| 1. BackgroundIn 1970's the scientists put forward new cell death conception: apoptosis. It is the initiative cell death process that cell die in itself program that is controlled by genes under some certain physiological or pathological conditions. Some recent investigation reports indicated not only in rumor but also in cardiovascular diseases abnormal apoptosis might play an important role. In the research of cardiovascular diseases it is a new viewpoint to explain some clinical problems by apoptosis phenomena. Atherosclerosis (AS) is the common disease and it is one of the main diseases that harm human health. Bennet etc firstly confirmed that vessel smooth muscle cell in AS plaque died in a pattern of apoptosis in 1995 From then on more and more scholars have been attracted to it. Their studies showed that theratio of apoptosis in AS VSMC was higher than in normal conditions .The plaque stability decreased because of the increase of VSMC apoptosis and shortage of the matrix excreted out of cell. In addition, apoptotic VSMC yielded thromboses and uncovered phosphatidyl serine with negative charge to evoke thrombosis. At last, many acute clinical complications such as AS plaque disruption, aneurysm, acute myocardial infarction, limb necrosis, and sudden death could occur. Thus if one drug can block the process of the VSMC apoptosis, it also can prevent the complication of AS. Chrysanthemum morifolium ramat is the flower of composite polyannual herbage with many medical components. Flavonoid that can remove free radical is one of the main components as an important physiological activity object.2. ObjectiveWe will observe whether Chrysanthemum morifolium ramat can inhibit the apoptosis of calf VSMC and the change of SOD and MDA in culture medium in order to clarify the possible mechanisms that Chrysanthemum morifolium ramat can prevent the occurrence of AS complications.3. Methods1) VSMC culture: Tissue patch implantation is used in the primary cell culture. The newly born calf thoracic aorta is taken out, adventitia and endometrium are peeled clean, then tissue patch is sheared into pieces about 1mm2 and inoculated into a 25ml culture bottle. 20%NCS 1640 culture medium is been added into the bottle, then the bottle is put into 5%C02 incubation bin with constant temperature of 37癈. Culture medium is changed once time every 3 to75 days, at last the dynamic growth of the cells are oberserved by invert microscope. With the growth the culture cells, "cusp and vallecula" constitution is formed, digestion transfer (with the rate of 1: 2) using 0.25% pancreas Protease is performed. Under phase contrast microscope the culture cells show fusiform or long fusiform, overlappi ng into many lamina, rising and falling like "cusp and vallecula" without contact inhibition. The fouth or fifith generation cells are chosen to use.2) Groups division: It is two groups: VSMC normal aptoptosis group and VSMC abnormal aptoptosis group. Dense unilaminar VSMC is inoculate into six-Aperture culture board (106/ Aperture), under 37癈 and 5%CC>2 for 24 hours. 20%NCS culture medium is added to normal group in succession but serum-free medium without blood serum growth factors is added into abonomal group to construct VSMC abonomal aptoptosis model. There are five subgroups among abonomal group . One is the vacuity control group without Chrysanthemum morifolium ramat and the other four subgroups are administrated with Chrysanthemum morifolium ramat extract of different Concentration^ 0, 100, 200, 400ug/ml). There are 4 Apertures for every subgroup. Cells culture last for 48 hours, then all the culture medium is collected and centrifugated for 10 minutes (2000r/min). Upper medium is as ample to be examined and other cells instantly are performed the measurement of the apoptotic cell.3) SOD, MDA measurement: Though spectrophotometre the concentration of SOD and MDA in above-mentioned upper medium are detected. By o-hydroxytris(di)Phenol self-Oxidation inhibition techniq...
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