The Study On The Morphology Changes Of Human Umbilical Vein Endothelial Cells After Lipid Peroxidation By TNF-a

Objective: Human Umbilical Vein Endothelial Cells were separated,incubated and identified to provide an important experimental method for the pathogenesis research of atherosclerosis in vitro.To observe the morphology character and their changes of human umbilical vein endothelial cells (HUVECs) after lipid perocidation induced by TNF-a. Light microscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used as imaging tools.Images obtained were analysised and compared. The advantages and disadvantages of SEM and AFM were discussed. Explore the method of observing cell membrane by AFM.Methods:⑴Take the uterine-incision delivery umbilical cord of healthy puerperants. Digest the endothelia cells from umbilical vein by 0.1% collagenaseⅠand go down to posterity by 0.05%parenzyme-0.02%EDTA. The DMEM medium containing 20 % fetal bovine serum, the DMEM medium containing 20%fetal bovine serum and 100mg/ml ECGS and the special medium of endothlia cells( M200-LSGS) were used to culture HUVECs seperately. Observe the growth and passage conditions of HUVECs in the medium above. The method of immuocytochemistry ofⅧfactor were used to identify the HUVECs.⑵Confluent cultures of HUVECs were exposed to 10ng/ml TNF-a for 24h to induce them lipid perocidation, with the control group without TNF-a.⑶The concentration of malondialde- hyde (MDA) and nitric oxide (NO) in cell medium were measured to judge whether the cells underwent lipid perocidation and its degree.⑷The expression of ICAM-1 on the HUVEC membrane was detected by flow cytometer(FCM).⑸Prepare HUVECs samples of SEM and AFM. Scan HUVECs by both AFM and SEM to obtain their surface topography.Results: 1 The part of culturing, separating and identfing HUVECs:⑴The primary cultured endothelia cells began to adhere to the flask in 4h after inoculation while it was 1h for passage cells. The ECs arrayed like pitching stone under light microscopy.⑵Ⅷfactors in the ECs showed positive reaction by immuocytochemistry which were identified as ECs.⑶The growing conditions of HUVEC in the three different medium: Cells ocubated in the DMEM medium containing 20%FBS can passage to 2～3 generation, about 4 generation in the DMEM medium containing 20 %FBS and 100mg/ml ECGS while 6～7 generation in the special medium of endothlia cells( M200-LSGS).2 The part of influences of TNF-a lipid perocidation on the Morphology changes:⑴TNF-a can injure HUVECs directly. The concentration of MDA in medium of the control group was 3.35±0.24nmol/ml while it was 5.46±0.11nmol/ml in the TNF-a group. The concentration of MDA in the TNF-a group significantly increased compared with it in the control group (P <0.01). The concentration of NO in medium was 101.33±1.06 umol/ml of the control group, 88.02±0.79umol/ml of the TNF-a group. Compared with the NO of the control group, it was significantly decreased in the TNF-a group (P <0.01).⑵TNF-a caused increase of the expression of ICAM-1 detected by FCM: The expression rate of ICAM-1 was 52.72±0.20% in the control group and 83.33±0.12% in the TNF-a group. The expression rate of ICAM-1 significantly increased in the TNF-a group compared to it in the control group(P <0.01)。⑶Images of HUVECs under light microscope(×200): The cells growed adherently and aligned like cobble-stone. They were in fusiform shape and the cell nucleus and nucleoli were clear. Cytoplasm was rich. The TNF-a group: The cells aligned in chaos. The intercellular space increased and the cell body shrinked, while some of the cells floated and fell off.⑷Images of HUVECs under SEM: The cells in the control group looked like balls. Their microvillus was rich and well-distributed. The cell membrane was integrated. The cell in the TNF-a group had erupted membrane. The erupted thing nearly covered the whole cells. However, microvillus decreased and its membrane broke.⑸Images of HUVECs under AFM: It was hard to obtain an integrated image of HUVECs because the degree of elasticity of AFM cantilever was not long enough(<8um).①Images of HUVECs(scanning scope: 50-70um): The cells were in fusiform shape and had integrated membrane. The length of the cell in macroaxis was more than 70um while it was about 25um in minoraxis. The cell nucleus was oval and it was about 22.69um in macroaxis while 16.56um in minoraxis. The cell nucleus was raised with a height of 0.65um. The cell membrane was not clear. However we cloud not obtain the integrated image of the cell in the TNF-a group, because it fluctuated so severely that it was beyond the degree of elasticity of AFM cantilever.②Comparisons of 1um images on the membrane between the control group and the TNF-a group: The membrane was smooth and fluctuated gently in the control group.Its Roughness was 12.32±1.00nm while Peak count was 15.11±0.85. In the TNF-a group, a lot of prominences were found on the membrane. They were different in shape and size. Its Roughness was 20.92±0.88nm while Peak count was 25.97±0.96. Compared with cells in the control group, both of them increased significantly(P <0.01).Conclusion: 1 Enzyme digestion can seperat HUVECs successfully. The special medium of endothlia cells can cause ECs proliferate. We provide an important experimental method for the pathogenesis research of atherosclerosis in vitro.2 TNF-a can be used as an inductor to make HUVECs lipid peroxidation and ultramicrostructure changed.3 AFM was considered as an upstanding tool of observing the cell membrane microstructure.4 SEM and AFM had their own advantages on observing the cell membrane morphous. They should be combined to utilize to fill up each other.