TGFβ1在低氧诱导肺血管内皮向平滑肌样细胞转分化中的作用

发布时间：2018-03-23 12:38

本文选题：低氧　切入点：肺动脉内皮细胞　出处：《华中科技大学》2006年硕士论文

【摘要】： 目的研究低氧条件下肺动脉内皮细胞(PAEC)向平滑肌样细胞的转分化及转化生长因子β1(TGFβ1)在此转分化过程中的作用。方法 1.胰酶消化法获取猪肺动脉内皮细胞,并用免疫磁珠法(以PECAM-1/CD31阳性表达作为分选标记)对原代肺动脉内皮细胞进行纯化; 2.形态学观察结合免疫组化检测Ⅷ因子和CD31对内皮细胞进行鉴定; 3.实验分组: 将纯化后的PAEC分成四组:(1)常氧组(N)(含21%O2,5%CO_2和74%N_2);(2)低氧组(H)(含1%O_2,5%CO_2,94%N_2);(3)常氧+TGFβ1抑制组(N+TGFβ1 Neutralizing Abs);(4)低氧+TGFβ1抑制组(H+TGFβ1 Neutralizing Abs)。各组分别培养1、4、7天; 4.平滑肌样细胞的鉴定:形态学观察结合免疫组化检测平滑肌标志性蛋白α-SM- actin,判定有无平滑肌样细胞的转分化; 5.用逆转录-聚合酶链式反应法(RT-PCR)检测常氧和低氧组细胞TGFβ-1mRNA.; 6.免疫印迹(Western-blot)检测常氧和低氧组细胞TGFβ-1蛋白的表达。结果 1.免疫磁珠纯化可剔除内皮细胞中的杂细胞,获得高纯度的内皮细胞。纯化后的内皮细胞相差显微镜下观察呈典型的铺路石样外观,免疫组化检测Ⅷ因子(FⅧ)和CD31呈阳性表达。 2.平滑肌样细胞的鉴定:形态学观察转化细胞呈梭形,免疫组化检测α-SM- actin呈阳性表达。 3.各组细胞转化率比较:第1天各组均未出现平滑肌样细胞的转化(图7、8);第4天和7天N组未出现转化细胞,H组出现转化细胞,且第7天H组转化率明显高于第4天H组转化率(P 0.05);阻断TGFβ1的作用后,第4天和第7天H+ TGFβ1 Neu Abs组的转化率分别低于其对应的H组(P 0.05),其余各组均未见转化细胞。 4.低氧可增加肺动脉内皮细胞TGFβ1 mRNA和蛋白的表达(P0.05)。结论 1.磁珠分选法是一种有效的细胞纯化方法,可高效快捷的分离纯化出高纯度的血管内皮细胞。 2.肺动脉内皮细胞中存在具有转分化为平滑肌样细胞潜能的细胞; 3.低氧可明显促进内皮-平滑肌转分化; 4. TGFβ1在此转分化过程中起促进作用。
[Abstract]:objective
To study the transdifferentiation of pulmonary artery endothelial cells (PAEC) to smooth muscle like cells and the role of transforming growth factor beta 1 (TGF beta 1) in the process of transdifferentiation under hypoxic condition.
Method
1. trypsin digestion method to obtain porcine pulmonary artery endothelial cells, and using immunomagnetic beads (with positive expression of PECAM-1/CD31 as a sorting marker) on primary pulmonary artery endothelial cells were purified;
2. morphological observation and immunohistochemical detection of factor VIII and CD31 were identified on endothelial cells;
3. group of experiments:
The purified PAEC was divided into four groups: (1) normoxic group (N) (containing 21%O2,5%CO_2 and 74%N_2); (2) hypoxia group (H) (containing 1%O_2,5%CO_2,94%N_2); (3) normoxia +TGF +TGF 1 inhibition group (N+TGF beta 1 Neutralizing Abs); (4) hypoxia hypoxia +TGF beta 1 inhibition group (Neutralizing beta 1 1).
4. identification of smooth muscle like cells: morphological observation combined with immunohistochemical detection of smooth muscle marker protein alpha -SM- actin to determine the transdifferentiation of smooth muscle like cells.
5. TGF beta -1mRNA. in normal oxygen and hypoxia group was detected by reverse transcription polymerase chain reaction (RT-PCR).
6. immunoblotting (Western-blot) was used to detect the expression of TGF beta -1 protein in the cells of normoxic and hypoxia groups.
Result
1. immunomagnetic purification complex cells removed in endothelial cells, to obtain high purity after purification of endothelial cells. The endothelial cells were observed under phase contrast microscope showed a typical cobblestone appearance, immunohistochemical detection of factor VIII (F VIII) and the expression of CD31 was positive.
2. identification of smooth muscle like cells: morphological observation of the transformed cells showed spindle shape, immunohistochemical detection of the positive expression of alpha -SM- actin.
3. each cell transformation rate: Transformation of smooth muscle cells were not found in first days in each group (Figure 7,8); fourth and 7 days N group did not appear transformed cell, H group and H transformed cells, seventh days group conversion rate was significantly higher than that of fourth day group H conversion rate (P 0.05); beta blocking TGF 1 for, H group of fourth days and seventh days H+ TGF beta 1 Neu conversion rate of Abs group were lower than those of their corresponding (P 0.05), the other groups were not transformed cells.
4. hypoxia can increase the expression of TGF beta 1 mRNA and protein in pulmonary artery endothelial cells (P0.05).
conclusion
1. magnetic bead separation is an effective method for purification of cells, which can efficiently and efficiently separate and purify high purity vascular endothelial cells.
2. there are cells that have the potential of transdifferentiation to smooth muscle like cells in the endothelial cells of the pulmonary artery.
3. hypoxia can obviously promote the transdifferentiation of endothelium - smooth muscle.
4. TGF beta 1 plays an important role in the process of transdifferentiation.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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