| Osteoporosis is characterized by bone mass decrease and bone microstructure degeneration, then results in bone fragility increase and fractures of the vertebrae ,hip or arm. Osteoporosis is metabolic bone disease,both primary osteoporosis and secondary osteoporosis are manifestation of osteopenia (reduced bone tissue volume).During the course of bone metabolism,osteoclasts play an essential role in bone absorption .The speed and lasting time of old bone absorption depend on the activity of osteoclasts in bone absorption and the unceasing complementation of osteoclasts. It is obvious that if osteoclasts bone absorption activity decreases the old bone absorption dimension will reduce and vice versa.It is thus clear that the quantity and the activity of osteoclast play an important role in the occurrence, progress and prognosis of osteoporosis.Gukang is based on my tutor's years of clinical experance.lt is instructed by the theory of " the kidney govens the bone" . Combined with "spleen and kidney are related" and "blood stasis",it is made up of Buguzhi, huang qi ,Danshen and so on. In a word, gukang has the effect of "supplement the kidney and strengthen the bone" , "replenish qi to invigorate the spleen" , "quicken the blood and free the connecting channels" .Correlative investigation shows that gukang can effetually increase the bone density ,bone mineral cotent and skeletal biomechanics strength of the osteoporotic animal models, and it also may give rise to a rise of serum estrogen,calcitonin level and osteocalcin (BGP) .It has a dual function of inhibitive of bone absorbption and acceleration of bone formation. Clinical study also suggests that gukang can promote the bone density of osteoporosis patientsIn order to furthermore study the mechanism of action of Traditional Chinese Medicine in treatment of osteoporosis and deeply investigate the therapeutic effect at cellbiology level ,at the same time, explore whether gukang realize its treatment goalthrough inhibiting osteoclast bioactivitv or inducing osteoclast apoptosis program,we set about the correlative study. - > Experimental study (- ) Experimental methods1- Isolation and cultivation of osteoclastUsing five clean grade male SD rats (1 month-age) , the limb long bone shaft were removed from experimental rats aseptically after the animals having been sacrificed. Draw out the culture medium with injection syringe and flush the marrow cavities , filter the collected marrow cells suspension with sieve and adjust cell density as 1X10 ml.Place them into 24 well and 6 well colture plate (5%CO2 ,37C) with carry sheet glass and ivory bone piece in it, then culture them in the following medium containing:FCS(fetal calf serum), 1,25-(OH)2D3 .2> Identification of osteoclastFor the identification of osteoclastings, we used the following methods :the observation of viable cell morphology ,HE staining,the staining for tartrate-resistant acid phosphatase (TRAP) and the erosion specula experiment.3 > Add medicine serumAfter the isolation and cultivation and identification of osteoclast , divid them into three groups:vacuity serum group, gubang serum group and gukang serum group.4> Detecting markerAccording to investigation goal, having finished the isolation and cultivation and identification of osteclast ,making of ivory bone piece and preparing of medicine serum and experimental grouping of osteoclast , we have measured the following experiments indices: the observation of viable cell morphology ,assay of the activity of TRAP cunting of absorption lacuna and computer-aiding image analysis,flow cytometer detecting and the observation of laser scanning confocal microscopy (LSCM).(H) Experimental results1 > The observation of viable cell morphologyThrough HE staining, it can be seen that the cytoplasm of osteoclast added with vacuity serum is full ,and there is much vacuoles structure in it , karyomegaly and round ,the nucleoli are clear .The cell plasm of osteoclast added with gubang serum and gukang serum is concentrate...
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