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Effect of Gene Heme Oxygenase-1 on Human Umbilical Vein Endothelial Cell Apoptosis Induced by Constant High Glucose and Intermittent High Glucose

Author: ZhuXuXin
Tutor: ChenYongSong
School: Shantou University
Course: Endocrine and Metabolic Diseases
Keywords: Heme oxygenase-1 Glucose Volatility blood sugar Endothelial cells Apoptosis
CLC: R587.2
Type: Master's thesis
Year: 2008
Downloads: 268
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Abstract


Objective: To the observed heme oxygenase -1 (heme oxygenase-1, HO-1) gene for high-sugar and high sugar volatility induced human umbilical vein endothelial cells (human umbilical vein of endothelial cells, HUVECs), apoptosis and apoptosis related genes bax, bcl-2 expression in experimental and theoretical basis for gene therapy of diabetic vascular lesions. Method: 1. Cultured in vitro not transfected HUVECs and transfected with the HO-1 gene HUVECs the (transfected HUVECs were). By reverse transcription polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR) to detect the transfected HUVECs HO-1 mRNA expression. Successfully cultured in vitro does not transfected HUVECs and transfected HUVECs as the research object to study the impact of HO-1 gene of the high sugar and high glucose-induced volatility HUVECs apoptosis and apoptosis-related gene bax, bcl-2 expression . Were divided into six groups: normal glucose group (untransfected HUVECs containing 5.5mmol / L D-glucose), the stability of the high-sugar group (untransfected HUVECs containing 20.0mmol / L D-glucose), the volatility of the high-sugar group (not transfected HUVECs with every 24-hour rotation to 5.5mmol / L or 20.0 mmol / L D-glucose), normal glucose / HO-1 group (transfected HUVECs containing 5.5mmol / L D-glucose), and the stability of the high sugar / HO-1 group (20.0mmol / L D-glucose) containing HUVECs after transfection with volatility high sugar / HO-1 group (transfected HUVECs with every 24-hour rotation of 5.5mmol / L or 20.0 mmol / L D-glucose ). Cells cultured for 7 days under the conditions of 37 ℃, 5% CO2. The application thiazole blue (MTT) determined the the HUVECs cell viability; application acridine orange / bromine ethidium (AO / EB) staining detected HUVECs apoptosis morphological changes; using RT-PCR detection of apoptosis-related genes bax, bcl- 2mRNA the expression. Results: 1.HO-1 gene mRNA in transfected HUVECs expression by RT-PCR results show: HO-1mRNA untransfected HUVECs very low expression in transfected HUVECs expression was significantly higher (P lt ; 0.05). 2 high-sugar and high sugar volatility HUVECs HUVECs survival in the high-sugar, high sugar and volatility environment for 7 days, HUVECs survival was measured by MTT method. The test results showed: decreased stability of the high-sugar group and the volatility of the high-sugar group HUVECs survival, were normal glucose group, 79% (P lt; 0.05) and 59% (P lt; 0.001), and the volatility of the high-sugar group HUVECs survival rate is less than the stability of the high-sugar group (P lt; 0.05). 3 high sugar and high sugar volatility HUVECs the HUVECs apoptosis of impact in the high-sugar, high sugar and volatility environment cultured for 7 days after the AO / EB staining results show: the stability of the high-sugar group and the volatility of the high-sugar group HUVECs withered marked increase in the number of apoptotic cells, respectively, for normal glucose group, 1.61-fold (P lt; 0.001) and 1.79-fold (P lt; 0.001), and the volatility of the high-sugar group HUVECs number of apoptotic cells is higher than the stability of the high-sugar group (P lt; 0.05). High sugar and high volatility sugar impact on the apoptosis genes bax, bcl-2mRNA expression of HUVECs in high sugar and sugar environment of high volatility cultured for 7 days after the extraction of total cellular RNA, RT-PCR detection of bax, bcl-2 mRNA expressed. The test results showed that: compared with normal glucose group, the stability of the high-sugar group expression of bcl-2mRNA with the volatility high sugar group significantly decreased (P lt; 0.05, P lt; 0.001), and the bcl-2mRNA volatility of the high-sugar group of to express higher sugar group weakened more significantly (P lt; 0.05). baxmRNA expression no significant changes, the difference was not statistically significance (P gt; 0.05). 5.HO-1 gene on the survival of HUVECs transfected HUVECs with high glucose environment in the high sugar and volatility of volatility untransfected HUVECs cultured for 7 days, MTT methods HUVECs survival. The test results showed that: compared with the stability of the high-sugar group and the volatility of high glucose group, the stability of the high sugar / HO-1 with the volatility high sugar / HO-1 group HUVECs survival increased. Stability of high sugar / HO-1 group was 1.37 times the stability of high-sugar group (P lt; 0.05); intermittent high glucose / HO-1 group was 1.47 times of volatility high glucose group (P lt; 0.05). 6.HO-1 gene on the role of the HUVECs apoptosis of high-sugar and high glucose-induced volatility transfected HUVECs with untransfected HUVECs cultured for 7 days in the high-sugar, high sugar and volatility environment, AO / EB staining results showed that: compared with the stability of the high-sugar group and volatility high glucose group, the stability of high sugar / HO-1 group with the volatility high sugar / HO-1 group HUVECs number of apoptotic cells decreased stability of the high sugar / of HO-1 group for the stability of high-sugar group 67% (P lt; 0.001); volatility high sugar / HO-1 group is the volatility of the high-sugar group of 81% (P lt; 0.001). The 7. HO-1 gene expression of apoptotic genes bax, bcl-2mRNA transfected HUVECs with untransfected HUVECs cultured for 7 days in the high-sugar, high sugar and volatility environment, extraction of total cellular RNA, RT-PCR detection of bax express bcl-2mRNA. The results showed that: compared with the stability of high-sugar group with the volatility of the high-sugar group, the stability high sugar / HO-1 group, the the volatility high sugar / HO-1 group enhanced the expression of bcl-2mRNA (P lt; 0.05). baxmRNA expression was no significant change (P gt; 0.05). Conclusion: The high sugar and high sugar volatility could induce apoptosis of vascular endothelial cells, and the volatility of the high sugar than the stability of high glucose-induced vascular endothelial apoptosis is far more serious, and its mechanism may be related to the anti-apoptotic gene The decrease in the expression of bcl-2. 2. HO-1 gene upregulation of vascular endothelial cell apoptosis caused by high-sugar, high sugar and volatility can be reduced, which play a protective effect on cells. 3. HO-1 gene may directly or indirectly raised the anti-apoptotic gene bcl-2 expression and reduce the high sugar and volatility high glucose-induced vascular endothelial cell apoptosis.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetic coma and other complications
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