本文選題：低氧　切入點：肺動脈內(nèi)皮細胞　出處：《華中科技大學》2006年碩士論文

【摘要】： 目的 研究低氧條件下肺動脈內(nèi)皮細胞(PAEC)向平滑肌樣細胞的轉(zhuǎn)分化及轉(zhuǎn)化生長因子β1(TGFβ1)在此轉(zhuǎn)分化過程中的作用。 方法 1.胰酶消化法獲取豬肺動脈內(nèi)皮細胞,并用免疫磁珠法(以PECAM-1/CD31陽性表達作為分選標記)對原代肺動脈內(nèi)皮細胞進行純化; 2.形態(tài)學觀察結(jié)合免疫組化檢測Ⅷ因子和CD31對內(nèi)皮細胞進行鑒定; 3.實驗分組: 將純化后的PAEC分成四組:(1)常氧組(N)(含21%O2,5%CO_2和74%N_2);(2)低氧組(H)(含1%O_2,5%CO_2,94%N_2);(3)常氧+TGFβ1抑制組(N+TGFβ1 Neutralizing Abs);(4)低氧+TGFβ1抑制組(H+TGFβ1 Neutralizing Abs)。各組分別培養(yǎng)1、4、7天; 4.平滑肌樣細胞的鑒定:形態(tài)學觀察結(jié)合免疫組化檢測平滑肌標志性蛋白α-SM- actin,判定有無平滑肌樣細胞的轉(zhuǎn)分化; 5.用逆轉(zhuǎn)錄-聚合酶鏈式反應法(RT-PCR)檢測常氧和低氧組細胞TGFβ-1mRNA.; 6.免疫印跡(Western-blot)檢測常氧和低氧組細胞TGFβ-1蛋白的表達。 結(jié)果 1.免疫磁珠純化可剔除內(nèi)皮細胞中的雜細胞,獲得高純度的內(nèi)皮細胞。純化后的內(nèi)皮細胞相差顯微鏡下觀察呈典型的鋪路石樣外觀,免疫組化檢測Ⅷ因子(FⅧ)和CD31呈陽性表達。 2.平滑肌樣細胞的鑒定:形態(tài)學觀察轉(zhuǎn)化細胞呈梭形,免疫組化檢測α-SM- actin呈陽性表達。 3.各組細胞轉(zhuǎn)化率比較:第1天各組均未出現(xiàn)平滑肌樣細胞的轉(zhuǎn)化(圖7、8);第4天和7天N組未出現(xiàn)轉(zhuǎn)化細胞,H組出現(xiàn)轉(zhuǎn)化細胞,且第7天H組轉(zhuǎn)化率明顯高于第4天H組轉(zhuǎn)化率(P 0.05);阻斷TGFβ1的作用后,第4天和第7天H+ TGFβ1 Neu Abs組的轉(zhuǎn)化率分別低于其對應的H組(P 0.05),其余各組均未見轉(zhuǎn)化細胞。 4.低氧可增加肺動脈內(nèi)皮細胞TGFβ1 mRNA和蛋白的表達(P0.05)。 結(jié)論 1.磁珠分選法是一種有效的細胞純化方法,可高效快捷的分離純化出高純度的血管內(nèi)皮細胞。 2.肺動脈內(nèi)皮細胞中存在具有轉(zhuǎn)分化為平滑肌樣細胞潛能的細胞; 3.低氧可明顯促進內(nèi)皮-平滑肌轉(zhuǎn)分化; 4. TGFβ1在此轉(zhuǎn)分化過程中起促進作用。
[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.

【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R363

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