血管平滑肌中都有表達,但在內(nèi)皮損傷后體外培養(yǎng)10天的血管表達明顯減弱,其中血清培養(yǎng)組血管基本不表達,加內(nèi)皮素受體阻斷劑BQ123后,各組SM22α的表達均有所上調(diào),這表明內(nèi)皮素分泌和血清培養(yǎng)促進了血管平滑肌的表型由收縮型向分泌型的轉(zhuǎn)化。 3.培養(yǎng)上清中ET-1的變化:內(nèi)皮損傷后血管ET-1分泌增加,其中血清培養(yǎng)組增加更為顯著(P0.01),加入ET-1受體阻斷劑BQ123后,各組ET-1分泌普遍減少,這一結(jié)果說明內(nèi)皮損傷和血清培養(yǎng)條件能促進血管ET-1分泌,而ET-1受體阻斷劑BQ123能減弱上述作用。 結(jié)論:大鼠主動脈經(jīng)體外培養(yǎng)能誘導(dǎo)平滑肌細胞異常增生并表型從收縮型向合成型轉(zhuǎn)化,ET-1和血清作用是該模型平滑肌增殖的主要因素。本模型為研究血管平滑肌增殖性疾病的機理和防治提供了一個較好的實驗平臺。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R-332
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R-332
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