Experimental Research On The Signal System Ca～(2+)/CaM,of Spleen-deficiency Syndrome

The spleen provides the materiel basis of the acquired constitution, is the source of growth and development, and occupies an important position in the course of life activity. For a long time, spleen-deficiency syndrome has been a hot subject people have been studying. Especially in recent years, the extensive development of the studies for animal models showing syndrome of TCM and the upsurge of molecular biology have laid a theoretical foundation for further probe into the essence of nsufficiency of the spleen?.Therefore, the experimental research on the signal system, Ca2~+/Cam, of spleen-deficiency syndrome is brought into the range of research and has been established as research subject. Purposes The inner link of dysfunction of the spleen in transport and insufficient nutrient essence caused-by spleen-deficieney with the signal system, Ca>ICaM in jejuna snaoolh muscle cells is sought for. Then, from the angle of gastrointestinal dynamics, annotations at the molecular level are made both on the essence of that the spleen has the function to transport and transform nutrients and on the producing mechanism of dysfunction of the spleen due to its deficiency. And the experimental materials are provided in order to clarify the mechanism of action of Sijunzi Tang. Materials and methods 1. Building of Animal Model of Deficiency of Spleen-energy. 30 wistar rats, both male and female, were randomly denied into three groups. The rats in the control group were fed normally and pleased with normal saline every day. While the rats in the model group were fed with half full and perfuse with Xiaochengqi Tang every day. The rats in the third group were perfused with Sijunzi Tang first, and then were given the 2 Xiaochengqi Tang one or two hours later. The period of building animal model is about 15 days. 2. Preparation of Jejunal Smooth Muscle Cells The rats were killed with decapitation. The jejunal tissues lying 1cm distal to the duodero jejunalis junction were obtained. After the serosa and mucous layer were stripped, the tissues were cut into pieces and were kept in a container with I Oml of digestive liquid. They were digested for 10 minutes under the adequate oxygen and at the constant temperature of 3000 .After that, the tissues were recovered by centrifugation (1 800r/min, 5mm) and washed with buffer HEPES-Ringer twice. After agitation for 20minites at the constant temperature of 30C. The jejunal smooth muscle cells were obtained through a nylon Sieae (500~.m). 3. Assay of rCa2I in Jejunal Smooch Musele Cells Fluorescerce Method Take 2ml of cell suspension, centrifugate it for 5mm at the speed of 1 800r/min , add 2m1 of the culture solution , IMDM . After agitation, add 1 Oul of DMSD solution containing Fura-2/AM (the final concentration is 5uM). This solution is loaded for 60mm in water bath cradle at the temperature of 3700. Throw the upper supernatant liquid. Then wash it twice with Hanks solution, centrifugate it for 5mm at the speed of 1 800r/min, throw the upper supematant liquid again, add 2m1 of culture solution. IIvI7DM. Before the assay. incubate the sample for 2 to 3mm at the temperature oL370C. Then put a magnetic rod in a color comparison tube to avoid cell preipitation. At last assay the fluorescein intensity with F-3 000 fluorophotometer. In its assay, excitation wave length is 340nm, emission wave length is SOOnm. the time of...