The Molecular Mechanism Of Maspin In The Occurence Of Preeclampsia Through The Regulation Of Human Umbilical Vein Endothelial Cell Injury

Part OneStudy of Maspin Expression in Human Umbilical Vein Endothelial Cells ObjectiveTo evaluate the expression of maspin in Human Umbilical Vein Endothelial Cells (HUVECs) both under normal and hypoxic conditions.MethodsThrough the construction of cell hypoxia environment by cobalt chloride(cobalt (II) chloride, CoCl2), we used real-time polymerase chain reaction (RT-PCR) and Nest-PCR to detect the mRNA expression of maspin in HUVECs. The protein expression of maspin both under normal and hypoxic conditions was detected by Westen Blotting method.Results1. The results of RT-PCR show that there was slightly amount of maspin mRNA expression in HUVECs both under normoxia and hypoxia.2. We observed see obvious bands at 196bp by agarose gel electrophoresis with the products of Nest PCR, which indicated that there was only slightly amount of maspin mRNA expression in HUVECs both under normoxia and hypoxia.3. We did not observe the protein expression of maspin in HUVECs both under normal and hypoxic conditions through the results of western blotting.ConclusionThese results indicated that there was only slight maspin expression at mRNA level both under normal and hypoxic conditions and we couldn’t detect the maspin protein expression. We believe that there is almost no expression of maspin gene in human umbilical vein endothelial cells.Part TwoEffect of Exogenous Recombinant Human Maspin protein on the Function of Human Umbilical Vein Endothelial CellsObjectiveTo investigate the effect of exogenous human recombinant maspin protein on the function of human umbilical vein endothelial cells.MethodsWe treated HUVECs with different concentration of r-maspin both under normal and hypoxic conditions. We detect the proliferation by CCK8 kit, the apoptosis rate by annexin V-FITC apoptosis detection kit with flow cytometry, the migration ability by the scratch assay, the tube formation of HUVECs with matrigel and the nitric oxide (NO) synthesis by NO kit respectively.Results1. The human umbilical vein endothelial cells maintained the trend of continuous proliferation for 72 hours under normoxia. However, the proliferation of human umbilical vein endothelial cells was inhibited significantly under hypoxia. R-maspin significantly promoted the proliferation of HUVECs under normoxia and the trend of this proliferation was peaked at the intervention of 48h. Only high concentration of r-maspin significantly promoted the proliferation of HUVECs under hypoxia.2. The apoptosis of human umbilical vein endothelial cells under hypoxia had no obvious changes compared with that of the normal control. R-maspin had no obvious effect on apoptosis of human umbilical vein endothelial cells both under normoxia and hypoxia.3. The width of the scratch between HUVECs narrowed gradually with the extension of time under normoxia; Under hypoxia, the width of the scratch had no obvious change along with the time, which indicated that hypoxia significantly inhibited the migration ability of HUVECs. After treatment of r-maspin,the scratch width between HUVECs had no obvious change with the extension of time both under normoxia and hypoxia, which indicated that there was no obvious migration of HUVECs.4. The results of tube formation assay showed that HUVECs gathered into groups and formed narrowed lumen under hypoxia, while the lumen number is almost 3 times that of the normal control. Treated HUVECs with different concentration of r-maspin under normoxia, the diameter of tube formed by HUVECs reduced by 50% compared with that of the normal control and the lumen number increased by 2 times. R-maspin could also obviously inhibit the tube formation of HUVECs under hypoxia. Treated HUVECs with high concentration of r-maspin, HUVECs could hardly form a complete lumen, and we could observe incomplete lumen branches.5. The synthesis of nitric oxide in human umbilical vein endothelial cells under hypoxia was reduced by 50% compared with the normal oxygen group (84.84±12.32 vs. 171.3±6.332, P＜0.01). R-maspin significantly reduced nitric oxide synthesis in HUVECs under normoxia. R-maspin also reduce the synthesis of nitric oxide in HUVECs under hypoxia while there was no statistical differenceConclusionR-maspin protein significantly promoted the proliferation of HUVECs and inhibited nitric oxide synthesis and the tube formation of HUVECs by narrowed lumen; While r-maspin had no significant effect on HUVECs apoptosis and migration ability. These results demonstrated that exogenous recombinant human maspin protein can regulate the biological function of HUVECs.Part ThreeThe Molecular Mechanism of Maspin on the Function of Human Umbilical Vein Endothelial CellsObjectiveTo explore the molecular mechanism of maspin on the function of human umbilical vein endothelial cells.MethodsWe treated HUVECs with different concentration of r-maspin both under normal and hypoxic conditions.we used real-time polymerase chain reaction (RT-PCR) and western blotting to detect the mRNA and protein expression of MMP2 in HUVECs. We also used gelatin zymography method to determine the activity of MMP2 in HUVECs.Results1. No obviously change of MMP2 mRNA expression was observed under hypoxia compared with the normal control. The level of MMP2 protein increased with increasing concentration of r-maspin during normoxia and the change was statistically significant at the highest concentration; However, the level of MMP2 protein was reduced with increasing concentration of r-maspin during hypoxia and the change was statistically significant at the highest concentration.2. The results of gelatin zymography results showed that the activated MMP2 (63-KD) and pro-MMP2 (72-KD) were both observed under normoxia. We could only observe pro-MMP2 (72-KD) under hypoxia. There was only pro-MMP2 (72-KD) with high concentration of r-maspin under normoxia.ConclusionR-maspin did not affect the mRNA expression of MMP2 both under normoxia and hypoxia. High concentration of r-maspin significantly promoted the protein expression of MMP2, while the protein expression of MMP2 was significantly inhibited under hypoxia. Under normal oxygen environment, r-maspin inhibited the activity of MMP2 under normoxia and hypoxia could directly inhibit the activity of MMP2.These results indicated that maspin may affect the function of human umbilical vein endothelial cells by altering the expression and activity of MMP2.